Recent advances in the antiphospholipid antibody syndrome

CE: Alpana; MOH/210504; Total nos of Pages: 9;

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REVIEW

CURRENTOPINION Recent advances in the antiphospholipid antibody

syndrome

Copyright © Lippincott W

1065-6251 � 2014 Wolters Kluwer

a b,c
Shruti Chaturvedi and Keith R. McCrae
Purpose of review

The antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by recurrentthrombosis and/or obstetrical morbidity in the presence of persistently positive antiphospholipid antibodies.Recent insights into the pathogenesis of APS have begun to elucidate pathophysiology and led to theidentification of potential therapeutic interventions. The objective of this review is to examine the advancesin this field and highlight the areas of further investigation.

Recent findings

Several mechanisms of thrombosis and pregnancy loss in APS have been proposed. These includeactivation of endothelial cells, monocytes, and platelets, and/or inhibition of natural anticoagulant andfibrinolytic systems by antiphospholipid antibodies. However, in many cases the underlying molecularmechanisms and their relevance to the human disorder remain uncertain. New therapeutic agents such asstatins, hydroxychloroquine, rituximab, complement inhibitors, and interventions aimed at disruption ofintracellular signaling pathways have shown promise in preclinical and clinical studies.

Summary

Indefinite anticoagulation remains the mainstay of treatment for thrombotic APS. Despite advances indiagnostic techniques, it remains difficult to predict thrombotic risk in asymptomatic patients withantiphospholipid antibodies. Further mechanistic and clinical studies are needed to predict thrombotic riskand develop improved therapies for this devastating illness.

Keywords

b2-glycoprotein I, antiphospholipid, lupus anticoagulant, thrombosis

aDepartment of Internal Medicine, bTaussig Cancer Institute andcDepartment of Cellular and Molecular Medicine, Cleveland Clinic,Cleveland, Ohio, USA

Correspondence to Keith R. McCrae, MD, Taussig Cancer Institute, R4-018, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.Tel: +1 216 445 7809; fax: +1 216 445 7809; e-mail: [email protected]

Curr Opin Hematol 2014, 21:000–000

DOI:10.1097/MOH.0000000000000067

INTRODUCTION

The antiphospholipid syndrome (APS) is a systemicautoimmune disorder characterized by recurrentthrombosis and/or obstetrical morbidity in thepresence of antiphospholipid antibodies (aPL),including lupus anticoagulant, anti-b2-glycoproteinI (anti-b2GPI), and/or anticardiolipin (aCL) anti-bodies [1]. Thrombosis in APS can affect any vascularsite, with the deep veins of the lower extremities andthe cerebral arterial circulation most commonlyaffected [2]. A small number of patients (<1%)develop catastrophic antiphospholipid syndrome(CAPS) [2], defined as small-vessel thrombosis inthree or more organs in less than 1 week in thepresence of aPL, with histopathologic confirmationof small-vessel thrombosis in the absence of inflam-mation [3]. CAPS is associated with high (50%)mortality, mostly because of cerebral and cardiacthrombosis, infections, and multiorgan failure [4

&

].Obstetrical morbidity in APS includes the unex-plained death of one or more morphologically nor-mal fetuses at or beyond the 10th week of gestation,

illiams & Wilkins. Unau

Health | Lippincott Williams & Wilk

the premature birth of one or more morphologicallynormal neonates before the 34th week of gestationbecause of either eclampsia or severe preeclampsia,and/or three or more unexplained, consecutive spon-taneous abortions before the 10th week of gestation[1]. Other clinical associations of aPL include throm-bocytopenia, livedo reticularis, transient ischemicattacks and skin ulcers [4

&

].

DIAGNOSIS AND THROMBOTIC RISKASSESSMENT

The diagnosis of APS rests on the presence of at leastone clinical and laboratory criterion (Table 1).

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KEY POINTS

� Antiphospholipid antibodies are associated with arterialand venous thrombosis as well as recurrent and latefetal loss.

� There have been numerous mechanisms proposed toaccount for the effects of these antibodies, although theprimary mechanistic pathways remain uncertain.

� Interactions of antiphospholipid antibodies withvascular cells, mediated through recognition of cell-bound b2GPI, may play a central role in pathogenesis.

� Guidelines recommend that patients withantiphospholipid antibody-associated thrombosis shouldbe treated with long-term anticoagulation.

Hemostasis and thrombosis

According to the revised classification guidelines(2006), the laboratory criteria for APS require thepersistence (for>12 weeks) of a lupus anticoagulant,detected according to the guidelines of the Inter-national Society of Thrombosis and Hemostasis [5]and/or high titers of IgG or IgM autoantibodiesagainst cardiolipin and/or b2GPI detected usingstandardized ELISAs [1]. Patients with other aPLssuch as antiprothrombin antibodies, IgA aCL oranti-b2GPI antibodies, or those with clinical mani-festations other than thrombosis or pregnancy mor-bidity cannot be formally diagnosed with APS usingthese criteria. It remains difficult to predict throm-botic risk in patients with aPL, and patients may beencountered who develop thrombi yet do not meetthe criteria for APS due to the presence of only low ormoderate levels of aCL or anti-b2GPI antibodies.

In most reports, lupus anticoagulants are morestrongly associated with thrombotic risk and preg-nancy complications than aCL or anti-b2GPI anti-

opyright © Lippincott Williams & Wilkins. Unautho

Table 1. Revised classification criteria for definite APS

Clinical criteria (one or more)

Vascular thrombosis

One or more objectively confirmed episodes of arterial, venous orsmall vessel thrombosis in any tissue or organ

Pregnancy morbidity

One or more unexplained deaths of a morphologically normal fetusat or beyond the 10th week of gestation; or one or morepremature births of a morphologically normal neonate before the34th week of gestation because of placental insufficiency,preeclampsia, or eclampsia; or three or more unexplainedconsecutive spontaneous abortions before the 10th week ofgestation

At least one clinical and one laboratory criteria must be present for the diagnosis ofaCL, anticardiolipin; APS, antiphospholipid syndrome; b2GPI, b2-glycoprotein I; ISTAdapted with permission [1].

2 www.co-hematology.com

bodies alone [6]. The risk of a first thrombotic eventin triple-positive patients, that is, those with posi-tivity for lupus anticoagulant, aCL, and anti–b2GPIantibodies, may be as high as 5.3% per year [7].Antibodies directed toward b2GPI domain 1 maycorrelate more strongly with thrombotic riskand pregnancy morbidity [8]. Antibodies againstprothrombin also are associated with thrombosis,but with lower odds ratios than those against b2GPI[9

&

]. A better understanding of the thrombotic riskprofile of patients with aPL is needed to help definethe need for, and the optimal type and duration of,antithrombotic therapy.

PATHOGENESIS

Antiphospholipid antibodies directed against phos-pholipid binding proteins bound to the surfaceof endothelial cells, monocytes and platelets arelikely central to the pathogenesis of APS. b2GPI isrecognized as the primary antigenic target of aPL[10] (Fig. 1). Affinity-purified human anti-b2GPIautoantibodies potentiate arterial and venousthrombus formation in a mouse model [11], andlupus anticoagulants whose effects are mediated viainteractions with b2GPI confer a higher risk ofthrombosis than those due to aCL or anti-prothrom-bin antibodies [6,7]. b2GPI is also expressed on thesurface of placental trophoblasts, where anti-b2GPIantibody binding to b2GPI results in inhibition ofgrowth and differentiation of trophoblasts, andinflammatory changes leading to fetal loss [12]. Dis-placement of annexin V from these cells may alsoallow exposure of anionic phospholipid, providing anidus for coagulation complex assembly [13].

b2GPI is a plasma glycoprotein containing five‘sushi’ domains, the fifth of which is atypical andmediates binding to anionic phospholipid [14].

rized reproduction of this article is prohibited.

Laboratory criteria (one or more should be present on two ormore occasions at least 12 weeks apart)

Lupus anticoagulant detected according to the ISTHguidelines

aCL antibody of IgG and IgM isotype, present in medium orhigh titer (>40 IgG or IgM phospholipid units or >99thpercentile) measured on standardized ELISA. Anti-b2GPIantibody of IgG and IgM isotype present in titer >99thpercentile measured on standardized ELISA

definite APS.H, International Society of Thrombosis and Hemostasis.

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Monocyte

Thrombosis

Platelet activationand aggregation

Complementactivation

Platelets

TXA2

Nuetrophil

VCAMICAM

E-selection

Microparticles

Endothelialactivation

Pro-inflammatorystate

Endothelialinjury

IL 6

IL 1

aPL

TNF-α

β2GPI

TLR4TF

B cell

BAFF

CTLA4

p38 MAPKNFKB

TF

TF

TF

C5a

TLR4

TLR2

AnnA2ApoER2

Intracellular signaling viaTLR4/MyD88, NFkBand p38 MAPK pathways

FIGURE 1. Potential mechanisms involved in the pathogenesis of thrombosis in the antiphospholipid syndrome. Theinvolvement of endothelium, platelets, and monocytes is postulated. aPL, antiphospholipid antibodies; BAFF, B cell activatingfactor; b2GPI, b2-glycoprotein I; ICAM, intracellular adhesion molecule; MAPK, mitogen activated protein kinase; MyD88,myeloid differentiation factor 88; TF, tissue factor; TLR4, Toll-like receptor-4; VCAM, vascular cell adhesion molecule.

Antiphospholipid antibody syndrome Chaturvedi and McCrae

A recent report suggests that b2GPI may exist in twoconformations. Circulating b2GPI has been foundto exist largely in a circular form in which domain1 is postulated to bind to domain 5, masking theputative anti-b2GPI antibody epitope in domain 1.Binding of b2GPI to anionic phospholipid or otherrelevant surfaces is proposed to induce unfolding ofb2GPI with conversion to a ‘fishhook’ conformationobserved in the crystal structure of b2GPI, in whichthe domain 1 epitope is exposed and induces for-mation and binding of anti-b2GPI domain 1 anti-bodies [15].

A ‘two hit’ model of thrombosis in APS has beenhypothesized in which a ‘first hit’ creates a pro-thrombotic state followed by a ‘second hit’, possiblyinflammatory, that perturbs the endothelium andinitiates thrombosis [16]. The pathophysiologicmechanisms that contribute to the prothromboticphenotype (first hit) include aPL-mediated activationof monocytes, endothelial cells and/or platelets,and/or inhibition of natural anticoagulant and fibri-nolytic systems by aPL [17

&

,18].

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Cellular activationAntiphospholipid antibodies have been reported tobind to and activate vascular endothelial cells,monocytes, and platelets in a b2GPI-dependentmanner [19–22]. Antiphospholipid antibodies aregenerally of low affinity and do not bind b2GPI insolution, but only after deposition at high concen-tration on an appropriate surface [23]. There is noconvincing evidence for circulating immune com-plexes containing b2GPI and anti-b2GPI antibodiesin patients with APS, and the clinical manifestationof APS are generally not those of an immune-com-plex-mediated disease. Thus, evidence suggests thatanti-b2GPI antibodies bind b2GPI previously boundto cells.

Endothelial cells play an important role inmaintaining blood fluidity through the expressionof anticoagulant proteins and the elaboration ofantithrombotic substances such as prostacyclin,glycosaminoglycans and nitric oxide. Endothelialcell activation via aPL–b2GPI interactions leadsto loss of these anticoagulant properties with


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transformation to a pro-adhesive, procoagulantphenotype characterized by increased expressionof adhesion molecules (E-selectin, intracellularadhesion molecule-1, and vascular cell adhesionmolecule-1) and tissue factor, enhanced secretionof pro-inflammatory cytokines and chemokines,and release of procoagulant and proinflammatorymicroparticles [24]. Annexin A2, a cell-surface recep-tor for plasminogen and plasminogen activator,mediates the binding of b2GPI to cells [20,25].However, annexin A2 does not have a transmem-brane domain; therefore, other co-receptors must beinvolved in cellular activation. A recent studysuggested that anti-b2GPI antibodies induce signal-ing through a multiprotein complex that includesannexin A2, calreticulin, nucleolin, and Toll-likereceptor 4 (TLR4) [25], with TLR4 inducing acti-vation of a TLR4/myeloid differentiation factor 88(MyD88)-dependent pathway culminating in NF-kBactivation [26,27

&&

]. The involvement of p38mitogen activated protein kinase in endothelialactivation has also been demonstrated [28]. In addi-tion to annexin A2, other potential receptors such asapoER2 [29] and TLR2 may also play a role in endo-thelial activation [30].

b2GPI co-localizes with annexin 2 and TLR4 onthe lipid rafts of monocytes and anti-b2GPI anti-bodies stimulate monocyte tissue factor expression[31]. Antiphospholipid antibody-induced tissue fac-tor expression in monocytes occurs through phos-phorylation of MEK-1/ERK proteins, and the p38mitogen activated protein kinase-dependent nucleartranslocation and activation of NF-kB/Rel proteins[32]. Tissue factor is the main initiator of the extrinsiccoagulation pathway and is likely a key mediator ofAPS-related thrombosis.

Recombinant dimerized b2GPI and preformed,immobilized complexes of b2GPI and anti-b2GPImonoclonal antibodies induce platelet adhesionto collagen-coated surfaces in a glycoprotein IBand apoER2-dependent manner [33,34]. Yet, despiteevidence for platelet activation in patients with APS,specific binding of monomeric b2GPI to unacti-vated platelets has not been convincingly demon-strated.

Anticoagulant and fibrinolytic systems

Antiphospholipid antibodies promote thrombosisvia interference with the anticoagulant activity ofprotein C, protein S [35], annexin V [36], and antith-rombin [37]. Antiphospholipid antibodies inhibitthe activation and activity of protein C, both inthe fluid phase and on cell surfaces, and in doingso may enhance thrombin generation [38]. Anti-prothrombin antibodies from APS patients have



lupus anticoagulant activity, inhibit the inactivationof thrombin by antithrombin, induce tissue factorexpression, and display prothrombotic propertiesin vivo [39]. Antiphospholipid antibodies may alsoinhibit the interactions of antithrombin with anti-coagulant glycosaminoglycans on cell surfaces [40].

Elevated levels of coagulation factor XI havebeen identified as a risk factor for thrombosis inthe general population. APS patients have higherlevels of the active free thiol form of factor XI thanage and sex-matched controls [41]. The free thiolform of b2GPI is formed by the action of thiore-doxin-I and protein disulfide isomerase. WhetheraPL and antib2GPI antibodies have direct effects onprotein disulfide isomerase is unknown.

Annexin A5 binds to phosphatidylserine on cellsurfaces and forms a shield that inhibits the for-mation of coagulation complexes. Anti-b2GPI anti-bodies complexed with b2GPI can disrupt thisshield, exposing procoagulant phosphatidylserineand promoting thrombosis [13]. Hydroxychloro-quine has been reported to protect the annexin Vshield and has shown efficacy against aPL-mediatedthrombosis in a murine model [42,43].

b2GPI may have intrinsic fibrinolytic activity,and several studies have suggested that aPL mayinhibit fibrinolysis through interactions with tissueplasminogen activator and/or plasminogen [18,44].

Complement activation

Activation of the complement system has beenimplicated in the development of thrombosis andfetal loss in APS [45]. Activated complement frag-ments bind to and activate cells through the C5b-9membrane attack complex or C5a-receptor-medi-ated effects. Complement activation by aPL maygenerate the potent inflammatory mediator C5a,which recruits neutrophils and monocytes and leadsto exposure of tissue factor by endothelial cells andneutrophils [46]. Mice deficient in C3, C4, C5, orC5a receptor are protected from fetal loss induced byaPL IgG [47]. Complement deficiency is also protec-tive against thrombosis in some murine models [48].However, although elevated levels of complementsplit products have been identified in patients withAPS, these have not been demonstrated to correlatewith thrombosis [49]. Several recent case reportsdocument the successful use of eculizumab (human-ized anti-C5a monoclonal antibody) in patientswith CAPS and APS complicating renal transplan-tation [50

&&

].

Origin of antiphospholipid

Loss of immune tolerance is thought to be respon-sible for the origin of pathogenic aPL, which appears




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to be predominantly antigen driven. Bacterial andviral infections have been implicated in the develop-ment of aPL and shown to induce pathogenic anti-bodies against b2GPI. Antiphospholipid antibodiesdevelop in mice immunized with a cytomegalovi-rus-derived peptide [51], and a recent study demon-strated that protein H of Streptococcus pyogenes canbind b2GPI and expose neoepitopes that induceproduction of anti-b2GPI antibodies [52]. Oxidationof b2GPI by reactive oxygen and nitrosative speciesalso increase in its immunogenicity [53].

Rauch et al. [54] demonstrated that TLR4 isinvolved in inducing a break in immune toleranceand production of aPL. Dysregulation of other TLRsincluding TLR7, TLR8, and TLR9 may also contrib-ute to the development of aPL [55]. Hydroxychlor-oquine inhibits TLR7 and is associated with reducedpersistence of aPL in patients with systemic lupuserythematosus (SLE) [56

&&

].

THERAPEUTIC ADVANCES

Anticoagulation with heparin followed by long-termanticoagulation with a vitamin K antagonist is themainstay of therapy for thrombotic APS. However, asignificant proportion of patients have recurrentthrombosis despite antithrombotic therapy [57].Vitamin K antagonists are also problematic becauseof food and drug interactions, bleeding compli-cations, and need for frequent monitoring. Fur-thermore, aPL interact differently with differentthromboplastin reagents affecting monitoring ofthe prothrombin time and international normalizedratio [58].

The oral direct thrombin inhibitors (dabigatran)and direct factor Xa inhibitors (rivaroxaban andapixaban) overcome some of these disadvantages– they are fixed dose, do not need routine monitor-ing, and have few drug or food interactions. How-ever, they are irreversible and there is limitedexperience in patients with APS. The Rivaroxabanin AntiPhospholipid Syndrome (RAPS) trial is anongoing, open-label, prospective, noninferiorityrandomized trial evaluating the efficacy of rivarox-aban in patients with thrombotic APS. Oral directinhibitors should be considered in APS patients witha first or recurrent venous thromboembolis (VTE)only when there is vitamin K antagonist intoleranceor poor anticoagulant control. There are no data torecommend their use in APS patients with recurrentVTE occurring on therapeutic anticoagulation or inAPS-related arterial thrombosis [59

&

].Advances in the understanding of patho-

genic mechanisms involved in APS have led tothe identification of new therapeutic approaches.These include inhibition of cellular activation and



intracellular signaling pathways, antiplatelet agents,and immunomodulatory therapies (Table 2).

Statins

Statins display anti-inflammatory properties andinhibit cellular activation by aPL. Fluvastatin pre-vents the expression of adhesion molecules andtissue factor by aPL-treated endothelial cells in vitro[60]. Simvastatin and pravastatin also reduced fetalloss in a mouse model [61]. In a recent prospectivestudy, Erkan et al. [62

&

] demonstrated that fluvasta-tin treatment reduced the levels of biomarkers ofinflammation and thrombosis in patients with aPL.Modulation of aPL effects on vascular cells by statinscould be a valuable approach in the management ofAPS. At this time, however, statins are not recom-mended in patients with APS in the absence ofhyperlipidemia [59

&

]. Clinical studies are neededto evaluate their role in adjuvant therapy andfor primary thromboprophylaxis in aPL-positivepatients.

Hydroxychloroquine

Hydroxychloroquine is an established treatment forSLE due to its anti-inflammatory and immuno-modulatory effects. Hydroxychloroquine has beenreported to protect against arterial and venousthrombosis in SLE patients with and without aPL[63]. This is likely mediated by a decrease in lupusactivity and cardiovascular risk factors as well asmodulation of aPL effects. Hydroxychloroquineinhibits platelet aggregation and arachidonic acidrelease from stimulated platelets. It also inhibitsbinding of anti-b2GPI antibodies to purified phos-pholipid membranes. Recent studies by Rand andcolleagues have shown that hydroxychloroquineprotects the annexin A5 shield on endotheliumand placental syncytiotrophoblast from disruptionby aPL and preserves anticoagulant activity [64].Observational studies have shown that hydroxy-chloroquine decreased aPL titers and lowered theodds of having persistently positive aPL [56

&&

].Hydroxychloroquine is currently recommended

for all aPL-positive patients with SLE [59&

]. There areno strong data to support the use of hydroxychlor-oquine in patients with aPL without systemic auto-immune diseases. A recently published prospectivetrial comparing oral anticoagulation (fluindione)with or without hydroxychloroquine in primaryAPS patients reported that there were six (30%)VTE events in the monotherapy group versus zeroevents in the hydroxychloroquine group at 6 and36 months [65

&

]. A multicenter, randomized controltrial (NCT 01784523) to determine the efficacy of



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MOH 210504

Tab

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6 www.co-hematology.com Volume 21 � Number 00 � Month 2014


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hydroxychloroquine for primary thrombosis pre-vention in patients with aPL without systemic auto-immune diseases is currently underway.

Complement inhibition

Complement is implicated in the pathogenesis ofthrombosis and fetal loss in APS via generationof C5a. Anti-C5a monoclonal antibodies and C5a-receptor antagonist peptides have demonstratedefficacy in attenuating thrombosis and preventingfetal loss in murine models of APS. Clinical studies ofcomplement inhibition in APS are limited to casereports describing the successful use of eculizumabin patients with refractory CAPS and APS-relatedpostrenal transplant thrombotic microangiopathy[50

&&

]. An ongoing phase II study (NCT01029587)is evaluating the efficacy of eculizumab to preventrecurrent CAPS after kidney transplantation inpatients with a prior history of CAPS. Eculizumabcarries a risk of infection with encapsulated organ-isms and patients should be immunized againstmeningococcus before starting treatment [59

&

].

Defibrotide

Defibrotide is an adenosine receptor antagonist withantithrombotic, profibrinolytic, and anti-inflamma-tory effects on vascular endothelial cells that alsoblocks neutrophil tissue factor expression. It is beensuccessfully used in hepatic veno-occlusive diseaseand multiorgan failure after stem cell transplant.Defibrotide could potentially decrease endothelialactivation in APS and has been used for the treat-ment of refractory CAPS [66].

B-cell-directed therapy

B cells contribute to APS pathogenesis through theproduction of pathogenic aPL. Rituximab (anti-CD20 chimeric monoclonal antibody) inducesB-cell depletion and has been used with success insome patients with refractory CAPS [67]. The Ritux-imab in AntiPhospholipid Syndrome (RITAPS) trial,a prospective, open-label, phase II trial of rituximabin primary APS patients, reported that rituximab iseffective in controlling some noncriteria manifes-tations of APS such as thrombocytopenia, hemolyticanemia, skin ulcers, and nephropathy [68

&&

].Two studies have shown a benefit of B-cell mod-

ulatory therapies in murine models of APS. The firstfocused on co-stimulatory blockage with cytotoxic Tlymphocyte antigen 4 immunoglobulin (CTLA4-Ig)and demonstrated that CTLA4-Ig could not treatAPS but was able to prevent B-cell activationand aPL production if administered prior to aPL



antibody development [69]. The second approachtargeted B cell activating factor (BAFF), a tumornecrosis factor (TNF)-like cytokine that supportsB-cell survival and differentiation. In contrast toco-stimulatory blockade, BAFF antagonists pre-vented APS onset and prolonged survival. Basedon these studies, B-cell modulation shows somepromise as a therapeutic approach in APS [70]. Aba-tacept, a CTLA4 blocker, and belimumab, a BAFFantagonist, are approved for use in rheumatoidarthritis and SLE, respectively, but have not beenused in patients with APS.

Inhibition of intracellular signaling pathways

As described above, several intracellular signalingpathways are involved in aPL-mediated pathogeniceffects. Several of these pathways pose potentialtherapeutic targets. For example, NF-kB inhibitioninhibits aPL-induced cellular activation and throm-bosis [71], and TLR4 inhibition decreases tissuefactor, MyD88, and TNF expression in in-vitro andin-vivo studies [72

&

].

CONCLUSION

Lifelong anticoagulation remains the mainstay oftherapy for thrombotic APS. The managementof asymptomatic patients with aPL is still contro-versial, as the pathogenesis of this disorder is stillnot well understood or supported by a unifyinghypothesis. Innovative therapeutic approaches suchas immune modulation, complement inhibition,and inhibiting intracellular signaling pathwayshave shown promising results in preclinical studies.Further mechanistic and clinical studies are neededto develop and implement improved therapies forthis devastating illness.

Acknowledgements

This work was supported by a Bridge Grant from theAmerican Society of Hematology (to K.R.M.) and NIHGrant P50 HL081011 (K.R.M. project leader).

Conflicts of interest

The authors have no conflicts of interest to disclose.

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Recent advances in the antiphospholipid antibody syndrome