UNDERSTANDINGaPLA

JacobHRand,MDDepartmentofPathologyandLaboratoryMedicine

WeillCornellMedicalCollege

DISCLOSURESTATEMENTSpeaker:JacobHRand,MD

IncompliancewithACCMEpolicy:Employment:NothingtodiscloseConsultancy:NothingtodiscloseEquityOwnership:NothingtodiscloseResearchFunding:NothingtodiscloseHonoraria:NothingtodisclosePatents&Royalties:InventorofUSPatents#6,284,475and7,252,959

assignedtoMountSinaiSchoolofMedicineSpeakersBureau:NothingtodiscloseMembershipinadvisoryboards:NothingtodiscloseOther:Nothingtodisclose

•  To review where we are now and how we got here

•  To describe the aPLA conundrum

•  Doing the best with the current empirically-derived lab tests

•  A path forward?

Planforthispresentation:

APS:clinicalmanifestationsandlaboratorytests

ClinicalDVT,PE,ATE,

stroke,pregnancyloss,placental

infarction,IUGR,oligohydramnios,preeclampsia

LaboratoryaCLIgG,IgM

aß2GPIIgG,IgM

LA:aPTT,dRVVT,others(aPTT,dPT,KCT,hexagonalphase…)

A“Classic”Case

30 year old woman referred for 4 spontaneous pregnancy losses •  Prior History: DVT and pulmonary embolism; imaging studies of

brain showed an old cerebral infarct •  Initial labs: Markedly abnormal antiphospholipid tests •  Diagnosis: APS Treatment: heparin and aspirin •  Course: At 18 wks gestation developed thrombotic microangiopathy

(catastrophic APS) with fetal loss Treated with plasmapheresis, anticoagulation and IV-IgG Subsequently developed recurrent lower extremity DVTs refractory to oral anticoagulant therapy and requires long term LMW-heparin

EmpiricOriginsoftheCurrentAntiphospholipidAssays

1906 Syphilis testing

1940 Isolation of cardiolipin

1950’s BFP

syphilis

1980’s Anticardiolipin antibody

assay; First description of syndrome

1990’s - present Recognition that

cofactors are targets: aß2GPI

antibodies

1950’s PTT inhibitor

1960’s Lupus Anticoagulant;

Association with thrombosis

1980’s Lupus

anticoagulants; aPTT, DRVVT, etc; Association with

APS)

•  Negative: Lower than the 95th percentile cutoff (generally <22 GPL units for IgG)

•  Low-positive/indeterminate: 95th-99th percentiles

•  Medium/moderate-positive: usually 60-80 GPL units

•  High-positive = >80 GPL units (Lakos et al, Arthritis & Rheumatism 2012)

InternationalConsensusGuidelinesonInterpretationofaCLantibodyassays

• Complementcontrol/shortconsensusrepeatsuperfamily

• BindsphospholipidviadomainVasmonomer,thenagglomeratesintoclusters

• Deficiencyisnotassociatedwithobviousdiseasephenotype

• Mayplayroleinclearingapoptoticcellsandmembraneparticles

• MayplayaroleinscavengingLPS

• Anti-ß2GPIIgGpromotethrombosisinanimalmodels

Gamsjaeger et al Biochem J (2005) 389:665

ß2GPI(apo-H)isamajortargetantigenforaPLA

Some aPLA Cofactors

• prothrombin • factor V • factor VII/VIIa • protein C • protein S • annexin A2 • annexin A5 • ox-LDL • high and low molecular weight kininogens • heparin plasmin • vimentin • sulfatides (acidic glycosphingolipids) and more ….

SomeadditionalaPLAcofactors

MultiplemechanismshavebeenproposedforAPS

•  Disruption of the annexin A5 antithrombotic shield •  Disruption of the annexin A2-mediated fibrinolysis •  Interferences with protein C and S pathway •  Inhibition of TFPI activity •  Effects of autoantibodies against: annexins, protein Z, plasmin • Inhibition of ß2GPI-mediated dampening of vWF-platelet interaction • Increase of plasminogen activator inhibitor-1 • Induction of receptors for cell adhesion molecules on endothelium •  Induction of tissue factor expression on endothelial cells and on monocytes •  Stimulation of platelet activation •  Direct injury to endothelium •  Induction of apoptosis in vascular cells •  Release of membrane-bound microparticles • Complement-mediated injury to trophoblast/endothelium • Antibody-mediated alteration of eicosanoid synthesis •  Increase of endothelin-1 • Ab-mediated concentration of prothrombin on phospholipid membranes •  Ab binding to intracellular membranes

Diagnostic Criteria Clinical•  VascularthrombosisForhistopathologicdiagnosis,thereshouldbeno

evidenceofinflamma9oninthevesselwall

•  Pregnancymorbidi9esa=ributabletoplacentalinsufficiency,including:a)threeormoreotherwiseunexplainedrecurrentspontaneousmiscarriages,before10weeksofgesta9on,b)oneormorefetallossesaEerthe10thweekofgesta9on,c)s9llbirth,andd)episodeofpreeclampsia,pretermlabor,placentalabrup9on,intrauterinegrowthrestric9onoroligohydramniosthatareotherwiseunexplained

Laboratory•  Mediumorhigh9teraCLoran9-β2GPIIgGand/orIgMan9bodypresenton

twoormoreoccasions,atleast12weeksapart,measuredbystandardELISAs

•  Lupusan9coagulantinplasma,ontwoormoreoccasions,atleast12weeksapart

DiagnosticCriteriaforAPS(investigational)

TheAPSConundrum

ClinicalThrombosis

andPregnancyComplications

L a bTestsaCL IgG/IgM,a n t i - β 2GP IIgG/IgM;LA

aPLposesauniquepuzzlebecause:§ Asofyet,thereisno“goldstandard”§ TheclinicalmanifestationsareneitherrarenorspecificforAPS§ Thelaboratorytestsoftendonotcorrelatewitheachother§ Patientswhotestpositivebuthavenothadanarelabeledas“falsepositives”whensomemaynotyethadaclinicalmanifestationbuthaveincreasedrisk

•  To review where we are now and how we got here

•  To describe the aPLA conundrum

•  Doing the best with the current empirically-derived lab tests

•  A path forward?

Planforthissegment:

Probability of developing a first VTE correlates with the number of positive aPL antibody tests

Modified from Pengo et al Blood 2011

Cumulative incidence of TE events in triple positives for aPL tests

Pengo et al Blood 2011

PROMISSE (Predictors of pRegnancy Outcome: bioMarkers In Antiphospholipid antibody Syndrome and Systemic lupus Erythematosus

Lockshin MD et al, Arthritis Rheum 2012 64:2311

%Adv

erseClin

icalEvents

%Adv

erseClin

icalEvents

Fig1

•  To review where we are now and how we got here

•  To describe the aPLA conundrum

•  Doing the best with the current empirically-derived lab tests

•  A path forward?

Planforthissegment:

PrincipleofLAAssay

AFM Working Principle

Advantages of AFM •  Minimal specimen preparation •  Able to image sample unfixed in fluid medium •  Resolution for biological samples in nm range

(from data obtained for Rand et al, Blood 112:1687 2008)

Atomic Force Microscopy: Structure of aPL IgG-ß2GPI complexes

Formation of aPL mAb-β2GPI Immune Complexes on Phospholipid Bilayers Visualized

by ImmunoAFM

Time (min)0 2 4 6 8 10 12 14 16 18 20 22 24 26

Bou

nd P

rote

in (µ

g/cm

2 )

0.0

0.1

0.2

0.3

0.4

0.5

0.6 APS IgG + fl2GPI

Control IgG + fl2GPI

Add IgG & fl2GPI ↓

Rapid adsorption of APS immune complexes to phospholipid bilayer

•  aPLAlabtestsareempiricallyderivedandcurrentdiagnosticcriteriaareinvestigationalmeanttoidentify“definite”APSpatientsforclinicalinvestigations

•  TheLAandmultipositivityappeartobethestrongestpredictorsofadverseclinicaloutcomes

•  AFMandSEMimagingdemonstrateformationofaPLAmacro-immunecomplexesonphospholipidmembranes

•  Thesearecomposedofimmunoglobulinsandmultipleplasma-derivedcofactors

•  Hypothesis:ThecoreabnormalityAPSistheformationofpolyclonalmultivalentmacro-immunecomplexesonvulnerablecellmembranesandvesiclesthattriggermultiplepathogeniceffects

Sometakehomepoints

Weill Cornell and Montefiore-Einstein Collaborators

Pathology Xiao-Xuan Wu Lucy Wolgast Ljiljana Vasovic Mojgan Raoufi Yanhua Wang Jessica Niakan Ervis Bezati Julissa Pena-Medina Mayra Almonte Laura Santambrogio

Pediatric Rheumatology Dawn Wahezi Norman Ilowite Einstein Biochemistry Steve Almo Ron Seidel, III Mark Girvin

Outside Collaborators

UVM - AFM Facility Douglas Taatjes Anthony Quinn Maastricht University Bas de Laat Saartje Bloemen NYU- Ob-Gyn Alan Arslan Seth Guller

Ques9ons?