SSC Meeting in Conjunction with ISTH 2015 Toronto Congress–

Meeting Minutes

Standing Committees

Bleeding Assessment Tool Committee ...................................................................2

Coagulation Standards Committee .........................................................................3

Subcommittees

Animal, Cellular and Molecular Models ..................................................................6

Biorheology ............................................................................................................7

Control of Anticoagulation ......................................................................................13

Disseminated Intravascular Coagulation ................................................................15

Exogenous Hemostatic Factors .............................................................................17

Factor VIII, Factor IX and Rare Coagulation Disorders ...........................................22

Factor XI and the Contact System..........................................................................27

Factor XIII and Fibrinogen ......................................................................................29

Fibrinolysis .............................................................................................................35

Genomics in Thrombosis and Hemostasis .............................................................43

Hemostasis and Malignancy ..................................................................................47

Lupus Anticoagulant/Phospholipid Dependent Antibodies ......................................48

Pediatric and Neonatal Hemostasis and Thrombosis .............................................51

Plasma Coagulation Inhibitors ................................................................................53

Platelet Immunology ..............................................................................................56

Platelet Physiology .................................................................................................58

Predictive and Diagnostic Variables in Thrombotic Disease ...................................61

Vascular Biology ....................................................................................................64

Von Willebrand Factor ............................................................................................68

Women’s Health Issues in Thrombosis and Hemostasis ........................................73

Working Groups

Working Group on Perioperative Thrombosis and Hemostasis ..............................77

ISTH-BAT Standing Committee – Business Meeting

21 June 2015 10:30 - 11:30

Francesco Rodeghiero and Barry Coller introduce the first part of the session that was focused

on the latest updates of the studies currently endorsed by the ISTH-BAT Standing Committee.

Frank Leebeek presented data about 113 children enrolled in the WiN Study: the ISTH score

was used to quantify bleeding severity in children. The score was higher in type 3 VWD than in

type 2 or 1 (17 vs. 10.5 vs. 6.5) and higher in children with severe VWD (VWF levels < 10 IU

dL).

Johanna Gebhart reported the characterization of patients with a mild to moderate bleeding

phenotype. 338 patients with a median age of 43 years were included. The median BS was 5 in

the overall patient population.

Alberto Tosetto reported the progress of the REBEL Study, that is enrolling cases with a

bleeding tendency and age and sex matched controls. 51 cases and controls are enrolled at this

moment, with a projected upper normal limit of BS ≤ 4.

Francesco Rodeghiero reviewed the achievements of the ISTH-BAT repository, particularly

focusing of the wave of interest in the last ten years but also in the light of currently ongoing and

promising developments of genomic studies. The ISTH-BAT mandate will expire on 2016.

Following this session, Willem Ouwehand presented the BRIDGE-BPD study, which is aimed at

evaluating the role of next-generation sequencing of a panel of candidate genes in the diagnosis

of bleeding disorders. The project uses a human phenotype ontology annotation to cluster

bleeding disorders with a similar presentation and may represent an attractive development in

phenotyping bleeding symptoms.

Finally, Barry Coller reviewed the modalities to access the ISTH-BAT repository and presented

data on active studies.

Approximately 50 people attended the session.

Coagulation Standards Standing Committee Minutes

21 June 2015 12:30 - 14:00

Chairman: Anthony R. Hubbard

Meeting Chairman: Craig Thelwell (on behalf of Anthony Hubbard)

Review of Lot #4 (C. Thelwell)

Lot #4 of the SSC/ISTH Secondary Coagulation Standard commenced dispatch in April 2012 at a cost of £4.00 (GBP) per vial. Between June 2014 and end May 2015 a total of 55 orders were fulfilled with 13,851 vials dispatched. Manufacturers in Europe received a total of 10,350 vials and External Quality Assurance schemes (UK NEQAS and College of American Pathologists, USA) received a total of 1,600 vials. Information on the availability of Lot #4 was distributed to 150 manufacturers in May 2014 and since then there have been 9 new customers. Five of these had received the mailshot (4 USA and 1 UK) and ordered 1,050 vials. Four were not included in the mailshot (1 Japan, 1 China, 1 France, 1 Germany) and ordered 650 vials. It was proposed to repeat the mailshot annually. No stability testing of Lot #4 was performed in the past year to retain samples for future time-points. The next time-point for the accelerated degradation study will be in January/February 2016. At the current rate of usage stocks of Lot #4 will be exhausted within 4 years. It was recommended to initiate replacement in 2016 to ensure continuity of supply by starting the tender process, inviting bids from manufacturers for the production of Lot #5.

Experience of EQA schemes with Lot #4

UK NEQAS (S. Kitchen)

Eight vials of SSC Lot #4 were used for "trouble-shooting” purposes between July 2014 and June 2015 relating to assay issues for FVIII, FIX, AT and VWF. SSC Lot #4 has also been included as an anonymous sample in one survey (UK NEQAS) which covered testing for FII, FV and FXI. For FII testing one manufacturer’s reference plasma (coded as reference B) returned a result of 105 IU/dl whereas the labelled potency for Lot#4 is 91 IU/dl, representing a difference of 15.4 % relative to the assigned potency. The result from two other reference plasmas used differed only by 5.5 % and 7.7%. Of the centers using reference B almost all used the same FII deficient plasma so the possibility that this contributed to the difference cannot be excluded, though different thromboplastin reagents were used so this is unlikely to be a contributing factor. For FV testing the median value returned for all commercial reference plasmas was 92.6 IU/dl, which is in good agreement with the assigned value of 89 IU/dl. All commercial plasmas were within 7 % of the assigned value. For FXI testing two commercial plasmas returned results with a 10.1 % and 18.0 % difference to the assigned value of 89 IU/dl (98 and 105 IU/dl for reference plasmas coded B and C). Different APTT reagents were used with reference plasma B, with good agreement between the results and so is unlikely to explain the difference. The contribution of the APTT reagent to the difference observed with plasma C is not clear. The reference plasma manufacturers have been informed by NEQAS and feedback on the outcome of any investigations has been requested.

College of American Pathologists (Dong Chen)

SSC Lot #4 was issued in the 2014 Thrombophilia Survey (CGS2B). Mean estimates for the thrombophilia associated analytes (Antithrombin activity and antigen, Protein C activity and antigen, Protein S activity and antigen) were close to the assigned values. Overall the survey results support the assigned values and stability of Lot #4.

Other business

S Kitchen raised the issue of inappropriate use of % when an IU is available. Values are often reported as a % that may or may not correspond to the IU value and it is often not made clear. This issue affects the reporting of results in peer reviewed publications as well as the labelling of reference materials in assay kits, with some manufacturers providing both IU and % values for a single reference material that conflict with each other. It was proposed that a recommendation is required from SSC on units for commercial reference plasmas and publications that the IU should be used when available.

There were 20 attendees.

Animal, Cellular and Molecular Models

21 June 2015 8:00 – 12:20

Chairman: Susan S. Smyth

Co-Chairs: Cecile V. Denis, Jose A. Diaz, Tom Knudsen, David Motto, Leslie Parise, Denise E. Sabatino

The Animal Models SSC session took place on June 21, 2015 and was a "Summit on

Preclinical Models of Venous Thrombosis”. The goal was to convene thought leaders in the

field to discuss the current state of venous thrombosis (VT) models, to understand novel

insight gained from VT models, and to identify gaps in their application that need to be

resolved to continue to move the field forward.

The first session was devoted to discussion of the most commonly employed preclinical VT

models. Jose Diaz provided an overview of considerations for selection of a particular

model. Then, Steve Grover, Brian Cooley, and Jose Diaz described the venous stenosis,

the electrolytic, and the venous stasis models and discussed strengths and weakness of

each. The second, educational session, featured presentations from Denisa Wagner,

Thomas Wakefield, Nigel Mackman, and Alisa Wolberg that highlighted the role of

inflammation, leukocyte and platelet accumulation, the formation of neutrophil extracellular

traps, tissue factor and its sources, fibrin and red blood cell accrual as essential components

to the pathophysiology of VT. A picture of the cellular and molecular events controlling the

initiation, progression / consolidation, and resolution of VT emerged. Questions and

comments from the audience and the speakers reinforced the differences in models and

their appropriate applications.

The Summit identified several current gaps, including the need to develop

Mechanism(s) to share detailed protocols from different laboratories

A repository of data from different laboratories obtained in VT models

A strategy for providing more hands-on training in VT models.

The SSC committee agreed to work the speakers and other leaders in the field to create a

consensus statement on animal models of VT.

Biorheology

20 June 2015 14:30 – 18:50

Chairman: Keith B. Neeves

Co-Chairs: Judith Cosemans, David N. Ku, Pierre Mangin, Owen J. McCarty, Mitsuhiko Sugimoto, Erik Westein

Saturday, June 20, 2015

14:30-18:50

Joint Biorheology and Platelet Physiology Education Session: Bioreactors for the study

of the biophysical mechanisms that regulate platelet function. Moderators: Keith Neeves

and Paolo Gresele.

1. Jose Lopez, United States

Dr. Lopez reviewed work from his lab on synthetic microvessels for studying VWF secretion

from endothelial cells, VWF self-association and platelet-endothelial cell-VWF interactions.

2. Shaun Jackson, Australia

Dr. Jackson reviewed work form his lab on single platelet studies using biophysical tools

including flow chamber studies with high resolution DIC and calcium dynamics imaging and

biomembrane force probe for measuring receptor-ligand kinetics.

Joint Biorheology and VWF session on acquired von Willebrand syndrome. Moderators:

Jorge Di Paola and Keith Neeves.

1. David Lillicrap, Canada, “Flow mediated interactions of VWF and ADAMTS13: shear

ecstasy,” 15:30-15:45

The interaction and cleavage of VWF by ADAMTS13 requires unfolding of the substrate (VWF).

In vitro flow studies show that VWF and ADAMTS13 interact increasingly as a thrombus grows

and the diameter of the remaining lumen gets smaller - resulting in increased flow. In addition,

tensile forces on VWF are further influenced by VWF ligands including platelet GpIb, GpIIb/IIIa

and P-selectin. Each of these ligands can contribute to the unfolding of VWF and influence

interactions and subsequent cleavage by ADAMTS13.

2. Barbara Ziegler, Germany, “Acquired von Willebrand syndrome in patients with VAD or

ECMO,” 15:45-16:00

Dr. Ziegler emphasized the expansion of Ventricular Assist Devices (VADs) over the last

decades. They are not only a transition form of therapy but are becoming more of a destination

therapy; therefore all complications may become more relevant. Dr. Ziegler discussed the 2

types of pumps in the market (centrifugal and axial). She also discussed anticoagulation and the

unusual rate of bleeding seen in patients likely due to AVWS with VADs. It is also possible that

hemolysis and free hemoglobin also influences the rate of AVWS. It is important to mention

that almost all recipients of LVAD (>90%) have higher ratios of VWF:RCo/VWF:Ag but not all of

them bleed clinically. She discussed ECMO and the high incidence of AVWS in this modality.

She also emphasized the poor correlation between VWF levels and bleeding suggesting that

many other factors may contribute to the bleeding observed.

3. David Schmidtke, USA, “Effects of high shear millisecond exposure on platelets”

The objective of this study was to replicate the VAD-induced shear-rate in a microfluidic device

and investigate the effect of the shear on platelet receptors, platelet adhesion and

aggregation. Platelet adhesion and platelet aggregate formation on collagen patterned surfaces

upstream and downstream of a transient (1-50 msec) exposure to high shear (40,000 - 100,000

s-1) were quantified. We observed that a single exposure to high shear was enough to inhibit

platelet aggregation while platelet adhesion was maintained. The defect in platelet aggregation

was dependent upon both the shear rate and exposure time. Treatment of blood with an

inhibitor to ADAMTS13 prior to the high shear exposure restored normal platelet aggregation

downstream suggesting a role of VWF in the downstream aggregation.

After these three presentations discussion ensued and several members of the SSC proposed

an AWS working group with members of the VWF and Biorheology subcommittees. The main

goal of this working group will be to study and standardize AVWS in an attempt to understand

better the presentation of disease and the mechanisms of disease as well as the factors that

contribute to bleeding. Drs. Barbara Ziegler, Augusto Federici and David Schmidtke will lead the

working group. Several members of the audience expressed interest in participating.

VWF-mediated mechanisms in complex and pathological flows. Moderators: Mitsuhiko

Sugimoto and Pierre Mangin.

1. Erik Westein, Australia, “Biomechanical regulation of VWF and thrombus formation under

complex flow conditions,” 16:50-17:05

Hydrodynamics regulate plaque development, rupture and thrombus formation. The flow in

these processes is complex in that there are areas of significant gradients in shear stresses and

recirculation flows. This work tests the hypothesis that complex blood flow induced by

intraluminal platelet aggregates is a key feature of thrombosis. The aim is characterize the

thrombotic mechanisms that are promoted by complex flows. The methods used include PDMS

microfluidic devices that model a stenotic vessel. It was found that VWF-platelet interactions are

exacerbated at the apex of the stenosis. These interactions are induced by gradients in shear

stress, which appear to be more important than the absolute magnitude of the shear stress.

Questions: What are the numbers of platelets quantified in rolling (Kinney, Dublin)? On the order

of thousands. What is the role of cellular sources of VWF (Lopez, Seattle)? Have cultured EC in

stenotic vessels, but these studies focus on plasma VWF and platelet interactions. How

activated are platelets in the poststonotic region (Ju, Sydney)? Likely intermediately activated,

working on measuring calcium dynamics. Is the poststenotic region a “black hole” due to

recirculation flows (Turitto, Chicago)? There are no recirculation flows due to low Reynolds

number in this system.

2. Keith Neeves, USA, “Update on Biorheology Subcommittee Project: Scaling in

Hemorheology” 17:05-17:20. Note this presentation replaced the scheduled talk by Matthias

Schneider (USA).

The mission and mandate of the Biorheology Subcommittee was presented. Results from our

current project on scaling in hemorheology was presented and discussed. The objective of this

project is to use the concepts from scaling analysis to allow for faithful comparisons between in

vitro models and develop new models based on in vivo blood flow/hemostasis/thrombosis.

Important dimensional and dynamic parameters were introduced and their relevance to flow

chamber studies was presented.

3. David Ku, USA, “Relative roles of VWF and platelets in formation of occlusive arterial

thrombosis,” 17:20-17:35.

The design criteria and results from a high shear microfluidic system was presented to model

occlusive thrombus formation in a stenosed vessel. Design constraints included <10 mL of

whole blood, wall shear rate of 6000 1/s in the stenosis, 5-30 minute duration and a geometry

where platelet-platelet interactions dominate (minimum height of 70 µm and aspect ratio of 5.5).

To avoid excessive shear gradients at the corners of the expansion/contraction a 15 degree

slope was used. Fabrication issues were presented using both soft lithography and

micromachining approaches. In this chamber, pulsatile flow did not affect thrombus formation.

Inter- and intra-donor variability was measured, with significant variation in occlusion times

between donors. The relative contribution of plasma VWF, platelet VWF, and platelets to

occlusive thrombus formation was measured by a series of depletion and rescue studies. In

these experiments the contribution of VWF was greater than platelets for pathologically high

shear occlusive thrombosis, and platelet VWF contributes to forming large occlusive thrombi.

Question: Previous animal studies by Bergmeir and colleagues found that the role of GP1b

exceeded its role as a receptor for VWF, do these studies contradict these studies? No, the

results are consistent with the requirement for GPIb that may be sufficient with 10% of platelets,

under the pathologically high shear conditions.

Novel flow-dependent mechanisms of platelet function, coagulation and

fibrinolysis. Moderators: Erik Westein and Judith Cosemans.

1. Elizabeth Gardiner, Australia, “Shear effects on platelet surface receptor shedding”, 17:45-

18:00.

Previous work from Gardiner and colleagues have examined the factors affecting GPVI function

and shedding including GPVI ligand binding mediated ADAM10 cleavage that yields soluble

GPVI. Pathophysiological levels of shear stress increase GPVI shedding. In these studies, the

platelet membrane levels of GPVI and GPIbα were measured during thrombus formation on

collagen in a custom flow chamber. Under steady flow (1800 1/s) the levels of GPIbα, but not

other primary receptors (GPVI, αIIb, CD9), correlates with thrombus volume. Under pulsatile

flow (mean shear 1800 1/s), platelet aggregates were smaller and GPIbα, GPVI and αIIb levels

correlated with volume indicating that levels of these receptors influenced thrombus properties.

Confocal microscopy analysis showed a loss of GPVI in the core of thrombi and a loss of

GPIbα in the thrombus shell region. A FRET substrate reporting on ADAM10 activity showed

that ADAM10 co-localized to regions of large shear gradients. These shear-dependent shedding

events have important consequences in extracorporeal devices such as LVADs and

quantification of sGPVI prior to LVAD implantation could identify patients at risk of bleeding and

aid clinical decisions relating to extent of anti-platelet therapy.

2. Paola van der Meijden, The Netherlands, “Assessment of platelet-dependent coagulation

and fibrinolysis under flow”, 18:00-18:15.

The aim of these studies was to determine how the type and density of adhesive surfaces

controls the composition and viscoelastic properties of thrombi formed under flow. The

methodology included perfusing citrated recalcified whole blood over collagen and collagen-TF

spots. Platelet and fibrin accumulation were measured by confocal microscopy. Mechanical

properties were measured by nanoindentation. Fibrinolysis was achieved by adding tPA or uPA

to the blood or rinse buffer. Lower collagen density (10 ug/mL) resulted in larger and more

dispersed thrombi than higher collagen density (50 ug/mL). Flow enhanced the accumulation of

fibrin with higher surface coverage under shear (150 1/s) compared to stasis. Thrombi formed

on collagen alone were more rigid (Young’s modulus from nanoidentation) compared to thrombi

formed on collagen/TF, where fibrin appears to decrease rigidity. Fibrin degradation under flow

showed a dose-dependence lysis for both tPA and uPA. Flow accelerates fibrinolysis compared

to static conditions, but the rate of lysis was independent to shear rate (150-1000 1/s).

Fibrinolysis is delayed proximal to the platelet surface. Questions: Why is fibrinolysis

independent of shear rate (Turitto, Chicago)? Perhaps it is reaction limited.

3. Pierre Mangin, France, “Platelet adhesion and aggregation under disturbed blood flow: A

focus on the role of pulsatility,” 18:30-18:50.

Pulsatile flow has been shown to increase, decrease, and have no effect of platelet adhesion

and aggregation in previously reported studies. This study examines steady versus pulsatile

flow in a custom flow chamber that includes a straight channel, which serves as an internal

control, and a severely stenosed (90%) channel. Microparticle velocimetry (mPIV) was used to

show in the absence of blood that the stenosis does not cause recirculation flows. A flow meter

upstream of the flow chamber was used to create flow rate waveforms that mimic those in the

common carotid artery, left coronary artery, and the portal vein. Hirudinated blood perfused over

collagen gave modest reductions in thrombus volume for both carotid and left coronary, but not

portal vein, waveforms. Pulsatility does not affect initial platelet recruitment, but does increase

platelet detachment from an evolving thrombus. Detachment occurs during the acceleration

phase of the waveform, similar observations were found in vivo. Occlusive thrombi formed in the

expansion zone of the stenosis. Under pulsatile flow there was increased thrombus formation in

the post-stenotic region relative to steady flow. ASA and cangrelor reduced thrombus formation

in the post-stenotic region. Questions: What happens in the presence of coagulation

(Cosemans, Maastricht)? Not clear, subject of future studies.

4. Discussion Session, “Challenges and needs for standardization of flow assays,” 18:30-

18:50.

An open discussion focused on the needs for standardization and best practices for flow assays

between co-chairs and audience members. There was a general consensus that a standardized

flow chamber was not a priority and could possibly stifle the creativity and mechanistic insight

that has been a result of the diverse flow chambers developed over the last 10 years. Instead,

there is a greater need to raise awareness about how rheological issues affect platelet function

and coagulation. Some of these issues are covered by the current “Scaling in hemorheology”

project. Other issues that could be addressed in a multi-center study include identifying the

sources of variability and development of internal controls that could be integrated into custom

chambers. A pamphlet or online guideline on rheological issues may be useful (Turitto,

Chicago). It is important that as we develop flow chambers and best practices to keep in mind

that ultimately we need these chambers to model what is happening in vivo, in humans (Brass,

Philadelphia). More scrupulous reporting of chamber dimensions and assays would be an

improvement in Methods sections. Co-chairs will take these comments into consideration for

future Biorheology Committee projects.

Joint Platelet Physiology and Biorheology Education Session: Bioreactors for

megakaryocyte studies and platelet production. Moderators: Paolo Gresele and Owen

McCarty.

1. Jonathon Thon, United States

Dr. Thon reviewed the evolution and state-of-the-art in the field of platelet production

bioreactors. The focus was on outlining the challenges and needs for translating recent

discoveries and devices (including his work on microfluidic reactors) into large-scale platelet

production.

2. Alessandra Balduini, Italy

Dr. Balduini reviewed the evolution and state-of-the-art in the field of megakaryocyte studies in

bioreactors. The focus was on recent advanced in bioengineering and biomaterials (including

her work on silk derived materials), phenotyping platelet produced in these bioreactors and

applications including studying diseases (myeloproliferative disorders), drug efficacy and drug

testing.

Control of Anticoagulation

21 June 2015 8:00 – 12:20

Chairman: Walter Ageno

Co-Chairs: Rebecca Beyth, Benilde Cosmi, Mark Crowther, Ismail Elalamy, Elaine M. Hylek, Pieter W. Kamphuisen, Peter Verhamme, Henry G. Watson

The meeting was introduced by the SSC chair, Walter Ageno, who presented the program and reminded the scopes of the Control of Anticoagulation subcommittee.

The first part of the morning was dedicated to the presentation of ongoing SSC projects.

The first session was chaired by Dr. Walter Ageno and was focused on registries. Dr. Alex Spyropoulos presented the proposal for a project on the perioperative management of patients treated with the direct oral anticoagulants (DOACs). Dr. Spyropoulos stressed the fact that, at present, there is little to no evidence for the periprocedural management of patients on DOACs undergoing elective procedures or surgeries. The objective of this large prospective registry is therefore to capture patient populations, management strategies, and outcomes in patients on DOACs undergoing elective or urgent/emergent surgeries or procedures. The registry is expected to accrue at least 50 centers with at least 2 patients/center per month over a 2 year time frame. Statistical analysis will be descriptive based upon type of DOAC used, patient indications, and type of procedure (major versus minor). Dr. Walter Ageno presented an update on the ongoing START-EVENTS SSC register. This observational prospective cohort study is collecting information on the management of major bleeding and thromboembolic events in patients treated with the DOACs. The registry is based on an electronic database which collects data on the index event, on the presence of risk factors, on the use of pro-hemostatic agents for the bleeding patients, on therapeutic strategies for patients with thrombosis, on the use of laboratory tests, and on the 6-month outcome of patients. An initial sample of 100 patients is planned, enrolment is currently half-way. Finally, Dr. Saskia Middeldorp presented the proposal for a registry on pregnancy outcomes in patients who become pregnant while on DOACs. This international, multicenter, prospective cohort study aims to gather experience on the outcome of pregnancy, birth defects in neonates, and late effects in children of mothers who unintendedly have been pregnant while being exposed to DOACs. Information will be collected on an electronic case report form. The study will start enrolling very soon.

The second session was chaired by Dr. Henry Watson and Pieter Kamphuisen and was focused on current SSC standardization projects. Dr. Scott Kaatz reported on the proposal for the standardization of the definition of clinically relevant non-major bleeding in studies of anticoagulants in atrial fibrillation and venous thromboembolism in non-surgical patients. The authors initially carried out a literature search for clinical trials in patients with atrial fibrillation and venous thromboembolism and extracted the definitions of clinically relevant non-major bleeding from the trial reports. Based on these definitions, they identified a number of criteria to be used, with a resulting recommended definition which was presented and discussed during the meeting, and that will be published soon on the Journal of Thrombosis and Haemostasis as an SSC communication. Dr. Nakisa Khorsand reported on the proposal for the definition of clinical outcomes to assess the effectiveness of major bleeding management. The authors carried out a systematic review of the literature to identify different criteria used in the trials and developed a definition that was agreed within the working group and with the SSC co-chairs. This definition was presented and discussed at the meeting, and will be published as an SSC

communication in the Journal of Thrombosis and Haemostasis. Dr. Geoff Barnes presented the results of a collaborative work on the nomenclature of the novel oral anticoagulants. The authors issued a survey to the leaders of 16 thrombosis and hemostasis organizations in Europe and North America. Based on the survey results, they have recommended that term Direct Oral Anticoagulant (DOAC) be used when describing oral anticoagulants with a direct target. They also recommended that a medications specific target be used when distinguishing between the various DOACs, such as for determining monitoring tools or reversal strategies. The recommendation was discussed at the meeting and is published as an SSC communication in the Journal of Thrombosis and Haemostasis. Finally, Dr. van den Besselaar presented an update on the progress of the project on the replacement of international standards for thromboplastin. There is still need to find additional contributing centers in order to complete the project in due time, and the presenter made a call for action.

The second part of the morning started with the education session, which was dedicated to the memory of Prof. Michel Meyer Samama and was chaired by Dr. Marc Samama and Dr. Jacqueline Conard. Professor Samama greatly contributed to the activities of the SSC Control of Anticoagulation and, most of all, to our knowledge on thrombosis and hemostasis. The first lecture was given by Dr. Jacqueline Conard who reviewed the contribution of Prof. Samama to the development of clinical assessment models to identify patients at increased risk of thrombosis who should benefit from thromboprophylaxis and discussed the state of the art on thromboprophylaxis in medical patients and its application in real world clinical practice. Finally, Dr. Conard reviewed the existing literature on risk assessment models for medically ill patients, their recommended use in international clinical guidelines, and their applicability in clinical practice. The second lecture was given by Dr. Ismail Elalamy on Prof. Samama studies on the laboratory measurement of antithrombotic drugs. Dr. Elalamy discussed the mechanisms of action of the DOACs, their pharmacodynamics and pharmokinetic properties, and the results of in vitro and in vivo assessment of their activity with a particular focus on the role of thrombin generation assays. This session gave us an important sense of the many achievements in basic and clinical research over the last decades in the field of anticoagulant drugs, many of which have been deeply discussed and actively promoted during the meetings of this SSC with the active contribution of Prof. Samama.

The last session was entitled "A look into future initiatives of the SSC Control of Anticoagulation” and was chaired by Dr. Elaine Hylek and Dr. Mark Crowther. The first speaker, Dr. Jan Beyer, presented the results of registries of patients treated with the DOACs in real world clinical practice, with particular focus on adherence, effectiveness, and safety issues, and discussed the strengths and weaknesses of observational studies, as well as their role in improving our knowledge on these drugs. Finally, Dr. Ismail Elalamy reviewed the role of laboratory testing for the management of patients treated with the DOACs and proposed a position paper prepared by this SSC to drive the correct use of all available tests.

Disseminated Intravascular Coagulation

21 June 2015 8:00 – 12:20

Chairman: Jecko Thachil,

Co-Chair: Marcello Di Nisio, Satoshi Gando, Takashi Ito, Bernd Jilma, Shinichiro Kurosawa, Sacha Zeerleder

This year’s SSC meeting started with an overview of DIC in obstetrics by Dr. Offer Erez from

Israel. He highlighted the difficulties in using the ISTH score in pregnancy due to the differences

in the laboratory values. A pregnancy-specific DIC score has been suggested and validated. A

call to work together in this area was suggested and was followed by a gathering of the SSC

committees for DIC and women’s health.

The next topic for discussion was the role of DAMPs or Damage Associated Molecular patterns

in DIC. Dr Patricia Liaw from Canada covered the role of various different DAMPs in sepsis and

its role in DIC. This was followed by the mechanism of generation of DAMPs by Dr. Sacha

Zeerleder from the Netherlands. It is expected that different groups will work more on this novel

marker for DIC and hopefully will identify mechanisms to use them as diagnostic markers and

therapeutic targets.

Dr. Bernd Jilma from Austria then discussed the unusual case of drowning causing

Hyperfibrinolytic afibrinogenemia in overt DIC. This was a novel concept and put forward the

need for more work on DIC from rarer causes. Prof. Toshiaki Iba from Japan related to us his

extensive experience in the use of anticoagulants, thrombomodulin and antithrombin. He

explained the timing and the dose of these agents and whether a different scoring system needs

to be used for their use.

The Educational session of DIC SSC had two fantastic and varied topics – details on basic

science from Dr. Jordan Shavit (USA) who focused on ‘zebra fish modelling in DIC’ and from the

clinical perspective from Prof. Jerrold Levy (USA) who gave his view on the management of DIC

in a critical care unit. Dr Shavit gave the presentation on zebrafish as a tool for high-throughput

genetic analysis. He showed that disruption of the zebrafish antithrombin resulted in

spontaneous venous thrombosis in larvae. Characterization of null fish revealed DIC in larvae,

which could be rescued by both human and zebrafish antithrombin complementary DNAs. Prof

Levy gave a practical overview of the clinical situations which is often encountered in critical

care from DIC point of view. He gave the diagnostic pathways and treatment algorithms which

needed to be followed in these cases to get the best outcome also elaborating on his extensive

experience with different anticoagulants and work with coagulation problems in the critically ill,

complex patients.

Prof. Satoshi Gando, a renowned DIC expert from Japan, reminded us about the

pathophysiological mechanisms of trauma-associated |DIC and stressed on the early

hyperfibrinolytic type of DIC in trauma and the differences in the coagulation activation between

inside and outside the blood vessel. Dr. Carl Erik Dempfle then covered the issues relating to

the use of D-dimer in clinical settings and the possibility of considering fibrin-related marker in

these areas.

The session ended with a call for an international registry from Dr Marcello DiNIsio who

explained the major differences in the DIC diagnosis and management in a survey under the

auspices of ISTH.

Exogenous Hemostatic Factors

20 June 2015 14:30 – 18:50

Chairman: Ivo Francischetti

Co-Chair: Kenneth J. Clemetson, Tur-Fu Huang, Manjunatha R. Kini, Francis S. Markland Jr, Robson D. Monteiro

Opening Comments and Welcome, Ivo Francischetti, USA

The mission and objectives of the SSC on Exogenous Hemostatic Factor were presented. The

educational component of the Session included talks on molecules from snake venoms and

hematophagous salivary glands affecting hemostasis, and theirs use in experimental

thrombosis. Speakers also presented recent discoveries in the areas of structural biology of

exogenous hemostatic components, and their use in drug development or as tools in

Biochemistry and Cell Biology.

Session I – Educational Session.

Manjunatha Kini, National University of Singapore, Singapore, “Factor XIa inhibitors from

snake venom” (14:30-15:00 pm).

Bleeding remains a major limitation of standard anticoagulant drugs that target the extrinsic and

common coagulation pathways. Recently, intrinsic coagulation factors are increasingly being

investigated as alternative targets for developing anticoagulant drugs with lower bleeding risk.

Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway

and (ii) characterize and further improve the properties of the identified anticoagulants. We have

isolated and sequenced a specific factor XIa (FXIa) inhibitor, henceforth named Fasxiator, from

the venom of the banded krait snake, Bungarus fasciatus. It is a Kunitz-type protease inhibitor

that prolonged activated partial thromboplastin time without significant effects on prothrombin

time. Fasxiator was recombinantly expressed (rFasxiator), purified, and characterized to be a

slow-type inhibitor of FXIa that exerts its anticoagulant activities (doubled activated partial

thromboplastin time at ~ 3 μmol L(-1) ) by selectively inhibiting human FXIa in in vitro assays. A

series of mutants were subsequently generated to improve the potency and selectivity of

recombinant rFasxiator. rFasxiatorN17R,L19E showed the best balance between potency (IC50

~ 1 nmol L(-1) ) and selectivity (> 100 times). rFasxiatorN17R,L19E is a competitive slow-type

inhibitor of FXIa (Ki = 0.86 nmol L(-1) ), possesses anticoagulant activity that is ~ 10 times

stronger in human plasma than in murine plasma, and prolonged the occlusion time of mice

carotid artery in FeCl3 -induced thrombosis models. We have isolated an exogenous FXIa

specific inhibitor, engineered it to improve its potency by ~ 1000 times and demonstrated its in

vitro and in vivo efficacy. These proof-of-principle data supported the further development of

Fasxiator as a novel anticoagulant candidate.

Ivo Francischetti, National Institutes of Health, USA, “Hematophagy, neutrophils, and

contact pathway” (15:00-15:30 pm)

Salivary glands from blood-sucking animals (e.g., mosquitoes, bugs, sand flies, fleas, ticks,

leeches, hookworms, bats) are a rich source of bioactive molecules that counteract hemostasis

in redundant and synergistic manners. Recent progress has been made in the identification of

salivary inhibitors affecting different aspects of hemostasis, through distinct mechanisms of

action. The contact pathways has been identified as an important player in thrombus formation,

having a peripheral role in hemostasis. Accordingly, salivary components are known to block

FIXa and FXIIa, thus affecting the intrinsic pathway and prolonging the aPTT, including a) a

FXIa anticoagulant from the Vampire Bat which inhibits arterial thrombosis without producing a

bleeding phenotype, b) a poly-phosphate binding protein from sandfly which inhibits FXI

activation by thrombin, in the presence of cofactor Poly-P, and which also prevent inflammation

in vivo, and c) a elastase inhibitor from a mosquito vector of malaria, which prevents elastase-

induced TFPI cleavage, platelet aggregation, neutrophil chemotaxis, and NETS formation, and

attenuates thrombosis. These proteins are notable examples of molecules from hematophagous

salivary secretions with antihemostatic properties. Exogenous hemostatic factors have been

employed as tools in biochemistry and cell biology and also display potential therapeutic

applications.

Session II – Subcommittee session.

Jim Huntington, University of Cambridge, Cambridge, United Kingdom, “Structure of

Pseutarin-C, a snake venom prothombin activator” (15:30-15:50 pm)

The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa,

efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the

complex assembles and its mechanism of prothrombin processing are of central importance to

human health and disease, because insufficient thrombin generation is the root cause of

hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the

crystal structure of the prothrombinase complex have been thwarted by the dependence of

complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable

prothrombinase complex preassembled in the venomgland of the Australian Eastern Brown

Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of

activated fXa from P textilis venom and the derived model of active pseutarin C. Structural

analysis supports a single substrate binding channel on fVa, to which prothrombin and the

intermediate meizothrombin bind in 2 different orientations, providing insight into the

architecture and mechanism of the prothrombinase complex-the molecular engine of blood

coagulation

Ana Marisa Chudzinski-Tavassi, Butantan Institute, Brazil, “Rational strategy applied to a

tick salivary FXa inhibitor in order to develop a potential antitumor drug candidate”

(15:50 – 16:10 pm)

Amblyomin-X is a recombinant protein, classified as a Kunitz type inhibitor, sharing 35%

homology with the TFPI Kunitz domain, identified through a transcriptome analysis of the

salivary gland from the Amblyomma cajennense tick. This recombinant protein is able to inhibit

FXa (Ki 2.2 uM) through a non-competitive pathway. In vitro assays demonstrated an increasing

time of coagulation of human, rabbit and mouse’s plasma on aPTT test using different

concentrations of Amblyomin X. The same way, aPTT increase was shown in the plasma of

rabbits and mice injected intravenously with Amblyomin X. The effect can be perceived after the

first30 minutes, has its major expression after completed 2 hours of test and ceases when

completed 4h of treatment. Besides the effect on the coagulation process, it has been shown

that Amblyomin X induces cell death by apoptosis pathway, being this action selective to tumor

cells. This way, dyne in and proteasome inhibition are targets for the Amblyomin X and play

central role in the cell death mechanism of action. In vivo assays demonstrated that the

treatment with Amblyomin X promoted regression of tumor growth and reduction of metastasis.

Acute safety pre-clinical assays in mouse and rabbits demonstrated low toxicity under 200 folds

of the effective dose. Similar results were obtained for preclinical repeated doses assays.

Robson Q. Montero, Federal University of Rio de Janeiro, Brazil, “Exogenous inhibitors

as tools for the study of new players in hemostasis and thrombosis” (16:10 - 16:30 pm)

Salivary glands from hematophagous animals constitute a major source of molecules capable of

modulating hemostasis. Neutrophil extracellular traps (NETs) have been described as web-like

structures of DNA and proteins formed through a process called NETosis. NETs have been

recently linked to thrombus formation by a variety of mechanisms which includes inactivation of

natural anticoagulants as well as platelet and contact phase activation. In this context, we have

demonstrated that salivary gland components may interfere with NETs pro-haemostatic function

by 1. Preventing thromboxane A2-dependent platelet-assisted neutrophil activation; 2. Inhibition

of elastase-mediated NETs formation or 3. Enzymatic degradation of formed NETs. Our results

provide new insight in elucidating the mechanisms of saliva to avoid host’s hemostatic

responses and innate immune system.

Kenneth Clemetson, Theodor Kocher Institute, Switzerland, “Exogenous Hemostatic

Factors as tools in platelet research” (16:50 – 17:10 pm)

Snake venoms are complex mixtures of biologically active proteins and peptides. Many affect

hemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting

endothelium. Snake venom components are classified into various families, such as serine

proteases, metalloproteinases, C-type lectin-like proteins, disintegrins and phospholipases.

Snake venom C-type lectin-like proteins have a typical fold resembling that in classic C-type

lectins such as the selectins and mannose-binding proteins. Many snake venom C-type lectin-

like proteins have now been characterized, as heterodimeric structures with alpha and beta

subunits that often form large molecules by multimerization. They activate platelets by binding to

VWF or specific receptors such as GPIb, alpha2beta1 and GPVI. Simple heterodimeric GPIb-

binding molecules mainly inhibit platelet functions, whereas multimeric ones activate platelets. A

series of tetrameric snake venom C-type lectin-like proteins activates platelets by binding to

GPVI while another series affects platelet function via integrin alpha2beta1. Some act by

inducing VWF to bind to GPIb. Many structures of these proteins, often complexed with their

ligands, have been determined. Structure-activity studies show that these proteins are quite

complex despite similar backbone folding. Snake C-type lectin-like proteins often interact with

more than one platelet receptor and have complex mechanisms of action.

Heyu Ni, University of Toronto, Canada, “Anfibatide and other exogenous platelet GPIb

antagonists” (17:10 – 17:30 pm)

Ischaemic stroke is a serious disease with limited therapy options. Glycoprotein (GP)Ib binding to von Willebrand factor (vWF) exposed at vascular injury initiates platelet adhesion and contributes to platelet aggregation. GPIb has been suggested as an effective target for antithrombotic therapy in stroke. Anfibatide is a GPIb antagonist derived from snake venom and we investigated its protective effect on experimental brain ischaemia in mice. Focal cerebral ischaemia was induced by 90 min of transient middle cerebral artery occlusion (MCAO). These mice were then treated with anfibatide (4, 2, 1 μg·kg-1 ), injected i.v., after 90 min of MCAO, followed by 1 h of reperfusion. Tirofiban, a GPIIb/IIIα antagonist, was used as a positive control. Twenty-four hours after MCAO, anfibatide-treated mice showed significantly improved ischaemic lesions in a dose-dependent manner. The mice had smaller infarct volumes, less severe neurological deficits and histopathology of cerebrum tissues compared with the untreated MCAO mice. Moreover, anfibatide decreased the amount of GPIbα, vWF and accumulation of fibrin(ogen) in the vasculature of the ischaemic hemisphere. Tirofiban had similar effects on infarct size and fibrin(ogen) deposition compared with the MCAO group. Importantly, the anfibatide-treated mice showed a lower incidence of intracerebral hemorrhage and shorter tail bleeding time compared with the tirofiban-treated mice. Our data indicate anfibatide is a safe GPIb antagonist that exerts a protective effect on cerebral ischaemia and reperfusion injury. Anfibatide is a promising candidate that could be beneficial for the treatment of ischaemic stroke.

Tur-Fu Huang, National Taiwan University, Taiwan, “The second generation of platelet

GPIIb/IIIa antagonists derived from snake venom disintegrins (17:30 – 17:50 pm)

Snake venoms contain unique components that affect cell-matrix

interactions. Disintegrins represent a class of low molecular weight, Arg-Gly-Asp (RGD)-

containing, cysteine-rich peptides purified from the venom of various snakes among the

Viperidae and Crotalidae. They bind with various degrees of specificity to integrins alpha IIb

beta 3, alpha 5 beta 1 and alpha V beta 3 expressed on cells. Snake venom metalloproteases

(high molecular mass haemorrhagins) also contain disintegrin-like domains, in addition to zinc-

chelating sequences. Membrane-anchored ADAMs (A Disintegrin and Metalloprotease domain),

multidomain molecules consisting of metalloprotease, disintegrin-like, cysteine-rich, and

epidermal growth factor domains, a transmembrane domain and a cytoplasmic tail, are a new

family of proteins. In the light of the large number and wide distribution of ADAMs, they may

participate in cell-cell fusion events, including sperm-egg binding and fusion, myoblast fusion

and other cell-cell and cell-matrix interactions. The structure-function relationship of these

molecules is discussed.

Factor VIII, Factor IX & Rare Coagulation Disorders

20 June 2015 9:00 – 13:30

& 21 June 2015 8:00 – 12:20

Chairman: Guy Young

Co-Chairs: Manuel Carcao, Michael Makris, Danijela Mikovic, Flora Peyvandi, Steven W. Pipe, Elena Santagostino

Project Reports

1. Standardization of post-registration surveillance: F. Peyvandi (IT)

Dr. Peyvandi updated the SSC on her project on post-registration surveillance for efficacy and

safety of novel factor products. She described all of the registries in the various world regions

that are collecting data. The goal of the group is to recommend standards for the data collection

within these registries such that data can be compared across those databases.

2. Prophylaxis in patients without inhibitors: V. Blanchette (CA) and K. Fischer (NL)

Dr. Fischer discussed the results of her project on prophylaxis in patients without inhibitors

describing when and how to start prophylaxis in FVIII and FIX deficiency in children. The

manuscript is nearly complete and will put up on the SSC website for comment in the coming

weeks.

3. Prophylaxis in patients with inhibitors: C. Escuriola (DE)

Dr. Nadia Ewing (in place of Dr. Escuriola) presented the working group’s progress on

recommendations for prophylaxis in inhibitor patients. The group has nearly completed its work

and will develop a manuscript over the summer.

4. Bleeding Score in Hemophilia: E. Mancuso (IT)

Dr. Mancuso presented her working groups progress on this project. Of note, 37 centers have

agreed to participate in a data collection project to validate the bleeding score that was

developed. So, far 99 patients with hemophilia and B have been enrolled.

5. ITI definitions: E. Santagostino (IT)

Dr. Santagostino presented an update of her group’s project. She reviewed the recommended

definitions for successful ITI based on both pharmacokinetic as well as clinical endpoints. The

group is working on developing a manuscript which is planned for completion by next year.

6. Mild hemophilia definitions: M. Makris (GB)

Dr. Makris discussed the final results of his group’s work on defining mild hemophilia.

Specifically, he discussed modifications of the existing SSC definitions of mild hemophilia

including defining some patients with >40% levels of factor VIII as having hemophilia if they

have a DNA change consistent with hemophilia and a family member with a level of <40%. He

also discussed issues with measuring factor levels with the one-stage versus choromogenic

assay that could lead to missing the diagnosis in some patients.

7. Inhibitor reporting standardization in previously treated patients: A. Iorio (CA)

Dr. Iorio provided an interim report of his group’s work on reporting of results from trials and

other studies on the inhibitor rate in PTPs. The group has developed recommendations

regarding how to report inhibitors in PTPs in the literature.

8. Standardization of genetic assays for diagnosis of hemophilia: V. Jenkins (IE)

Dr. Jenkins reported on his group’s work on standardization and quality assurance as it applies

to genetic testing in hemophilia. First, a survey was sent out to various laboratories to determine

the types of tests that are done, the logistics of test performance, the methods of determining

the genotype. The group will use this data to develop recommendations for standardization and

quality assurance for genetic testing in hemophilia.

Education Program

1. From Alexie Romanov to Stephen Christmas to 2015: What is different about factor

IX deficiency: E. Santagostino (IT)

Dr. Santagostino provided a review of the differences between FIX and FVIII deficiency focusing

on whether or not FIX deficiency is a less severe bleeding disorder. The presentation evaluated

the literature on factor VIII and IX deficiency with respect to the frequency of prophylaxis,

hemophilic arthropathy, and bleeding rates, and in general as opposed to FVIII deficiency,

patients with FIX deficiency were less likely to be on prophylaxis, had less hemophilic

arthropathy, and had the first joint bleed at a later age, however it was not clear if this is a real

difference in terms of disease severity or epidemiologic findings that may be due to the notion

that FIX deficiency is less severe.

2. What is the “true” incidence of inhibitors in hemophilia: M. Soucie (US)

Dr. Soucie provided an epidemiological overview of inhibitor incidence including a detailed

discussion on the definitions of incidence and prevalence and elaborating on the limitations of

the statistical methods for detection of inhibitors in a low prevalence disease.

Rare Bleeding Disorders

1. Update on afibrinogenemia and dysfibrinogenemia: P. de Mooerloose and A. Casini

(CH)

Dr. Casini provided an overview of fibrinogen disorders including a detailed discussion on

genotype-phenotype correlations. Dr. Casini discussed congenital afibrinogenemia, congenital

hypofibrinogenemia, and congenital dysfibrinogenemia. All 3 conditions are associated with a

bleeding disorder with congenital afibrinogenemia clearly being the most severe of the three.

2. PRO-RBDD Project: Prospective evaluation of the intensity of bleeding episodes in

patients with fibrinogen and FXIII deficiency: F. Peyvandi (IT)

Dr. Peyvandi updated the SSC on her project of these two rare bleeding disorders including

data from a multisite registry study on bleeding events in these two rare disorders as well as

treatments given by the institutions that participated. Specifically for FXIII deficiency, she

discussed that patients with a mild deficiency exhibit bleeding symptoms as well including

mucus membrane bleeding and menorrhagia. Treatments varied throughout the world, however

most developed nations now have factor concentrates available for treatment of both and many

patients with FXIII deficiency are on prophylaxis.

Laboratory Issues 1

1. Update on Nijmegen group’s high sensitivity inhibitor assay: B. Verbruggen (NL)

Dr. Verbruggen described a novel increased sensitivity inhibitor assay based on the Nijmegen-

modified Bethesda assay. He reviewed a small validation study that his team performed which

did demonstrate that the assay correlates with the half-life, however the number of patients was

small and further validation is warranted.

2. Inhibitor measurement using SMIA: a new approach in inhibitor measurement: S.

Raut (GB)

Dr. Raut described a novel assay (South Mimms Inhibitor Assay) which is a modification of the

Classical Bethesda Assay which is aimed mostly at reducing the inter-laboratory variation of the

Bethesda assay and the Nijmegen-modified Bethesda assay. It may also improve the sensitivity.

His next step is to do a multi-laboratory study to determine the inter-laboratory coefficient of

variation.

3. Establishment of the 5th International Standard for FIX: E. Gray (UK)

Dr. Gray described the establishment of the 5th international plasma standard FIX and the

1stinternational standard for recombinant FIX. There is general agreement to have this new

standard move towards endorsement by WHO. There is less agreement about the need for an

international standard for recombinant FIX and plans for that have been deferred for now.

4. Update on EAHAD Coagulation Factor Variant Genotype Database: K. Gomez (UK)

Dr. Gomez presented the newly developed EAHAD supported database of mutations in FVIII,

FIX, and VWF. He demonstrated the utility and ease of use of the database.

5. IU vs %: Clearing up the confusion and a proposal for a position paper: S. Kitchen

(GB)

Dr. Kitchen described the issues and concerns with the use of the terms units versus IU versus

%-age. He discussed that %-age is not always equal to IU and he felt that a position paper or

recommendation from the FVIII/FIX/RBD subcommittee. Dr. Kitchen will send me a proposal for

a position paper on this topic in the next 2 months for further discussion.

6. WAPPS Project (Web-accessible population pharmacokinetics service): A. Iorio

(CA)

Dr. Iorio presented the development of his web-accessible pharmacokinetics service and

reviewed how clinicians and researchers can access it and utilize it for clinical decisions or for

developing research projects. Once the application is ready to go live, anyone will be able to

access it as a free web-based application. Once this is available, he will let me know and we

can put an announcement out on the SSC webpage.

Clinical Issues

1. Moderate hemophilia update: K. Fischer (NL)

Dr. Fischer provided an update on the clinical aspects of moderate hemophilia based on some

recent observations and large studies mostly from The Netherlands. She demonstrated data

from a large database in the Netherlands that a subset of patients with moderate hemophilia will

develop joint disease if not treated aggressively as they act more like patients with severe

hemophilia, and even those who don’t bleed like severe hemophilia are prone to developing

severe bleeds on occasion.

2. Clinical use of extended half-life factor products: G. Young (US)

Dr. Young presented a series of questions regarding the use of extended half-life factor

products to the audience and solicited audience opinions which led to an open forum discussion

on this topic. Several audience members spoke of their opinions regarding the use of these

drugs in PUPs indicating that they would not treat PUPs with these drugs until there was more

data though one person indicated that she thought PUPs with their poor venous access would

be the ideal patient group to initiate these drugs in. There was a general feeling that for patients

with poor venous access or those who have not been on prophylaxis due to the frequency of the

IV infusions are the best candidates for extended half-life drugs.

Laboratory Issues 2

1. Extended half-life factors and laboratory assays--overview of the problem: S. Pipe

(US)

Dr. Pipe provided an overview of the issues surrounding laboratory analysis of the extended

half-life factors demonstrating that this is a relevant clinical issue when treating patients with

these agents as it relates to measuring levels of the infused factor.

2. Overview of chromogenic assay basics: K. Friedman (US)

Dr. Friedman provided an overview of the mechanism for the factor VIII and factor IX

chromogenic assay. He described in detail the differences between the assays stating that the

chromogenic assay has less variability and is more reliable as it truly focuses on the action of

FVIII or FIX rather than evaluating the entire coagulation cascaded.

3. Implementation of the chromogenic assay into the clinical lab: R. Ko (US)

Dr. Ko described the implementation of the chromogenic assays for factor VIII and factor IX in

his clinical coagulation laboratory. He described the steps that he went through to make this

assay available in his institution. Briefly, the steps included making a request to the clinical

laboratory, identifying vendors for the assays, choosing a vendor, performing laboratory

calibration followed by validation, and then once ready, developing a mechanism for ordering

the laboratory tests.

Factor XI and the Contact System

21 June 2015 8:00 – 12:20

Chairman: Jonas Emsley

Co-Chairs: Jose W. Govers-Riemslag, Christine Mannhalter, Joost Meijers, James Morrissey, Ophira Salomon, Evi X. Stavrou

The factor XI and contact system was attended with 200 attendees.

Bernd Engelmann (Germany): Propagation of blood coagulation by extracellular

nucleosomes/neutrophil extracellular traps was presented. This talk emphasized the importance

of neutophils/platelets/contact system in thrombus formation; particularly in venous thrombosis

where FXIIa seems to be more important than neutophil elastase. This talk also showed the

association of DNA from NETs with factor XII and DNA as an activator of the contact system.

Jonas Emsley (UK): Data on the structure of PK and the interaction with HK was presented

together with

Ammar Majeed (Sweden): The Factor XII Registry Database. This talk reported a new

website FXII registry website URL is (www.factor12.net) where patient information will be stored

on FXII deficient. There are several reasons why this important. 1. There are few reports

characterizing factor XII deficient patients and currently no systematic collection of data. This is

important as FXII is considered an important novel target for the treatment of thrombosis and yet

there is no epidemiological data on the effects of deficiency in humans. This contrasts with the

case of Factor XI where and established website is available and >200 cases of FXI deficiency

have been characterized together with the genes sequenced. It was discussed that it would be

beneficial to have sequencing carried out for the FXII genes and that patients with a defect but

normal FXII plasma levels (CRM+) be entered into the database. The audience was encouraged

to submit information on FXII deficient patients.

Nicola Mutch (UK): Driving plasminogen activation by factor XIIa. This talk described new data on

FXII association fibrinolysis - polyp modulated the fibrinolytic side of the FXIIa activity and polyp

enhances fibrinolysis. PolyP binding characterized to FXII and plasminogen. The contribution of

platelets to fibrinolysis was characterized as platelets secrete PolyP. PolyP accumulates in fibrin

knots. A pathway was described for to contribute to fibrinolysis. FXII and fibrinogen were imaged

localized on the surface of platelets at the cap with polyp.

Keith McCrae (USA): This talk presented an update on kininogen in thrombosis and stroke models

and as an anti-angiogenic protein.

Toshitaka Sugi (Japan): Autoantibodies to FXII and kininogens in patients with recurrent

pregnancy loss were described. This talk presented data on a series of FXII deficient patients

characterized with recurrent pregnancy loss and also antibody interactions with FXII and

kininogen in the present of phospholipid. A mechanism of FXII-antibody complexes with platelets

is proposed for promoting thrombosis.

Theme: Inhibitors of FXI and the contact system: Safer anticoagulation

Maria Luiza Vilela Oliva (Brazil): Effect of plant inhibitors of the contact system on a mouse

thrombosis model. CTI is a well know plant protein that is used as an inhibitor of the contact

system. A further plan kunitz type inhibitor was presented which inhibits kallikirein and its

properties were characterized as inhibiting thrombus formation in a mouse model. It was also

proposed the inhibitor is bi-functional affecting the collagen receptor.

David Gailani (USA): New data were described for FXI anion binding sites and the effects of

mutations here on PolyP and nucleic acid activation.

Sanjay Bhanot could not attend so David Gailani presented a second talk (USA): Antisense

Reduction of FXI for Thromboprophylaxis: A Novel Therapeutic Approach. This data showed the

first clinical trial of and antisense RNA targeting FXI (N Engl J Med. 2015 Jan 15;372(3):232-40.)

This showed effective reduction in circulating FXI levels over a period of weeks such that

thrombosis was mitigated with no bleeding side effect. Data on factor XI contribution to venous

thromboembolism was also presented.

Open discussion José Govers-Riemslag (the Netherlands), and Joost Meijers, (the

Netherlands), presented in an open discussion several topics to the audience. Since a factor XII

registry was proposed, the first question was if each contact factor needs its own registry, which

was nearly unanimously accepted. A combination of registries for the 4 contact proteins was found

to be logistically unrealistic.

Furthermore, an inventory was made of the interest of the audience for the mechanisms that can

be activated by the contact system. In order of interest, the audience voted for the coagulation

system, the complement system, the kallikrein-kinin system and the fibrinolytic system. Finally,

the development of assays for the contact system was discussed. A general test that could

determine outcome of all the affected mechanisms was not found to be practical, but there was

great interest in the design and execution of specific tests that could measure contact system

mediated activation of the coagulation, kallikrein-kinin system, fibrinolytic system and complement

system. The audience felt that there was a role for the SSC subcommittee in developing such

assays.

Factor XIII and Fibrinogen

20 June 2015 14:30 – 18:50

Chairman: Helen Philippou

Co-Chairs: Zsuzsa Bagoly, Matthew J. Flick, Vytautas Ivaskevicius, Marlien Pieters, Verena Schroeder, Alisa Wolberg

Saturday 20 June 2015 | 14:30 - 18:50

At the start of the session it was estimated that there approximately 300-400 attendees. Dr

Philippou opened the session welcoming the audience.

Educational session

14.30-15:00 “Roles of the alpha chains of fibrinogen" Leonid Medved (Baltimore, USA)

Apologies were received from Prof Medved, who due to unforeseen was unable to attend the

conference.

15:00-15:30 “A Historic Recollection of Factor XIII Research" Akitada Ichinose

(Yamagata, Japan)

Prof Ichinose gave a historical overview of the discovery of FXIII and some key highlights in the

early discoveries related to FXIII and its characterization, as follows:

1) Evolution of transglutaminases, 2) Nomenclature of a plasma transglutaminase (FXIII) and

the annual numbers of its publication, 3) Discovery of FXIII through clot insolubility, 4) Real

function of FXIII; Hereditary deficiency and autoimmune hemorrhagic disease, 5) Purification of

plasma FXIII, 6) Legendary players in biochemical studies of FXIII, 7) Legendary players in

clinical studies for FXIII, 8) Cloning and molecular biology of FXIII, 9) Molecular pathological

analyses of hereditary FXIII deficiencies, 10) 3D structure of FXIII, 11) Polymorphisms of FXIII

and its relationship with thrombosis, 12) Rec. FXIII-A subunit; a very long way to the market, 13)

FXIII KO mice, 14) Modern times; active distinguished researchers and their cutting-edge

projects, 15) Remaining enigmas/issues regarding FXIII.

SSC session

15:30-15:50 “Standardization of turbidity and fibrinolysis measurements” Marlien Pieters,

South Africa

Marlien Pieters gave an update on the progress of the standardization of the fibrin clot turbidity

and lysis assay. The purpose of standardizing the assay is to improve comparison of results

between labs and to attempt to move in the direction of developing normal ranges for these

variables. In essence, the progress thus far is as follows: the protocol is being finalized by the

working group. They will also submit a funding application to the ISTH to obtain funding to

supply the necessary reagents to all participating labs. Once the protocol has been finalized and

funding obtained, labs will be invited to participate. It is envisioned that the experimental work

will take place in the second half of 2015 and that the project will be completed before the 2016

SSC meeting.

15:50-16:10 “FXIII-B standardization update” Éva Katona (Dubrecen, Hungary), Verena

Schroeder (Bern, Switzerland)

Verena Schroeder nicely introduced the session by describing the procedure necessary for

working with the NISBC and WHO for the introduction of a new standard.

Éva Katona subsequently introduced the progress made on the generation of selective

antibodies and ELISAs specific to the B-subunit measurement. The work has progressed well

and will require publication of the new ELISAs prior to being able to continue with the

standardization procedure.

16:10-16:30 “New Insights into Automated Fibrinogen Clauss Assays” Sanj Raut

(Potters Bar, UK)

The CLOTr (clot removal) method is currently the E P recommended method for assessing

fibrinogen activity in therapeutic concentrates/fibrin sealant products (used by Manufacturers

and Regulatory laboratories for Lot release purposes). This method was also used to value

assign the current 2nd International Standard for Fibrinogen Concentrate (09/242). However,

this method is slow, cumbersome, time consuming & labor intensive; not at all ideal as a high

throughput assay. There is therefore a real need for a more simple/automated alternative

method.

Clauss assay is one alternative functional assay used routinely for measuring fibrinogen in

plasma and is based on time for fibrin to clot. High concentrations of thrombin are used

(typically 100 IU/ml) to ensure clotting times are independent of thrombin concentration over a

wide range of fibrinogen levels. Fibrinogen samples are incubated with thrombin and calcium at

37ºC, and time taken to clot is compared vs reference standard. Most laboratories have an

automated Clauss method for measuring plasma (method based on photo optical end point

determination). Although, Clauss assay was shown to be suitable for measuring fibrinogen in

plasma, previous studies have shown Clauss assay to be unsuitable and highly variable for

measuring fibrinogen in concentrates.

This study assessed whether Clauss assay could be optimized to be sufficiently suitable for

measuring fibrinogen in therapeutic concentrates. Data from Claus assays (measuring

Fibrinogen concentrates) using mechanical end-point instrumentation (e.g. KC4) was found to

be comparable to data from CLOTr method and Absolute Methods (Kjeldahl & Clot

Weight). However, significantly higher (~30%) potencies were obtained from Clauss assays

using photo-optical instrumentation or when attempting to automate the assay (e.g. using auto

analyzers) & using specific commercial kits. This was still the case when pre-diluting fibrinogen

concentrates in fibrinogen deficient plasma (in order to mimic assays of plasma) to help

standardize turbidity issues, although some improvements were noticed.

As all these assays produced perfectly valid clotting curves for all dilutions, the various pre-

defined mathematical algorithms provided by the analyzers were investigated. As most auto-

analyzers are designed for assessing fibrinogen in plasma, these analyzers use predefined

“Threshold” algorithm by default, which is based on time to clot, measured in seconds. As the

analyzers were making measurement in Absorbance, a more appropriate algorithm for

measuring using photo-optical instrumentation would be one that measures in Absorbance

related units. Such algorithms exist in these analyzers, such as the “Delta” algorithm, which

uses the 2nd derivative of the clotting curve & measures in ∆ mAbs and if this algorithm is

selected then we get a very good agreement in fibrinogen potency between the CLOTr method

and the automated Clauss assay (<2% difference) and with CV <3%.

The study concluded that automated Clauss assays can be suitable for measurement of

fibrinogen concentrates using photo-optical instrumentation, as long as pre-defined algorithm

selected is based on optical (∆ mAbs) readout.

Further work is on-going to assess different fibrinogen therapeutic concentrates,

assess different Instrumentation (different algorithms), assess different Kits/reagents (specific

for analyzers) with the ultimate aim to carry out Collaborative Study to assess the suitability and

variability (between laboratories) of this assay for measurement of fibrinogen in therapeutic

concentrates.

16:50-17:15 “The Role of Fibrin(ogen) in Inflammation" Matthew Flick, Cincinnati

Children’s Hospital and Medical Center, USA

Fibrinogen has been previously implicated as a key factor in mediating host defense against

peritonitis infection by Staphylococcus aureus, a common and re-emerging gram-positive

bacterium that is a leading cause of both community and hospital-acquired infections. Dr Flick’s

laboratory has investigated the hypothesis that thrombin-mediated fibrin polymer formation is a

critical mechanistic component of the host antimicrobial response during of S. aureus

peritonitis. To establish a system to distinguish fibrinogen- and fibrin-dependent processes in

vivo, FibAEK mice were generated that have normal levels of circulating fibrinogen but lack the

chain thrombin

cleavage site. Thrombin failed to release fibrinopeptide-A from fibrinogenAEK and failed to induce

polymer formation with FibAEK plasma or purified fibrinogenAEK in 37°C mixtures regardless of

incubation time. Platelet-rich plasma from FibAEK mice supported normal platelet aggregation in

vitro, highlighting that fibrinogenAEK retains the functional capacity to support interactions with

platelets. FibAEK mice displayed both an absence of fibrin polymer formation following liver

injury, as assessed by electron microscopy, and a failure to generate stable occlusive thrombi

following FeCl3 injury of carotid arteries. Notably, FibAEK mice exhibited a profound impediment

in S. aureus clearance following intraperitoneal infection similar to fibrinogen-deficient mice, yet

FibAEK mice displayed a significant infection dose-dependent survival advantage over fibrinogen-

deficient mice following peritonitis challenge. Collectively, these findings establish for the first

time that fibrin polymer is the molecular form critical for antimicrobial mechanisms while

simultaneously highlighting biologically meaningful contributions and functions of the soluble

molecule.

17:15-17:40 “Fibrinogen Density Decreases Slightly and Fiber Stiffness Decreases

Strongly with Increasing Fibrin Fiber Diameter” Martin Guthold, Wake Forest University,

USA

The major structural component of a blood clot is a meshwork of fibrin fibers with an average

diameter of about 130 nm. The longitudinal assembly of fibrin monomers into protofibrils is well

understood; however, the radial (lateral) assembly of protofibrils into mature fibers is poorly

understood. It has long been thought that the internal structure of fibrin fibers is regular and

homogenous; that is, the protein density and the bond density between protofibrils are

uniform. Prof Guthold and colleagues performed two types of experiments to investigate the

internal structure of fibrin fibers. They formed fibrin fibers with fluorescently labelled fibrinogen

and determined the light intensity of a fiber, I, which is proportional to the number of monomers,

as a function of fiber diameter, D. They found that the intensity, I, scales as I~D1.43±0.03. This

implies that the cross-sectional monomer density also scales as D1.4, and not as D2, as would

be expected for fibers with a solid, homogeneous cross-section. They also determined the

Young’s modulus, E, as a function of fibrin fiber diameter, and found that E strongly decreases

with increasing D, as E~D-1.4. The modulus data suggest that the number of bonds per cross-

section scales as D0.6. The data are consistent with a fiber model that has a dense core and a

very loosely connected periphery. In contrast, electrospun fibrinogen fibers, used as a control,

do seem to have a homogenous cross-section, where the density scales as D2.

17:40-18:05 "Novel functions of fibrin(ogen) in liver repair and fibrosis" James P.

Luyendyk (Michigan, USA)

Experimental acute and chronic liver toxicity is associated with activation of the coagulation

cascade in rodents. This is marked by increased thrombin generation and hepatic fibrin(ogen)

deposition. Although the association between hepatotoxic responses and coagulation has been

appreciated for decades, the role of fibrin(ogen) in acute and chronic liver toxicity has not been

investigated in detail. Utilizing a combination of tools including pharmacologic modulation of

fibrinolysis and mice completely lacking fibrin(ogen) or expressing mutant fibrin(ogen) with

defective integrin-binding capacity, Dr Luyendyk and colleagues have identified previously

undescribed mechanisms whereby fibrin(ogen) can promote liver repair and inhibit liver fibrosis.

The take-home message from the presentation was that the role of fibrin(ogen) in experimental

settings of liver disease and toxicity is context and time-dependent.

18:05-18:30 “The Role of Crosslinking in Fibrin Structure and Function” Robert Ariens

University of Leeds, UK

Dr Ariëns discussed the role of cross-linking by FXIIIa in fibrin clot structure and function. He

began with explaining how FXIII activation is closely regulated by the conversion of fibrinogen to

fibrin by thrombin, and how activated FXIIIa cross-links the fibrin when it is being formed. He

described all the known FXIII cross-linking sites in fibrin and set out how gamma chain cross-

linking stabilizes the protofibril structure while alpha chain cross-linking stabilises the lateral

aggregates of protofibrils and contributes to fibrin stability through the incorporation of inhibitors

of fibrinolysis. Then he went on to discuss the binding sites for FXIII on fibrinogen, which include

sites on the fibrinogen gamma prime splice variant chain, gamma 390-396, alpha 233-425 and

alpha 389-402. These binding sites localize and direct FXIII to the reactive cross-linking sites.

Finally, Dr Ariëns discussed the effects of cross-linking by FXIIIa on fibrin clot structure and

function. Cross-linking by FXIIIa has subtle but significant effects on clot structure, by increasing

fibre number, reducing pore size and straightening fibres. Functionally, cross-linking by FXIIIa

increases stiffness of the fibrin clot, increases resistance to fibrinolysis (largely through

incorporation of inhibitors of fibrinolysis) and helps the retention of red blood cells in the clot.

18:30-18:40 “Proposal of ISTH/SSC Diagnostic Criteria 2015 for Autoimmune

Hemorrhaphilia Due to Anti-FXIII/13 Antibodies” Akitada Ichinose (Yamagata, Japan),

Hans Kohler (Bern, Switzerland), Laszlo Muszbek (Dubrecen, Hungary) and Helen

Philippou (Leeds, UK).

Professor Ichinose presented an overview of a proposal of ISTH/SSC diagnostic criteria 2015

for autoimmune hemorrhaphilia due to Anti-FXIII/13 Antibodies, as follows:

＜Possible AH13＞

1. Recent onset of bleeding symptoms mainly in the older adult.

2. No family history of congenital/inherited deficiency of FXIII/13.

3. Lack of previous bleeding symptoms especially in association with previous

hemostatic challenges (e.g., surgery, invasive tests, trauma, etc.).

4. Not explained by medication such as anticoagulants and antiplatelet drugs.

5. Abnormality of FXIII/13 parameter(s) on laboratory testing (FXIII/13 activity and/or antigen

<50%).

＜Probable AH13＞

6. Items 1-5 plus the presence of FXIII/13 inhibitors** (positive by cross-mixing tests between

patient’s and healthy control’s plasma using standard functional tests, such as an ammonia

release assay and an amine incorporation assay, after 2 hours incubation at 37°C).

＜Definite AH13＞

7. Items 1-5 plus the presence of anti-FXIII/13 autoantibodies (positive by immunological

methods, such as dot blot, ELISA, and immunochromatography, etc.).

*; Autoimmune hemorrhaphilia due to anti-FXIII/13 antibodies

**; are not always autoantibodies because non-immunoglobulin (antibody) inhibitors were

reported before.

The audience were asked whether there was an objection to this proposal, of which there none.

All members were in support of the proposal.

Fibrinolysis

20 June 2015 9:00 – 13:20

Chairman: Nicola Mutch

Co-Chairs: Jonathan H. Foley, Paul Y. Kim, Krasimir Kolev, Craig Thelwell, Shirley Uitte De Willige, Waander Van Heerde

Standardization of fibrinolytic proteins

Chairs: Dr Jonathan Foley & Dr Waander van Heerde

09:05-09:15 Dr Colin Longstaff, NIBSC, UK

‘Standardization of D-dimer’

CL updated the subcommittee on on-going activities to prepare a standard for D-dimer.

Previous work had identified problems of stability in D-dimer preparations, both in patient

plasma pools and when using cross-linked FDP made by lysing fibrin, after freeze drying. This

would make the development of a WHO standard impossible. However recent studies on long

term stability had identified formulations that included trehalose as more stable. It is hoped that

future work will include a collaborative study in a small number of labs to test of potential

standards, and these studies will include some patient samples as a way of addressing

questions of commutability. The results from these studies will inform future work on the

development of a definitive WHO International Standard for harmonization of D-dimer

measurements.

09:15-09:25 Dr Craig Thelwell, NIBSC, UK

‘Development of standards for plasmin, streptokinase, ancrod and Batroxobin’

Plasmin: The completed study on the calibration of the proposed WHO 4th International

Standard for Plasmin (13/206) was presented to the Fibrinolysis subcommittee in Milwaukee,

2014. The proposal was endorsed by the SSC/ISTH and the 4th IS was established by the

ECBS of WHO October 2014, with a potency of 8.0 International Units. The 4th IS is now

available to order from the NIBSC catalogue.

Streptokinase: Stocks of the WHO 3rd IS for Streptokinase (00/464) are low and a replacement

is needed. A proposal to calibrate the 4th IS as a direct replacement was presented. Potency

estimates of a candidate material, which will be a native therapeutic streptokinase preparation,

will be made in a multi-center study relative to the 3rd IS using chromogenic and fibrin-based

assay methods. Laboratories with appropriate assay methods were encouraged to contact

NIBSC to register an interest in participating in the study.

Ancrod and Batroxobin: A joint collaborative study was proposed to calibrate replacements for

the WHO 1st International Reference Reagent (IRR) for Ancrod (74/582) and the 2nd British

Standard for Batroxobin (93/526). Ancrod and Batroxobin are thrombin-like serine proteases

derived from snake venoms. Unlike thrombin ancrod and batroxobin cleave fibrinogen removing

only A-fibrinopeptides (not B-fibrinopeptides). This exerts an anticoagulant effect through the

removal of circulating fibrinogen and both are indicated for treatment of a range of thrombotic

diseases. Ancrod has been investigated in several clinical trials for treatment of acute ischaemic

stroke, with mixed results and is currently being investigated in a clinical trial for sudden

sensorineural hearing loss (SSHL). Batroxobin is used clinically to determine if prolonged

thrombin clotting times are a result of heparin contamination or abnormal fibrinogen, referred to

as "Reptilase time”. WHO/ECBS has endorsed the replacement of the 1st IRR for ancrod as the

2nd IS, and the 2nd BS for Batroxobin as the 1st IRR. Both will be calibrated relative to the

previous standard in a collaborative study using clotting and defibrinogenase assay methods.

The study is being run through the Exogenous Factors Subcommittee, but may be of interest to

members of the Fibrinolysis Subcommittee and interested laboratories are encouraged to

contact NIBSC.

09:25-09:40Dr Craig Thelwell, NIBSC, UK

‘Standardization of TAFI (CPU)’

Thrombin-activatable fibrinolysis inhibitor (TAFI, also known as proCPU or pCPB2) is a 56 kDa zymogen. TAFI is activated to TAFIa physiologically by the thrombin/thrombomudulin (TM) complex and plasmin. TAFIa is a basic carboxypeptidase that cleaves C-terminal lysines from partially degraded fibrin inhibiting fibrinolysis. TAFI/a levels have been shown to correlate with thrombotic diseases and inflammation. There are a large number of commercial and in-house methods for measuring TAFI/a in plasma. ELISA based assays are commonly used and are relatively simple to perform, however these methods rely on antibodies to distinguish between TAFI, TAFIa and TAFIai and do not allow measurement of active TAFIa. Furthermore detection for some antibodies differs between Thr/Ile325 isoforms. Functional activity based assays are also available. Direct activity based assays are complicated in plasma by carboxypeptidase N (CPN); indirect measurements require quantitative activation of TAFI and standardized methods are required due to instability of TAFIa and increased stability of Thr/Ile325, although kinetic activity is independent of 325 isoform. The activity of CPN must also be accounted for in these assays. Estimates of plasma TAFI concentration range from 73-275 nM (4-15 µg/ml) and there are a range of possible contributing factors to this apparent variation. Genotype differences in the regulatory region affect gene expression and mRNA stability and non-genetic factors can also affect gene expression. Different reactivities of antibodies in commercial kits e.g. detection of Thr/Ile325 and different plasma pools and purified TAFI used as calibrators in different methods also contribute to the apparent difference. A common reference of a normal plasma pool could help to identify the source of, and reduce some of this variability. A traditional WHO approach to calibrate a standard in International Units (IU) could follow the convention for coagulation plasma standards with 1 IU being equivalent to the amount of TAFI in normal plasma. A candidate reference plasma would be calibrated relative to local normal plasma pools arbitrarily assigned a potency of 1 IU. The study would also include local materials and a common freeze-dried plasma to determine if the variation is reduced by the inclusion of a common reference. This approach is compatible with kits currently using a normal plasma reference with a % labeled potency, where 100 % would be equivalent to 1 IU, however calibrators assigned ‘ng’ values would require a change of unit, which could be confusing. Assigning a ‘consensus ng’ value relative to local standards would not be acceptable to WHO since this is an SI unit which requires an absolute measurement with metrological traceability.

Quantitative analysis of TAFI in plasma is possible using isotope dilution mass spectrometry (IDMS) but is complicated by the high abundance plasma proteins. A tryptic peptide analysis of purified TAFI was demonstrated and two approaches to quantitate plasma TAFI were proposed. The first is to enrich TAFI by immunoprecipitation, and data was presented using a TAFI antibody magnetic bead approach. The second approach is to use multi-affinity columns to deplete the high abundance plasma proteins to improved TAFI detection. A collaborative study was proposed to include a candidate plasma reference material and a common plasma sample, both quantified for TAFI content by IDMS, to be measured by TAFI antigen and activity assays relative to local plasma pools and local reference standards. Interested laboratories were invited to contact NIBSC to participate, and collaborators were invited to contribute to the IDMS quantitation of TAFI in plasma.

Diverse roles of plasmin

Chairs: Dr Paul Kim & Dr Nicola Mutch

09:40-10:00 Dr Claudia Tersteeg, University Medical Center Utrecht, the Netherlands

‘Plasmin and Von Willebrand Factor in Thrombotic thrombocytopenic purpura’ Patients with thrombotic thrombocytopenic purpura (TTP) suffer from the presence of von Willebrand factor (VWF)/platelet-rich thrombi in the microvasculature, resulting in thrombocytopenia, hemolytic anemia, organ failure and death when left untreated. TTP is associated with a deficiency in VWF-cleaving protease ADAMTS13. However, ADAMTS13 activity levels not always predict acute TTP episodes and therefore suggest a role for other VWF-cleaving enzymes in the regulation of TTP episodes. We have investigated the role of plasmin in the cleavage of VWF in TTP. We have demonstrated that plasmin activated via uPA/uPAR is able to degrade VWF/platelet-rich microthrombi in vitro and that endogenous plasmin levels are able to control acute TTP episodes in vivo in a mouse model for TTP. Additionally, patients with acute TTP demonstrate increased levels of plasmin-alpha2-antiplasmin and PAI-1, and decreased uPA activity, suggesting regulation of plasmin activation during acute TTP. Furthermore, thrombolytic agents were demonstrated to be a possible future treatment strategy for TTP. Together, this demonstrates that plasmin cleavage of VWF is important in TTP pathology, and future research will open new doors towards new treatment strategies for this severe and life-threatening disease.

10:00-10:20 Ms. Marijke Peetermans, Katholieke Universiteit Leuven, Belgium

‘Plasmin generation by Staphylokinase during skin infections with Staphylococcus aureus’

Staphylococcus aureus (S. aureus) is a major etiologic agent of a very broad range of infections, such as skin and soft tissue infections and intravascular catheter infections. Interestingly, S. aureus produces not only coagulases that trigger fibrin formation, but also staphylokinase (SAK), a fibrin-specific plasminogen activator. The SAK-plasmin complex is protected against rapid inhibition by alpha-2-antiplasmin (α2AP). We aimed to elucidate the role of SAK-induced human plasminogen activation in infections with coagulase-positive S. aureus and understand the dynamics of the apparent opposing activation of both the coagulation and the fibrinolytic systems by S. aureus.

Skin infection: Skin infection was induced by subcutaneous injection of S. aureus Xen36 in mice

containing an adenoviral vector for human plasminogen. We observed higher bacterial loads

and increased spreading of infection in human plasminogen-expressing mice compared to wild-

type mice, indicating SAK-mediated human plasminogen activation in local invasion. In α2AP

knock-out mice (α2AP -/-) local spreading in later stages of infection was attenuated. However,

close to the initial abscess site the SAK-plasminogen complex is protected from α2AP action by

either the bacterial surface or by fibrin produced by coagulase-positive S. aureus. Fibrin-specific

SAK action and downstream gelatinase activation allows the local spreading of S.

aureus through tissue barriers and decreases bacterial clearance.

Catheter-related infection: We have previously shown the importance of S. aureus coagulase

activity in adhesion to foreign bodies and formation of biofilm matrix. However, in later stages

of in vitro biofilms, scanning electron microscopy revealed dispersal of biofilms. We analyzed

the role of SAK in biofilm structure, using S. aureus strains with different levels of SAK

production grown in diverse media with or without added prothrombin, fibrinogen or

plasminogen. High production of SAK by S. aureus LS-1 spasak interfered with bacterial-

induced fibrin formation and adhesion to foreign body surfaces. If plasminogen was omitted

from the culture medium, a thick fibrin-rich biofilm with almost no evident dispersal was seen. A

jugular vein catheter infection model was performed in human plasminogen expressing mice

using the same congenic S. aureus strains with different levels of SAK production. Indeed, the

high-SAK producing S. aureus spasak strain showed lower virulence in biofilm infection. In S.

aureus foreign body infections, SAK production leads to dispersal of biofilms and release of

bacteria from the fibrin-containing biofilm structure. The expression of SAK in S. aureus biofilms

is regulated by quorum sensing, shifting the balance from coagulase activity to fibrinolysis in

later-stage biofilms, when bacterial density increases.

Impact of cells on fibrinolysis

Chairs: Dr Krasimir Kolev & Dr Jonathan Foley

10:20-10:40 Dr Paraskevi Untiveros, University of Aberdeen, UK

‘Effect of hematocrit on clot structure and resistance to lysis’

Red blood cells (RBCs) have been considered a relatively inert bystander in coagulation and

thrombus formation, yet their sheer abundance in blood means they dominate the resulting clot.

Here we examine the effects of haematocrit (HCT) on coagulation parameters, clot firmness and

resistance to fibrinolysis. Whole blood was separated into plasma and RBC constituents by

centrifugation. Samples were reconstituted with 35% platelet rich plasma and RBCs to produce

20%, 40% and 60% final HCT. Thrombus formation and dissolution were assessed using

Thromboelastography (TEG) and the Chandler Loop Model. TEG revealed significantly shorter

clot time, clot formation time and increase in the α-angle at 20% HCT compared to 60% HCT;

indicative of faster clot formation at lower HCT. An increase in maximum clot firmness was

detected at 20% HCT, consistent with enhanced mechanical stability. Chandler model thrombi

formed with 20% HCT were significantly longer than those formed at 60% HCT (p<0.05) and

demonstrated increased resistance to lysis by both tPA (p<0.005) and uPA (p<0.005). Inclusion

of a transglutaminase (TG) inhibitor, to inhibit factor XIIIa, significantly increased lysis of 20%

HCT thrombi (p<0.001). Similarly, the TG inhibitor increased lysis of 40% HCT thrombi (p<0.01),

albeit to a lesser degree, but no change in lysis was observed at 60% HCT (p=0.113).

Interestingly, when the TG inhibitor was present all thrombi lyse at an equivalent rate,

suggesting that inhibition of factor XIIIa overcomes the stabilizing effect of HCT on lysis. IN

conclusion HCT has a dramatic impact on coagulation parameters, with lower HCT enhancing

clot formation, resulting in thrombi with increased firmness and resistance to fibrinolysis. Factor

XIIIa helps to maintain thrombus integrity and stabilizes against fibrinolytic degradation at lower

HCT, an effect that is lost at higher HCT. These observations may help to explain the increased

risk of thrombosis in anemic patients.

10:40-11:00 Dr Imre Varjú, Semmelweis University, Hungary

‘Neutrophils as key modulators of fibrin structure and lysis in thrombi’

The therapeutic modality of thrombolysis relies on administration of substances capable of

converting plasminogen to plasmin, which in turn digests the primary fibrin scaffold holding the

thrombus together. Since this approach is often ineffective, and is associated with bleeding

complications, a need for the exploration of factors influencing the effectiveness of thrombolysis

is emerging. Thrombi may contain considerable amounts of neutrophil granulocytes, which are

capable of neutrophil extracellular trap (NET) formation by catapulting their DNA with associated

histones and granular enzymes to the extracellular space as a response to thrombosis-

associated inflammatory signals. Since, according to recent studies, NETs are mandatory

components of arterial and venous thrombi, assessment of their thrombolytic consequences is

crucial. Our previous studies have shown that DNA and histones, the major components of

NETs alter the structure of clots rendering them more resistant to fibrinolysis, and recently we

have confirmed these effects using neutrophil-derived NETs in plasma clots. Mounting data

from our and others’ studies suggest that the currently used fibrin-targeted thrombolytic

protocols might be augmented by the addition of enzymes capable of NET degradation (such as

DNAses), which could open a new area of designing thrombolytic agents of the near future.

Educational Session

Chair: Dr Nicola Mutch & Dr Paul Kim

11:20-11:25 Dr Tetsumei Urano, Hamamatsu University, Japan

TU introduced the 1st joint meeting of the International Society of Fibrinolysis and Proteolysis

and the plasminogen activation workshop to take place on Oct. 17-21, 2016 in Shizuoka, Japan

11:25-11:50 Prof Guy Reed, University of Tennessee, Tennessee

‘Influence of a2AP on the pathogenesis of stroke’

Thrombotic vascular occlusion is the primary cause of ischemic stroke. Higher blood levels of

a2-antiplasmin are associated with increased stroke risk in epidemiologic studies, yet a2-

antiplasmin depletion during plasminogen activator therapy has been linked to coagulation

protein depletion and increased risk of bleeding. Therefore, we examined the contribution of a2-

antiplasmin to ischemic stroke outcomes in a model of middle cerebral artery thromboembolism

that closely simulates human stroke. We evaluated the dose-related effects of a2-antiplasmin on

stroke outcomes in mice with increased, normal or no circulating a2-antiplasmin, as well as in

mice given an a2-antiplasmin-inactivating antibody. Higher a2-antiplasmin levels were

correlated with greater ischemic brain injury, brain swelling and reduced middle cerebral artery

thrombus dissolution. In contrast, a2-antiplasmin deficiency enhanced thrombus dissolution,

increased cerebral blood flow, reduced brain infarction and decreased brain swelling. In a2-

antiplasmin-deficient mice, middle cerebral artery thromboemboli that were deficient in a2-

antiplasmin dissolved more extensively than those containing a2-antiplasmin; however, the

presence of a2-antiplasmin did not further reduce brain infarction, swelling or hemorrhage. By

comparison to tissue plasminogen activator, a2-antiplasmin inactivation hours after

thromboembolism still reduced brain infarction and hemorrhage. Microvascular thrombosis, a

process that enhances brain ischemia, was markedly reduced in a2-antiplasmin-deficient or a2-

antiplasmin-inactivated mice compared with tissue plasminogen activator-treated mice or mice

with increased a2-antiplasmin levels (all p<0.001) Matrix metalloproteinase-9 expression, which

contributes to acute brain injury, was profoundly decreased in a2-antiplasmin-deficient or a2-

antiplasmin-inactivated mice vs. tissue plasminogen activator-treated mice or mice with

increased a2-antiplasmin levels. Alpha-2-antiplasmin inactivation markedly reduced stroke

mortality vs. tissue plasminogen activator. Taken together, these data indicate that alpha-2-

antiplasmin has profound, dose-related effects on ischemic brain injury, swelling, hemorrhage,

and survival following cerebral thromboembolism. By comparison to tissue plasminogen

activator or controls, a2-antiplasmin inactivation treatment after the onset of ischemic stroke

significantly increased survival and it reduced ischemic brain injury and hemorrhage. In part, the

protective effects of a2-antiplasmin inactivation or deficiency appear to be mediated through

reductions in microvascular thrombosis and matrix metalloproteinase-9 expression.

11:50-12:20 Dr Manuel Yepes, Emory University, Georgia

‘Plasminogen activators in the brain’

Tissue-type plasminogen activator (tPA) is a serine proteinase found not only in the

intravascular space but also in a well-defined sub-set of neurons in the brain. We show that

overexpression of tPA in neurons protects the brain from the deleterious effects of ischemic

stroke. Early after the onset of cerebral ischemia neuronal tPA is rapidly released into the

synaptic cleft where it activates specific cell signaling pathways that promote the detection and

adaptation to metabolic stress. More specifically, the non-proteolytic interaction of tPA with N-

methyl-D-aspartate receptors (NMDARs) and a member of the LDL-receptor family on the

surface of dendritic spines activates the adenosine monophosphate-activated protein kinase

(AMPK; the sensor of cellular energy status) which leads to the recruitment to the neuronal

membrane of the glucose transporter GLUT3, and GLUT3-mediated uptake of glucose.

Together this promotes survival in neurons exposed to cerebral ischemia. These data indicate

that tPA is an endogenous neuroprotectant in the central nervous system that promotes the

detection and adaptation to metabolic stress.

Inhibitors of fibrinolysis in disease

Chair: Dr Craig Thelwell & Dr Shirley Uitte de Willige

12:20-12:40 Prof Paul Declerck, Katholieke Universiteit Leuven, Belgium

‘Thrombolysis induced by a diabody against PAI-1 and TAFI’

Endogenous fibrinolysis is hampered either through inhibition of plasmin (e.g. alpha2-

antiplasmin), through inhibition of PAs (e.g. plasminogen activator inhibitor-1, PAI-1) or through

modification of the fibrin surface (e.g. activated thrombin activatable fibrinolysis inhibitor, TAFIa).

PAI-1 and TAFI are widely studied antifibrinolytic proteins and have been linked to various

thrombotic disorders. PAI-1 is a serine protease inhibitor (SERPIN) whereas TAFIa is a

carboxypeptidase. The concerted action of PAI-1 and TAFIa by which protection of the fibrin clot

is provided, has led to the idea of dual targeting strategies. Development of antibody-

engineered inhibitors against TAFI and PAI-1 have been shown to stimulate fibrinolysis

efficiently. A heterodimer diabody, Db-TCK26D6x33H1F7, cross-reactive with human, mouse

and rat PAI-1 and TAFI was developed and was demonstrated to enhance fibrinolysis in in

vitro clot lysis and thromboelastometric experiments. Its profibrinolytic properties were further

confirmed in vivo in mouse models of thromboplastin-induced thromboembolism and in various

mouse models of stroke. Taken together all data demonstrate that dual targeting of PAI-1 and

TAFI results in a pronounced profibrinolytic effect with superior properties to those of t-PA and

minimal bleeding risks.

12:40-13:00 Mr. Travis Gould, McMaster University, Ontario

‘Cell-free DNA modulates clot structure and impairs fibrinolysis in sepsis’

Though several studies on the effects of cell-free DNA (cfDNA) and coagulation in sepsis have

been performed, little is known about the effects of cfDNA on fibrinolysis. Our studies are the

first to suggest that cfDNA impairs fibrinolysis in sepsis by preventing plasmin-mediated

cleavage of fibrin via the formation of a non-productive ternary complex of cfDNA-plasmin-fibrin.

Our studies identify cfDNA as a potential pro-fibrinolytic therapeutic target in sepsis treatment.

13:00-13:20 Ms. Dorien Leenaerts, University of Antwerp, Belgium

‘Measurement of active CPU (TAFIa) in samples from patients with acute coronary syndrome’

The presentation focused on the measurement of carboxypeptidase U (CPU, CPB2 or TAFIa) in

clinical samples and more specifically in those of patients with acute myocardial infarction (AMI).

The first part of this presentation covered the role of the proCPU (proCPB2 or TAFI) system in

arterial thrombosis. Despite the large body of literature available on proCPU, a lack of clarity

remains with regard to the exact role played by active CPU during thein vivo process of

thrombosis. This is mainly due to the difficulty of measuring ultra-low CPU concentrations in the

circulation. However, recently these challenges were overcome by the development of assays

that quantify CPU activation, instead of measuring the zymogen proCPU. During this

presentation, existing assays were discussed and particular emphasis was devoted to the

pitfalls associated with the measurement of CPU.

Secondly, data on CPU measurement in clinical samples were shown. Using a sensitive and

specific enzymatic assay, we investigated whether it is possible to detect the active enzyme CPU

in the circulation of patients with AMI. We found that CPU activity levels were significantly higher

in patients with AMI than in controls (P=0.0035). However, elevated CPU levels were not found

in all AMI patients, but in a subpopulation.

With respect to the clinical significance of these elevated CPU levels, it must be noted that CPU

activities are low, especially when comparing to the total amount of available proCPU in plasma.

However, this is probably the result of an important dilution in the circulation and rapid decay.

Most likely, detectable levels of active CPU reflect a process of ongoing generation and as a

consequence, future research (e.g. serial blood sampling on different time points) will clarify the

relevance of these findings.

13:20 Close of Meeting.

With thanks to all the speakers for excellent presentations and keeping to time. We were happy

to have such a large audience and to have such good participation which made for an

outstanding subcommittee session.

Genomics in Thrombosis and Hemostasis

21 June 2015 8:00 – 12:20

Chairman: Willem Ouwehand

Co-Chairs: Paul F. Bray, Kathleen Freson, Anne Goodeve, Michele P. Lambert, Pieter Reitsma

Part 1: NGS IN DIAGNOSTICS

Moderators: Professor Willem H. Ouwehand (United Kingdom) and Dr Michele P Lambert (United

States)

Welcome by Professor Willem H. Ouwehand

Professor Willem H. Ouwehand presented an overview of the SSC Genomics in Thrombosis and

Hemostasis.

Key people involved in the project and key activities have been presented.

Activities include:

1. Development and validation of a NGS test for rare inherited T & H disorders 2. Identification and annotation of pertinent genes and variation in these genes to clinical

standards 3. Development and maintenance of a database to provide a stable and sustainable frame

of reference for sequence information 4. Development and application of Human Phenotype Ontology terms (HPO) to patients

Speaker: Dr Char Witmer – Philadelphia, USA

Title: Inherited bleeding /coagulation disorder

Dr. Witmer has presented the limitations to coagulation testing to diagnose patients with bleeding

and coagulation disorders. She talked about some of the current applications of genetic testing

for VWF and Hemophilia A and B underlying the limitations of the available tests including the

high costs and the importance of having a new comprehensive test such as a NGS platform with

higher sensitivity and specificity.

Speaker: Professor Anne Goodeve – Sheffield, UK

Title: Hemostasis NGS panel in routine diagnostic use

The NGS gene panel currently available at Sheffield Diagnostic Genetics Service to screen

patients with bleeding and platelet disorders has been presented. Sample workflow and GATK

bioinformatics pipeline currently in use have been described including the way variants are filtered

according to frequency and pathogenicity to identify the potential causative variant.

The platform has been validated with control samples and performance assessed, and has been

in diagnostic service use for 6 months for sample from the UK and worldwide. Availability of

several genes on the panel facilitates discrimination between disorders where necessary; e.g.

hemophilia A and B, hemophilia A and type 2N VWD and also provides more rapid analysis for

disorders resulting from more than one gene e.g. FIX deficiency, Glanzmann thrombasthenia and

fibrinogen disorders.

Speaker: Dr Ilenia Simeoni, Cambridge, UK

Title: ThromboGenomics platform

An update about the ThromboGenomics (TG) project has been presented. The TG platform is

now fully validated and will become a NGS platform for routine clinical use from July 2015. A

single, comprehensive DNA based test will be offered to all the UK Hemophilia Centers to screen

patients with bleeding, thrombotic and platelet disorders. The gene panel includes so far 86 genes

and in the validation phase about 400 samples have been enrolled, sequenced and analyzed.

Results are discussed in MDT meetings and reports generated for the referring clinicians using

Sapientia, a new software developed by Congenica Ltd.

Part 2: NGS IN DIAGNOSTICS

Moderators: Professor Anne Goodeve (United Kingdom) and Dr Walter Kahr (Canada)

Speaker: Dr Dan Hampshire – Sheffield, UK

Title: Coagulation Factor Variant Databases - an update

An update about the EAHAD-DB (European Association for Haemophilia and Allied Disorders

Coagulation Factor Variant Databases) initiative has been presented. The intention of this

initiative is to gather together single gene variant databases involved in clinical bleeding disorders,

principally hemophilias A and B and von Willebrand disease, as well as other rarer coagulation

factor variants. So far F7, F8, F9 and VWF databases have been included. In future, databases

for fibrinogen and factors FII, FV, FX, FXI and FXIII will also be included. The database is free,

easily accessible and open to any new variant submission, including submission of previous

reported variants.

Speaker: Dr Andrew Paterson, Toronto, Canada

Title: Challenges in the interpretation of variants from next generation sequencing

Many false positive variants are present in current databases. NGS and the availability of whole

exome and whole genome sequencing (WES and WGS, respectively) on a large number of

controls will help to remove several of the misinterpreted variants in addition to the identification

of new variants likely to cause an inherited disease. Advantages and limitations of the Exome

Aggregation Consortium (ExAC) variant database have also been mentioned.

EDUCATIONAL SESSION

Moderators: Professor Pieter H. Reitsma (the Netherlands) and Professor Paul Bray (United

States)

Speaker: Dr Ernest Turro Cambridge, UK

Title: Methodological challenges of gene discovery by genome sequencing

An overview about sequencing strategies, variant calling and filtering of sequencing data has

been presented. One of the challenges of WGS is variant prioritization. The process includes

multiple steps and takes advantage of the use of the Human Phenotype Ontology (HPO) terms,

pathogenicity score and a novel statistical methodology call “phenotype similarity regression”.

WGS enables detection of variations also in the non-coding regions of the genome adding a

further level of complexity to the bioinformatics analysis.

Speaker: Dr Walter Kahr, Toronto, Canada

Title: Inherited platelet disorders

An overview about inherited bleeding disorders and their classification have been presented.

Glanzmann Thrombasthenia, Bernard Soulier, Hermansky Pudlak and Arthrogryposis - Renal

dysfunction – Cholestasis (ARC) syndromes have been presented in details. The recent

discovery of germline mutations in ETV6 as a cause of autosomal dominant thrombocytopenia,

red cell macrocytosis and ALL was also presented.

REPORTING OF CLINICAL VARIANTS

Moderators: Professor Kathleen Freson (Belgium) and Dr Daniel Bellissimo (United States)

Speakers: Dr Daniel Bellissimo, Pittsburgh, USA & Professor Kathleen Freson, Leuven,

Belgium

Title: International Guidelines to annotate pathogenic variants

This was a joint presentation between Dr. Dan Bellissimo, who was the first speaker, and

Professor Kathleen Freson.

The first presentation described the recently published ACMGG/AMP Guidelines for the

interpretation of sequence variant in US (Richards et al (2015 Genet in Med 17(5): 405-423).

This guideline for interpretation of sequence variants is an evidence-based scoring system that

considers the strength of the following pieces of evidence when classifying variants as

pathogenic, likely pathogenic, uncertain significance, likely benign and benign:

Population Data Computational and Predictive Data Functional Data Segregation Data De novo Data Allelic Data Other Database

Other Data

The second talk was about the Guidelines for the Interpretation of sequence variants in Europe.

NGS not only influences diagnostic outcome but also the complete organization of genome care

that can no longer be the sole responsibility of clinical geneticists but of Multi-Disciplinary Team

(MDT) that includes research specialists, clinicians, bioinformaticians and clinical geneticists.

Conclusions include the importance of variant databases, wide data sharing within and between

countries, the importance of a careful re-classification of variants of unknown significance (VUS)

and MDT for variant interpretation and reporting.

Speakers: Dr Karyn Megy, Cambridge, UK & Dan Hampshire, Sheffield, UK

Title: New large control data: opportunities & pitfalls

The outline of this joint talk included an update about the current variation databases (dbSNP,

dbVar, ClinVar, Human Gene Mutation Database (HGMD) and Locus specific databases

(LSBDs)) and the large volume of datasets coming from WES and WGS. Opportunities and pitfalls

of the available large datasets were presented. Opportunities of having large datasets to help in

classifying a variant include availability of controls, phenotypes and ethnicity data but there are

pitfalls to consider: false positive variants present in public databases, the type of sequencing

which might not be sufficient to identify the causal variant and a lack of standardization for variant

description, the reference sequence and phenotype coding.

To avoid these pitfalls, the use of multiple datasets, a standardization of the variant description in

addition to the use of a stable database such as Locus Reference Genomic (LRG) which includes

a unique stable sequence record of transcripts using IDs and no versioning.

Final Remarks

Progress

The ThromboGenomics (TG) project, which was informally initiated by Kunicki and Ouwehand at

the ISTH in Kyoto (2011) is now after extensive validation moving to the clinical stage. The global

TG network continues to expand to include new collaborators, clinicians and researchers from all

around the world.

Hemostasis and Malignancy

20 June 2015 14:30 – 18:50

Chairman: Alok A. Khorana

Co-Chairs: Marc Carrier, Howard A. Liebman, Nigel Mackman, Simon Noble, Ingrid Pabinger, Joseph S. Palumbo

Education Session

The education session was chaired by Drs Khorana and Carrier and included Drs Carrier and

Noble as speakers. Dr. Carrier spoke about occult malignancy screening after unprovoked

VTE, particularly in light of two trials to be presented at ISTH 2015. Although results of these

studies were not yet available, Dr Carrier identified study design and expectations for outcomes.

He proposed a guidance statement from SSC of ISTH after results of these studies are known

to better inform hematologists on practice issues. Dr. Noble provided an overview of qualitative

research in hematology, how it differs from generally accepted standards and sample sizes in

quantitative research and how it can inform patient care.

SSC Working Session

The session was first chaired by Drs Mackman and Pabinger. The second half was chaired by

Drs Ay and Zwicker. Drs Palumbo, Mackman and Pabinger described recent results linking

platelet activation with hemostatic factors and tumor biology. Dr. Mackman posed the question

of how therapeutic targeting of platelets could affect malignancy outcomes and potentially even

prevent thrombosis in this setting. Dr. Khorana reviewed recent data regarding clinical

predictors and biomarkers of recurrent VTE in malignancy, in context of the recent completed

CATCH study. Dr. Falanga described early findings and study design of an Italian study of

biomarker screening for cancer-associated thrombosis, the HYPERCAN study. Dr. O’Connell

described emerging data regarding prevalence and outcomes related to incidental thrombosis in

malignancy. She was followed by Dr. Di Nisio who presented the new ISTH guidance statement

on incidental thromboembolism in malignancy, recently published in JTH. Dr. Ay described how

network analysis could evaluate anticoagulants in different clinical trials indirectly. Dr. Othman

presented data suggesting a strong hypercoagulable state in prostate cancer. Dr. Zwicker

presented data on intracranial bleeding in patients with brain metastases and a guidance

proposal to better categorize such bleeds in future studies. Dr. Palumbo described draft

guidance statement proposal encompassing venous thromboembolism in pediatric cancer

patients. Finally, Dr Kamphuisen provided an update on the LONGHEVA trial which has now

transformed into an online registry and asked for SSC members to participate. The session was

adjourned at 6.40 PM.

Lupus Anticoagulant/Phospholipid Dependent Antibodies

20 June 2015 9:00 13:20

Chairman: Bas De Laat Co-Chairs: Tatsuya Atsumi, Maria Laura Bertolaccini, Katrien Devreese, Rolf T. Urbanus, Denis G. Wahl

Dr. Bas de Laat opened the session and proposed to change the title of the SSC subcommittee

from ‘Lupus coagulant / phospholipid-dependent antibodies’ to ‘Lupus

anticoagulant/antiphospholipid antibodies’.

Dr. Kumano postulated that, although the SSC recommends to use silica-based APTT assays

for LA screening over ellagic acid-based assays due to a higher sensitivity of the former, these

assays were never adequately compared with the same phospholipid concentration. In his study

he compared home-made silica-based and ellagic acid-based reagents with the same

composition and low concentration of phospholipids, with four commercial APTT reagents. Their

results on 63 LA positive and negative samples showed that the home-made ellagic acid-based

reagent was more sensitive to LA than the silica-based reagent in a low phospholipid condition

and had adequate sensitivity to detect LA. They concluded that the sensitivity of APTT reagents

for LA is dependent on the phospholipid concentration rather than the activator. In response to

the question ‘will the SSC change their guidelines based on these results?’ Dr. de Laat

emphasizes that larger multicenter studies are required and that requests for such studies can

be proposed to the SSC.

Dr. Devreese in a first presentation elaborated on the importance of the determination of cut-off

values in solid phase and automated assays used in the diagnosis of APS as these determine

the clinical performance of the tests. In this study she focused on the refinement of the cutoff

value using the HemosIL Acustar in a multicenter study in which 5 laboratories participated with

a total of 626 samples. Compared to original work in which 250 samples were used to

determine the cut-off, the multicenter approach resulted in a lower cut-off value with a stricter

confidence interval, a higher sensitivity, slightly reduced specificity and comparable predictive

value for thrombosis. Prof. Devreese concluded that the multicenter approach is a valid

alternative for the determination of cut-off values.

In a second presentation, Dr. Devreese addressed the need for a multicenter study to unravel

the importance of IgM antibodies in the diagnosis of APS. Indeed, together with Dr. Hilde

Kelchtermans she performed a meta-analysis of the available literature on the relevance of

measuring IgM antibodies. The analysis is still ongoing, but so far results are disappointing

because many of the included articles do not show individual data, nor correlations with

thrombosis or show combined data. Therefore, in a new clinical study, a minimum of 1000

patients, including APS patients, diseased controls, auto-immune diseases and healthy controls,

will be tested using solid-phase and automated assays from different suppliers. She concluded

with inviting centers to collaborate in this multicenter study. In response to the questions, she

mentions that it may be interesting to add infection disease controls, investigate the role of IgA

antibodies and to compare with LA positivity although for these determinations we will be

dependent on the values of the local labs.

Although currently the diagnosis of APS is mainly done by endpoint and quantitative assays,

Leonie Pelkmans emphasizes the need for the development of more global functional assays

to avoid false positive results due to non-pathogenic antibodies. Based on the promising results

obtained by the β2GPI-dependent LAC and earlier thrombin generation data from Katrien

Devreese, she showed an ellagic-acid triggered thrombin generation test in the absence or

presence of cardiolipin. She applied this test on normal pooled plasma supplemented with

monoclonal anti-β2GPI antibody as well as on healthy controls and APS patient plasma samples

positive or negative for reactivity against the pathogenic domain I epitope of β2GPI. Although the

results look promising, they need to be confirmed in a larger study. Therefore she asked the

SSC for help in collecting APS plasma samples.

Dr. Bertolaccini elaborated on the relevance of measuring anti-β2GPI IgA and anti-PS/PT

antibodies for the diagnosis of APS. Given the low frequency of isolated IgA positivity, she

concluded that for APS, the utility of IgA antibodies should be restricted to patients with a strong

suspicion for APS and that are negative for other antiphospholipid antibodies. As to the anti-

prothrombin antibodies, the ones that are directed against the complex PS/PT have been shown

to correlate better with thrombosis. Combined positivity for LA, anti-β2GPI and anti-PS/PT was

shown to result in a better accuracy for the diagnosis of APS and the prediction of thrombosis

and pregnancy morbidity as the current Sydney criteria. Positivity for anti-PS/PT has been

added to the GAPSS score. Furthermore, she provided evidence that the anti-PS/PT antibodies

are pathogenic in a mouse model.

Dr. Wahl aimed to determine the value of APC resistance measured by thrombin generation to

predict thrombosis in platelet-rich plasma of APS patients. In a prospective multicenter study

consisting of 137 persistent anti-phospholipid positive patients, the IC-50 APC concentration

was determined. This concentration proved to be a significant predictor for thrombosis, both in

asymptomatic patients as in patients that already showed clinical symptoms. He concluded that

there is a need for standardized protocols and cut-off determinations.

Dr. Urbanus aimed to solve the LA paradox. By using a Taipan snake venom that already

contains an FXa-FVa-like complex, he provided evidence that anti-β2GPI antibodies no longer

affect the coagulation time when the prothrombinase complex is already formed. Further

research demonstrated that anti-β2GPI antibodies inhibit the activation of FV by FXa at limiting

concentrations of phospholipids. Interestingly in patients positive for anti-β2GPI antibodies and

LA he discovered that these anti-β2GPI antibodies are not responsible for the observed LA

activity.

Dr. Rand presented data to support that resistance to annexin A5 anticoagulant activity can be

used as a biomarker for adverse clinical outcomes in APS. The value of this mechanistical

assay in the prediction of clinical events was already demonstrated in multiple clinical studies. In

a real world population of 966 patients undergoing routine thrombophilia testing, he additionally

demonstrated that annexin A5 resistance inversely correlated with anti-phospholipid antibody

assays. An increased resistance was also observed with increasing number of events. Dr. Rand

concluded that the development of such mechanistical assays may be interesting to better

predict the occurrence of thrombosis.

Dr. Bas de Laat presented the work of Dr. Pengo on Domain I and risk stratification. After an

overview on the evidence of the pathogenicity of anti-domain I antibodies, he explained the

competitive inhibition ELISA developed by Prof. Pengo to demonstrate the presence of anti-

domain I antibodies. Using this assay, a strong correlation was found between anti-domain I

antibodies and triple positivity, as well as thrombosis. Interestingly, anti-domain I antibodies

proved to be stable over time. Bas de Laat ended the presentation with the question if these

anti-domain I antibodies should be included in the APS criteria. Prof. Devreese mentioned that

this may be interesting for risk stratification, but that concerning the reduced sensitivity of the

assay, the anti-domain I assay can’t replace the anti-β2GPI assay.

Dr. Wahl presented results of clinical studies to determine if scores should be used for risk

assessments in APS and if these scores should be based on laboratory tests only or should

also include clinical parameters. In the RATIO study and a prospective multicenter study, the

GAPSS score, including both the antiphospholipid tests and clinical risk factors, proved to be

useful in the prediction of clinical events. However, the anti-domain I reactivity that doesn’t take

the clinical risk factors into account was as predictive as the GAPSS score.

Dr. Atsumi further elaborated on APS scoring and compared the anti-PL-S and GAPSS score

for their predictive value for thrombosis. In 295 patients with autoimmune diseases, 46

thrombotic events occurred during the follow up of 5 to 10 years. Both scores were predictive for

future thrombosis but the anti-PL-S score proved to be more suitable than the GAPSS score in

this cohort of patients. The question remains how to standardize each anti-phospholipid test and

to minimize the number of tests.

Dr. Arachchillage investigated if rivaroxaban influences LA assays and if anti-phospholipid

antibodies affect the activity of rivaroxaban. In vitro and ex vivo studies showed false positives

with two commercial DRVVT assays at the peak concentration of rivaroxaban. However, the

Taipan venom time/Ecarin clotting (TVT/EC) time proved to be unaffected, allowing a reliable

detection of LA. Both by thrombin generation and rivaroxaban anti-Xa levels, she demonstrated

that anti-phospholipid antibodies do not influence the activity of rivaroxaban. In reply to a

question she discourages the use of DRVVT tests in patients taking rivaroxaban but rather

advises to incorporate the alternative TVT/EC test in each laboratory.

Dr. Cohen investigated if the superior results of rivaroxaban and other direct oral anticoagulants

for the treatment of venous thrombosis can be extended to APS. The primary aim of the

prospective RAPS study was to demonstrate, in patients with APS and previous venous

thromboembolism that the intensity of anticoagulation achieved with rivaroxaban is not inferior

to that of warfarin. Secondary aims were to compare rates of recurrent thrombosis, bleeding and

the quality of life in patients on rivaroxaban with those on warfarin. Tissue factor-triggered

thrombin generation was used to compare the inhibitory effects of the anticoagulants. Results

will be presented in the late breaking abstract session on Monday.

Pediatric and Neonatal Hemostasis and Thrombosis

20 June 2015 14:30-18:50

Chairman: Anthony K. Chan

Co-Chair: Mariana Bonduel, Leonardo R. Brandao, Elizabeth A. Chalmers, Neil Goldenberg, Shoshana Revel-VilK, Heleen Van Ommen

The focus of the Subcommittee is to address issues in the area of thrombosis and hemostasis in children and neonates by developing clinical standards for evaluation, foster international collaboration in research and clinical trials, establish/maintain registries and to generate, publish and distribute reports and recommendations relating to patient care for the pediatric population. Overall, work within the Subcommittee is done through the Working Groups lead by one of the Co-chairs.

1. Position Papers:

Three position papers have been published:

Recommendations for the development of a dedicated pediatric anticoagulation service: communication from the SSC of the ISTH. Newall F, Jones S, Bauman M, Bruce A, Massicotte MP, Monagle P; Subcommittee on Perinatal and Paediatric Haemostasis. J Thromb Haemost. 2015 Jan;13(1):155-9.

Recommendations for the assessment of non-extremity venous thromboembolism outcomes: communication from the SSC of the ISTH. Rajpurkar M, Sharathkumar A, Williams S, Lau K, Ling SC, Chan AK; Subcommittee on Pediatric/Neonatal Hemostasis and Thrombosis. J Thromb Haemost. 2015 Mar;13(3):477-80.

Recommendations for the development of new anticoagulant drugs for pediatric use: communication from the SSC of the ISTH. Male C, Monagle P, Chan AK, Young G; Subcommittee on Pediatric/Neonatal Hemostasis and Thrombosis. J Thromb Haemost. 2015 Mar;13(3):481-4.

2. Administration:

Two co-chairs (Dr, Elizabeth Chalmers and Dr Mariana Bondel) will step down. Dr Fiona Newall and Dr Christoph Male are nominated to the co-chair positions. The nomination is based on their past contribution to work of the subcommittee and to avoid country or continent over-representation.

3. Ongoing Projects:

Below is the list of ongoing projects with the intention of developing a position statement or a guidance paper (to be determined by the Working Group lead by the Co-chair):

1. Diagnostic criteria for thrombosis in children: (Responsible Co-chair: Leonardo Brandao): Progress Presented at SSC meeting

2. Management of coagulopathy in liver disease. (Responsible person: Paul Monagle working with Maria Magnusson) Progress Presented at SSC meeting

3. VTE prophylaxis. (Responsible co-chair: Neil Goldenberg) Progress Presented at SSC meeting

4. Congenital Severe Purpura Fulminan Registry (Responsible person: Vicky Price , Adrian Minford ) Progress Presented at SSC meeting

5. Management of pulmonary embolism (Responsible co-chair: Neil Goldenberg) 6. Management of arterial thrombosis (Responsible co-chair: Neil Goldenberg working with

Manuela Albisetti) Progress Presented at SSC meeting 7. Antithrombin Registry with a focus on genotype and phenotype correlation ( Responsible

person : Riten Kumar) 8. Guidance paper on Catheter-related thrombosis prevention and treatment in the

pediatric population (Responsible person : Ketan Kulkarni)

Collaborations with other Subcommittees (DIC, Women’s Health Issues in T&H, Hemostasis and Malignancy) have been discussed and with some projects initiated.

1. DIC Survey (initiated) 2. Position statement on Standard and Development of Bleeding Disorder Clinic for

Women (ongoing discussion) 3. Guidance paper on Perinatal Management of hemophilia carrier and baby (ongoing

discussion) 4. Guidance paper Prevention of venous thromboembolism in pediatric cancer patients

(paper in draft form)

Anthony K C Chan Chairman, Pediatric/Neonatal Thrombosis and Hemostasis Subcommittee

Plasma Coagulation Inhibitors

20 June 2015 14:30 – 18:50

Chairman: Richard A. Marlar

Co-Chair: Ian Jennings, Jun Teruya, Hiroko Tsuda

Educational Session: Plasma Coagulation Inhibitors: Relationship of Phenotype, Plasma

Levels and Clinical Phenotype (Thrombosis).

Pieter Reitsma (The Netherlands): Molecular Defects in Protein S

Protein S is a very complex protein. It is a vitamin K-dependent protein significantly different

from the other vitamin K proteins in that it has a unique region that allows the interaction with

C4B bp Binding Protein. This binding interaction creates very complex molecular and

physiological interrelationships, hence difficulty in assessing the plasma levels and determining

the laboratory phenotype of the deficiency. Possible protein S deficient individuals were studied

from the Dutch population using a multitude of genetic methodologies to assess genetic

deficiencies. The majority of deficient patients did have identifiable genetic defects. Genetic

defects span the complete gene and have all genetic types of defects.

Anna Pavlova (Germany): Comparison of Phenotype and Genotype for Antithrombin,

Protein C and Protein S.

Clinical phenotype and laboratory values were compared to the genetic defects in AT, PC and

PS deficient patients. Evidence presented demonstrated that genetic defects were more likely to

be found in patients with lower plasma levels of the factor. In patients with only mildly abnormal

levels the potential for finding a genetic defect was significantly decreased. Patients with 40-

60% plasma levels of AT, PS or PS decreased the potential for detecting defects in only 20-30%

of the patients.

Working Session #1: On-going Projects

Project: Update and maintain genetic mutation databases for AT, PC and PS.

The committee was mandated 6 years ago to update the mutation databases for AT, PC and

PS. Over the last 6 years this has been attempted. However using only volunteer participants

with no funding, there was no significant progress toward the completion of this project. With the

creation of the Subcommittee on Genomic in Thrombosis and Hemostasis, the updating and

maintenance of a database for AT, PC and PS will be transferred to the new Subcommittee on

Genomics.

Project: Investigation into discrepancies in Protein S activity assays results.

The protein S activity assay was has been shown in External Quality Control studies to have

very significant variation among laboratories and these issues extend to patient results as well.

This study will attempt to elucidate the causes of the variation observed in the clinical PS activity

assays. Up to 40 laboratories will participate in this study. The design of the study has been

completed. The difficulty is obtaining the appropriate samples for distribution to the participating

laboratories. Both lyophilized and frozen plasma will be distributed. The results will be analyzed.

It is anticipated to have the data by the next meeting.

Project: Investigation into racial differences in genetic risk factor (AT, PC, PS) for venous

thrombosis.

The design for the study is developed and request and receipt of specimens is on-going. At this

time, the East Asian samples and some European have been received and processed. It is

anticipated that the collection of specimens and the analysis of data will be completed by the

next meeting. Then the complete set of data will be presented.

Project: Manuscript on Guidance for Clinical Testing for AT, PC, PS and APC-Resistance.

Clinical laboratory assessment of plasma levels of AT, PC, PS and APC-R is difficult and is

associated with many problems and pitfalls. The committee is producing four manuscripts on

the state-of-the-art in assessing plasma levels of the common thrombophilic risk factors. The

project leaders for these four manuscripts are:

Antithrombin: Piet Meijer

Protein C: Peter Cooper

Protein S: Richard Marlar

APC-R: Gary Moore and Dorothy Adcock

The manuscripts will be completed by December, 2015 with submission planned for May, 2016.

Working Session 2:

Jun Teruya: Other Plasma Coagulation Inhibitors

The first part of the presentation dealt with all of the plasma inhibitors that might play a role in

the coagulation process.

Heparin Cofactor II data was presented to show the possibility of the clinical relevance of this

inhibitor. Levels of this inhibitor were discussed in both adult and children. This inhibitor appears

to not play a role as a risk factor for the development of thrombosis. It may play roles in other

physiologic systems not related to coagulation.

Thrombomodulin is the cofactor for thrombin activation of protein C. The soluble form of

Thrombomodulin is being investigated as a potential drug for the treatment of sepsis. It appears

that levels of Thrombomodulin may be of benefit for increasing the survival of septic patients.

Since it is not a plasma protein, it is difficult to assess the naturally occurring protein. However

plasma levels will probably need to be assessed if the soluble form is used as treatment of

sepsis.

Tissue Factor Pathway Inhibitor (TFPI) is a regulator of factor VII and factor X. The physiology

and biochemistry of TFPI is complex which may lead to potential bleeding issues. No genetic

deficiencies of this protein have been identified. Acquired decreases and increases have been

seen in a number of clinical conditions. With this assessment, TFPI may play a role in causing

bleeding or thrombotic pathology.

Thrombophilia Testing Schemes:

Thrombophilia testing schemes vary by country and/or region. This session reviewed three

different countries standards for thrombophilia testing schemes (United Kingdom, Japan and the

United States). Testing variations occur for a variety of reasons, including mandated testing or

restricted testing (from insurance companies or national health services), racial differences (no

factor VLeidenor prothrombin 20210 testing in Japan), and clinician driven or testing availability.

The information presented showed that there are multiple approaches to thrombophilia testing

with no unified approach to testing schemes. The criteria for who to test and when the patient

should be tested are not consistent. Further studies into the variability are necessary. Other

countries and regions will also be presented at the next meeting to allow.

Platelet Immunology

20 June 2015 9:00 – 13:20

Chairman: Yves Gruel (France) Co-Chairmen: Donald Arnold (Canada), Tamam Bakchoul (Germany), Sentot Santoso (Germany), Yoshiaki Tomiyama (Japan), Chris Ward (Australia) Wednesday, 25 June (8:00-12:00) Part 1. Top Rated abstract The effects of different B-cell activating factor receptors on lymphocyte function and secretion of cytokines in immune thrombocytopenia. Presenting author: Yanan Min (China) Unfortunately, the author apparently did not attend the meeting and this presentation was therefore cancelled. Part 2. Drug-induced and autoimmune thrombocytopenia 1- During this session, the usefulness of the measurement of immature platelet fraction (IPF) by a Sysmex autoanalyzer for the diagnosis of primary ITP was discussed by Y. Tomyiama (Japan). The preliminary results revealed that XN-1000 showed better CV to measure IPF. 2- T. Bakchoul (coauthor D. Arnold) also discussed the methodological aspects related to the detection of drug-dependent antibodies. Several questions remain to be further defined such as the number of platelets, and the concentrations of drug to be used for testing. In any case, a confirmation by another laboratory is requested to ensure the responsibility of a specific drug in DITP. 3- On the other hand, T. Bakchoul (coauthor J. Fuhrmann) also discussed the optimal conditions to be used when evaluating the pathogenic effect of a platelet antibody using a NOD.SCID mice model. The methods for isolate and inject the platelets as well as the conditions of injection of the antibody have been briefly discussed. 4- Finally, A. Pecci from Italy gave a lecture on the diagnosis of inherited thrombocytopenia and recalled that many affected patients are often misdiagnosed as having ITP. In this regard, he therefore outlined how essential is the questionnaire looking at a familial history of bleeding and low platelet count and the careful analysis of platelet size and morphology. Part 3. : Alloimmune thrombocytopenia The key messages delivered during this session were: 1- The diagnosis of NAIT is confirmed when a genetic paternal/maternal incompatibility for a Human Platelet Antigen is identified and a corresponding antibody is detected in the maternal serum. 2- HPA typing should be hold using at least two methods: phenotyping and genotyping or genotyping using at least two different primers to avoid mistyping due to new variants in HPA.

3- Reference laboratories are able to diagnose only about 30% of all referred suspected cases of NAIT. The following reasons for this diagnostic gap should be taken into consideration: 1) maternal immunization against low frequency HPA antigens; 2) Low avidity antibodies; 3) some "conventional” antibodies (such as HPA-3, -15, -2) are difficult to detect, and 4) HLA antibodies may cause some cases of NAIT. 4- Modified Epitope-specific monoclonal antibodies represent a promising therapeutic approach in the management of FNAIT. While safety has already proven in human, protective efficiency still need to be verified in clinical trial.

Part 4. : Heparin-induced thrombocytopenia The key messages delivered during this session were: 1- The team of Y. Gruel (France) has developed 5B9, a new monoclonal antibody (MoAb) to PF4/heparin complexes with a human Fc fragment that fully mimics human HIT antibodies. A comparative evaluation of this antibody with 5A1, another MoAb developed in Japan and that is also a potential standard for HIT diagnosis assays showed that 5B9 was the only one to activate platelet in a heparin-dependent manner. 2- The performances of flow cytometric analysis of platelet activation for the diagnosis of HIT has also been evaluated in France in a large cohort of patients (B Tardy). Combined with and IgG-specific assay, this approach appeared as sensitive and specific than SRA. 3- The use of Platelet Microparticle Generation Assay for the diagnosis of HIT has also been discussed by V. Minet (Belgium) 4- Finally, C. Ward presented an update on the practical use of whole blood impedance assay for HIT diagnosis, but this procedure has to be prospectively evaluated.

Final discussion and perspectives Based on the presentations and the discussion of this SSC meeting, the following projects are planned for the next future. The first is to prepare an official communication of the SSC related to the laboratory testing in patients with suspected drug-induced thrombocytopenia (DITP). The second project is about to be finalized by Tamam Bakchoul (Greifswald, Germany) and will propose recommendations on the use of NOD/SCID mouse as a model for studying the pathogenesis of immune thrombocytopenia. The third ongoing project under the coordination of Chris Ward (Sydney, Australia) aims to propose a standardized procedure about the use of the whole blood impedance aggregometry (using the Multiplate analyzer) for the diagnosis of HIT. Finally, Y Gruel will propose in the next week a study coordinated by the SSC aiming to evaluate the use of 5B9 as a standard in HIT immunoassays.

Platelet Physiology

21 June 2015 8:00 – 12:20

Chairman: Paolo Gresele

Co-Chairs: Hans Deckmyn, Andrew L. Frelinger III, Paul Harrison, Diego Mezzano, Andrew D. Mumford, Patrizia Noris

The Platelet Physiology SSC Session included two 1 hour Educational Sessions, one of which was a joint session with the Biorheology SSC, and a 3 hour business SSC session.

The first educational, Saturday June 20th 2015 at 14:30-15:30, Room 716, was on “Bioreactors

for the study of the biophysical mechanisms that regulate platelet function?”

Attendance was good, scientific level of the talks excellent, and discussion from the audience

active and participated. Further details are reported in the minutes from Keith Neeves, chairman

of the Biorheology SSC.

The second educational session, Sunday, June 21st 2015, 10:00-11:00, Room 801, was on

“Bioreactors for megakaryocyte studies and platelet formation: Where do we stand?”

Jonathan Thon (United States) reviewed the evolution and state-of-the-art in the field of platelet

production bioreactors. The focus was on outlining the challenges and needs for translating recent

discoveries and devices (including his work on microfluidic reactors) into large-scale platelet

production.

Alessandra Balduini (Italy) reviewed the evolution and state-of-the-art in the field of

megakaryocyte studies in bioreactors. The focus was on recent advances in bioengineering and

biomaterials (including her work on silk derived materials), phenotyping platelet produced in these

bioreactors and applications including studying diseases (myeloproliferative disorders), drug

efficacy and drug testing.

Sunday, June 21st 2015, 8:00-12:20, Room 801

The subcommittee session started at 8:00 and was introduced by Paolo Gresele (Italy) who gave

an overview of the mission statement of the ISTH-SSC and of the specific mandate of the Platelet

Physiology SSC. The subcommittee web page was shown and audience encouraged to register

as members. A list of the concluded projects and relative publications was given (Diagnosis of

suspected inherited platelet function disorders: results of a worldwide survey - J Thromb Haemost

2014;12:1562-9; Diagnosis of inherited platelet function disorders: guidance from the SSC of the

ISTH - J Thromb Haemost 2015;13:314-22; A review of platelet secretion assays for the diagnosis

of inherited platelet secretion disorders - Thromb Haemost 2015 Epub ahead of print).

Ongoing projects and projects about to be started were listed:

Evaluation of the Bleeding Assessment Tool (BAT) for the assessment of inherited platelet function disorders (P. Gresele, P. Harrison – enrolling)

Consensus/guidance on the methods for the study of platelet secretion (D. Mezzano – work in progress)

Laboratory monitoring of P2Y12 inhibitors: a position statement (A. Frelinger – work in progress)

Generation of guidance on the measurement of platelet dimensions: methods and clinical use (P. Noris – work in progress)

Prospective evaluation of the bleeding phenotype in PT-VWD to support evidence-based diagnosis and management (M. Othman – work in progress).

Generation of guidance on the use of platelets in regenerative medicine (P. Harrison – to be started)

Validation of the diagnostic algorithm for the diagnosis of inherited platelet function disorders (P. Gresele, A. Mumford – to be started).

Paolo Gresele (Italy) then presented the preliminary, provisional results of the ongoing “Evaluation

of the Bleeding Assessment Tool (BAT) for the assessment of inherited platelet disorders” study.

The data obtained in 243 patients so far enrolled show that inherited platelet function disorders

seem to have a higher bleeding score than von Willebrand disease type 1. The data raised interest

and a call to participate in the study was made.

Diego Mezzano (Chile) then presented the ongoing project to produce a position statement on

the methods for the study of platelet secretion for the diagnosis of platelet function disorders. A

questionnaire on the evaluation of the currently available methods, to be circulated to a number

of experts outside the SSC in order to produce a consensus statement based on the RAND

methodology, was presented.

Andrew L. Frelinger III (United States) then presented a draft position statement on the laboratory

monitoring of P2Y12 inhibitors. This was preliminary discussed among the SSC members.

Literature data show that in ACS patients on dual antiplatelet therapy high on-treatment platelet

reactivity (HPR) is associated with a greater risk for ischemic outcomes while low on-treatment

platelet reactivity (LPR) with a greater risk for bleeding events, raising the possibility that altering

antiplatelet therapy based on P2Y12 monitoring would reduce ischemic and bleeding events.

Small randomized and non-randomized cohort studies demonstrated a reduction in ischemic

events when P2Y12 inhibitor therapy was altered on the basis of platelet function testing (guided

therapy). Larger randomized controlled trials, using different platelet function tests and different

therapeutic strategies, demonstrated no difference in ischemic events with vs. without guided

therapy. However, whether patient selection, choice of test, timing of test, test cut-off, and

treatment strategy in these trials was optimal has been questioned. Thus, the utility of

P2Y12 monitoring to guide antiplatelet therapy is unclear. In contrast, the limited available data

suggests that P2Y12monitoring, because of its high negative predictive value, may be useful to

identify patients who can undergo operation without elevated bleeding risk less than 5 days after

discontinuation of antiplatelet medications. Interesting discussion from the audience on the

relative weight to be given to small vs large-size guided therapy studies and about the arguments

in favor and against the questions raised on the design of the negative, large-size, guided therapy

studies took place during the session.

Maha Othman (Canada) then shortly presented the progress of the project “Prospective

evaluation of the bleeding phenotype in PT-VWD to support evidence-based diagnosis and

management”. She highlighted the importance of continuing to study PT-VWD among rare

platelet function defects and provided a brief review of current knowledge and update on the

registry. So far only 20 responses, most incomplete, were submitted. These provisional data

suggest an under-reporting of the disease worldwide; prospective information about PT-VWD

is still missing; physicians/specialists worldwide are encouraged to report information to ISTH via

the survey; survey will remain open for participation.

Patrizia Noris (Italy) gave an overview on “The measurement of platelet dimensions: Methods and

clinical use”; this will serve as the basis for the generation of a position statement on the methods

and design of clinical studies for the measurement of platelet dimensions in disease.

Finally, a talk on RNA signatures in platelets from cancer patients was given by Thomas

Wurdinger (The Netherlands) showing that platelets take up RNAs, from cancer cells that can be

measured as a reliable biomarker of tumor presence and activity in man. This subject may

represent a topic for a possible future project.

In the second part of the session, starting at 11:20 after the joint Educational session with the

Biorheology SSC, one topic, i.e. the use of platelets in regenerative medicine, was dealt with by

Harald Langer (Germany), who spoke about the pathophysiological bases of the participation of

platelets in tissue regeneration, and by Paul Harrison (United Kingdom), who discussed the state

of the art of the clinical use of platelets in regenerative medicine, underlying the lack of

standardization in the platelet preparations used and in the design of clinical trials. He also

presented the new “Platelet Rich Plasma in Achilles tendon healing” clinical study (PATH-2 trial)

he is coordinating together with Keith Willett (United Kingdom). These overviews will serve as a

basis to generate a position statement from the Platelet Physiology SSC on the use of platelets

in regenerative medicine: methods of platelet preparation and design of clinical studies.

Attendance to the session was reasonably good, ranging from approximately 150 to 350

attendees in the different phases of the SSC meeting. Discussion from the audience was active

and lively. The room session facilities were insufficient (position of the chairmen rendering

impossible to see the projection screen and lack of a monitor on the table to follow the

presentations, lack of a timer to check for adherence to the allotted times).

Predictive and Diagnostic Variables in Thrombotic Disease

20 June 2015

9:00 – 13:20

Chairman: Paul A. Kyrle

Co-Chairs: Suzanne Cannegieter, Shinya Goto, Karel Moons, Marc C. Samama, Alex C. Spyropoulos

Welcome, information on the new scope of the subcommittee, introduction of co-chairpersons and update on ongoing projects (P Kyrle)

Diagnostic variables in thrombotic disease

Chairmen: G Le Gal, P Kyrle

Dr. Geersing addressed the importance of a personalized approach to VTE diagnosis and

treatment. According to interviews in nursing homes, the majority of patients presenting with

symptoms highly suspicious for VTE do not undergo objective testing. Many patients then

receive anticoagulant treatment without VTE verification. The reason could very well be the

reluctance of doctors and elderly patients to be admitted to a hospital or to undergo

cumbersome diagnostic strategies. Dr. Geersing stressed the importance of age-adjusted D-

Dimer cut-offs and the availability of mobile ultrasound equipment that makes investigating the

patient on an out-patient basis easier.

Dr. Klok addressed issues regarding diagnosis of recurrent PE and DVT. In these instances D-

Dimer is less sensitive as compared when diagnosing a first event. There are several studies

successfully ruling out recurrent PE by algorithms including D-Dimer measuring. Therefore, a

first and a recurrent PE can be managed equally. As regards diagnosis of recurrent DVT, CUS

is less reliably and a definite diagnosis is possible in only approx. 70% of patients. MRI could be

an alternative, but it is not readily available in many hospitals and outcome studies are lacking.

The next 2 presentations were devoted to the diagnosis of “small clots”: subsegmental PE and

distal DVT. Dr. Carrier pointed out that the diagnosis of PE became more frequent over the last

years whereas PE- associated mortality remained the same. This might indicate that with the

introduction of multi-detector CTPA more PEs of unclear clinical significance are diagnosed.

There is no agreement among experts whether to treat SSPE or not and there is now an

ongoing study in France and Canada in which patients with SSPE and a negative CUS are left

untreated. A similar situation exists for distal DVT, which was covered by Dr. Righini. Studies

showing a similar frequency of recurrent VTE when the whole leg is investigated by

ultrasonography as compared to studies investigating only the proximal veins indicate, that

distal DVT might be of little clinical importance and possibly does not require anticoagulant

treatment

At the end of this session Drs. Kearon, Klok, Stevens, Di Nisio and Righini presented planned or

ongoing clinical studies: D-Dimer and diagnosis of VTE (4D, Peged), MRI to rule out recurrent

DVT (THEIA), CUS in pregnancy (CLOT3), an age-adjusted D-Dimer cutoff to rule out DVT

(ADJUST-DVT), and D-Dimer together with a modified Wells score to diagnose VTE in cancer

patients. All presentations were followed by a lively discussion.

Coffee break (20 minutes)

Education session

Chairmen: MC Samama, AC Spyropoulos

Dr. Cannegieter eluded first on the important distinction between prediction and cause of a

disease, explained confounding and bias in clinical studies and then addressed differences

between RCTs and observational studies with regard to strengths and limitations.

Dr. Moons addressed the issue of clinical prediction models, the importance to use existing data

rather than generating new ones, and how to proceed with a prediction model until it is ready for

use in clinical practice.

Predictive variables in thrombotic disease

Chairpersons: S Cannegieter, S Ghoto

Dr. Cabrera-Fuentes discussed the role of extracellular nucleic acids as potential biomarkers for

venous thrombosis. From his presentation it became evident that studies regarding its clinical

applicability are still missing although there is considerable progress in understanding the

interaction of extracellular DNA with the coagulation system.

Dr. Spyorpoulos presented a new statistical method for planning and analyzing clinical trials

which consists of combining bleeding and thrombosis as a common endpoint. This would allow

a better data interpretation and would also facilitate the conduct of clinical trials because of a

lower number of patients required.

Dr. Samama addressed the observation that over the last years due to improved surgical and

anaestheological techniques the duration of many operations has become shorter and some of

them can now be performed on a day care basis. This means that the risk of post-interventional

VTE becomes lower and in some circumstances thromboprophylaxis may no longer be required

or should be shortened. Studies addressing shorter durations of thromboprophylaxis or even

questioning the need of thrombosis prevention are needed.

Closing remarks by P Kyrle once again stressing the new scope of the subcommittee covering

now both predictive as well as diagnostic variables in arterial and thrombotic disease.

End of meeting: 13:30

Vascular Biology

20 June 2015 9:00 – 13:20

Chairman: Rienk Nieuwland

Co-Chairs: Alexander Brill, Elizabeth E. Gardiner, Chris Gardiner, Anna Randi, Florence Sabatier, Pia R. Siljander

Circulating endothelial progenitor cells; chairs Anna Randi (UK) and Florence Sabatier (France) From VSELs to endothelial progenitor cells: What is the “perfect” vasculogenic cell? David Smadja (France) In the setting of cardiovascular disease, the vascular compartment undergoes complex molecular and cellular changes that determine the range of perfusion recovery within the ischemic tissue. Thus, stimulation of vessel growth and/or remodeling has emerged as a new therapeutic option in patients with ischemic diseases. In particular, strategies based on the administration of endothelial progenitor cells (EPCs), or cell population thought to contain these vascular progenitor cells, have been shown to augment neovascularization in experimental models of ischemia and in patients with cardiovascular ischemic diseases. Several methods to obtain a better way to isolate and expand them have been developed. However, cardiovascular risk factors constitute an inhibitory environment and induce an impairment of their vasculogenic cell functions. Moreover, precise origin and identity of EPCs are controversial. Very small embryonic-like stem cells (VSELs) are multi-potent stem cells localized in adult bone marrow (BM) that may be mobilized into peripheral blood (PB) in response to tissue injury. We aimed to quantify VSELs in BM and PB of patients with critical limb ischemia (CLI) and to test their angiogenic potential in vitro as well as their therapeutic capacity in mouse model of CLI. We quantified VSELs in BM and PB from CLI patients compared to healthy controls. Our results suggest that ischemia may trigger VSELs mobilization in this patient population. Sorted BM-VSELs cultured in angiogenic media acquired a mesenchymal phenotype (CD90+, Thy-1 gene positive expression). VSEL-derived cells had a pattern of secretion similar to that of endothelial progenitor cells, as they released low levels of VEGF-A and inflammatory cytokines. Noteworthy, VSELs triggered post-ischemic revascularization in immune-deficient mice (p< 0.05 vs PBS treatment), and acquired an endothelial phenotype either in vitro when cultured in the presence of VEGF-B (Cdh-5 gene positive expression), or in vivo in Matrigel implants (human CD31+ staining in neo-vessels from plug sections). In conclusion, VSELs are a potential new source of therapeutic cells that may give rise to cells of the endothelial lineage in humans. Given the potential usefulness of a cell therapy product, real ontogeny of vascular cells, their thorough characterization as well as a deep understanding of the molecular and cellular mechanisms involved in their pro-angiogenic capacities is of major importance. Blood outgrowth endothelial cells offer novel insights into von Willebrand disease. Richard Starke (UK) Blood out growth endothelial cells (BOEC) are endothelial cells that can be derived from circulating progenitors in blood. BOEC provide access to the normally inaccessible endothelium of patients. We have extensive experience in the isolation, expansion and culture of BOEC from

a variety of patients with vascular disorders, including Von Willebrand disease (VWD). VWD describes a deficiency or dysfunction of VWF in blood. Although there is some heterogeneity, BOEC from type 1 VWD patients confirmed the long-held belief that type 1 VWD is due to quantitative defects in the ability of the endothelium to produce VWF. VWF synthesis and release from type 2 VWD patients was mostly normal supporting the belief that this subtype of VWD is mainly due to dysfunctional VWF. Type 3 VWD patients are defined by the absence of plasma VWF and we demonstrated a similar absence of VWF in a type 3 patient’s BOEC. Although a good consistency is seen between repeat isolations from the same donor, some patients with the same genotype show variations in endothelial VWF production consistent with their different plasma VWF levels, suggesting that VWF production by BOEC from these patients is influenced by factors in addition to the mutation. In conclusion, BOEC can provide novel insight into the cellular biology of VWD and could be useful in the testing of novel treatment approaches on the patients’ endothelium prior to their use in the patient. Derivation of endothelial colony forming cells from pluripotent stem cells. Nattan Prasain (US) Since vascular endothelial growth factor ligand (VEGF)/receptor 2 (KDR) signaling pathway is critical in the emergence of endothelial cells during development, we have developed a novel pluripotent stem (PS) cell differentiation protocol and found neuropilin-1 (NRP-1), a VEGF co-receptor, as an early marker for identifying the emergence of ECFCs. Compared to other sub-sets of sorted cells, only NRP-1+CD31+ cells exhibited umbilical cord blood (CB) ECFC-like properties and produced a clinically relevant number of cells. We identified NRP-1+CD31+ selected cells that displayed a stable endothelial phenotype exhibiting high clonal proliferative potential, formation of human vessels that inosculated with host vasculature in vivo, and made significant contributions to vascular repair of the ischaemic limb, but lacked teratoma formation potential in vivo. This protocol advances the field by generating highly replicative but stable endothelial cells for use as a potential cell therapy for human clinical disorders. Receptor shedding from platelets in the inflamed vasculature; chairs Elizabeth Gardiner (Australia) and Alexander Brill (UK) Platelets and the inflamed vasculature. Paul Kubes (Canada) Paul gave an elegant presentation showcasing State-of-Art imaging approaches to study the role of platelets in inflammation responses. Whilst platelets are traditionally recognized for their central role in hemostasis, numerous studies now highlight how platelets can be potent immune modulators and effectors. Platelets directly recognize, sequester and kill pathogens to activate and recruit leukocytes to sites of infection and inflammation, and to modulate leukocyte behavior, enhancing their ability to phagocytose and kill pathogens. Paul highlighted a ‘touch and go’ surveillance property of platelets in liver and then demonstrated first responder roles for platelet chemokines and chemotactic gradients in mice exposed to Staphylococcus aureus where the majority of bacteria were sequestered immediately by hepatic Kupffer cells, resulting in a robust neutrophil infiltration into the liver. A marginal zone proximal to these Kupffer cells and devoid of neutrophils was described as a “necrotactic” zone. This zone displayed severe blood vessel constriction and platelet aggregation. Future work will more fully describe the molecular properties of this zone. Mechanistic insights into platelet receptor shedding. Yotis Senis (UK) Yotis provided an update on signaling properties of platelets from mice deficient in G6b-B. Mice lacking this ITIM-containing receptor exhibit macrothrombocytopenia and aberrant platelet function. The loss of response to collagen was explained by an enhanced ADAMs mediated shedding of the platelet collagen receptor GPVI. Yotis was able to demonstrate enhanced

phosphorylation of protein-tyrosine kinase Syk (a key component of platelet activation signaling pathways) in these mice and could rescue significant platelet function by crossing G6b-B deficient mice with mice bearing an arginine to alanine point mutation within Syk which modulates Syk phosphorylation events, possibly reducing the pathway that activates ADAMs-mediated removal of GPVI. Yotis also outlined some opportunities to unify and standardize protocols that measure platelet biomarkers of activation, in particular focusing on the unique platelet/megakaryocyte receptor GPVI. Glycoprotein Iba shedding and platelet clearance. Renhao Li (US) Renhao described some of the unique metalloproteinase blocking properties of an anti-GPIb antibody that recognizes the ADAM17 cleavage sequence within GPIbα. Clone 5G6, and its monomeric Fab fragment specifically bound purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα, but did not affect platelet activation and aggregation. 5G6 blocked GPIbα shedding induced by calmodulin inhibition or mitochondrial ageing reagent CCCP with similar potency to broad shedding inhibitor GM6001 in human platelets. 5G6 does not recognize mouse GPIbα or inhibit shedding of other platelet receptors. 5G6 was shown to have utility in increasing the longevity of platelets stored for transfusion in bags, by reducing GPIbα metalloproteolysis observed as part of the platelet storage lesion. This new focus of research is significant as it could potentially extend the life of platelet transfusion bags beyond 4-5 days, and enhance platelet function in people in receipt of platelet transfusion. Microparticles and standardization; chairs Pia Siljander (Finland) and Chris Gardiner (UK) Vesicle size approximation enables inter-laboratory comparison of vesicle measurements by flow cytometry. Rienk Nieuwland (Netherlands) Measuring single extracellular vesicles by flow cytometry is a challenge. Vesicles scatter light, which is detected by flow cytometry and expressed in arbitrary units. These light scattering signals / arbitrary units are incomparable between instruments, and interpretation is hampered by differences in sensitivity, the unknown refractive index of vesicles, etc. To standardize these measurements, we (1) selected traceable reference materials (www.metves.eu), (2) developed a method to determine the refractive index of single particles < 500 nm diameter in suspension (Nano Letters 2014, E. van der Pol et al.), (3) developed reference materials and software to correct for differences in optical configurations of commercially available flow cytometers (www.exometry.com), (4) distributed reference materials, software and biological samples to 33 laboratories worldwide, (5) determined the vesicle size gate by measuring reference materials, and (6) measured and compared vesicle concentrations. Overall, 34% of included flow cytometers was too insensitive to measure any vesicles in the largest size gate (1,200-3,000 nm). In addition, marked differences in sensitivity are observed between similar instruments (brand, type) in different laboratories. Finally, a comparison of vesicle measurements in the AMC on 4 different instruments results in a CV of 20% for the largest size gate. Taken together, beads and software have been developed to set vesicle size gates in absolute units (nm) on most types of flow cytometers, the CV is improved compared to previous strategies, and 33% of included flow cytometers are too insensitive to detect vesicles. Importantly, further standardization of vesicle measurements will be done also in collaboration with ISEV (www.isev.org) and ISAC (www.isac-net.org). See also www.evflowcytometry.org for additional information. The goal of this collaboration is to develop standards and reference materials, protocols, rules for minimal requirements, education, etc. At present, a follow-up of METVES (www.metves.eu), entitled METVESII, is being written and Letters of Support are needed. Additional information is available at http://msu.euramet.org/health_2015/SRTs/SRT-h03.pdf and please contact Rienk Nieuwland (r.nieuwland@amc.uva.nl) for questions.

Studies of the microparticle cargo and its functional components. Eric Boilard (Canada) Platelets are anucleated blood element highly potent at generating extracellular vesicles (EV) called microparticles (MPs). Whereas EVs appear as an important means of intercellular communication, the complexity of the platelet MP cargo is starting to be delineated. Studies show that platelet MPs contain functional enzymes and a broad repertoire of nucleic acid (e.g. microRNA). A subset of platelet MPs also bears organelles, such as respiratory-competent mitochondria. Including specific mitochondrial markers for MP assessment thus permits to reveal the diversity of platelet MPs. Furthermore, mitochondria are thought to originate from the endosymbiosis of the Alphaproteobacterium Rickettsia prowazekii during the development of eukaryotic cells. As mitochondria are released concomitantly with MPs from activated platelets, they can trigger highly potent pro-inflammatory signals. The platelet MP cargo is thus complex, and its contribution to physio(patho)logical conditions must be considered. Progress of endothelial microparticle detection and characterization by flow cytometry. Romaric Lacroix (France) Endothelial-derived microparticles (EMPs) are a useful marker of endothelium injury in vascular disorders. However, measuring EMP by flow cytometry remains a challenge, which explains the diversity of (CD) markers used in the literature for identification. Therefore the objective of the presented work was to evaluate the capacity of antibodies used in the literature to detect EMPs. Several clones of antibody were selected for each specificity, and their detection capacity was evaluated on EMPs from primary endothelial cells of different territories both in unstimulated and inflammatory conditions. This work showed that among published markers CD31 (PECAM), CD54 (ICAM1) and CD146 have the best capture efficiency by immune-magnetic separation and detection capacity by flow cytometry to detect EMPs. Interestingly, despite the expression on leukocytes, these markers were not detected on leukocyte-derived MPs in the conditions of the study. It was also demonstrated that combining markers is an interesting approach to improve the signal to noise ratio, the repeatability and lowered the detection limit of the EMP detection. Finally, a strategy was proposed to evaluate and select antibodies for the different subpopulation of MPs whose performances cannot be extrapolated from cellular data.

von Willebrand Factor

20 June 2015 14:30 – 18:50

Chairman: Jorge A. DiPaola

Co-Chair: Sandra Haberichter, Daniel J. Hampshire, Koichi Kokame, Johanna Kremer Hovinga, Frank W. Leebeek, Alberto Tosetto

1) EDUCATION SESSION

Peter Lenting, PhD (France)

Factor VIII and VWF, rekindling a longstanding relationship

In his presentation Dr. Lenting discussed the cellular origin of FVIII and VWF. Interestingly

DDAVP response may be independent from the liver pool. He also presented the controversy of

the requirement of FVIII for ADAMTS 13 cleavage. He also presented the overexpression of

FVIII in a wild type mouse that shows that the FVIII/VWF molar ratio >6 is associated with

bleeding. Discussed clearance. About 95% of FVIII is bound to VWF. Knowing VWF, VWF pp

and blood group you can calculate the half-life of infused FVIII in hemophiliacs. LRP 1 mediates

clearance of both FVIII and VWF. Other receptors also do. And most of them bind both VWF

and FVIII. This information in the future may help to improve the half-life of FVIII. Interestingly,

pegylated VWF had a 4-5 X longer half-life.

Johanna Kremer Hovinga, MD (Switzerland)

ADAMTS13 deficiency, TTP and beyond

Dr. Kremer Hovinga discussed pathophysiology of TTP and its diagnostic criteria. She pointed

out that the dramatic increased in survival with plasmapheresis. Clinical cases were presented

and different clinical situations associated with ADAMTS13 deficiency. Also carriers of

mutations in ADAMTS13 (heterozygote state) might present with AHA associated with clinical

situations of stress (pregnancy, sepsis, etc.). The relapse rate appears to be less in Rituximab

treated patients. Long-term survivors appear to have increased hypertension, depression and

autoimmune diseases. This long-term chronicity might be associated with the presence of

immune complexes of ADAMTS13/ADAMTS13Ab. These immune complexes may persist up to

6 years. Spleen from long term chronic patients show that specific memory cells can generate

anti ADAMTS13 Ab.

2) JOINT BIORHEOLOGY & VWF SESSION ON ACQUIRED VON WILLEBRAND

SYNDROME

David Lillicrap (Canada)

Flow mediated interactions of VWF and ADAMTS13: shear ecstasy

The interaction and cleavage of VWF by ADAMTS13 requires unfolding of the substrate

(VWF). In vitro flow studies show that VWF and ADAMTS13 interact increasingly as a thrombus

grows and the diameter of the remaining lumen gets smaller - resulting in increased flow. In

addition, tensile forces on VWF are further influenced by VWF ligands including platelet

GpIb, GpIIb/IIIa and P-selectin. Each of these ligands can contribute to the unfolding of VWF

and influence interactions and subsequent cleavage by ADAMTS13.

Barbara Zieger (Germany)

Acquired von Willebrand syndrome in patients with VAD or ECMO

Dr. Zieger emphasized the expansion of Ventricular Assist Devices (VADs) over the last

decades. They are not only a transition form of therapy but are becoming more of a destination

therapy; therefore all complications may become more relevant. Dr. Zieger discussed the 2

types of pumps in the market (centrifugal and axial) in continuous flow VAD. She also discussed

anticoagulation and the unusual rate of bleeding seen in patients likely due to AVWS with VADs.

It is also possible that hemolysis and free hemoglobin also influences the rate of AVWS. It is

important to mention that almost all recipients of VAD (>90%) have lower ratios of

VWF:CB/VWF:Ag and ratio of VWF:RCo/VWF:Ag, respectively, but not all of them bleed

clinically. The ratio of VWF:CB/VWF:Ag (in-house test with collagen type 1) was more sensitive

to the loss of HMW multimers than the ratio of VWF:RCo/VWF:Ag. She discussed ECMO and

the high incidence of AVWS in this modality. She also emphasized the poor correlation between

VWF levels and bleeding suggesting that many other factors may contribute to the bleeding

observed. In addition, she showed that patients with VAD or ECMO develop thrombocytopenia

and thrombocytopathy.

David Schmidtke (USA)

Effects of high shear millisecond exposure on platelets

The objective of this study was to replicate the VAD-induced shear-rate in a microfluidic device

and investigate the effect of the shear on platelet receptors, platelet adhesion and

aggregation. Platelet adhesion and platelet aggregate formation on collagen-patterned surfaces

upstream and downstream of a transient (1-50 msec) exposure to high shear (40,000 - 100,000

s-1) were quantified. We observed that a single exposure to high shear was enough to inhibit

platelet aggregation while platelet adhesion was maintained. The defect in platelet aggregation

was dependent upon both the shear rate and exposure time. Treatment of blood with an

inhibitor to ADAMTS13 prior to the high shear exposure restored normal platelet aggregation

downstream suggesting a role of VWF in the downstream aggregation.

After these three presentations discussion ensued and several members of the SSC proposed

an AWS working group with members of the VWF and Biorheology subcommittees. The main

goal of this working group will be to study and standardize AVWS in an attempt to understand

better the presentation of disease and the mechanisms of disease as well as the factors that

contribute to bleeding. Drs. Barbara Zieger, Augusto Federici and David Schmidtke will lead the

working group. Several members of the audience expressed interest in participating.

3) Update of VWF functional assays

Sandy Haberichter (USA)

Update on VWF functional assays

Dr. Haberichter presented the current nomenclature for activity assays recently published in the

Journal of Thrombosis and Haemostasis as an SSC communication (Bodo et al.). She

discussed advantages and disadvantages of this nomenclature. One of the advantages is that

some of the assays prevent the issues raised by genetic variants and their influence on

ristocetin based assays. New automated techniques show higher precision than the ones

previously used. Still she emphasized the fact that standardization is needed. In the clinical

laboratory not all the tests that report VWF activity are the same. She mentioned that influence

of collagen binding discusses specifically 1-3 and 4-6 including the fact that some genetic

variants appear to exhibit lower collagen binding.

Koichi Kokame (Japan)

Update on ADAMTS13 functional assays

Summarized 3 studies on ADAMTS13 assays including the Italian and Swiss studies. He

emphasized that this is an ideal time to perform a new multicenter study on ADAMTS13 assays

since many assays are being used. The WHO 1st ADAMTS13 standard is ready to be used;

however other studies are needed since for example it is unclear the role of immune complexes

on these assays. A new standardization sample study was proposed. Dr. Federici suggested

the use of lyophilized plasma to decrease cost. Also Dr. Leebeek proposed the use of antigen

and activity.

4) Development and validation of assessment tools for clinical trials in VWD

Paula James (Canada)

Dr. James presented the evolution of the BATs over the last decade. She discussed a newer

and shorter version of what is called a “self-BAT” which is administered to people (in lay

language) and compared to the ISTH BAT in a crossover study. Actually individuals received

the questionnaire and self-administer it. There was excellent agreement between scores.

Normal ranges were determined and those were in agreement with the ISTH-BAT. It was also

tested for VWD screening. NPV was 100% for women but lower for men. Carriers of hemophilia

were tested and the NPV was 0.75. Dr. Leebeek emphasized the issue of quality of life and

correlation with the bleeding score.

Alberto Tosetto (Italy)

There was a proposal by Dr. Tosetto for the formation of a new working group on the

standardization of diagnostic criteria of VWD. This will attempt to (re) define VWD since the last

communication on VWD classification from the SSC dates back to 2005. He argues that several

advances in the field including more comprehensive genetics, better assays and the use of

bleeding scores have changed the way we categorize the disease and therefore we might be

ready for a new classification. The working group will spend 2 years attempting this task with the

intention of reporting to the SSC in Berlin in 2017. The use of bleeding assessment tools

emerged 10 years ago, but the considerable amount of diagnostic and prognostic data available

have never translated into clinical guidelines. As a first step he proposed the preparation and

distribution of a first survey as an SSC activity in the next months.

5) Studying VWF under flow

Maria Brehm (Germany)

Studying VWF mutations under flow

Dr. Brehm discussed VWF mutants she studied under flow. Most of these mutations have been

traditionally associated with VWD 2A. Dr. Brehm showed the intracellular processing of these

mutants by immunofluorescence. Then she studied these mutants under flow. The shear rate

for most of the experiments was between 500s -1 and 10000s-1 on a VWF surface. The onset of

string formation occurs at 2500s -1. Dr. Brehm showed three different phenotypes of platelet

adhesion/aggregation for the specific mutations. She finally emphasized on the importance of

studying VWF under shear flow conditions.

Chris Ng (USA)

Studying VWD under flow

Dr. Ng described the use of Microfluidic (MF) devices to study platelet adhesion under flow in

individuals with VWD. He presented data from approximately 40 individuals either diagnosed

with VWD or with mucocutaneous bleeding. A microfluidic device was used in a cohort of

individuals with von Willebrand disease and mucocutaneous bleeding to determine if it could

better characterize VWD/mucocutaneous bleeding in the context of currently available clinical

labs and potentially predict clinical bleeding. This quantitative outputs from this device correlate

with common laboratory assays and demonstrates a more linear relationship with VWF:Ag and

VWF:RCo than the PFA100 in all patients and those with Type 1 VWD. Output of this device did

not correlate with clinical bleeding as measured by the ISTH Bleeding assessment score.

6) Visualizing VWF

Walter Kahr (Canada)

VWF in megakaryocytes

Dr. Kahr focused his presentation on VWF in megakaryocytes. First he discussed the formation

of Weibel Palade bodies containing VWF in endothelial cells. He then mentioned the unique

characteristics of megakaryocyte (MK) and platelet alpha granules when compared to Weibel

Palade bodies. He explained that 20% of plasma VWF originates from platelets, that there is no

uptake of plasma VWF into MK or platelets, that platelet VWF is characterized by enrichment of

HMWM, and that platelet VWF has altered VWF glycosylation. He went ahead and discussed

several molecules involved in alpha granule formation including VPS33B, VPS16B and

NBEAL2. He presented a Nbeal2-/- mouse model where VWF instead of being packaged into

developing alpha granules in MKs, is being externalized to the outside. Finally he showed the

results of a collaboration with Dr. P. James of a patient with type 3 VWD containing a

homozygous frameshift mutation at position c.8418_8419 resulting from a TCCC insertion

therewith adding 17 amino acids to the C-terminal of VWF. This patient had no VWF in their

plasma but did have VWF in their platelets, where using high resolution confocal IF microscopy

the VWF was at the periphery of the platelets instead of being localized in platelet alpha

granules.

Simon Webster (United Kingdom)

Optimization of immunocytochemical staining and high-resolution imaging of VWF in

HEK293 cells

Simon Webster discussed the issues he had previously trying to get HEK293 cells to make WPB

when we transfected them with VWF. This was optimized by reducing the amount of DNA used

for transfections, increasing the post transfection culture time and using different batches of

HEK293 cells. Fluorescent microscopy imaging was improved initially by using a widefield

deconvolution microscope. He then presented recent imaging using a super resolution Structured

Illumination Microscope (SIM), which was able to resolve WPB membranes and show distinct

Rab27a localization. He aimed to highlight the importance of generating high quality images in

order to make any useful comparisons between wild-type and mutant VWF biosynthesis and

storage.

Jeroen Eikenboom (the Netherlands)

Visualizing VWF exocytosis

Dr. Eikenboom described new studies using correlative light and volume scanning electron

microscopy to determine the budding of Weibel Palade bodies from the Golgi network. Multiple

connections of the WP bodies with the Golgi were identified facilitating content delivery indicating

that the Golgi apparatus may provide a framework that determine size and content of WP bodies.

Women's Health Issues in Thrombosis and Hemostasis

20 June 2015 9:00 – 13:20

Chairman: Rezan A. Abdul-Kadir

Co-Chair: Claudia Chi, Hannah Cohen, Ida Martinelli, Saskia Middeldorp, Maha Othman, Rochelle Winikoff

Welcome and introduction (Rezan Abdul-Kadir)

Rezan Abdul-Kadir opened the session and welcomed the attendees, new co-chairs and members. Rezan Abdul-Kadir presented history of women subcommittee and reported that the subcommittee membership increased over the last year from 26 members to 138. Rezan Abdul-Kadir also summarized the Mandate of the subcommittee; produce guidance, promote collaboration, identify gaps in knowledge. Rezan Abdul-Kadir encouraged further participation.

Benjamin Brenner gave a report on WHITH Berlin 2015 summarizing number of delegates and range of topics spoken on. Tributes to Victor Marden and Meyer Michel Samama. Invited all to attend next WHITH meeting in February 2017.

Updates on registries and projects (Moderated by Saskia Middeldorp and Maha Othman)

DOAC and pregnancy (Saskia Middeldorp) - Registry was first presented to ISTH in Milwaukee 2014. Protocol has been written over past year and steering committee appointed. Guidance document currently being drafted. Question of can we reassure women who conceive whilst taking DOAC? Outline of web-based international registry. Project under auspices of Women's and Anticoagulation ISTH SSCs. Protocol has been submitted to Amsterdam REC. Primary objective to record fetal and maternal outcomes from DOAC exposure in pregnancy. Secondary objective is to record late effects in children. Data collection has started, web based registry to launch soon. Invitation extended for volunteers for national coordinators.

Thrombophilia screening after repeated failures in assisted reproductive techniques (Elvira Grandone) - FIRsT registry introduced. A prospective registry aiming to collect data on whether there is benefit in giving LMWH to improve ART outcomes. Plan to collect and evaluate data on the 1st cycle after 2 or more ART failures. In addition aim to collect and evaluate thrombophilia screening data if available. OTTILIA registry update presented. OTTILIA is an observational study on antithrombotic prevention in thrombophilia and pregnancy loss. To date 114 centers involved with 122 women recruited (143 pregnancies). The attendees were invited for participation in both.

Thrombophilia and outcomes in assisted reproduction technologies: Summary of systematic review and meta-analysis (Marcello Di Nisio) - Systematic review of 33 studies including around 6000 patients. Quality assessment procedure explained. Criticism of poor overall methodological quality in studies discussed. No robust evidence of benefit of using LMWH in any condition. Further criticisms of research were highlighted to include underpowered, heterogeneity, lack of correction for other risk factors. One study even suggested a protective effect for Factor V Leiden and lupus anticoagulant.

YEARS diagnostic study in PE (Menno Huisman) - Current algorithms for diagnosis of PE not validated for pregnancy. Ongoing debate of CTPA vs VQ and respective risks of radiation exposure to both mother and fetus discussed. YEARS involves D-dimer and clinical algorithm for use in pregnant population. Evidence of 11% reduction in use of CT. Currently 78 patients registered, invitation for participation extended to all.

The use of thrombo-elastography in pregnancy: Registry (Maha Othman) - Fact that currently guidance only exists for use of TEG/ROTEM in hemophilia (ISTH), insufficient evidence to produce other guidelines. Aims of registry included: recording access to technology problems, protocols used, problems encountered. Questionnaires have been distributed, so far 71 responses but only 18 complete. From these: ROTEM used more than TEG; 43% had no access; 50% clinical use only; 50% access in local lab; 47% no training; most using manufacturers reference ranges (not pregnancy specific). Mostly used in DIC/PPH/transfusion. Main barrier to use is expense.

Obstetric issues in mothers of children with severe protein C deficiency (Adrian Minford) - Severe protein C deficiency explained with significant long-term complications for children (visual and neurological). Potential benefit of elective early delivery to prevent complications if fetus known to be affected. Based on carrier rates should be 135 cases in UK but actually only 10 known - hypothesis that affected couples suffer excess rates of fetal loss and neonatal death. Suggestion that screening couples with recurrent miscarriage, unexplained stillbirth or neonatal death may be of benefit. Aim to collect data on obstetric history of couples with affected children.

Thrombosis issues (Moderated by HC and I. Martinelli)

Management of Direct Oral Anticoagulants (DOAC) in women of childbearing age (Deepa Arachchillage) - Potential reproductive toxicity is unknown. Animal model studies have suggested increased miscarriage, skeletal and cardiac vessel abnormalities. No effect on fertility. Radiolabeling studies show lower level in fetal tissues compared to maternal but high levels are expressed in milk. Individual drug properties (e.g. molecular weight, drug binding and pKa) are likely to affect extent of transfer across placenta. Gestational age at which exposure occurs also affects type risk likely. Comment that human data is limited and incomplete (reliance on data from drug companies). Current recommendations outlined including that in pregnancy DOAC should immediately be switched to LMWH, anomaly scan and fetal echo by fetal medicine specialist, regular scan and specialist follow-up. Inadvertent exposure not grounds for TOP. In women of childbearing age importance of counselling and contraception highlighted. Recommendation is to avoid DOAC in breastfeeding. ISTH guidance document is in progress.

Management of obstetric anti-phospholipid syndrome: New developments (Beverley Hunt) - Lack of data on the prevalence of specific complications highlighted. Risk factors identifying women with APS likely to suffer obstetric morbidity were discussed (evidence from PROMISSE study, Rufatti et al). Treatment regime used at St Thomas's explained including use of aspirin, LMWH and steroids. Steroids used in women with recurrent first trimester loss. New potential treatment option of hyroxychloroquine discussed. Dr Hunt planning prospective study on use of hydroxychloroquine in pregnant women with APS (HYPATIA); will be presented at ISTH 22/6/15. Invitation is open for all to participate.

Management of thrombotic thrombocytopenic purpura in pregnancy (Marie Scully) - Most recent UK registry shows 10% cases TTP associated with pregnancy and of these 56% are late onset congenital TTP. Overlap between patients with diagnoses of TTP, PET, HELLP and HUS. UK registry shows higher number of presentation in 3rd trimester/postpartum. Good evidence of

benefit of treatment in reducing obstetric morbidity in congenital TTP, acquired TTP presenting in pregnancy less consistent benefit of treatment seen. Treatment regime used by Dr Scully for each type of TTP in pregnancy explained. Case study of use of Rituximab in TTP in pregnancy was also presented.

Education Session (Moderated by Rezan Abdul-Kadir and Andra James)

Preeclampsia - the role of hemostasis in its pathophysiology (Chris Gardiner) - Origins of PET in placenta. Differentiation of characteristics of early- vs late-onset PET was presented as well as role of increased tissue factor in placenta and platelet activation in PET. Overview of current and ongoing research projects looking at novel treatments. Upcoming study is to report on use of Pravastatin in PET. Good evidence for low dose aspirin in reducing risk of early-onset PET, study looking at 150mg dose ongoing. PRESERVE-1 study (antithrombin to treat established PET) ongoing. Some evidence of benefit of activated protein C for early-onset PET. Case study of successful use of Eculizumab to prolong pregnancy in HELLP, price of drug is barrier.

Women and bleeding disorders - the role of bleeding assessment tools (Paula James) - Overview of BATs currently in use was presented. Case study used to illustrate importance of appropriate selection of BAT to reach correct diagnosis. Validation of self-BAT BS (assessment tool developed by her group) against ISTH BAT-BS in carriers of hemophilia was discussed. Project highlighted group of women with normal factor levels but increased bleeding score, and of these 11% reported experiencing haemarthrosis. Need for further research in this area.

Hemostasis issues (Moderated by Rochelle Winikoff and Peter Kouides)

Women and platelet function defects (Sarah O'Brien) - Detailed her unit's experience of using electron microscopy in diagnosis of bleeding disorders in teenagers referred with HMB. Correlation with platelet aggregation tests and analysis of distribution of EM results in her unit showing over-diagnosis and prompt for her unit's lab to establish own reference ranges. Conclusions: importance of site-specific reference ranges for EM, larger studies needed to determine value of repeat testing for diagnosis, difficulty of diagnosis in patients with discordant results for EM and platelet aggregation.

Can carriers of hemophilia with normal factor levels bleed? (Robert Sidonio) - Plug I et al 2006 showed women carriers with levels at 40-60% showed increased bleeding. Severity of genetic mutation correlates with severity of phenotype in carriers (Miesbach study). Paroskie and Sidonio BJH 2015 FVIII level not good predictor for bleeding in carriers. Olsson et al 2014 BAT score does not correlate with factor levels. Hypothesis is that perhaps these carriers are not able to mobilize factor appropriately.

Postpartum hemorrhage in carriers of hemophilia and women with VWD (Jeroen Eikenboom) - International guidelines suggest factor replacement in 3rd trimester if levels <0.50, but dose is not specified. Dr Jeroen Eikenboom presented his study showing that VWD and hemophilia B carriers had excess rates of PPH despite factor replacement when indicated. Proposed changes in guidelines for management of these patients: use of antifibrinolytics, double uterotonics, and studies to look at what is the appropriate dose of factor replacement

Abnormal uterine bleeding in adolescents: Patterns, diagnoses and clinical outcomes (Ayesha Zia) - Concern that underdiagnosing bleeding disorders in adolescents with HMB as very often just labelled as due to anovulatory cycles. Study focused on these patients, management algorithm explained. Interim results shared - 36% seen in the emergency department, 20%

needed transfusion, 50% iron deficient, 1/3 anemic. 44% found to have VWD. Evidence that for girls with HMB and inherited bleeding disorder management algorithm discussed leads to significant improvement in quality of life. Invitation for collaborators to enter data to the SSC registry.

Foundation for women and girls with blood disorders (FWGBD) (Ann-Marie Nazzaro) - Need to educate healthcare providers about consequences and effects of blood disorders in women. FWGBD devised at ISTH 2009. Strategy includes presenting at medical society meetings. Website (fwgbd.org). Assistance with establishing 'Clinics of Excellence' in USA, census of specialist services provision in USA. International collaboration is needed to reach out all women with bleeding disorders and improve their lives wherever they live.

Meeting closed - Rezan Abdul-Kadir thanked all the attendees and then speaker and emphasized the importance of collaboration to achieve the goals of the subcommittee in producing good quality data for aspects of care with lack of evidence. Rezan Abdul-Kadir also invited the attendee to provide feedback on the today session on the website and send proposal for what should be discussed in future meetings. In addition, Rezan Abdul-Kadir asked for the members and all attendees to participate by leading / send suggestions and proposals for projects and guidance papers.

Working Group on Perioperative Thrombosis and Hemostasis

21 June 2015 8:00 – 10:00

Chairman: James Douketis

1) Review of the Session:

This 2-hour session began with an Introduction by Jim Douketis, outlining the goals of the Perioperative Working Group, its multidisciplinary make-up and ways for ISTH members to become involved.

The first presentation by Pierre Albadalejo outlined the challenges in the perioperative management of patients who are receiving dual antiplatelet therapy. This was followed by a presentation by Jerry Levy, outlining the biological basis for managing anticoagulant-associated bleeding.

After a discussion period, the third presentation by Marc Samama identified gaps in knowledge relating to laboratory monitoring and administration of different pro-hemostatic agents in the perioperative period, outlining a call-to-action for the Working Group to lead. The final presentation, by Alex Spyropoulos, reviewed and critiqued recently completed and ongoing randomized trials addressed perioperative anticoagulation.

Overall, the session was very well-received, with standing-room only attendance of ~150 people.

2) Future leadership and activities of (applied for) Subcommittee on Perioperative

Thrombosis and Hemostasis:

Originally, group was formed with a non-hierarchical structure with Jim Douketis as the de facto chair due to linkage with the SSC.

With proposed application to a bona fide Subcommittee of the SSC, there is a need for a more structured organization, with a Chair and Co-chairs.

There was unanimous agreement that Marc Samama would be the proposed Subcommittee Chair and other current Working Group members would be Co-chairs.

3) Guidance and guideline development:

There was consensus that the field of perioperative thrombosis and hemostasis was a fertile area for guidance document development, including guidance on the topics presented during the educational session.

The new Chair, with the assistance of the Co-Chairs would identify and prioritize potential guidance projects, also identifying more junior ISTH members as co-authors.

There was general agreement that development of formal practice guidelines was beyond the scope of this group and, indeed, not a direction (at this this time) that was being pursued by the ISTH.

4) Research activities:

The Working Group identified 2 broad areas of research involvement:

1) Facilitating research led by Working Group members or other colleagues by

providing input and expertise. For example, members could be called up to serve as

Steering Committee members or act in another advisory capacity. In addition, members

would serve a conduit to collaborative research networks in their home country.

2) Developing de novo multi-center clinical trials would be an important goal of this

group, building on research networks developed by individual member; there would be the

potential for concurrent applications to funding agencies across countries.

5) Potential future studies:

Yaron Shargall, a thoracic surgeon at McMaster University, Hamilton, Canada discussed a proposed randomized trial assessing VTE prevention after thoracic surgery, with feedback provided. The group encouraged Dr. Shargall to continue this research, where evidence is lacking about best practices, and would provide ongoing feedback and, if needed, participation in the study organization.