1.1 – INITIATION OF COAGULATION

1. Tissue factor is the primary initiator of coagulationStructure of TF

47kDA integral membrane protein Is the only pro-coagulant factor that does not require proteolytic cleavage for activation

Physiology of TF normally located on extravascular sites, namely on CSMs of VSMCs & Fibroblasts Is at higher concentrations on cell surfaces in certain organs to provide extra haemostatic

protection (lungs, brain, gonads, placenta)

TF has 2 functions Acts as a cellular receptor is the cofactor for factor 7/7a

2. Factor 7/7a has the typical Gla-egf-Ser domain structureStructure of F7

48kDa zymogen Gla domain – binds (Vitamin K dependent factors) to activated phospholipid membranes (i.e.

PPDserine on outer surface) Serine protease domain – contains catalytic triad of his/asp/ser which cleaves after arg or lys

Physiology of F7 secreted by the liver into plasma at concentration of 10nM ~10% of this is (for reasons unknown) circulating as F7a

o the circulating F7a is largely inactive unless bound to TF

3. the TF-7a complex is key for the initiation of coagulationStructure/Function of TF-7a

TF binds to F7a on subendothelial CSM and allosterically activates serine protease domaino the reaction has a very high affinity (Kd < 1nM)

all 4 F7 domains interact with TF due to low F7a concentrations, only a small amount of TF can form the TF-7a complex at first

o TF-7a can auto-activate (via proteolysis) other TF-F7 complexes 10a can also do this

Initiation of coagulation involves generating small amounts of thrombin by TF-7a TF-7a activates (via proteolysis) F9 by cleaving after Arg 145 and Arg 180 TF-7a activates F10 by cleaving after Arg 191 F10, without its cofactor (F5) generates trace amounts of thrombin by cleaving prothrombin

o Thrombin cleaves 8 8ao Thrombin cleaves 5 5a

F9a-8a activate F10; F10a-5a cleaves prothrombin much more efficientlyo hence propaogation of coagulation becomes independent of TF-7a

4. there are different forms of TFPI, each with their own roles in the regulation of coagulationStructure of TFPI

43kDa molecules acidic (-ve) N terminus and alkaline (+ve) C terminus Has 3 kunitz domains

o Kunitz 2 inhibits 10a

o Kunitz 1 inhibits TF-7a

Physiology of TFPI The majority (80%) of TFPI is cellular:

o extravascular (VSMCs etc)o endothelialo platelet

Cellular TFPI is either stored and released upon demand or bound to cell surfaces. TFPI-beta is a type of membrane bound TFPI made by alternative mRNA splicing

The remaining (20%) circulates free in the plasma. of this proportion, most is truncated by binding to lipoproteins, and only ~0.25nM is at its full length and active

Functions of TFPI TFPI uses its own Arg and Lys residues at ‘P1’ regions on the Kunitz domains to act as mock

substrates for 10a and TF-7a respectively The interaction with 10a comes first after it has dissociated from subendothelial membrane.

this interaction is usually slow as the concentration of active TFPI in the plasma is lowo Protein S increases the rate of the first step by binding 10a to phospholipid surfaceso TFPI-beta can then bind 10a to phospholipid surfaces, which reduces dependence on

protein S the subsequent interaction between TFPI-10 and TF-7a forms an inactive quarternary

complex

Roles of TFPI during coagulation Extravascular TFPI regulates the very 1st step of coagulation initiation at subendothelial sites endothelial TFPI localises coagulation to the site of injury and prevents spread of the plug to

neighbouring/distant sites Platelet TFPI regulates TF in a developing haemostatic plug (and possibly microparticles) Plasma TFPI regulates TF-7a at all sites (and possibly microparticles)

5. TF microparticles are a controversial issue, but if they do exist they offer a different model of coagulation propagation

Idea behind microparticles It is very difficult for circulating factors to get through the fibrin meshwork in a growing plug Experiments have shown that [TF] increases at site of injury as the haemostatic plug forms

o Hence circulating TF may be another mechanism which propagates coagulation it is thought that microparticles, derived largely from monocytes, express TF and circulate in

the plasma Microparticles bind to P-selectin on activated platelets and/or endothelial cells via PSGL binding is thought to ‘decrypt’ this TF, initiating coagulation all over again Evidence suggests cancer patients have higher circulating microparticles, which could

account for their pro-thrombotic state if being produced by tumour cells.

6. Knockout studies in mice have shown that factors in initiation coagulation are key to survival

Factor knocked out ResultTF Death in uteroF7/10 haemorrhagic death after birth F7 and F10 can cross the placentaTFPI death in utero

1.2 – FIBRINOGEN, FIBRIN & FIBRINOLYSIS

1. Fibrinogen is the product of multiple genesFibrinogen Structure

fibrinogen is encoded by 3 coding genes on chromosome 4 (gamma, alpha, beta in direction of transcription)

The resulting molecule is symmetrical; 2 of each chain connect terminal D domains to a central E domain

the whole protein is roughly 350kD big fibrinogen is soluble (polymerised and cross linked fibrin are

not)

Fibrinogen physiology It is an acute phase protein so transcription is induced by pregnancy, inflammation, steroids The central E region contains the key peptides, FpA & FpB which thrombin cleaves

2. cleavage of fibrinogen by thrombin produces monomers that spontaneously polymerize Thrombin cleaves the alpha chain between arg16 and gly17 (at FpA) to expose the

polymerisation domain 17-20 Thrombin also cleaves the beta chain between arg 14 and gly15 (at FpB) to expose its

polymerisation domain (15-18) D domains on fibrinogen always have an exposed polymerisation domain hence, the cleaved E domains polymerise with D domains on other molecules to form a ‘half-

staggered’ fibrin clot gamma chains also interact linearly

3. Thrombin activates F13a, which

is key in cross-linking fibrin polymersStructure of

F13 Only transglutaminase enzyme involved in coagulation exists as a tetramer (320kDa)

Physiology of F13 Circulates in plasma bound to the d-domain of fibrinogen thrombin cleaves the activation peptide of F13 on the surface of the fibrin clot

o this is much more efficient than thrombin-F13 in vitro F13a uses calcium ions to create covalent (isopeptide-gamma-glutaminyl-epsilon-lysyl)

bonds between adjacent fibrin polymerso this cross linking usually occurs between lysine and glutamine residues (lysine acting

as an acid/ electron donor) cross linking between gamma chains occurs first, then alpha chains

4. One of the functions of fibrin it to provide the trigger for its own dissolution

Functions of fibrin provides mechanical meshwork/matrix for wound repair supports platelet adhesion/aggregation provides trigger for its own dissolution (fibrinolysis)

Initiation of Fibrinolysis is driven by fibrin by two major mechanisms Plasminogen is a zymogen synthesised in the liver. tPA is expressed by endothelial cells. It circulates as an active enzyme with low intrinsic

activity. o Both plasminogen and tPA bind to fibrin via lysine residues on fibrin kringle domainso this brings plasminogen & tPA into close contact and thus reduces the (Km)

minimum concentration of plasminogen required for a reaction to occur Fibrin also directly enhances tPA (increased Vmax for reaction) Fibrin enhances tPA to cleave plasminogen on the surface of fibrin to produce plasmin

o Another plasminogen activator, uPA is involved extravascularly and is not influenced by fibrin. uPA is involved in extraceullar matrix degradation (which contributes to cell motility) and growth factor activation via plasmin

5. Plasmin is the key molecule in fibrinolysis. Its broad specificity highlights the need for antiplasmin and other anti-fibrinolytic molecules

Plasmin has broad substrate specificity and cleaves after lysine and arginine residues Plasmin breaks fibrin into ‘D-dimers’; it has the ability to break 50-60 bonds at once Plasmin generated by uPA can activate other proteases and cellular growth factors hence plasmin is implicated in a wide arrange of disease

o tPA plasmin – CVS disease, atherosclerosiso uPA pasmin - cancer

Anti-fibrinolytic molecules Antiplasmin Plasimogen-activator-inhibitors 1 & 2 (tPA inhibitors)

o these are both serine proteases Thrombin-activated-fibrinolysis-inhibitor (TAFI)

o carboxypeptidase enzyme which removes lys or arg residues from C-termini of fibrin to prevent it from stimulating tPA

o hence, when thrombin binds to thrombomodulin, it is not only an anti-coagulant (protein C) but also an anti-fibrinolytic (TAFI)

6. Synthetic plasminogen activators have therapeutic uses thanks to their fibrinolytic properties tPA (alteplase) used in MI, stroke, PE Streptokinase (bacterial product which stimulates plasminogen and causes plasmingoen to

act like tPA) used in MI, VTEs and arterial thrombi TNKase (tenecteplase) is a synthetic tPA with a longer half life and greater bioavailiability

used in MI Desmodus rotundus PA (from bats which use it to keep sucked blood liquid) for stroke uPA used for MI/PE

1.3 – ANTITHROMBIN & HEPARIN

1. Antithrombin is the key anti-coagulant molecule in the termination of coagulationStructure of antithrombin

8kDa serine protease Has 3 main ‘parts’ to the molecule – the main body, a bait loop, and a helical area for

GAG/heparin attachment

Physiology of Antithrombin Circulates in plasma at higher concentration than most coagulation factors effectively inhibits free circulating serine proteases, but principally Thrombin and F10a Its action is enhanced by endogenous heparan sulphate (GAGs) and exogenous heparin,

both of which have a similar structure

2. Antithrombin inactivates thrombin by use of a ‘moustrap’ mechanism Antithrombin is inactive when its ‘bait loop’ is partially contained within the main body of

the molecule The active conformation exposes P1 residues on the bait loop P1 residues bind to thrombin covalently and irreversibly

o the ‘captured’ thrombin has its structure changed so it loses functionalityo the T-AT complex is subsequently cleared from the bloodstream

Some thrombin molecules can cleave the bait loop, if this is successful they are not captured and the antithrombin molecule is rendered inactive

3. Heparan Sulphate and Heparin have similar structures and both stimulate antithrombin Heparan sulphate is present on the surface of endothelial cells Heparin is stored in mast cells (and rarely ‘sees’ bloodstream) and can be administered in a

number of different formulations relating to molecular weight Both molecules are compromised of repeating disaccharide residues of D-glucosamine and

one of uronic or iduronic acid. the major difference between the two is that Heparin contains an essential pentasaccharide

sequence, which is not fully developed (/sulphated) in heparan sulphate

Mechanism of action of heparin heparin pentasaccharide binds to helical area and

causes antithrombin to adopt active conformationo this is enough to inhibit 10a

Longer heparins (>18 saccharides) form a bridge between the helical area and a thrombin molecules = inhibition of thrombin

Heparin dissociates after 10a/thrombin is bound; catalyst

4. The length/Mr of heparins determines their anticoagulant function and hence clinical use Unfractionated heparin (Mr>6000) can inhibit both thrombin and 10a Low molecular weight heparins (1500-6000) will inhibit both 10a/thrombin in a ratio

determined by the specific length (shorter ones inhibit more 10a and vice versa) Heparins with a Mr<1500 (eg Fondaparinux) are essentially just the pentasaccharides i.e

indirect anti-10a molecules

Low Molecular weight heparins are useful in clinical practice They have a high anti 10a and anti-thrombin activity (better than heparins <1500) LMWH have better bioavailability than Unfractionated heparin hence are more predictable

in their course of action

LMWH have reduced interaction with neutralising proteins which leads to less heparin-induced thrombocytopenia

They are mainly used prophylactically and therapeutically in VTE, as well as:o treatment of arterial thrombosiso cardiovascular catheterisationo haemodialysiso DIC

1.4 – INTRODUCTION TO THROMBOPHILIA

1. Venous thromboembolism is a disorder predisposed to by a number of genetic and environmental factors

The key notion is that most people with recurrent VTE disease have a transient risk factor which pushes them over their thrombotic threshold in the context or pre-existing genetic and/or environmental predispositions to clot.

Genetic Environmental Mixed TransientAT deficiency Antiphopholipid syndrome Hyperhomocysteinaemia SurgeryProtein C def Myeloproliferative disease High F7/9/ fibrinogen Long distance travelProtein S def Increasing Age OCPF5 leiden Previous Thrombosis HRTPT G20210A Cancer pregnancy

2. An important polymorphism is Factor V leiden confers resistance to activated protein CNormal physiology

APC cleaves the 5a heavy chain in two places on phospholipid membranes of platelets and endothelial cells to inactivate it - the two places where APC splices are Arg 506 & Arg 306

Arg 506 is rapidly cleaved and cleavage makes 5a moderately less active Arg 306 is slowly cleaved and cleavage makes 5a completely inactive

F5 leiden A common single polymorphism occurs at 506 Arg Gln this polymorphism is likely to have been a ‘founder’ polymorphism that has since spread

amongst Europeans in particular this stops rapid cleavage of 5a and confers resistance to activated protein C slow cleavage at Arg306 still occurs

F5 leiden polymorphism interacts with other genetic/environmental pro-thrombotic predispositions Genetic – F5 leiden (homozygosity), Protein C/S def, AT def, PT G20210A Environmental – OCP

3. An important polymorphism in prothrombin confers increased thrombotic potentialThe G20210A polymorphism increases prothrombin production, which subsequently increases thrombin production = thrombosis.

4. Polymorphisms are more common and have less thrombotic potential than single gene mutations

Single gene mutations are implicated in AT def, Protein C def and Protein S defHomozygotes for single gene mutations tend to be severely disabled or die in utero. Heterozygotes do not have increased mortality but may experience the following symptoms due to increased risk of thrombosis:

Common – DVT/PE/ pregnancy associated thromboembolism Rare – warfarin induced skin necrosis, clotting in other veins (i.e not legs) and arteries

o warfarin inhibits protein C/S so if the person is already deficient then the drug makes the problem worse

F5 leiden and PT G20210A are atleast twice as common but have half the thrombotic risk

1.5 – UNDERSTANDING ENZYMES IN HAEMOSTASIS

1. Km & Kcat are important measure of enzyme function (Michaelis Menten mechanism)Km = the [substrate] at which the reaction is catalysed at ½ Vmax

also known as the dissociation constant of an enzyme substrate complex (i.e. at equilibrium/ Km; half substrate will have reacted and half will not)

A low Km value denotes a high affinity If the Km value is >[substrate] the reaction will not occur Units = M (nm microM etc)

Kcat = is the number of times the enzyme reacts with the substrate per second

also known as turnover No; proportional to Vmax Units = s -1

Kcat/Km = specificity constant and is a measure of catalytic efficiency

Units = s -1 M -1 in haemostasis this should be of the order >106

2. Surfaces and cofactors have predictable effects on Km & KcatSurfaces bring reagents together.

This reduces the substrate concentration for the reaction to occur (less reaction-by-chance) This reduces Km = increases affinity Shifts reaction curve left but same Vmax is reached

Cofactors cause conformational changes in substrates which make them more reactive This increases Kcat = increased Vmax This may also reduce Km = increased affinity

AN INCREASE IN ENZYME CONCENTRATION DOES NOT CHANGE Km This change only increases Vmax hence the 1/2Vmax value is faster, but occurs at the same

substrate concentration as before

2. Activation of Prothrombin, Plasmin and antithrombin are all important examples of how enzymes make sure reaction only happen where they are supposed to

Prothrombin Thrombin is catalysed by 10a 5a is the cofactor for 10a (increases Kcat) Platelet phospholipid is the surface (reduces Km)

o Prothrombin concentration is low in plasma. this reaction would not occur if it weren’t for phospholipids Km would be too high.

Plasminogen Plasmin is catalysed by tPA Fibrin is the cofactor for tPA (increases Kcat) and the surface (reduces Km)

Thrombin antithrombin/thrombin complex is catalysed by Antithrombin Heparan Sulphate is the surface (reduces Km), but also increases Kcat slightly

COFACTORS AND SURFACES ACT AS ENHANCING MECHANISMS FOR REACTIONS AND AS REGULATORY MECHANISMS MAKING SURE REACTIONS HAPPEN WHERE THEY ARE SUPPOSED TO

1.6 – WON WILLEBRAND FACTOR

1. vWF factor is the largest circulating protein in human plasmaStructure of vWF

Large (upto 20000kDa) multimeric glycoprotein Largest molecules bind to subendothelial collagen with greater affinity and have greatest

pro-thrombotic ability

Physiology of vWF Majority is synthesised by vascular endothelial cells

o Expression is highest in brain, lungs and heart (greater haemostatic protection)o expression seems to be higher in venous endothelium than in arterial

Minority is released from alpha granules on platelet activationo thought to be a compensatory mechanism if there is a lack of endothelial vWF

Plasma levels vary greatlyo low in blood group O (ADAMTS13 susceptibility)o increased during preganancy/ birth and possibly with ageingo Wiebel-Palade body release from endothelial cells is stimulated by stress,

inflammation, DDAVP

2. the vWF monomer has a number of important domains relating to its function

Domain FunctionD1-D2 Propeptide, multimerisationD’-D3 F8 binding, multimerisationA1 Gp1b (platelet)A2 ADAMTS13 (regulation)A3 Collagen 3 (subendothelium)C1 GP2b3a (activated platelet)CK dimerisation (ER)

3. vWF undergoes extensive post-translational modificationIntracellular Modification

ER – N linked glycosylation & dimerisationo Dimerisation is achieved by disulphide knots via CK domains. the propeptide acts as

a catalyst for this because it has disulphide isomerise activity.o diemerisation is the cue for peptide to leave ER (not enough space in ER for

multimers) Golgi – O linked glycosylation & start of multimerisation

o multimerisation achieved via d-domains Post-golgi – addition of ABO blood sugars (vWF is the only circulating protein that expresses

ABO)

Wiebel-Palade Bodies store large vWF Most vWF are transiently stored in these and subsequently released, a small proportion

remain intracellular to be released on a stimulus (eg stress, DDAVP) WPBs are only found in endothelial cells (hence can act as a marker for these [+CD34]) vWF’s propeptide induces WPB formation (+ve feedback) WPBs contain other molecules involved in adhesion (p-selectin) and F8

Extracellular modification Furin removes the propeptide (some of this happens intracellularly) The resulting vWF can adopt either a globular or linear structure. the linear structure

exposes binding domains.

4. The primary role of vWF is in primary haemostasis – making GP1b its most important proteinShear Stress and endothelial binding forces globular vWF to unfold

Shear stress from blood flow will cause vWF to spontaneously unfold/refold whilst circulating in the plasma due to varying blood speeds (?threshold for unfolding in 5000s -1)

Similarly, when vWF binds to sudendothelial collagen, one end is anchored and shear stress subsequently unfolds the rest of the molecule, exposing binding sites

o vWF binds to subendothelial collagen type 3 via its A3 domain (A1 binds to different collagens).

A1-GP1b binding slows platelets down and RGD-2b3a binding fixes them vWF-A1 domain binds to Gp1b on platelets – forcing them to slow down in circulation

o this bond is rapid on-rapid off (high dissociation rate) platelets roll along vWF under torque of blood flow and become activated

o hence platelet-GP2b3a becomes activatedo platelets also degranulates – initiating aggregation

vWF uses RGD sequence on its C1 domain to bind to 2b3a and fix platelets

5. the secondary role of vWF is in coagulation as a carrier of F8 F8 and vWF circulate as a complex in plasma. complex formation is rapid.

o the complex dissociates once F8 is activated by thrombin vWF protects F8 from degradation from activated protein C, prevents premature association

with F10 & hence increases its half life defect of vWF-F8 binding = Type 2N vWD

6. The functions of vWF are regulated by the MMP ADAMTS13By cleaving large vWF molecules, ADAMTS13 prevents excessive clotting that would be otherwise associated with lots of large circulating vWF molecules. TTP is associated with low ADAMTS13

Proteolysis is enhanced by elevated shear stress, type 2A vWD and deanaturants like urea because these all unfold vWF and lead to exposure of the A2 binding site for ADAMTS13.

Blood group O is more sensitive to ADAMTS13 cleavage because vWF expresses no ABO sugars, which provide a degree of resistance to cleavage in blood groups A/B/AB.

7. vWD is a quantitative/qualitative deficiency on vWF The hallmark is prolonged bleeding time and ?APTT (haemophilia would not prolong

bleeding time) it is a defect of primary haemostasis (impaired platelet binding) and coagulation (F8

‘deficiency’)

High circulating vWF may be implicated in cardiovascular disease (stroke, MI).

Anti 2b-3a is used therapeutically in CVD in reproductive medicine.

1.7 – POST-TRANSLATIONAL MODIFICATION OF COAGULATION FACTORS

1. Glycosylation is an important post-translational modification Involves covalent attachment of a glycan to a protein

different cell lines produce different glycosylation patterns, which makes it difficult to make synthetic fully functioning coagulation factors

alterations in glycosylation patterns are observed in a variety of pathologies there are two types of glycans:

o Linear (GAGs)o Branched

N-linked O-linked

N-linked O-linkedattaches to aspargine as part of a N-X-S/T triplet where X is any AA except for proline

attaches n-acetylgalactosamine to threonine or serine residues

not truly post-translational; occurs in ER occurs in Golgi – true post translationalinvolved in protein folding, bond stability, solubiolity, protection for proteolysis, clearance

less well understood – provide stability and may have roles in clearance

2. N & O linked glycosylations play important roles in the function of vWFImportance of N Linked glycosylations (NLG) – 12sites on vWF

Formation of vWF dimers/ multimerso inhibition of NLG = retention of monomeric vWF in endothelial cells

Disulphide bond formation = maintenance of globular structure of vWFo inhibition of NLG = ‘early’ exposure of binding sites = tendency to clot

Presenting ABO blood sugarso inhibition of NLG = increased cleavage of ADAMTS13

Importance of O linked glycosylations (OLG) – 10sites on vWF OLG has a tetrsaccharide t antigen whose plasma concentration is inversely proportional to

vWF Inhibition of vWF A1-Gp1b binding

o Inhibition of OLG = increased binding and tendency to clot Steric hindrance increases stability of molecule Protection of ADAMTS13

o Inhibition of OLG = increased clearance vWF without a terminal sialic acid is cleared more quickly from bloodstream because of

increased affinity to ASGRP receptor; sialic acid protects VWF against proteolysis by serine and cysteine proteases but specifically enhances susceptibility to ADAMTS13 proteolysis

3. importance of glycosylatons in other clotting factors F& - has 2 N- & 2O glycosylation sites which are important for normal secretion F8 – glycans are important for interaction with chaperone proteins in ER F9 – recovery, activity, efficacy

4. importance of gamma-carboxylation occurs on glutamic residues of Gla domains on F2,7,9,10, Protein C/S the catalysing enzyme, gamma-glutamyl carboxylase is vitamin K dependent warfarin blocks recycling of Vit K = impaired gamma carboxylation = impaired binding of

factors to platelet phospholipids due to inability to bind calcium

1.8 – MOLECULAR MODELLING WORKSHOP

1. Why protein structures are important

SHAPE DETERMINES ACTICITY. KNOWLEDGE OF SHAPE = Understanding the effect of gene mutations

o Protein structures allow you to see the location of a missense mutation.o A mutation in the core of a domain often leads to misfolding, a failure to secrete protein

from the cell and therefore low plasma level. (Type I mutations: most common)o Some mutations cause disease because they are in a functionally important part of the

protein (Type II mutations: less common). Understanding how drugs affect enzymes Understanding research papers Essential tool in research

2. How protein structures are derived X-ray crystallography

• high resolution but need large sample size, time consuming and gives static picture NMR (nuclear magnetic resonance) Spectroscopy

• high resolution and gives information about mobility but protein needs to be soluble and not good for large molecules (size limited)

Homology modelling (prediction of structure)• involves using homolgous proteins as templates as a last resort basically• medium resolution (limited accuracy)and structure of homologus protein needed

using computer algorithms to predict structures when all else fails

3. What is being shown in protein molecular graphics pictures of different types4. What you can do with molecular graphics Show representations of molecules in different presentation styles, colours &c Explain functional features (e.g. enzymatic activity or receptor binding) at molecular level Qualitatively interpret amino acid substitutions (missense mutations) in disease states Hypothesise about unknown structures or interactions Explain functional features (e.g. enzymatic activity or receptor binding) at molecular level

4 types of molecular graphics:• Ball and Stick

• can view interior of molecules but only gives partieal view (no sense of density)• Secondary Structure

• good view of domains but no representation of surface• Spacefill

• good view of surface but cant see inner structure• Surface representation

• accurate and realistic• can show charge/ surface hydrophobicity

5. What you need to produce your own molecular graphics6. To download a structure from the PDB Database and produce a graphic in Pymol.

1.9 – RARE COAGULATION DISORDERS

1. Coagulation disorders apart from haemophilia are very rare and are classified based on severity

Haemophilia A/B and vWD account for 97% of all inherited coagulation disorders Incidence - Haemophilia A 1/10k

o Haemophilia B 1/50ko F7 def is the most common of the others – order is 1/500ko The others are about 1/million

Inherited disorders of primary haemostasis eg Glanzmanns are of the order 1/million

Most rare bleeding disorders are classified based on severity An assay reading of less than 1 IU/dL is deemed as an absence Heterozygotes (>20) may still have a bleeding problem <20IU = homozygote/compound heterozygotye

o a compound heterozygote is someone who has inherited to different mutant polymorphisms

<10IU = homozygote

Type 1 disorder = absence. Type 2 disorder = dysfunction

The concentration of coagulation factor required for haemostasis to occur is much lower than the lowest boundary of the normal range. Hence bleeding severity vary rarely correlates with deficiency (except for fibrinogen, f10, f13), bleeding patterns are variable, and heterozygotes/carriers may still have bleeding problems.

2. The majority of rare coagulation disorders follow an autosomal recessive inheritance and demonstrate largely unpredictable bleeding patterns

Factor Deficiencies Result of absence

Bleeding ‘pattern’ PT APTT TT

Fibrinogen (AD)

Type 2> Type 1; majority of Type 2 are acquired eg Liver dx

Not fatal Umbilical cord, oral, muscle/joint, nose, periods. Thrombosis if mutation near to thrombin cleavage site

- - +

PT/F2 Rarest of them all Always fatal Nose, periods, oral, muscle/joint

+ + -

F5 Type 1> type 2 Always fatal Nose, periods, oral + + -F5+F8* Nose, periods, oral (mild) + + -F7 Commonest of them all

(Arg353G mutation & environmental influences cited)

Nose, periods, oral + - -

F10 Always fatal Joints, GI/urinary + + -F11 (AI) Particularly common in

Ashkenazi jews (f7 levels low in liver dx, preg)

Genitourinary, oral, nose, tonsils (injury related; mild)

- + -

F13 Umbilical, intracranial, miscarriages, impaired wound healing

- - -

*Combined F5/F8 deficiency is not due to mutations in the genes but due to problems in intracellular traifficking

3. The management of rare bleeding disorder is difficultRare disease = limited expertise and resources

Haemophilia centres will usually cover these patients FFP/Promthrombinase concentrates (PCC )are usually the only resource, recombinant

factors are being developed but they are expensive and the market is very narrow. Adjuvants like TAFI, DDAVP, OCP, IUCD can be used Offer advice to communities where consanguinuity is more common (AR diseases)

1.10 – LABORATORY TESTS OF COAGULATION

1. Patients usually bleed because of defects in their procoagulant activityi.e. having an inherited excess anticoagulant/ fibrinolytic activity is rare

Low Procoagulant activity can be qualitative or quantitative Type 2; Qualitative – poorly functioning protein - usually inherited Type1; Quantitative – low circulating protein – usually inherited but acquired causes

included liver failure, excess consumption (DIC), drugs (warfarin)

2. PT & APTT are practical screening tests but not unphysiologicalPT initiates coagulation via Thromboplastin (TF) until thrombin is generated.

Normal range 9.6-11.6 Elongated in extrinsic/ common pathway deficiencies

APTT works in the same way but uses partial thromboplastin in the presence of a contact activator eg kaolin, silica, elagic acid, glass

Normal range 26-32 Elongated in intrinsic/common patway deficiencies

TT uses a small amount of thrombin to measure the time taken to convert fibrinogen into fibrin. Normal range 15-19 Elongated in a/dysfibrinoginaemias, heparin (and other thrombin inhibitors)

Unphysiological nature of coagulation tests aptt seems to be sensitive to F12 which we know plays little part in coagulation in vivo –

false positive they do not have appropriate sensitivity for factors that we know play bigger roles – false

negatives they do not detect a number of disorders eg vWD, platetlet disorders, vessel wall/

fibrinolytic disorders different reagents in different labs mean that the results are not standardised

3. Antigenic assays and functional assays are being used for specific factor deficiencies

Antigenic assays are precise but give only quantitative (not qualitative) data ELISA is the assay usually used – the principle is catching the antigen with one antibody and

measuring the titre using another, labelled antibodyo Time consuming

Immunoturbidimetric assays use reactions that create agglutination and shine a light through the solution to measure a change in light intensity at a sensor

The specificity of antigen-antibody reactions means that antigenic assays are not prone to interference

Functional assays are imprecise but are more reflective of coagulatory function to an extent One stage clot based assays compare the test plasma to a standard one by adding each

solution to a factor-deficient plasma. Time to clot vs Dilution is plotted on a graph and then you can say how ‘dilute’ the test is in comparison to the standard for that given clotting factor.

Chromogenic activity assays use a chromogenic substrate which is cleaved by the factor of interest to produce colour. These may be more reproducible than clot based assays but are not always physiological

Subject to interference

PFA100 is a useful assay in measuring primary haemostasis

Bleeding time used to be used – but this is no longer the case because it has poor sensitivity, specificity, reproducibility and poor predictive value

PFA 100 forces blood through a collagen-lined aperture. Blood flow is terminates when a haemostatic plug is formed.

o Sensitive to platelet number, function, vWF function & Hcto But not sensitive to Thrombin, drugs (clopidogrel)

i.e. haemophiliacs would have a normal PFA-100 test; you would need another test for coagulation disorders in conjunction with this

4. Laboratory tests can be misrepresentations of the clinical sample

Variables that are due to the sample Some clotting factors (8, vWF) are acute phase proteins that can be induced by stress Collection of blood may have been inadequate

o Fill blue bottles to the top – need to ensure anticoagulant volume does not dilute sample

o Haemolysis Transport to laboratory should not exceed half life of clotting factor Centrifugation & storage are different depending on where you are

Variables that are due to computers/analytical machines Computers usually use electrical- or light-transmission based methods to analyse

concetrations Automation is good because it increases throughput of samples, has good reproducibility The bad parts of automation is that they are susceptible to end point errors (i.e if the end

point takes part in the initial reaction) and can introduce artefact

1.11 - HAEMOPHILIA

1. There are broad patterns of bleeding concerning primary and secondary haemostatic defectsPrimary haemostatic defects essentially cover platelet disorders and vWD

Vessel wall & collagen disorders are also in this bracket, but are comparatively rare Bleeding is from small vessels, involves no delay and doesn’t stop

Secondary haemostatic defects are usually due to procoagulant deficiencies Haemophilia is the commonest Heaprin therapy may produce a similar type of bleeding Bleeding is from deep structures, involves a delay (the time in which the plug is stable;

before it breaks apart) and doesn’t stop from then on

Fibrinolytic disorders are rare

2. The genetic basis of haemophilia concerns an inversion mutation of chromosome XFemale carriers are not safe

Circulating factor levels in women are extremely variable A carrier may display very low circulating F8 and then experience symptoms Conversely, a female may have high circulating F8 but is obviously not homozygous for the

disease.

The inversion mutation causes 45% of severe haemophilia A cases The mutation tends to occur in spermatogenesis Essentially, two homologus regions on the X chromosome recombine to give a gene that is

subsequently un-transcribable A number of other polymorphisms have been implicated in haemophilia A & B (B is not

associated with the inversion phenomenon) Some patients produce antibodies against F8/9

3. The pathogenesis of haemophilia involves the inability to generate a thrombin burstLack of either factor 8/9 means that the propagation stage of coagulation cannot occur

Failure of a thrombin ‘burst’ in haemophilia slow thrombin generation = weak, unstable platelet aggregate The platelet aggregate will break due to lack of fibrin production NO factor 13 cross linking = any fibrin generated is insecure NO TAFI activation = any fibrin generated is more prone to fibrinolysis

4. Haemophilia is particularly prone to bleeding in joints and musclesHaemophilia A & B are clinically indistinguishable. It was discovered thatthey were different diseases when two patients (one A one B) could cure each others’ clotting abnormality.

Classification of bleeding in Haemophilia Severe (factor level <1%)

o Spontaneous bleeding, from joints/muscles, which is delayed but prolonged. Moderate (factor level 2-5%)

o Prolonged/excessive bleeding after minor trauma Mild (factor level 6-40%)

o Prolonged bleeding after major trauma and/or surgery HAEMOPHILIA CAN BE FATAL (surgery in undiagnosed patients)

Haemarthrosis is a vicious cycle and a hallmark of haemophilia Joint bleeding may be spontaneous (preceded by ‘tingling sensation’) and ceases when the

joint pressure gets too high for bleeding to continue Following bleeding, the synovium ‘mops up’ blood and undergoes hypertrophy. Hypertrophied synovium is friable and prone to subsequent bleeding

Recurrent bleeding leads to loss of cartilage and joint space Because joints become immobile – muscles begin to undergo disuse atrophy

Muscle bleeds in haemophilia may result from exertion. The psoas muscle is commonly involved – bleeding in this muscle may result in compression of the femoral nerve and/or a compartment syndrome.

5. The treatment of haemophilia is switching to a prophylactic schemeThe major advantage of a prophylactic scheme is decreased bleeding!

‘saves’ patients from haemophilic arthopathy No need for patients to carry around their own FFP Brings mortality rate upto normal Major disdavtages concern frequency of injections. Cost is also an issue

o Half life of F8 = 8-12h = 3 injections a weeko Half life of F9 = 18-24h = 2 injections a week

Adjuvant treatments may be used in haemophilia treatment Tranexamic acid reduces clot lysis but may be teratogenic DDAVP stimulates circulating vWF-F8 concentration via its V2 receptor

New developments in the management of haemophilia Anteneatal diagnosis allows prophylactic treatment from birth

o Pre-implantation genetic diagnosis Designer molecules like recombinant F8 which have longer half lives & lower immune-

reactivityo A therapy linking F9 to the Fc portion of immunoglobulin has been shown to

increase the half life to 60hrso Pronlonging half life of vWF may prolong F8 half life indirectly?o Non-peptide cofactors may stimulate existing reservoirs

Gene therapy – has a role in haemophilia B The main concerns about novel therapies are their thrombogenic potential and their cost

1.12 – THE CONTACT ACTIVATION SYSTEM

1. The contact activation system is the part of the intrinsic pathway which does not really contribute to coagulation

The CAS occurs primarily on polyphosphate surfaces ( platelet dense granules ) Collagen is also touted as another -ve surface where this may occur

The activation phase involves production of alpha12ao F12 binds with the help on zinc ions and a conformational change occurso This leads to a cleavage at R353/V354 to produce alpha12a

Alpha12a cleaves F11 & PK The fluid phase is driven by kallikrein (K)

o K autostimulates 12a12a cleavageo K also cleaves 12a into smaller forms like beta12a.

beta 12a is involved in PK/ C1 esterase activation but not F11 activationo K cleaves HMWK to release bradykinino K cleaves c3/c5 into their active forms

2. The main products of the CAS are 12a, kallikrein, 11a and BradykininF12a may have pro-inflammatory function

Promotes (Il1/6-) monocytes and neutrophils B12a cleaves C1 in the complement cascade 12a cleaves plasminogen slowly Is mostly regulated by C1 esterase (negative feedback control)

Kallikrein/fletcher factor 75% is bound to HMWK Apart from what has been mentioned within the CAS, K also cleaves plasminogen Inhibited by c1 esterase

HMWK is a source of bradykinin and may be anti-thrombotic HMWK binds to -ve surface with the help of Zn via their D5 domains. When doing so it

displaces fibrinogen HMWK can cleave PK & F11 via its D6 domains When bradykinin is released, HMWK binds to surfaces more tightly and prevents other

cells/molecules from doing so

Apart from releasing bradykinin, HMWK inhibits neutrophil adhesion, enhances fibrinolysis Kininogens tend to have a number of antithrombotic domains and can achieve this function

by:o Preventing thrombin binding to and activating plateletso Inhibiting calpain, which is a molecule released by thrombino Inhibiting cleavage of PAR1 by thrombin

HMWK is upregulated in pregnancy/ HRT

3. The CAS has a number of biological functions but it is unclear how far its role in coagulation is in humans

a) Bradykinin formationa. Inmportant for vasodilation, platelet inhibition, inhibition of SMC proliferation, pain.

(?excessive CAS shock)b. Promotion of tPA release from endothelial cells (fibrinolysis)

b) Complement activationa. Pro-inflammatoryb. C1 esterase deficiency = excessive complement activation = heridtary angioedema

c) Thrombin inhibition by HMWKd) Fibrinolysis

a. Cleavage of plasminogen by F12a and Ke) Anti-adhesive

a. HMWK inhibits protein/cell bindingf) Angiogenesis

a. HMWK & F12 bind to the uPA receptor on endothelial cellsg) ?coagulation

a. F12, HMWK & PK deficiencies both increase APTT but are not associated with bleeding

b. F12 deficient mice taken longer to form clots but this is not seen in humans. (This could be because humans lack the PAR3 receptor which 12a otherwise activates)

1.13 F5 & F8

1. F5 & F8 have similar domain structures and similar physiological rolesF5 physiology

Synthesised only in hepatocytes

Plasma F5 is taken up (not synthesised) by platelets, where they are stored in a partially active form that confers some APC resistance in platelet granules.

Circulates as a single chain molecule Binds to and stimulates TFPI Acts as the cofactor for F10 within the

prothrombinase complex

F8 physiology Synthesised in hepatocytes and in some

groups of endothelial cells It is stored with vWF in WPBs in

endothelial cells and circulates as a heterodimer in a complex with vWF in plasma

o vWF increases its t1/2, protects it from APC and prevents premature association with F10

o removal of vWF leads to change in conformation and circulation as an unstable heterotrimer

circulating levels vary considerably because F8 is an acute phase protein

acts as the cofactor for F9 within the intrinsic tenase complex

F5/F8 have homologous domain structures The common template is A1-A2-B-A3-C1-C2

o This domain sequence is also homologous to ceruloplasmin and hephastin A domains are responsible for inter-protein interactions C domains are responsible for phospholipid binding B domains have very low sequence similarity between F5 & F8 and play different roles

between the two F8 has additional acidic peptides which are responsible for its additional functions eg a3

binds vWF

2. The B domain is a key difference in the structure/function relationships between F5/F8The F8 B domain is important for intracellular trafficking but NOT coagulation

B domain allows F8 to bind to chaperone proteins within the ER Examples of such chaperone proteins are CNX/CRt and LMAN1/LMAN2; chaperone proteins

facilitate folding, quality control etc for transport into the Golgi and, ultimately, secretion Removal of B domain does not alter F8 half life/ cofactor activity and hence has no role in

pro-coagulant function Thrombin cleaves the B domain as well as the bond between A1/A2 to activate F8

The F5 B domain is important for coagulation F5 B domain also interacts with chaperone proteins for trafficking, but it is conserved in the

circulating protein Some polymorphisms in F5 lead to truncation of the B domain - truncations that lead to the

loss of a key regulatory sequence leads to a pro-thrombotic activity

Like F8, thrombin activates F5 by cleaving the B domain

Inactivation of F5/F8 is another key difference in their physiology Active F8 is an unstable molecule - the majority of F8 inactivation is spontaneous

dissociation of the heterotrimer APC can inactivate remaining F8 by cleaving the A2 domain in 2 places (336,562) F5 is only inactivated by APC cleavage at 3 sites in the A1/A2 domains (306,506,679)

o FV Leiden is the Arg506Gln mutation that prevents ‘fast cleavage’

3. Combined F5/F8 deficiency occurs because the factors share trafficking pathways Both F5 & F8 have heavily glycosylated B domains which allow interactions with ER

chaperone proteins Combined ‘deficiency’ is a rare AR disease associated with mutations in LMAN1/LMAN2 Blood levels vary but are never severely deficient - this is not a problem of

transcription/translation; merely a problem in transport

4. F5 & f8 are both implicated in important disease processes concerning coagulationThe major disease concerning F8 is haemophilia

Low levels = Haemophilia Ao Can be ‘mimicked’ by Type 3 vWD

High levels are associated with increased risk thrombosis

F5 & disease Low levels = ‘parahaemophilia’ (rare) High levels are not associated with increased risk of thrombosis FV leiden = APC resistance = pro-thrombotic

SUMMARYF8 F5

Mr Mr 3mill Mr 3.3mill[] 0.4nM 20nMDomains A1-A2-B-A3-C1-C2 + acidic peptides A1-A2-B-A3-C1-C2B domain Not important for coagulation Important for coagultionProduction Liver & endothelium LiverCirculation Heterodimer+vWF Single chainDistribution Plasma & VPBs Plasma & PlateletsT1/2 8-12h 20hActivation Thrombin cleavage of B domain heterotrimer Thrombin cleavage heterodimercomplex Intrinsic Tenase ProthrombinaseInactivation Dissociation of heterotrimer + APC APC cleavageHigh levels Thrombosis No ThrombosisLow levels Haemorrhage Haemorrhage

1.14 - ACTIVATED PROTEIN C

1. Protein C is the key anti-coagulant pathway in regulating the propagation of coagulationSummary of Protein C activity

Produced in liver and circulates in plasma ~65nM Activated by T-TM complex on EC to inactivate F5a & F8a

o Inactivation of 5a is by fast (but not inactivating) cleavage at 506 and slow (but completely inactivating) cleavage at 306

o Inactivation of F8a is via cleavage of Arg 336/562 APC is generates adjacent to the site of vessel injury; hence its physiological function relates

to regulating coagulation, and in particular thrombin generation, to the site of injury. APC has a half life of about 30mins and is inactivated (slowly) by PC inhibitor, alpha1

antitrypsin and alpha2 macroglobulin

2. In usual circumstances, protein C binds to phospholipid membranes with poor affinityDomain Structure of Protein C

Gla - EGF2 - SPo Gla domain requires Vitamin-K dependent gamma carboxylation (in 9 places) to bind

to calcium ions and forming an omega loopo Gla domain facilitates binding to PL, EPCR & protein So On its own, protein C binds PL poorly

Gla - EGF2 - SPo EGF1 is beta-hydroxylated to bind to Calcium ions tooo EGF2 is glycosylated (?function)

3. Thrombomodulin and EPCR are useful in bringing protein C into proximity with thrombinThrombomodulin ‘alters’ the function of thrombin

TM is a transmembrane protein (105kDa) expressed on all endothelial cells Thrombin binds to it with high affinity Binding to TM means that thrombin loses all its pro-coagulant functionality and acquires

anti-coagulant (APC) and fibrinolytic (TAFI) properties

EPCR binding brings protein C into closer proximity with TM EPCR is similar to MHC I and binds to protein C with high affinity

o PC-EPCR affinity is considerably more than PC[Gla]-PL affinity

EGF domains on TM are sufficient for protein C activation Domain structure: Transmembrane - Serine/threonine - 6EGF- lectin

o Serine-threonine binds the GAG chondroitin sulphate which allows thrombin to bind with TM much more efficiently (providing a surface; reducing Km)

Transmembrane - Serine/threonine - 6EGF- lectino EGF 4 binds to the SP of protein C (low affinity)o EGF 5/6 bind to exosite ?1 on thrombin, which aligns its active site in such a way that

protein C can be cleavedo Thrombin cleaves Arg 169 to release activation peptide and create APC

Transmembrane - Serine/threonine - 6EGF- lectino Lectin domain has no haemostatic role

4. Protein C is probably entirely dependent on its cofactor, protein S, to function properlyPhysiologically - PC is probably useless without PS; there is no change in rate of coagulation regardless of PC concentration, in plasma that is deficient of PS

Structure of Protein S 69Kda protein synthesised in the liver

Domain structure: Gla - thrombin sensitive - 4EGF - SHBGo 11 Gla residues (2 more than protein C) gives protein S the highest affinity for PL of

all vitamin-K-dependent proteinso Gla residue also contributes somewhat to protein C binding

Gla - thrombin sensitive - 4EGF - SHBGo Thrombin sensitive region is essential for co-factor binding & function

Gla - thrombin sensitive - 4EGF - SHBGo EGF1 contributes to protein C bindingo All EGFs are heavily beta-hydroxylated

Gla - thrombin sensitive - 4EGF - SHBGo SHBG binds to complement4b binding protein c4b-bp; bound protein S cannot act as

a cofactor for protein C (approx. 60% is bound) Hence the domains important for protein C binding are Gla, thrombin sensitive & EGF1

The major function of Protein S may be to increase protein C’s affinity for Pl binding Protein S increases affinity for PC/APC to bind PL and hence interact with thrombin/cleave

5a and 8a respectively Inactivation of 5a:

o Protein S relocates APC active site - particularly important because this means the usually slow 306 cleavage for F5a becomes fast

o Protein S may enhance inactivation of the prothrombinase complex, which otherwise confers F5a ‘resistance’ to APC by keeping the factor bound.

Inactivation of 8a:o Protein S enhances APC inactivation of F8a (3fold) by enhancing the 562 cleavage

and making the inactivation of F8a less dependent on its spontaneous disscoiation

5. Deficiencies in the Protein C pathway are associated with thrombophilia

Protein deficiency

Allele frequency Clinical result

PC 3/1k Purpura fulminans in homozygotes, VTE in heterosPS 1/20k Purpura fulminans in homozygotes, VTE in heterosEPCR ? Unknown; lethal in miceTM V rare PEs; MIsF5 leiden ~10% European

caucasiansOnly increases thrombotic risk slightly because usually only one allele is affected meaning that protein C mediated inactivation is only slowed, not terminated. Does increase thrombotic risk in context of other genetic/environmental risk factors

1.15 - ADAMTS13

1. The multimeric structure of vWF usually protects it from the MMP ADAMTS13Structure of vWF relevant to ADAMTS13

Ck domains create dimers in ER

D3 domains are key in multimerisation - larger vWF molecules have more binding sites so they adhere to collagen better and ‘catch’ platelets more effectively - more pro-coagulant activity

o Larger multimers are more unstable so become linear with less difficulty A3 domain (collagen binding) is exposed in globular structure. A1/C1 (platelet binding) and

A2 (ADAMTS13 binding) only become exposed in linear structureo Hence in its globular circulating form, vWF is usually protected from ADAMTS13

2. The domain structure of ADAMTS13 confers step-wise interactions with vWF, ultimately leading to proteolysis

Summary of ADAMTS13 structure 190kDA zinc-dependent MMP which circulates as an active enzyme Very long half life (days) Only has one substrate and one cleavage site- the Tyr1605-Met1606 bond on the A2 domain

of linear vWF Has no natural inhibitor

Domain structure of ADAMTS13

CUB domains may be implicated in binding globular vWF TSP1 repeats 5-8 may also be implicated in binding globular vWF

o Hence the idea is that globular vWF can carry ADAMTS13 via its c-terminal domains in the circulation without proteolysis occurring

Spacer domain is essential to ADAMTS13 functiono Has no disulphide bonds (only part of the molecule; hence ‘spacer’)o Arg 660, tyr 661 & tyr 665 bind to linear A2 vWF with high affinityo Hence the spacer domain is the first domain that plays a role in recognizing unfolded

vWF Cysteine rich domain has an unknown function but is important for normal ADAMTS13

activity Dis(integrin) domain is the next domain that recognizes linear vWF

o R349 and L350 bind linear A2 vWF (at D1614) MMP

o 3 histidine residues (in a highly conserved HEXXHXXGXXH sequence amongst all MMPs) bind Zn to the active site

o Zn binding leads to a shape change that ‘exposes’ or ‘activates’ the key catalytic glutamic acid residue (much like how serine is the key residue in SPs)

o The activated conformation contains a ‘cleft’ for vWF to lie in; glutamic acid hydrolyses the scissile Tyr1605-Met1606 bond

o Calcium ions are required for normal MMP function - but don’t know why

The stepwise interactions with vWF of ADAMTS13a) Low affinity C-terminal bonding keep ADAMTS13 in proximity with globular vWFb) collagen binding and/or shear stress unfolds globular vWF and exposes A2 domainc) Spacer domain binds linear A2 domain of vWF with high affinityd) Dis domain further binds A2 domaine) MMP binds and cleaves A2 domain at scissile bond tyr1605-met1606

3. ADAMTS13 may have its major role as soon as vWF is released form the endothelium In WPBs , vWF is in globular form and elongates under shear stress as soon as it is released -

ADMATS13 may act here to prevent circulation of ultra large vWF multimers In other times when unstable globular vWF spontaneously unravels under shear stress,

ADAMTS13 may also act ADAMTS13 probably acts during platelet plug development to limit its size and prevent

production of an occlusive thrombus.

The ADAMTS13-vWF specificity is achieved in 3 ways vWF MUST unravel before it can be cleaved there are multiple sites of interaction in order to get ADAMTS13 & vWF to fit together

properly vWF must fit into the active site cleft or it will not by hydrolysed by glutamic acid

4. Aberrant vWF proteolysis correlates to diseases of coagulationExcessive vWF proteolysis is implicated in Type2a (group II) vWD

A2 domain mutations = easy unfolding = increased cleavage by ADAMTS13 The phenotype relates to easy haemorrhage

Impaired vWF proteolysis results in TTP; thrombotic thrombocytopenic prupura Rare disease characterised by microthrombi which are rich in platelet and UL-vWF (little

fibrin)o High circulating unstable Ul-vWF more spontaneous unravelling inappropriate

primary haemostasis (thrombotic)o Platelets are used up (thrombocytopenic)o MAHA occurs because red cells get damaged against micro0thrombi/ occluded

vasculature Disease process is seen when ADAMTS13 activity drops to <5-10% of normal Congenital TTP:

o Most mutations impair secretiono Heterozygotes tend to be asymptomatic

Acquired TTPo Usually due to IgG antibodies produced against the spacer domain, in particular the

sequence which allows vWF A2 binding Untreated = mortality >90%. Principle of treatment lies with plasma exchange every 2-3

weeks to give a source of ADAMTS13 and remove the auto-antibodies in acquired disease

1.16 - PATHOPHYSIOLOGY OF vWD

1. Diagnosing vWD requires a number of laboratory tests assessing vWF quantity & functionThere are a number of problems when it comes to assessing vWF - it is difficult to say what is normal

It has multiple binding interactions and hence multiple functions; one or many of which may be lost

It has a multimeric structure There are different pools of human vWF (plasma, WPB, subendothelium, platelets) The ABO blood group plays an influence on circulating vWF concentration. In addition vWF

levels increase with age Stress induces vWF release from stored WPB In vivo function is shear dependent, and this is difficult to replicate in a lab

Antigenic assays only measure vWF quantity Methods include Laurell rocket (not in use), ELISA & Immunoturbidometric (more commonly

used) analyses Although accurate, sensitive and reliable - gives no reflection of functional activity and hence

is not applicable to Type2 vWD

Rco and RIPA are two tests that assess vWF-platelet binding Both tests use the antibiotic Ristocetin, which mimicks shear stress and thus causes globular

vWF to unfold and interact with platelets The vWF-platelet agglutinate causes turbidity The Rco test uses an excess Ristocetin in the presence of a low concentration of vWF [dilute

plasma] to test whether vWF can bind to platelets at allo It is labour intensive, has poor reproducibility but is generally reliableo Failure of a response suggests lack of large vWF multimers or an aberrant GP1b

The RIPA test uses minimal Ristocetin. In normal patients this concentration (0.7) is not enough to stimulate vWF, so this test is used to assess whether vWF is hyper-active

o Hence RIPA is diagnostic for T2B vWD

The other three main tests of vWF function relate to collagen & F8 binding, and ability to form multimers

Collagen assays use a labelled anti-vWF Ab to pick up vWF binding on a collagen mediumo Poor binding suggests lack of large multimer or an aberrant A1 domain (collagen 6)

Multimer analyses compare multiemr profiles to a standardo Abnormal profile can be suggestive of a number of disease processes

Only small multimers = early clearance/cleavage of vWF-Plt complex Only large multimers = failure of ADAMTS13 Loss of multimer ‘flankers’ = failure of ADAMTS13

F8 assays use a labelled rF8. vWF is in excess so there are ample F8 binding siteso Poor binding is diagnostic of T2N vWD

2. The classification of vWD relates to structure and function

vWD Physiology Diagnosis1 (AD) Partial quantitative deficit Difficult - bleeding score2A (AD) Absence of large multimers defective platelet

bindingRco with abnormal multimer analysis

2B (AD) Hyper active vWF strong platelet binding RIPA2C Loss of collagen binding ability Collagen binding assay2M (AD) Aberrant GP1b defective platelet binding Rco with normal multimer analysis2N (AR) Defective F8 binding F8 assay3 (AR) Complete quantitative deficit Antigenic assay

T1 & T2 vWD are differentiated by their function:antigen ratio T1 vWD will demonstrate a concordant loss of function as antigen levels drop T2 vWD can be suspected if the loss of function is <0.7 than the loss of antigen

o Function:antigen <0.7

3. The normal variation in circulating vWF makes it difficult to conclusively say whether a patient just has low normal levels or T1 vWD, and has led to production of the ‘bleeding score’

Because vWF levels vary so much amongst the population (blood group, ethnicity) and in individuals too (age, stress), it is difficult to distinguish people who have a low vWF level which is normal for them, from those who have a true partial deficiency – T1 vWD.

Of people who actually have T1 vWD, only 50% have demonstratable mutations. in most cases, the defect is caused by interactions between mutant and normal subunits [dominant negative effect]

The questions asked in a history might be key in highlighting T1 vWD Epistaxis lasting > 10min Large bruising, in more than one site Haematomas

A Bleeding score >4 may be the most appropriate diagnostic tool in T1 vWD The bleeding score repeats the following grading for bleeding from oral cavity, minor wounds, GI, epistaxis, menorrhagia etc

Blood source -1 0 1 2 3Dental extraction

none

Bleeding occurs <25% of the time, doesn’t require intervention

Bleeding occurs >25% of the time, requires intervention

Bleeding requires suturing/ packing

Bleeding requires transfusion and/or DDAVP

4. The other types of vWD are not as common as T1 vWDType 2A = quantitative inability to bind platelets [because of loss of large multimers]

Group 1 patients lose large multimers because of mutations that prevent normal secretion of vWF and hence retention of vWF in the ER

o Mutations occur anywhere in the vWF gene, and large multimers are more likely to contain the mitation

Group 2 patients lose large multimers because of enhanced ADAMTS13 activityo Mutations only occur in the A2 domain

Some other mutations implicated are defects in di-/multimerisation & susceptibility to intracellular proteolysis

Type 2B = hyperactive vWF Increased affinity for platelets = early clearance of vWF-Plt complex = loss of large multimers

and thrombocytopeniao Mutations are in A1 domain

Type 2M = qualitative inability to bind platelets [because of defective GP1b] All the normal multimers are there Mutations [in GP1b] are commonly found in A1 domain

Type 2N = inability to bind F8 All the normal multimers are there, in the adequate quantity and can bind to platelets

normally, but loss of F8 binding dramatically reduces half life of F8 Gives haemophilia-type bleeding pattern Mutations seen in D’/D3 domains

Type 3 = complete loss of vWF Loss of vWF = impaired primary haemostasis and concomitant reduction in F8 half life gives a

coagulation deficit too A number of mutations are implicated Heterozygotes do not suffer significant bleeding

5. Management of vWD depends on the sub-type because of different responses to treatmentOptions for therapy:

Nothing Tranexamic acid DDAVP Replacement

o Cryoprecipitate and platelets are commonly usedo rF8 is being developed (human concentrates used to be used but transmitted

HIV/HBV/HCV)

DDAVP stimulates WPB vWF release – who responds? T1 – most patients respond

o A T1 subtype named T1 ‘Vicenza’ make normal concs of vWF but cleave it rapidly, and so they display an unsustained response to DDAVP

T2 – response is limited because most patients fall into a rapid clearance/ defective vWF group anyway

o 2N patients will not respond because F8 just can’t bind to vWF T3 – no response

1.17 – STROKE

1. Epidemiology of stroke 3rd commonest cause of death and commonest cause of severe adult disability

£3billion pounds per annum in direct costs alone in UK (versus 2bill for CHD)o 4.5bill on indirect costs?

80% of strokes are ischaemic; 20% are haemorrhagic hypertension is a strong risk factor

2. Pathogenesis of stroke Stroke is a focal neurological deficit or presumed vascular origin

lasting >24hrs; the diminished blood supply leads to loss of o2, accumulation of co2, and loss of ionic gradients across membranes

synaptic transmission is lost after 3 mins, no electrical excitability occurs after 5 mins

the core of the lesion will not recover; the surrounding ‘penumbra’ region has the potential to

mechanisms of iscahemic stroke:o carotid artery stenosis (atherosclerosis)o emboli of a cardiac origin (AF)o small vessel thrombosis/occlusiono watershed infarct in presence of systemic hypertension?

mechanisms of haemorrhagic stroke:o primary/small vessel intracerebral bleedo Rupture of [berry] aneurysm subarachnoid haemorrhage

3. Risk factors for stroke AF & hypertension are strong risk factors poor diet etc – CVS/ CAD risk factors Polymorphisms that lead to a thrombophilic state (eg F5 leiden, Prothrombin G20210A)

4. Acute management of stroke as time passes, the size of the core increases and the penumbra decreases (potential to

recover diminishes over time) tPA benefit is seen particularly within the first 3 hours

1.18 - PLATELETS & ANTIPLATELET THERAPY

1. Platelets have extracellular receptors and intracellular granules which are key to their functionExtracellular glycoproteins are adhesive - they make the platelet ‘sticky’

GP2b3a (integrin like) binds to fibrinogen and vWFo It also binds to a number of other proteins with the key ‘RGD’ AA sequenceo In its rested state it can not take part in binding; calcium influx causes it to unfold

GP1b exists as a complex with 2alpha, 2beta, 2 GP9 and 1 GP5 complex (2:2:2:1). Its main ligands are vWF, thrombin and F11(a?)

GP1a and GP6 complex are both involved in adhesion to collagen (GP1a- collagen 6)

G protein linked receptors play a role in positive feedback after platelet degranulation ADP binds to P2Y receptors Thrombin cleaves the PAR (protease-activated-recptor) receptor 1 & 4 Thromboxane binds to its own receptor and there is also a receptor for prostacyclin Prostacyclin (and NO) produced and released by endothelial cells keeps platelets quiescent

until they meet an appropriate ligand

There are 2 main types of platelet granules, as well as lysosomes Dense granules contain molecules involved in positive feeDback - mainly ADP &

catecholamines but also ATP, calcium ions and serotonin Alpha granules contain coagulAtion molecules - platelet factor 4, vWF, fibrinogen, F5 Lysosomes contain a number of enzymes, most of which are proteases

2. Platelet activation involving intracellular signalling pathways that result in a calcium influxStep 1 involves induction of positive feedback mechanisms

a. GP1a binds to exposed collagen at low shear stress or GP1b binds exposed (subendothelial) vWF at high shear stress [probably a degree of both]

b. Both activate the second messenger PLC gammac. PLC gamma induces the IP3/DAG signalling pathway which leads to a calcium influxd. The calcium influx causes degranulation, which importantly releases ADP vWF and

fibrinogen, and stimulates thromboxane A2 synthesisi. At the same time GP2b3a begins to unfold

ii. Also at the same time, microtubules begin to change the structure of the platelet into a ‘spreading’ cell. The change is ultimately irreversible

Step 2 causes aggregation of plateletse. ADP, thrombin and Thromboxane A2 bind to their respective receptors

i. These mediators bind to platetlets they were released from and/or other local platelets

f. The secondary molecule in this case is PLC beta - which also causes a calcium influxg. At this point (i.e. after thrombin and collagen binding) there is enough calcium to cause

‘inversion’ of the phospholipid membrane - which speeds up the coagulation cascadei. This is achieved by calcium inhibition of flippase (which keeps PPDserine &

ethanolamine on the inner membrane) and stimulation of scramblase h. The main players in platelet aggregation are Thromboxane A2 & Gp2b3a

3. There are a number of ways that platelet number and function can be assessed in the laba. Number - assessed using impedance studies, the same as for RBCs - technique varies

from lab to lab so normal ranges (150-400e9) may differb. PFA-100 – aspiration of blood through collagen lined aperture and measuring time for

plug formation and occlusion of the aperture – upto 190s is normalc. Aggregation tests - shines light through platelet rich plasma and measure change in

transmisiion following addition of an agonist eg ADP, adrenaline or collagen

i. ADP & adrenaline are weak agonists for platlets so tend to show a 2 wave increase in transmission

ii. A lack of secondary wave suggests receptors are ok but there is some fault downstream of this (strage pool disease) or use of aspirin

iii. Lack of aggregation in all agonists except for ristocetin suggests a GP2b3a problem – Glanzmanns

iv. requires large volume of blood, is poorly reproducible and is sensitive to platelet interaction with food etc

d. Storage pool & release function - 3 methods:i. Measuring release reaction (Lumi - similar to aggregation tests)

ii. EMiii. Measuring ATP:ADP ratio (usually <2) - this is increased in storage pool diseases

because of a lack of ADPiv. very labour intensive

e. Flow cytometry – sensitive, specific and only requires a few milliliters of blood. diagnostic for receptor defects

f. Genetic analyses

4. Antiplatelet agents play a key role in the prevention of arterial thrombosisAspirin irreversibly inhibits COX1 (and COX2)

the mechanism of action is acetylation of serine near the active site platelets, being anucleate can not synthesise anymore COX1 low dose (75mg) aspirin is good for anti-platelet effects

o half life = 15-20mins and as it is almost completely metabolised first pass, it inhibits platelets that pass in the portal circulation

o mean life span of platelets ~10days so knocking them all out keeps you ‘safe’ for about this time until new ones are synthesised

very high doses of aspirin would be needed for anti-inflammatory affects via inhibition of COX2 – because COX2 is induced by shear stress on endothelial cells. this is not ideal because of side effects associated with high dose aspirin (bleeding, GI ulcers)

o COX2 makes prostacyclin (high shear COX2 PGI2 vasodilation less shear) Aspirin ‘resistance’ terms compliant patients who still form thrombi when on aspirin. this is

mainly due to failure to suppress TXA2 production due to alternative production mechanisms in the individual:

o production of TXA2 by monocyteso an unusually high/fast platelet turnovero high vWF?

ADP receptor blockade Clopidogrel is a prodrug which is activated by 2 step cytochrome P450 metabolism.

o it irreversibly binds to P2Y12 receptors (disulphide bond)o it causes 50-60% inhibition of ADP-induced aggregation & this lasts for several dayso commonly used with aspirin post ACS (CURE study)o polymorphsims in the liver enzymes/ P2Y receptor genes can give variable responses

Prasugrel is similar to clopidogrel but is more rapid (1 step) and consistent (perhaps more bleeding risk)

Ticagrelor is a reversible inhibitor of P2Y12 – onset takes 2 hours and its half life is 12hours dipyridamole dimishes intracellular adenosine by preventing reuptake and inhibiting

adenosine deaminase. it also inhibits phosphodiesterase which causes high intraceullar cAMP – this impairs platelet aggregation

GP2b3a receptor blockade Abciximab can completely abolish aggregation if 80% of GP2b3a receptors are blocked Tirofiban is a tyrosine derivative eptifibatide is another alterntative GP2b3a blockers have been licensed for ACS/PCI but they all cuase increased bleeding

Novel targets incluce P-selectin, thromboxane recptor blockade

Some protocols: Aspirin + Clop; 1year post ACS with metal stent Aspirin + dipyridamole – secondary prevention of stroke Warfarin + aspirin – PCI + AF

1.19 – HERIDITARY PLATELET DISORDERS1. Clinical picture of platelet disorders* Muco-cutaneous bleeding * in the history is suggestive of platelet disorder

spontaneous skin petechiae/ purpura

bleeding from mucous membranes, epistaxes prolonged bleeding after trauma/surgery bad menorrhagia at menarche for girls family history

Bleeding is unusual if platelet count is >30e9; if there is a discrepanacy between bleeding history and platlet count, this may suggest a functional platelet defect.

2. classification of heridtary platelet disorders – production and functionInherited Disorders of platelet production are rareThese are almost always part of a wider problem or a pre-leukaemic disease. some examples are: Congenital aplastic anaemia syndromes amegakaryocytic thrombocytopenia

o mutations in TPO receptor = no megakaryocytes in BM; platelet count <20o develops into aplasia/leukaemia, requires BM transplant

thrombocytopenia with absent radii syndromeo AR disease with absent radii & phalanges although the thumb is always normalo the platelet count is <50 but normalises with ageo TPO signalling pathway is probably defective; megakaryocytes (defective CFU-meg) are

absent from BM MYH9 related disease

o spectrum of disease associated with non-muscle myosin mutationso macrothrombocytopenia, (count <20), WBC inclusions, renal failure, hearing loss and

cataracts are some clinical featureso genetic analysis key to diagnosis; Dohle bodies on microscopy are pathognomonic

a number of other eponymous syndromes (digeorge, , paris-trousseau)

Functional disorders (covered below) can be defects in GPs, storage pool or release

3. Plasma membrane defects Bernard Soullier syndrome is due to decreased/ defective GP1b GP1b complex contains 2 disulphide linked subunits, covalently bonded to GP9 as a heterodimer.

there are roughly 25k complexes per platelet this is an AR disease which has a variable but often severe bleeding pattetn mild thrombocytopenia with increased mean platelet volume and bleeding time are seen absent/reduced agglutination with ristocetin and flow cytometry may also diagnose it

Pseudo-vWD is the opposite half of T2b vWD Hyper active GP1b (not hyper active vWF) is the problem in this case as with t2b vWD, low conc ristocetin (Rco) test will diagnose it, but you must treat it with

platelets, not vWF

Glanzmanns thrombasthenia is due to decreased/ defective GP2b3a GP2b3a complex is also a heterodimer; there are roughly 80k complexes per platelet this is also an AR disease associated with severe bleeding. there are 3 subtypes:

o type 1 = Absent GP2b3a or no fibrinogen in alpha granuleso type 2 = partial deficiency in GP2b3a or fibrinogen in alpha granuleso Variant Glanzmanns is due to a functionally defective GP2b3a

Flow cytometry and genetics are key to diagnosis Platelets to treat. formation of antibodies to normal platelet GP2b3a is associated with a

worse prognosis (because you cant give them any more treatment!)

Scott Syndrome is due to defective phospholipid inversion pathogenesis is a failure of platelets to generate microparticles on activation; hence

PPDserine is not expressed normally and clotting factors can not bind bleeding is moderate to severe; this disease is very rare though

collagen receptor defects often only causes mild bleeding because platelets have a number of membrane proteins which can bind collagen

3. intracellular disorders are subclassified by what storage granule is problematicintracellular disorders are more common but only give mild bleeding. platelet count and lifespan are usually normal and ATP:ADP ratio is an important diagnostic tool.

storage pool disease - Dense granule deficiency Idiopathic (non-albino type) hermansky-pudlak syndrome

o AR disease associated with mild bleedingo due to membrane and content abnormalities for dense granuleso oculocutaneous albinism and very bad nystagmus are features – treat with

tranexamic acid chediak higashi syndrome

o AR disease associated with increased risk of infections; treat with bone marrow translpant

wiskott-aldrich syndromeo due to defective WASP protein expressiono associated with eczema, immune deficiency and requires splenectomy or BMT

storage pool disease = alpha granule deficiency Grey platelet syndrome

o AD rare disease where alpha granules are absent/empty (so platelets appear grey on blood film)

quebec platelet disordero AD disease associated with alpha granule multimerin deficiency; reduced contents

combined storage pool disease

defects of thromboxane generation are due to COX/ thromboxane synthetase deficiencies

defects in signal transduction can be suspected if you defective aggregation responses to all agonists

4. treatment of hereditary platelet disorders requires specialist care Supportive measures (avoid aspirin, NSAIDs, give tranexamic acid) DDAVP (but not in kids as it gives hyponatraemia) platelet transfusion – HLA match if they will need lifelong transfusion to avoid Ab synthesis rF8 for Haemophil/glanzmanns patients who have developed Abs BMT – usually just for Bernard soullier and glanzmanns; 10% mortality at least

1.20 – PREGNANCY OESTROGENS AND THE COAGULATION SYSTEM

1. Oestrogens are associated with an increased risk of VTEThrombotic changes associated with oestrogens

Increased procoagulant activityo stimulation of Fibrinogen, F7-11, vWF, protein C resistance

Decreased anticoagulant activityo Inhibition of protein S and antithrombino the pill reduces transcriptional synthesis of protein S

But there is some increased anticoagulant activity tooo Heparin cofactor 2, protein C & alpha2 antitrypsin are stimulated

Fibrinolytic changes associated with oestrogens [net reduction in fibrinolysis] Increased antifibrinolytic activity

o stimulation of F13 decreased profibrinolytic activity – inhibition of tPA there is also some stimulation of fibrinolysis (via plasminogen, F12 and prekaallikrein) as well

as inhibition of anti-fibrinolysis by PAI-I, there is a net anti-fibrinolytic activity though

The oestrogen dose of the pill has decreased amidst evidence of VTE risk The drop from 100microg to 50 of ethinyl oestradiol gave the biggest drop in risk all contraceptive pills seem to increase risk of VTW between 4-6times

o 3x risk if <30years old; higher risks with increasing ageo the highest risks are seen when first starting the pill, and post partum

different ‘generations’ of progesterone component of the COC were designed to reduce this risk, the 3rd generation (eg desorgestrel) however gave more risk than the second generation (eg levonorgestrel)

o But note - the progesterone only pill and mirena do not increase VTE risk COCP also seems to increase risk of arterial thrombosis by 2-3x; this is a cloudy area though hence you need to balance risks with benefits (avoids unplanned pregnancies, regulates

dodgy periods, controls PMS/migraine, decreases ovarian/enodemetrial cancer)

2. The pill interacts strongly and synergistically with other thrombophiliasthe degree on interaction is unpredictable

risk increases upto 30times in F5 leiden, but only about 16x in PT G20210Ao although the risk of cerebral vein thrombosis is particularly high in Prothrombin

mutation patients who take the pillo also interacts with High F7 [and other factors], antithrombin/PC/PC deficiencies

screening is an option but between 400k and 2mill women would have to be screened to prevent one death – not viable; better to offer patients counselling and leave treatment decision on a good history and assessing risks/ benefits – some thrombophilias can be asymptomatic

3. HRT is used in an older patient cohort – this translates to a higher relative risk of VTE than OCP HRT is now used in small doses to alleviate symptoms of menopause. it still has beneficial

effects in preventing osteoporosis but does not seem to be protective for CVD risk is greatest in first year of use; otherwise relative risk seems to be about 2x transdermal HRT patches have not been shown to increase VTE risk HRT in asymptomatic thrombophilic women should be discouraged

4. Pregnancy puts women in a procoagulatory state to prevent excessive blood loss when utero-placental separation occurs at birth

Major coagulatory changes in pregnancy pre – hypercoagulable & hypofibrinolytic increased thrombin generation

o vWF (and F8) increase 2-3x progressively throughout termo fibrinogen increases 2xo F7 increases 1.5xo protein S falls by half

?increased fibrin cleavage reduced fibrinolysis

o PAI1 increases 5 foldo PAI2 is produced by the placenta

interactions with other maternal factors

85% of pregnancy VTEs occur on the left due to anatomy of common iliac arteries

Risk factors of VTE in pregnancy 1/1k pregnant women suffer VTE; the risk is highest in the first two trimesters and the post

partym period women with high BMI are at an increased risk dehydration, pre-eclampsia, emergency caesareans, old age and multiple parity are all

associated with increased risk

Management of VTE in pregnancy improve assessment of which women are at a higher risk recognise symptoms like leg pain as early warning signs and diagnose quickly

o TEDs should be worn for 2 years if a diagnosis is made; and thromboprophylaxis given for subsequent pregnancies

If women are at increased risk then give thromboprophylaxis (LMWH + TEDs) and encourage mobilisation and hydration

o aim for 6 months treatment in totalo warfarin is teratogenic in first trimester (small molecule; crosses placenta)

5. management of obstetric haemorrhage Preconception counselling for women who have a bleeding tendency/increased risk antenatal counselling fetal sexing/ USS diagnosis/ fetail DNA sampling/ CVS/ amniocentesis upto 17weeks

gestation to diagnose if the unborn child has the hereditary problemo 1% risk of miscarriage with these procedures

avoiding instrumental deliveries etc if child is found, for example, to be haemophilic

1.21 - AUTO-IMMUNE DISEASE IN HAEMATOLOGY

1. Autoimmune diseases are generally acquired in haemoatology; some targets are:

Target Disease Phenotypeplatelet ITP

BLEEDINGF8c acq Haemophilia AvWF acq vWDF13c acq F13 defF9/10/11c acq factor defGP2b3a acq GlanzmannsADAMTS13 TTP

THROMBOSISHeparin-PF4 HITTProthrombin APSbeta2GP1 APS

2. Idiopathic TTP is a serious disease thought to be related to ADAMTS13 deficiencyTTP is a fairly rare disorder with a high mortality incidence ~4mill/year, affecting young females commonly mortaility >90% if untreated idiopathic TTP presents without a predisposing condition or oliguric renal failure

o familial disease is associated with ADAMTS13 deficiencyo isolated disease is thought to be autoimmune destruction of ADAMTS13

secondary TTP is a thrombotic microangiopathy with a predisposing condition; eg:o cancer, sepsis/ HIV infection, pregnancy, pill, eclampsiao drugs eg Ticlopidine, clopidogrel, quinine, ciclosporin, mitomycin C, pentostatin

HUS is a thrombotic microangiopathy with acute loiguric renal failure and sparing of the other organs – shiga toxin from E-coli seems to be causative

TTP presents as a classic pentad of signs MAHA*[shistoc ytes on blood film]+ Low Plt* are key Neurological dysfunction renal failure fever Investigations may show low Hb, high creatinine, high

LDH PT/APTT are normal

Plasma exchange is the management of choice in initial TTP plasma exchange still has 20% mortality associated with it (within 5 weeks)

o plasma esxchange 30-60ml/kg daily +methylprednisolone for 3days until there is a complete response – normalisation of platelets and LDH with stable neurological signs

o continue exchange after response every other day for four dayso complications of exchange include blockage of catheter or reaction against plasma/

introduction of infectiono avoid platelets and anti-platelet drugs

80% respond in roughly ~16days; 40% of these have exacerbations within a week and 30% definitely relapse within 2 years

o [20% dead within 5 weeks]o relapsing disease may warrant immunosuppression – rituximab/vincristine/splectomy

3. Heparin induced thrombocytopenia is a rare disease that must be diagnosed clinically

non immune HIT causes mild transient thrombocytopenia(>100e9) which doesn’t require treatment; it is however a diagnosis of exclusion

clinically significant HIT only occurs in 3% of people:o platelet count drops on heparin admission to <30-50% of previous or <100e9 within

5dayso thrombo-embolic complications begin to occur eg VTE/ heparin induced skin lesions/

venous gangreneo prior history and use of UFH before major surgery are associated with higher risko rapid onset/delayed onset HIT occur either side of the 5day ‘cut-off’

the differential diagnosis for HIT includes ITP, DIC. if the disease process starts <5days suspect DKA/SBE/PNH

HIT score can aid diagnosis: higher the score, more likely it is that you have HITFinding 2 1 0thrombocytopenia fall of ~50% fall of ~30-50% fall of <30%timing day 5-10 after day 10 before day 5thrombosis new clot, skin necrosis progress/recurrence noneoTher cause none seen possible definite

The pathogenesis of HIT involves widespread platelet activation PF4 normally binds to activated platelets and forms complexes with heparin autoantibodies (IgG) bind to the PF4/heparin complex platelets get activated via their fc receptors to release procoagulant microparticles

Management = stop heparin, initiate alternative anticoagulant and test for antibodieso can also add direct thrombin inhibitors eg Lepirudin

4. Haemophilia A and vWD like syndromes can be acquired due to autoimmune diseaseAcquired Haemophilia A occurs because of antibodies against F8 presents in middle-aged/elderly patients with extensive cutaneous purpura/ internal bleeding antibody titre seems higher in pregnancy polyclonal IgG4 Abs formed against A2/C2 domains of F8

o antibodies are time-dependent (type 2 kinetics?) treat with immunosuppression eg prednisolone +/- cyclophosphamide.

o some studies say 71% of people respond; with about 20% relapsing roughly 8months after stopping immunosuppresion

Acquired vWD occurs in the context of a background disease process

Immune mediated disease is associated with background Lymphoproliferative or autoimmune disease. DDAVP/IVIg/F8 concentrate may lead to response

Proteolysis-mediated disease is associated with background thrombocytosis or conditions leading to high shear eg aortic stenosis – you must treat the underlying cause

1.22 – NOVEL METHODS OF ASSESSING HAEMOSTATIC FUNCTION

1. The traditional ‘reductive’ methods of assessing haemostatic function are limited because about 50% of patients with thrombosis do not have a specific deficit

The pros of the reductive approach are specificity of diagnosis and treatment that is directly targeted to a deficiency

traditional methods are less attractive for complex diseases – leading to increased popularity of global assessments of haemostasis

global assessments take into account that coagulation is an integrated system

2. Some global assessments of haemostasis are not specific = high false positive rateAntibodies to measure enzyme-activation markers are not in general use

Activation peptides [these are released on zymogen activation] and enzyme inhibitor complexes [the intermediate] can both be measured

o it is a difficult process, has limited sensitivity in thrombophilia and is largely ineffective in predicting thrombosis – not in general use

D-dimer is useful, but not specific, in ruling out thrombosis d-dimers are only released by plasmin when fibrin has been crosslinked by F13 They offer an indirect measure of thrombin generation (and F13 activity) Low/normal d-dimer = low chance of thrombosis (clinical application is ruling out [not

diagnosing] VTE)o used to diagnose DIC, but, as with VTE, only by ruling it out (poor specificity)

3. Measures of coagulation potential thromboelastogram/ROTEM

o involves measuring changes in clot strength on a blood sample exposed to celite

o does not give a specific diagnosis but gives idea of coagulation and fibrinolyutic ‘strength’

endogenous thrombin potentialo Uses TF to give real time thrombin

generation using a fluorescent substrate on platelet rich/poor plasma

o poorly standardised so it is primarily a research tool at the moment

o may have future roles in assessing responses to therapies

4. Measures of primary haemostasis onlyPFA/bleeding time

1.23 – MALIGNANCY ASSOCIATED THROMBOGENESIS

1. Cancer is a big risk factor for development (and recurrence) of VTE annual incidence is roughly 1:200

o although it could be higher than this because asymptomatic VTE is common and unaccounted for, and clinical manifestations are non-specific due to background disease

There is a 4-7x increased risk of VTE in cancer patients VTE is the second biggest killer in cancer patients; 1 in 7 cancer px die of a PE

o the longer the time between cancer and VTE diagnosis (presumably because of undetected, asymptomatic disease) –t he greater the risk of dying

Other VTE risk factors are age, stasis/surgery, family history of VTE, varicose veins, CHF/MI/Stroke, lower leg fractures, OCP/HRT/pregnancy

Idiopathic VTE may be a predictor of occult malignancy Tumour type/site influences risk of VTE:

RR sitebaseline risk lung, colon, breast, ovary,

prostateintermediate risk >17x leukaemia, liver,

cervical/uterushighest risk >25x pancreas, lymphoma, brain

Anti-tumour therapy may play a role in increasing VTE risk too

o chemo (eg Thalidomide and Lenolidomide in Multiple Myeloma)

o hormoneso surgery/immobility

2. The pathogenesis of VTE in cancer is complex and probably poorly understood It is possible that malignant cells induce

monocytes/macrophages to produce TF Worth noting that cancer patients can often have

other co-exisiting thrombophilias eg F5 leiden some tumours eg adenocarcinoma produce pro-thrombotic mucin

3. The management/secondary prophylaxis of VTE in cancer may switch to long term use of LMWH Usual management of VTE in cancer involved 5-7days LMWH until INR reaches 2 and then

warfarin for 3-6months as secondary prophylaxis

The CLOT study 2003 suggested long term LMWH is associated with decreased risk of VTE recurrence with no change in bleeding or mortality

1.24 – ACQUIRED HAEMOSTATIC DEFECTS (DIC/CPB/HUS)

1. DIC is an excessive and inappropriate activation of coagulation which is always secondary to an underlying disorder

The pathogenesis of DIC probably involves a confluence of pro-coagulant mechanisms TF/ other procoagulant material is directly released into circulation

o this can be secondary to trauma/malignancy TF expression can be increased

o Either due to endothelial damage (sepsis, burns) or provoked by bacterial toxins/other cytokines (TNFalpha, IL1,6,10)

Decreased expression of thrombomodulin o secondary to endothelial damage

Increased degradation (by neutrophils) of antithrombin +/ impaired synthesis of antithrombin high levels of PAI-1 which depresses fibrinolysis [snake venoms activate factor 10/2]

There are four main causes of DIC Infections (g-ve sepsis, malaria) Cancer (acute leukaemia) obstetric (septic abortion, placental abruption,

eclampsia) tissue necrosis (burns, trauma, liver disease)

The clinical picture of DIC is BAD Widespread fibrin deposition within circulation =

o MAHA (shistocytes on blood film)o Compromised blood supply to organs multiple organ failure

particularly wary of kidney, brain, heart, liver, lung damage Widespread consumption of platelets and clotting factors = bleeding Prolonged PT,APTT, TT with Increased FDP and D-dimer low fibrinogen, protein C, antithrombin, platelet A diagnosis of DIC is difficult but an ISTH score >=5 can be suggestive

1 2 3Platelet <100 <50FDP ‘moderate

increase’‘strong increase’

PT prolonged by 3-6s prolonged by >6sfibrinogen <1

DIC is always secondary to another disease – you must treat the cause supportive measures can be given in the mean time

o blood transfusion if blood loss/anaemia is severeo replacement coagulation factors (FFP/PCC) [FFP15ml/kg body weight]o platelet transfusions 1unit/10kg body weight when it drops below 50o cryoprecipitate when fibrinogen <0.8g/lo Protein C/ TFPI have some use in sepsis induced DIC [PC causes haemorrhage]

2. Cardio-pulmonary bypass is used to prevent excessive blood loss in cardiac surgery The principle is diverting the circulation (which is heparinised) over a non-biological surface in an

oxygenator. The surface is lined with polyurethane, polypropylene and PVC.

Preventing excessive blood loss in cardiac surgery reduced mortality and decreases need for transfusions

The risks of bleeding in CPB are largely associated with platelet dysfunction administration of preoperative aspirin turbulence/stress in the machine (extra-corporeal circuit) = MAHA & similar damaging affect on

platelets [drop in platelet count by upto60%] contact with ‘alien’ surface = activation of fibrinolysis, inflammatory mediators, complement hypothermia may be experienced in surgery – this is associated with defective thromboxane A2

synthesis by platelets = impaired aggregation The dysfunction usually reverses within 3 hours of coming off the machine aprotinin is an inhibitor of plasmin and kallikrein. it used to be used to decrease bleeding risk but

was associated with increased risk of MI, renal failure and stroke. [These risks are not seen with other antifibrinolytics eg tranexamic acid]

if patients bleed in CPB– avoid transfusion (associated with renal failure, infection) and give platelets

3. Bleeding associated with uraemia is again largely due to platelet dysfunction The clinical picture including bruising, epistaxis, Gi bleeding and intracranial haemorrhage

o uraemic haemorrhage can be fatal Bleeding is contributed to by:

o platelet dysfunction impaired aggregation storage pool defects decreased thromboxane A2 production increased platelet calcium content

o defective platelet/vessel wall adhesiono low haematocrit (anaemia)

platelet dysfunction is caused by guanidosuccinic acid – which is produced by an abnormal ammonia detoxification pathway that occurs in uraemia

Management of uraemic haemorrhageo correct anaemia (transfusion)o DDAVPo Cryoprecipitateo Conjugated oestrogens

1.25 – METABOLIC DISORDERS ASSOCIATED WITH THROMBOSIS

1. There are 7 well known disorders associated with an increased risk of thrombosis Homocyteinuria malignancy pregnancy post-operative drugs haemolytic anaemias hyperviscous blood

o plasma mediated (Ig’s, myeloma, Waldenstroms macroglobinuria)o cell mediated (myeloproliferative disease, leukaemia)

2. High levels of homocysteine are associated with an increased risk of atherosclerosisPhysiology of Homocysteine produced during synthesis of methionine It is usually metabolized in two pathways:

o transulphuration to cysteine (vitamin B6 & cystathione beta synthetase dependent)o remethylation to form methionine (dependent on vitamin B12/folate amd the enzymes

MS, MTHR) normal level 5-15microM/l; severe levels are >100microM

Homocysteine promotes development of atherosclerosis by 3 mechanisms Production of oxidants

o hydrogen peroxide damages endotheliumo hydroxyl radicals oxidize lipids in LDL

Endothelial dysfunction o inhibition of TM, heparan sulphate & NO

synthaseo induction of TF

SMC proliferation is stimulated

Homocysteinuria is associated with defects in normal homocysteine metabolism Cystathione b-synthetase deficiency is an AR disease where homocysteine level is >100microM A polymorphism in MTHFR (C677T) makes the enzyme more thermolabile – this variant is more

common in CHD/ coronary stenotic patients Homocysteinuria is associated with skeletal deformities, lens dislocation and intellectual

handicap alongside increased arterial, and possible venous, thrombosis Cyanide-nitroprusside test [brand’s test] can diagnose it by detecting urinary H-cytstine

3. Thrombosis in PNH is largely due to complement-mediated RBC lysis which leads to excessive free circulating haemoglobin Normal protection from complement mediated lysis The PIG-A gene codes for a protein that anchors GPI to red cells GPI has a number of surface molecules including CD55, CD59 and CD87 CD55 increases the rate of dissociation of the complement complexes c4b2a and c3bbb CD59 prevents c8-c9 interaction and this inhibits the classical complement pathway CD87 usually binds u-PAR to stimulate plasmin production

PNH is an acquired somatic mutation in PIG-A lack of protection GPI makes red cells vulnerable to complement mediated lysis

o this leads to intravascaular haemolysis at night and haemoglobinuria in the morningo loss of CD87 function also means there is less fibrinolysis

Increased cell lysis gives high levels of circulating Hb (and arginase) which increases thrombotic risk

o free Hb activates endothelium, inflammation, thrombosis and irreversibly reacts with NO to form metHb & nitrate

o less NO = dystonia in VSMCs, endothelial dysfunction, platelet aggregation and platelet activation

Haptoglobins usually bind free Hb and are degraded by monocytes. another safeguard is haemopexin which takes Hb to the liver

Thromboembolism accounts for upto 2/3 deaths in PNH and commonly occurs in hepatic, portal, and mesenteric veins and the sagittal sinus

eculizumab stops assembly of MAC and thus reduces the number of thrombotic events

3. Myeloproliferative diseases associated with the JAK2 mutation increase thrombosis risk by activation of platelets and cell-mediated blood hyper-viscosity

Thrombosis is the leading cause of death/morbidity in myeloproliferative disease PRV = increased red cell mass; 12-39% patients present

with thrombosis ET = increased platelet count >600 for >6weeks; 11-25%

patients present with thrombosis

The JAK2 V617F mutation increases thrombotic risk in myeloproliferative disease This JAK2 mutation increased Hct, WBC, TF, TM and

adhesion molecule production (p-selectin) The mutation also seems to make platelets hyper-active

by inducing c-mpl expression High Hct not only increases blood viscosity but increases

platelet adhesion to endothelium Higher WBC contributes to thrombosis mainly via activated neutrophils and monocytes:

o neutrophils aggregate with plateletso monocytes make TFo production of pro-inflammatory cytokines

Large vessel thrombosis is more common in myeloproliferative disease Cells migrate axially so platelets are displaced to vessel wall

o inter-platelet and platelet-vessel wall interactions increase Upto half of sufferers of hepatic vein thrombosis (Budd-Chiari) have underlying MPD thrombosis risk increase with age, previous thrombosis, raised HCT, clonality in white cells

(seen in ET) Low dose aspirin can decrease the number of thrombotic events

1.26 – THE MULTIPLE ROLES OF THROMBIN IN COAGULATION

1. Thrombin has multiple procoagulant and anticoagulant roles

Procoagulant

Role Cofactor ExositeFibrinogen fibrin

none needed

1F5 F5a ?F8 F8a ?F13 F13a Fibrin 1F11 F11a GP1b 2Cleavage of PARs & GP5 GP1b 2

Anticoagulantstimulates production of F10a (via 5a) and hence the 10a-antithrombin complex

Heparan Sulphate

2

Activates Protein C TM 1Anti-fibrinolytic Activates TAFI TM 1

2. The main structural features of thrombin and 2 hidden exosites generation of thrombin from prothrombin exposes hidden

exosites exosites bring substrate into the active site cleft the active site serine is number 195

3. Thrombin uses exosites to bind to different cofactors and extend the range and increase specificity of its interactions

The initial procoagulant role of thrombin do not require cofactorso during fibrinogen cleavage thrombin uses exosite I, a sodium loop and its active site

exosite 1 binds to the fibrinopeptideso it uses numerous different residues for recognition and interaction of F5 and F8

Thrombin uses the same residues to recognize Antithrombin, Protein C and F13 The key residues are W50, E229 and R233 to recognize antithrombin It uses exosite II to bind heparin/heparan sulphate when forming the thrombin-antithrombin

complexo i.e exosite II binds the cofactor for the reaction which is catalysed by antithrombin

It uses exosite I to bind TMo i.e. exosite I binds the cofactor for the reaction catalysed by thrombin

By cleavage of fibrinogen, it generates its own cofactor (fibrin) for the activation of F13

4. From initial generation – the main regulation of thrombin activity is driven by exosite competition

the cofactor sites on thrombin for fibrin and TM overlap – this suggests competition for exosite Io this competition halts unregulated platelet plug expansion

GP1b complex assists thrombin in binding to PAR/GP5 on platelet surfaces. It competes with heparin/heparan sulphate for exosite 2

o this competietion halts unregulated platelet activation Lastly, antithrombin and heparin cofactor 2 mop up any excess thrombin

1.27 - DEVELOPMENTAL HAEMOSTASIS IN THE NEONATE

1. Differences in fetal coagulation factor and platelet quantity and quality account for coagulation disorders seen in the newborn

coagulation factors do not cross the placenta; synthesis starts in utero from about 10weeks and levels increase as term progresses

o hence PT, APTT and TT all decrease as term progresses haemostasis is ‘impaired in the newborn because of:

o decreased synthesis & accelerated clearance of coagulation factorso synthesis of proteins with decreased functional capacityo fetal/newborn megakaryocytes are small and less matureo platelet responses to certain stimuli may be impaired in the fetus/newborn

2. Factor 8 & fibrinogen reach adult values at term, otherwise newborns are largely vitamin K-dependent factor deficient

Factor % of adult value reached at termF7 53%F9 32%F10 40%Prothrombin 44%F8 94% Fibrinogen =>100%

3. Acquired coagulation/platelet disordersAcquired haemostatic disorders in the newborn: haemorrhagic disease of the newborn (HDN) DIC liver disease Neonatal thrombocytopenia

HDN is due to vitamin K deficiency poor placental transfer of vitamin K low fetal vitamin K stores low vit K content of breast milk absent bacterial vit K synthesis in neonatal gut immaturity of fetal liver

HDN usually presents within a week Haemorrhages in the brain, GIT, post circumcision and excessive bruising/purpura Diagnose with prolonged PT Manage with prophylactic and/or episodic Vitamin K or FFP if bleeding is severe

HDN classification Time of presentation causesearly within 1 day maternal drugsclassic within 1 week physiological [see above]late 2-12weeks liver disease, breast feeding

The principle of treating neonatal DIC is the same as always – treat the cause Infection Necrotizing enterocolitis Asphyxia

Meconium aspiration Placental abruption Retained dead second twin Diagnose with prolonged PT, APTT, TT, low Plt + high FDPs. Treat underlying cause and may need to give platelets/FFP if bleeding is severs

Neonatal thrombocytopenia is the commonest abnormality in newborn babies Reduced platelet production

o transient - maternal HTN/ DM and fetal IU growth restrictiono rare – BM failure, fanconi anaemia

Increased platelet clearanceo immune mediated - maternal ITP, neonatal alloimmune thrombocytopeniao non-immune mediated – DIC, infections (CMV, rubella)

The mechanism of NAITP involves HPa1a antibodies Mother is HPA1a negative Fetus inherits HPA1a from father Mother produces antibodies against fetus which can cross the placenta this occurs 1in1500 preganacies – it is the commonest cause of severe thrombocytopenia in

newborn infants Manage with HPA1a-negative platelet transfusion. most cases are transient and resolve

within 3-4weeks as the antibodies are cleared.

4. Inherited coagulation disordersInherited haemostatic disorders in the newborn: Haemophilia A/B T3 vWD Rare coagulation factor deficiencies

Haemophilia is the commonest inherited coagulation disorder and is frequently diagnosed post circumcision

affected boys may present with excessive bleeding after circumcision, cephalohaemoatomas, ICH of bruising after blood sampling

long APTT and suspect factor assays give oral vitamin K

1.28 - ARTERIAL THROMBOSIS [ this lecture was not actually given]

1. The intiation of atherosclerotic lesion formation starts with fatty streak formation Streaks begin to form by end of first decade Sites that are predisposed to a pattern of flow that affects endothelial permeability (high shear/

low shear bifurcations etc) are particularly affected The change in permability leads to retention of LDL which is oxidised in the ECM Oxidised LDL production leads to endothelial dysfunction:

o Expression of adhesive molecules eg selectins, VCAM, ICAM and chemokines eg MCP1o Reduced NO production

Diets etc which predispose to high circulating LDL/low HDL (which usually inhibits LDL uptake into endothelium) accelerate plaque formation

2. Platelets adhere to plaque prone sites because of dysfunctional endothelium Platelets do not usually bind to endothelium (prostacyclin, NO) Decreased NO production & selectin expression allows platelets to bind to dysfunctional

endotheliumo Firstly platelets bind and roll, just like when they normally bind subendothelium, to

endothelial p-selectin via GP1ao Secondly, Firm adhesion is mediated by GP2b3a which binds to fibrinogen or vWF - this

causes degranulation of platelets Activated platelets achieve the following roles:

o Release of vWF etc and subsequent thrombus formationo Recruitment and activation of monocytes via p-selectin receptoro SMC proliferation via TGF/PDGFo Further LDL retention via PF4

3. Macrophages play a key role in forming foam cells Enzymes released from macrophages may speed up LDL oxidation and cause tissue damage Macrophages eat oxidised LDL to form foam cells, which subsequently die and release of lot of

TF The local tissue damage leads to fibrous cap formation on the surface of a core of dead

macrophageso Necrotic lesions become calcified by pericyte-like cells in the intima

Occlusion is mediated by plaque rupture and release of TFo Plaque rupture is to do with instability rather than plaque sizeo Plaque progression is stimulated by HTN, hormones, infection

4. The role of vWF in atherosclerosis vWF stimulates WPB production, and WPBs contain inflammatory and angiogenic mediators elevated vWF is associated with acute occlusions experiemnts in vWF deficient mice show less leukocyte recruitment and reduced atheroma

formation other murine stroke models have shown vWF increases infarct size in stroke (and ADAMTS13

reduces infarct size) aptamer (anti vWF A1) and abciximab (anti GP2b3a) are both potential targets for vWF, aptamer

seems to have a higher therapeutic index

5. the role of coagulation factors in atherosclerosis F5leiden prothrombin G20210A and anticoagulant deficiencies have weak associations for

IHD/strokeo this may be because available data is poor

warfarin is more effective than aspirin but you need to aim for INR3-4 or combine it with aspirin to prevent second MI

o ACCP and NICE recommend aspirin because warfarin is more costly and has higher bleeding risk

1.29 - BLANCING THE RISK IN MANAGING THROMBOSIS

1. There are many problems associated with administrating long-term anticoagulation Fatal haemorrhage Non-fatal but clinically relevant haemorrhage

o Bleeding tendency increases with ageo Risk of bleeding is highest when treatment is initiated

Regular blood testing Lifestyle/ diet restrictions Drug interactions

2. However, there are also clear risks in not administrating long term anticoagulation First VTE (PE can kill you. DVT won’t) Recurrent VTE

o DVTs predispose to DVTs, and PEs predispose to PEs (idea that some people are just prone to forming embolic thrombi)

o F5liden people tend to form DVTs if untreated Post-phlebetic syndrome

o Pain, swelling, ulcerations; requires 2 years of TEDs after first clot Patient anxiety

3. They key is knowing who is at an increased risk of forming a clot, and of those, who is at an increased risk of VTE recurrence

Risk factors for first VTE - asymptomatic thrombophilic patients F5 leiden is a less strong risk factor for thrombosis than PC/PC/AT deficiency Common polymorphisms can combine to increase risk of developing spontaneous VTE Warfarin does more harm than good in people with just pure AT deficiency >50% of thrombophilic people get their first VTE because of a transient risk factor

o So the key might be preventing exposure to these factors Cancer also predisposes to thrombus formation

Risk factors for recurrence Previous PE has higher risk of recurrence than DVT Idiopathic VTE has higher risk of recurrence than VTE due to transient risk factor (surgery) Male APS Some thrombophilias Increased levels of d-dimer Increased ETP (endogenous thrombin potential) Presence of Residual vein thrombosis

4. Simple guidelines have been developed for management There is no need to anticoagulate asymptomatic thrombophilic patients without prior clot Laboratory tests are generally poor predictors of recurrence in thrombophilic patients

o Although d-dimer may be useful You should consider anticoagulation for thrombophilic people who have had a clot if the

predictive risk of recurrence is >12.5% and relative risk >5% [but availability of data is poor] It seems that most people remain at a high risk of thrombosis between 3-6months after a

first clot, it is unclear whether this risk plateaus after 6 months or begins to decline Genotype-based profiling may be more accurate in predicting risk of recurrence

First VTE due to transient risk factor 3months VKACancer associated VTE LMWH 3-6months

First idiopathic VTE VKA 3-6months (?lifetime)First VTE in thrombophilic patient VKA 3 months (?lifetime)Recurrent VTE Lifetime VKA

Aim for an INR between 2.0-3.0

1.30 – NORMAL ENDOTHELIAL FUNCTION1. The endothelium is an organ with a complex structure and a variety of functions

Structure of endothelium it is a single flat layer of cells that forms the interface between tissues and blood endothelial cells line the entire circulatory system cells appear in a cobblestoned morphology in vitro cells appear in an aligned morphology in vivo - and this is due to the effect of blood flow/

shear stress; with cells aligning parallel to flow overlaps between cells in vivo occur at junctional areas – this helps them to regulate barrier

function

The endothelium has 5 main functions regulation of vascular tone barrier function pro-inflammatory anti-coagulant angiogenesis

2. the regulation of vascular tone is achieved by balancing production of vasoconstrictive and vasorelaxing mediators

The immediate response of the newborn is to upregulate vasorelaxing agents and downregulate constrictive agents in small pulmonary arteries. failure to do so would result in death

Nitric Oxide has differing effects on endothelial cells and SMCs It is produced in endothelial cells from arginine by NOS eNOS is constitutively expressed in endothelial cells – it maintains a constant production of

NOo eNOS can be induced by shear stress, O2, ACh and bradykinino iNOS is induced in inflammation, nNOS is only produced by neurons

NO improves endothelial barrier function, growth, motility and promotes angiogenesis (by promoting VEGF)

NO diffuses to SMCs to bind to solugble guanylyl cyclase. this produces cGMP which in turn produces PKG

o Hence NO causes SMC relaxation but inhibits SMC growth and motility NO also has anti-inflammatory and anti-thrombotic (platelet adhesion inhibition) effects

Prostacyclin causes cell membrane hyperpolarization it is produced in endothelial cells from arachidonic acid via COX it elevates cAMP in SMCs. this activates potassium channels and inhibits calcium channels –

net effect is hyperpolarization of the membrane and vasodilationo it also inhibits SMC proliferation

Prostacyclin also inhibits platelet aggregation

Natriuretic peptides there are 3main types, synthesized in the atria (ANP), brain (BNP) and endothelium (CNP) they bind to particulate guanylyl cyclase (not soluble) on SMCs to induce cGMP formation

and achieve vasodilation they are stimulated by arterial/ventricular dilation and neurohumoral factors

Endothelin-1 has 2 types of receptors; only one of which is expressed by endothelial cells

it is produced in the endothelium under the influence of hypoxia, shear stress, certain cytokines and toxins

it binds to Eta receptors on SMCs to cause vasoconstriction and proliferation It also binds to ETb receptors on SMCs, but this only causes vasoconstriction

o it has the opposite effect if it binds to ETb receptors on endothelial cells themselves, because this induces production of prostacyclin and NO

o there are no Eta receptors on endothelial cells

Angiotensin II also has two receptors; both of which are expressed on endothelial cells It is produced by the rennin-angiotensin system It binds to AT1 receptors to achieve the following functions:

o TF & thromboxane production – procoagulanto Endothelin1 & ROS production – vasoconstrictive

ROS inhibits NO when it binds to AT2 receptors – bradykinin receptor expression is upregulated and this

achieves a vasorelaxing function by induction of eNOS.

Summary

Molecule mechanisms

vasorelaxingNO soluble GC cGMP; opposite effects on EC/SMCProstacyclin cAMP hyperpolarizationnatriuretic peptides bound GC cGMP

vasoconstricting

Endothelin1 Eta receptor – vasoconstrict & SMC prolifETb receptor – opposite effects on EC/SMC

Angiotensin2 AT1 – procoagulant & vasoconstrictiveAT2 – upregulation of bradykinin receptor

Thromboxane increases ROS, decreases NO

3. Barrier function is achieved by inter-ceullar junctions, and can be modified by changes in intracellular cytoskeletal properties

There are 3 types of endothelial junctions 2 of them play a role in barrier function:

o adherens junctions (proteins, cadherin, PECAM1)o tight junctions (occluding, claudin, JAMs)

1 of them plays a role in signaling by being permable to cyclic nucleotides (<1kDa)o gap junctions (connexins)

junctional proteins adhere to intracellular actin which gives junctions stiffness and adequate positioning

remodeling of actin causes leaky junctions thrombin, histamine, endotehlin, TNF and hypoxia all induce Rho GTPases

o Rho GTPases inhibit MLCphosphatase and this leads to greater phosphorylation of myosin light chain

o calclium influx also achieves this by stimulating MLCkinaseo greater phosphorylation leads to remodlling of actin microfilaments into

actinomyosin fibres; which causes cellular contractility and barrier dysfunction Barrier function is maintained by Rac1 GTPases – these confer cellular tethering

o Rac1 is induced by sphingosine1phosphate and HGF

4. angiogenesis is a multi-step procedure; each step can be interfered withthe main steps are:

breaking cell-cell adhesions (to allow diffusion of growth factors from blood into tissue) basement membrane degradation cell migration morphogenesis of new vessels

5. The endothelium is naturally anticoagulant

Anticoagulant ProcoagulantProstacyclin TFNO vWFTM PAI1ProteinSHeparan sulphatetPAannexin5 (competes with factors to bind PL)TFPIEcto-ADPase (inhibits ADP-mediated platelet aggregation)

Inflammation, shear stress, metabolic stress (LDL) and oxidative stress (superoxide) all shift the endothelium to a pro-coagulant state

6. the endothelium produces pro-inflammatory molecules which makes it pro-coagulant the production of pro-inflammatory cytokines (IL1, TNFa) suppresses TM and stimulates TF, PAF

and adhesion moleculeso leukocyte adhesion triggers release of superoxide which causes endothelial damage

hence inflammation leads to a pro-coagulant stateo this is good because it can restrict spread of infection

e.coli and salmonella have adapted by recruiting plasmin to their surface strep have adapted by the ability to produce streptokinase (tPA like)

o this is bad because it can increase the risk of developing cardiovascular disease

1.31 – THROMBOSIS & EMBOLISM: GENETICS AND DETECTION

1. VTE is a single disease entity comprising both DVT & PEDVT & PE share many characteristics

Identical pathophysiologyo predisposing risk factors leads to thrombus formation in veinso thrombi form legs/pelvis embolise to pulmonary circulation

90% of PEs attributable to leg thrombi; 10% pelvic about 50% of people with DVT develop a subsequent PE

o increasing thrombus size leads to destruction of venous valveso destruction of venous vlaves gives rise to post-phlebitic syndrome

Similar risk factorso pregnancy, HRT, orthopaedic surgery, thrombophilia, flights etco Basically, Virchow’s triad

Identical therapeutic goals similar treatment strategies

both DVT & PE present with non-specific symptoms and a clinical diagnosis is hence unreliable symptoms of inflammation

o pain, swelling, redness, warmth unilateral signs

o leg – pitting oedema, tenderness, muscle indurationo lung – pleuritic chest pain, haemoptysis

systemic upset – purexia, tachycardia

2. Diagnosis of DVT can make use of d-dimers and ultrasound

The simplified wells score is used to assess pretest probability for DVT

Risk factor pointCa, paralysis, bed>3d, surgery within 4wk, vein tendereness, swollen leg, calf swollen>3cm, pitting oedema, collateral veins, previous DVT

(any one) 1

alternative diagnosis -2

Low risk/unlikely <1 Moderate 1-2 High/likely >2

If someone has a low risk from history, a negative d-dimer means they probably don’t have a DVT because d-dimers have a high negative predictive value

o the cut off for d-dimer reading is proportional to its specificity if someone has a high risk from history, negative d-dimer does

not mean no DVT. high risk patients should always have an ultrasound

if someone has low risk from history, negative ultrasound probably confirms the diagnosis; but ultrasound is only sensitive 97% of the time – they may still require venogram/serial ultrasound within 1 week if the history points to very high risk.

Venograms are rarely used these days in DVT invasive, expensive, dangerous contrast mediated effects

on kidney (especially in HTN, T2DM)

reserved for people with high risk from history and normal USS – this may suggest recurrent DVT (which USS is poor at picking up)

o recurrence is diagnosed if vessel diameter has enlarged by 2mm or more from previous venogram

3. Diagnosis of PE can make use of d-dimers and V/Q or CTPASimplified wells score for PE

Feature ScoreDVT, tachy >100, immobility or surgery 4 weeks ago, previous VTE, haemoptysis, Ca, most likely than alternative diagnoses

(any one) 1

PE is deemed unlikely if score is <1

V/Q is only diagnostic in 30% of cases, but it should be used before CTPA in the interest of decreasing radiation exposure

V/Q may be influenced by any coexisting lung disease

TTE is the test of choice in a massive PE you are looking for signs of RV stress/dilation in the emergency situation with TTE then confirm diagnosis with VQ of spiral CT avoid CTPA because it increases risk of major bleeding in massive PE patients

1.32 – ANTIPHOSPHOLIPID SYNDROME1. The antiphospholipid syndrome is an autoimmune disorder associated with a pro-coagulant

stateDefinition & classification of APS

APS is an AI disease, where Abs react with phospholipids/ CSM glycoproteins, leading to in-vivo increased risk of arterial & venous thrombosis

primary APS occurs in the abscence of other AI disease secondary APS occurs in conjunction with diseases eg SLE, RA, scleroderma etc catastrophic APS is a very rare variant which causes multi organ thrombosis and is always

fatal

Most patients develop low titre APS Abs without actually having thrombosis Lupus anticoagulant antibodies

o target prothrombin and beta2glycoprotein 1o have differing effects in vitro (anticoagulant) as opposed to in vivo (pro coagulant)o rareo beta2GP1 Abs have the highest thrombogenic potentialo PT Abs associated more with arterial thrombosis rather than venous

anti-cardiolipin antibodieso cardiolipin is a highly charged part of the PL bilayero these are 5x more common than lupus anticoagulant Abso only thrombotic when in moderate-high titreso usually IgG and/or IgM

other APS Abs target lipoprotein surface antigens and components of the coagulation/fibrinolytic systems

2. the pathogenesis of antiphospholipid syndrome is unknown inhibition of protein C/fibrinolysis? inhibiting the normal anticoagulant effects of beta2GP1 stimulation of platelets, endothelial cells, monocytes

o stimulation of TF production by monocyteso upregulation of adhesion molecules on endothelial cellso stimulation of thromboxane A2 synthesis by platelets?

3. antiphospholipid syndrome is diagnosed based on clinical and laboratory findingsThe revised Sapporo classification diagnoses APS when one clinical and one laboratory finding are met, between 12weeks and 5years of each other.

Clinical criteria – vascular and obstetric events Vascular events – a history of one or more arterial/venous thrombosis episodes, objectively

diagnosed by imagining, with no evidence of vessel wall inflammation Obstetric events – requires only one of the following:

o >3 spontaneous and unexplained abortions prior to 10weeks gestationo >1 miscarriage beyond 10weeks gestationo >1 premature birth due to pre-eclampsia, eclampsia or placental insufficiency

placental insufficiency can be due to HTN/ T2DM in ACS, placental insufficiency leads to intra-uterine growth restriction

Laboratory criteria ELISA to detect anti-cardiolipin antibodies at medium or high titres, at least twice in more

than a 12 weeks periodo illness etc can induce transient APS Abs production in normal people

Prolonged coagulation in dilute Russell’s viper venom time demonstrates lupus anticoagulant only when

o normal plasma addition does not correct the prolongationo addition of phospholipids (dilution of antibody) does correct the prolongation

4. although most patients prevent with VTE, APS patients clot in unusual placesCommon clinical findings:

vascularo VTE prevalence is roughly 30%o recurrence rate of untreated patients is 10%; this carries a higher mortality rateo cerebral thrombosis is most common arterial thrombosis evento digital necrosis and gangrene may also be seen

cardiac – valvular heart disease neuro – TIA/stroke, vascular dementia skin – livedo reticularis (rash that appears reticular) renal – vasculopathies chronic reanl ischaemia

rare clinical findings Thrombocytopenia (splenic sequestration following Abs binding to Plt PL) pulmonary HTN peripheral thrombosis catastrophic APS

5. treatment of APS in obstetric medicine Lupus anticoagulant patients with high titre anti-cardiolipin bodies have greatest thrombotic

potential and are at much higher risk of obstetric complications APS is also consistenly associated with intra-uterine fetal ?disease, pre-eclampsia

development and the HELLP syndrome (haemolysis, eleveated liver enzymes, low platelets) heparin and low dose aspirin are being used as prophylaxis to prevent (recurrent)

miscarriages steroids should only be used in treatment of coexisting AI disease, not to treat APS (side

effects).

1.33 – WARFARIN & HEPARIN; USE AND MONITORING

1. Warfarin & heparin have very different mechanisms of action warfarin prevents recycling of vitamin K and hence prevents gamma carboxylation of vitamin K

dependent factors so that they can no longer bind PL via their gla domains.o hence vitamin K (or FFP and PCC in urgent situations) can reverse ito resistance arises due to compliance (false

resistance), vitamin K diet, cytochrome p450 induction/polymorphisms

heparin is a polysaccharide of repeating N-acetylgalactosamine and n-acetylglucosamine

o enhancement of antithrombin = inhibition of all SPs, mainly thrombin and 10a

o release of TFPI from GAGs on endothelial cell surfaces

o iactivation of thrombin via heparin cofactor2o interactions with fibrinogen/TAFI?

2. Warfarin & heparin both have side affects apart from haemorrhage

warfarin o skin necrosis (sever protein c deficiency, commonly manifests in adipose tissue)o Chondrodysplasia punctata in utero (teratogenic in 1st trimester; failure of epiphysis

fusion)o Purple toe syndrome (disruption of atheromatous plaques leading to cholesterol emboli

lodging distally) heparin

o bleeding (protamine is the antidote)o heparin induced thrombocytopeniao hyperkalaemia, osteoporosis, alopecia

3. different tests are use to monitor warfarin/heparin warfarin; INR

o Target 2-3 for patients afte first VTE, AF, caridomyaaothy and vavular heart diseaseo target 3-4.5 for recurrent VTE, valvular heart disease and some thrombophilias

heparin – APTTo aim for APTT ratio of 2.0o LMWH gives a more predictable response than UFH

1.34 – NEW ANTICOAGULANT AGENTS; DESIGN & PRACTICE

1. New oral anticoagulants are deisgned to act at specific targets in coagulation

0

50

100

150

200

250

300

0 0.5 1 1.5 2

Drug (U/ml)

APT

T (s

)

UFH

LMWH

Therapeutic Range1.5 – 2.5

potential targets in anticoagulanbtso TFPI/PC/APC analogueso f9, f10a, thrombin, tafi inhibitorso rThrombomodulin

requirements of new agentso efficacy – proven in arterial/venous thrombosiso safety – wide therapeutic window, low bleeding risk, predictable response, antidote

presenceo convenience – oral, fixed dosage, few interactions, rapid on-&offset of action, no need

for monitoringFeature warfarin UFH LMWHrapid onset - + +rapid offset - + ?predictable response - - +lmited interactions - - +antidote + + -cost + + +oral administration + - -

2. Some examples of new agents in clinical practice synthetic pentasaccharides – can be used in VTE & HIT;

o fondaparinux vte prevention but requires parenteral (SC) admin excreted by kidney so cant be used in renal failure not reversible by protamine

o idraparinux longer half life than fondsparinux

factor 10a inhibitors – few functions outside coagulationo natural options – tick anticoagulant protein and antistasino rivaroxaban

licensed for orthopaedic vte prophylaxis, stroke prophylaxis in AF no antidote

thrombin inhibitorso dabigatran

licensed for orthopaedic vte prophylaxis, stroke prophylaxis in AF protein C analogues

o drotrecogin-a is rAPC – used in sepsis that causes multiple organ failure but very expensive

1.35 - ENDOTHELIAL DYSFUNCTION

1. The pathogenesis of endothelial dysfunction is closely linked to low/turbulent shear stressMechanical injury (shear stress) is one of four broad causes of endothelial dysfunction

Inflammation (infectious or immune-mediated) Apoptosis & necrosis Oxidative stress

o Rapid reoxygenation following hypoxia generates reactive radicals MECHAINCAL INJURY

o Particulary low or turbulent blood flowo Blood pressure leads to circumferential stretch too

Normal Shear stress is important for the healthy endothelium Shear stress is higher in arteries than veins (upto 15-30x greater) Shear stress is normally required for:

o NO productiono Pro-coagulant molecule productiono Growth factor & adhesion molecule expression

Shear stress affects endothelium by activating signalling cascades via mechanosensors eg integrins, stress-activated cation channels, junctional proteins

o Early effects = IP3, DAG = Ca influx = protein kinase activationo Later effects = TF/gene regulation; both pro-and anti-thrombotic genes are affected

as well as some kinases and genes regulating the cytoskeletono Late effects = cell alignment and migration with blood flow

Failure of normal shear-stress endothelium interaction is implicated in pro-thrombotic disease At arterial branching points, shear stress is low or turbulent - this makes endothelial cells:

o Cause vascular remodelling: display a cobblestone morphologyo Cause vasoconstrictiono Cause inflammation and thrombosis

Similarly, dysfunctional endothelium that cannot adapt properly to shear stress will appear this way

Stretch leads to junctional loss and monocyte adherence (role of HTN in atherosclerosis)

2. Endothelial oxidation of LDL is a key process in atheroma formationRecap of atherosclerosisExcessive inflammatory-fibroproliferation by the endothelium in response to vascular insult

o Step 1 - fatty streak formation & adhesion of monocytes (reversible)o Step 2 - intermediate lesion with layers of macrophages & SMCs in the wallo Step 3 - fibrotic plaque covering a necrotic core (irreversible)o Step 4 - plaque cap ruptures, exposing the thrombus to blood flow

Four things can happen to a thrombus after a plaque ruptures Completely/partially resolves Progresses and grows because of failure of normal anticoagulant/fibrinolytic mechanisms to

cope. This can lead to vessel occlusion. Incorporation into vessel wall following fibroblast/ SMC migration Fragmentation and embolization

Endothelial oxidation of LDL has the following functions Adhesive glycoprotein expression

chemokine production to attract monocytes & lymphocytes pro-proliferative growth factor/ cytokine production contributes to fibrous plaque formation

The dysfunctional endothelium also prevents senescense/repair, loses anti-thrombotic function and may express receptors for viruses, particularly Kaposi sarcoma and herpes viruses.

Treatment = statins, anticoagulants, vasodilators

3. The problem in pulmonary HTN is increased vasoconstrictor production by the endotheliumThe Dysfunctional endothelium begins to cause vaso-active imbalance in pulmonary HTN

Increased vasoconstrictors (endothelin, angiotensin) Decreased vasorelaxants (prostacyclin, NO) Reduced expression of voltage gated K channels [reduced response to prostacyclin] Pro-proliferative and pro-fibrotic cytokine productions (VEGF, PDGF) Increased cell permeability and inflammatory cell migration

Vascular changes in pulmonary HTN SMC proliferation = intimal thickening/luminal narrowing Fibroblast proliferation = fibrosis, ECM deposition

o Vasculature adopts Plexiform lesion appearance due to disorganised angiogensis Vasoconstriction

o Some vasoconstrictors have dual effects as growth factors and hence play a part in proliferation too

Thrombosiso Pulmonary HTN patients show higher levels of TF, fibrinopeptides, PAI1, vWF

A few patients with pulmonary HTN develop chronic thromboembolic pulmonary HTN the problem here is embolised DVTs interactions occur between fibrin/thrombin from the embolus and the pulmonary

endothelium the endothelium begins to produce more vasoconstrictors and down regulated

thrombomodulin (pro-coagulant) in the context of concomitant hypoxia/inflammation, the net result is vessel occlusion and

remodelling

Management = inhaled NO, IV prostacyclin/prostacyclin analogues, anticoagulants, anti-inflammatories, calcium channels blockers and phosphodiesterease inhibitors ( no 1 effective treatment )

4. Endothelial-and platelet derived microparticles are prothrombitc and play roles in arterial disease and cancer

Microparticles are often released following apoptosis or activation of platelets/endothelial cells. Release secondary to cell activation is due to cytoskeletal disruption

MP release is induced by all four broad causes of endothelial dysfunction MPs show extracellular proteins of the parent cell, particularly TF and PPDserine, as well as

intracellular signalling molecules eg DNA fragments/ micro RNA Chronic thromboembolic pulmonary HTN patients show increased microparticles in plasma - as

well as pro thrombotic, they wre pro-angiogenic and pro-inflammatory in this disease Endothelial MPs are implicated in arterial disease, sepsis and cancer

o Tumour MPs can prime distant sites for metastasis in cancero MPs may be initially protective in sepsis

dysfunctional endothelium

persistence of pro-

inflammatory and pro-

coagulant processes

accumulation of foam cells, LDL,

Ca

increased vessel wall mass

lumen occlusion

turbulence over the atheroma

1.36 - COAGULATION, INFLAMMATION & SEPSIS

1. Coagulation and inflammation are closely intertwinedInflammation influences coagulation in 4 main ways thanks to the dysfunctional endothelium

Increase in TF release (from tissues & monocytes) Down regulation of anticoagulant molecules (TM, EPCR, APC, TFPI, AT) Increased vWF production to stimulate platelet adhesion Decreased fibrinolytic capacity (increased PAI1 and downregulated tPA)

2. Further recap on the role of the endothelium in pathogenesis of atherosclerosisThe starting point is induction of VCAM expression on endothelial cells, influenced by oxidised LDL

Monocyte binding on VCAM leads to microparticle release In normal people, this step is required for monocytes to become activated to macrophages and

mop up LDL - keeping the endothelium in balance In hypercholesterolaemia - excessive monocyte uptake leads to the formation of foam cells and

release of TF microparticles by activated monocytes TF microparticles express PSGL and bind to activated platelets where they are decrypted to allow

binding of 7a and initation of further coagulationo The net result is a pro-inflammatory, pro coagulant state

T-cell binding on VCAM is key in activating monocytes Th1 and Th2 cells usually do this and release cytokines to activate uptaken monocytes into

macrophages Excessive t-cell uptake gives excessive monocyte activation

o The net result is a pro-inflammatory, pro-coagulant state

Atherosclerosis demonstrates the pro-coagulant, pro-inflammatory cycle associated with dysfunctional endothelium

The dysfunctional endothelium loses CSM proteins (including TFPI, EPCR, TM) due to activation of ‘sheddase’ enzymes

It also increases transcription and release of vWF

There is upregulation of PAI1, and downregulation of tPA

Adhesion molecules are upregulated Cell-cell contacts are broken and the layer

becomes ‘leaky’o This leads to inappropriate

haemostatic plug formationo This contributes further to

extravasation

Management of atherosclerosis Statins are lipid lowering and hence

prevent endothelial dysfunction Anti-inflammatory agents may work with

statins to prevent plaque progression/rupture

Anti-platelet drugs prevent formation of occlusive thrombi post plaque rupture

Atherosclerosis

infection SIRS sepsis severe sepsis

septic shock death

3. The same inflammation/coagulation cycle is implicated in sepsis

The disease continuum in infection can progress very quickly Criteria for SIRS requires >2 of the following:

o Temperature >38oC or <36oCo Heart rate >90 beats/mino Respiratory rate >20/mino White blood cell count >12,000/mm3 or <4,000/mm3 or >10% immature neutrophils

Sepsis terms SIRS which is due to infection (not trauma, burns etc) Severe sepsis is sepsis with atleast one organ failure Septic shock (which can be fatal) is severe sepsis with hypotension that is resistant to fluid

resuscitation

Thrombin may be implicated in sepsis pathogenesis Normally, WBC activation, pro-inflammatory cytokine production & coagulation are effective in

combatting the pathogen and limiting its spread However, in sustained infection, these three processes, couple with excessive endothelial

activation - lead to the same pro-inflammatory, pro-coagulation cycle seen in atherosclerosis The result is production of thrombin, which cleaves PAR-1 receptors on endothelial cells and

platelets to:o Cause cellular contractiono Upregulate adhesion molecule production

In sepsis the release of TF microparticles from monocytes is about 800x more than normal

o Promote vascular permeatbility by upregulating S1P3 In sepsis this can contribute to dilutional coagulopathy

o Promote NfKB

4. Neutrophils may play an important role in pathogensis of sepsisFunctions of neutrophils in innate immunity against sepsis

Phagocytosis Reactive oxygen species generation Enzyme release from granule Production of neutrophil extracellular traps (NETs)

NETs can cause endothelial dysfunction NETs are webs of neutrophil DNA and associated histones, elastases, myeloperoxidases, that

trap microbes within the vascular wall following uptake of neutrophils from bloodstream NET release is stimulated by pro-inflammatory cytokine release from activated endothelial

cells/platelets Excessive cytokine production in sepsis causes neutrophil to release NETS whilst in the

circulation to the following effects:o Trapping RBCso Activation of plateletso Activation of contact system of coagulation

In addition, histone H4 is cytotoxic and contributes to endothelial dysfunction

Novel targets in sepsisAnti-histone H4, DNAase (cleaves nets) & APC (cleaves PAR1 but has opposing effects to thrombin)

1.37 – GENE THERAPY IN HAEMOPHILIA

1. The principle of gene therapy lies with introduction of a vectorGene therapy is deliberate transfer of genetic material into cells for therapeutic purposes there are two main methods:

Correction of genetic deficiency by replacing a defective gene introducing a new gene that adds a new function eg increased sensitivity/resistance to a

tertiary agento the latter method is what gene therapy is moving towards

gene therapy uses viruses as vectors disassembly of the viral genome incolves removal of infective (proliferatrive) parts what is left is the plasmid and accessory genes

o the gene to be added is then inserted virus replication occurs inside eukaryotic cells copies of the recombinant gene are extracted when viruses are released from cells

Retroviral vectors integrate into DNA, rAAV vectors do not

2. retroviral vectors have been used in disease but may have malignant side effectsRetroviral vector use in Severe combined Immunodecificy XI

the problem in SCID is defiecieny of gammaC chain of IL2 = T&B cell deficiencyo most children die within 12 months if unteated

bone marrow transplant used to be the treatment in SCID, but this had a high mortality gene therapy uses CD34+ HSCs, which are cultures, have the deficient factor added and

transplanted back into patiento Most patients trialled have good T cell recovery with partial B & NK cell recoveryo 5 year survival approaches 95%

the problem is that the gene is an oncogene – increased incidence of t cell lymphoblastic leukaemia

Chimeric antigen receptors are used in melanoma/CLL the principle here is extracting the host t cells that recognise the malignancy and redesigning

their antigen receptor to recognise other molecules ego CD19 (BCR)o CD137 (costimulatory molecule on T cells)o CD3-zeta (signal tranducer)

the modified t-cells are cloned and reinfusedo unfortunately the response rate is only 22% but this is better than the previous

treatment the solution is to keep making the antigen receptor more specific

3. rAAV vectors are of use in haemophilia BrAAV vectors are more attractive than retroviral ones

They are non-pathogenic and only become pathogenic in the presence of a helper adenovirus

they have decreased immunogenicity they have more stable responses in the long term

there is increased flexibility in administration regimens BUT

o once the cell with the vector inside dies – the vector is lost (because it doesn’t integrate with DNA)

o rAAV vectors can onlu hold small genes <5kb

The advantages of using gene therapy in haemophilia B: it is a single gene defect, and rAAV therapy would would to 24/7 endogenous F9 production

following a single gene transfer (avoids the 2-3 injections a week) the therapeutic goal is modest

o the goal is to get from <1% to >1% F9 – this reduces morbidity and mortality signinifcantly

the response to treatment is easily measurable via ELISA

Current therapy involves prophylactic/recombinant F9 administration requires frequent injections given via central venous access very expensive (>110k/pt/yr) risk of bleeding is not completely eliminated

o all groups (severe, moderate, mild disease) still have reduced life expectancy 80-90% of the world’s haemophiliacs do not have access to this treatment

Nathwani et al are trialling AAV8 vectors in haemophilia B AAV8 avoids the immune response

o it has low human-prexisting immunity, unlike the previously studied AAV2 it is more potent than AAV2

o addition of ‘self complimentary’ AAV8 makes the molecule ‘fold in’ on itself and makes rate of transcription faster

it is highly specific for the livero addition of a liver specific codon means only heaptocytes can recognize it – synthesis

of F9 by hepatocytes is very efficient it can be administered peripherally (because of the codon)

o this avoids the need for hepatic-artery injections (which were needed for AAV2), which are dangerous

BUT – It takes a long time to make, is very expensive, and will be difficult to get worldwide the trial was made less risky by enforcing use of contraception ( to prevent germline

transmission of vector), exclusing use of patients with F9 inhibitors/ previous immunity (inflammation), and using a low dose vector

there seemed to be t-cell mediated IgM responses @20days and IgG responses at 50days to AAV8’s capsid in hepatocytes (demonstrated by deranged LFTs and high IFNgamma levels).

patients on high dose AAV8 with short term steroids seem to be cured of haemorrhage and the need for prophylactic/eoisodic F9 treatment – WIN!!

4. gene therapy may have further use in haemophilia A and other diseasesDifficulties in gene therapy for haemophilia A

F8 gene is much bigger than F9 and exceeds AAV capacity F8 undergoes a lot of pos-translational modification so getting a hold of ‘wild’ F8 is difficult

o the solution being tried is codon optimisation (taking out codons that are less important) of the F8 gene to make it small enough to fit into rAAV

o seems to work in mice

gene therapy may also be used in other coagulation disorders, lysosomal storage diseases, haematological and solid malignancies.