The antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by recurrent thrombosis and/or obstetrical morbidity in the presence of antiphospholipid antibodies (aPL), including lupus anticoagulant, anti-β2-glycoprotein I (anti-β2GPI), and/or anticardiolipin (aCL) antibodies . Thrombosis in APS can affect any vascular site, with the deep veins of the lower extremities and the cerebral arterial circulation most commonly affected . A small number of patients (<1%) develop catastrophic antiphospholipid syndrome (CAPS) , defined as small-vessel thrombosis in three or more organs in less than 1 week in the presence of aPL, with histopathologic confirmation of small-vessel thrombosis in the absence of inflammation . CAPS is associated with high (50%) mortality, mostly because of cerebral and cardiac thrombosis, infections, and multiorgan failure [4▪]. Obstetrical morbidity in APS includes the unexplained death of one or more morphologically normal fetuses at or beyond the 10th week of gestation, the premature birth of one or more morphologically normal neonates before the 34th week of gestation because of either eclampsia or severe preeclampsia, and/or three or more unexplained, consecutive spontaneous abortions before the 10th week of gestation . Other clinical associations of aPL include thrombocytopenia, livedo reticularis, transient ischemic attacks and skin ulcers [4▪].
DIAGNOSIS AND THROMBOTIC RISK ASSESSMENT
The diagnosis of APS rests on the presence of at least one clinical and laboratory criterion (Table 1). According to the revised classification guidelines (2006), the laboratory criteria for APS require the persistence (for >12 weeks) of a lupus anticoagulant, detected according to the guidelines of the International Society of Thrombosis and Hemostasis  and/or high titers of IgG or IgM autoantibodies against cardiolipin and/or β2GPI detected using standardized ELISAs . Patients with other aPLs such as antiprothrombin antibodies, IgA aCL or anti-β2GPI antibodies, or those with clinical manifestations other than thrombosis or pregnancy morbidity cannot be formally diagnosed with APS using these criteria. It remains difficult to predict thrombotic risk in patients with aPL, and patients may be encountered who develop thrombi yet do not meet the criteria for APS due to the presence of only low or moderate levels of aCL or anti-β2GPI antibodies.
In most reports, lupus anticoagulants are more strongly associated with thrombotic risk and pregnancy complications than aCL or anti-β2GPI antibodies alone . The risk of a first thrombotic event in triple-positive patients, that is, those with positivity for lupus anticoagulant, aCL, and anti–β2GPI antibodies, may be as high as 5.3% per year . Antibodies directed toward β2GPI domain 1 may correlate more strongly with thrombotic risk and pregnancy morbidity . Antibodies against prothrombin also are associated with thrombosis, but with lower odds ratios than those against β2GPI [9▪]. A better understanding of the thrombotic risk profile of patients with aPL is needed to help define the need for, and the optimal type and duration of, antithrombotic therapy.
Antiphospholipid antibodies directed against phospholipid binding proteins bound to the surface of endothelial cells, monocytes and platelets are likely central to the pathogenesis of APS. β2GPI is recognized as the primary antigenic target of aPL  (Fig. 1). Affinity-purified human anti-β2GPI autoantibodies potentiate arterial and venous thrombus formation in a mouse model , and lupus anticoagulants whose effects are mediated via interactions with β2GPI confer a higher risk of thrombosis than those due to aCL or anti-prothrombin antibodies [6,7]. β2GPI is also expressed on the surface of placental trophoblasts, where anti-β2GPI antibody binding to β2GPI results in inhibition of growth and differentiation of trophoblasts, and inflammatory changes leading to fetal loss . Displacement of annexin V from these cells may also allow exposure of anionic phospholipid, providing a nidus for coagulation complex assembly .
β2GPI is a plasma glycoprotein containing five ‘sushi’ domains, the fifth of which is atypical and mediates binding to anionic phospholipid . A recent report suggests that β2GPI may exist in two conformations. Circulating β2GPI has been found to exist largely in a circular form in which domain 1 is postulated to bind to domain 5, masking the putative anti-β2GPI antibody epitope in domain 1. Binding of β2GPI to anionic phospholipid or other relevant surfaces is proposed to induce unfolding of β2GPI with conversion to a ‘fishhook’ conformation observed in the crystal structure of β2GPI, in which the domain 1 epitope is exposed and induces formation and binding of anti-β2GPI domain 1 antibodies .
A ‘two hit’ model of thrombosis in APS has been hypothesized in which a ‘first hit’ creates a prothrombotic state followed by a ‘second hit’, possibly inflammatory, that perturbs the endothelium and initiates thrombosis . The pathophysiologic mechanisms that contribute to the prothrombotic phenotype (first hit) include aPL-mediated activation of monocytes, endothelial cells and/or platelets, and/or inhibition of natural anticoagulant and fibrinolytic systems by aPL [17▪,18].
Antiphospholipid antibodies have been reported to bind to and activate vascular endothelial cells, monocytes, and platelets in a β2GPI-dependent manner [19–22]. Antiphospholipid antibodies are generally of low affinity and do not bind β2GPI in solution, but only after deposition at high concentration on an appropriate surface . There is no convincing evidence for circulating immune complexes containing β2GPI and anti-β2GPI antibodies in patients with APS, and the clinical manifestation of APS are generally not those of an immune-complex-mediated disease. Thus, evidence suggests that anti-β2GPI antibodies bind β2GPI previously bound to cells.
Endothelial cells play an important role in maintaining blood fluidity through the expression of anticoagulant proteins and the elaboration of antithrombotic substances such as prostacyclin, glycosaminoglycans and nitric oxide. Endothelial cell activation via aPL–β2GPI interactions leads to loss of these anticoagulant properties with transformation to a pro-adhesive, procoagulant phenotype characterized by increased expression of adhesion molecules (E-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1) and tissue factor, enhanced secretion of pro-inflammatory cytokines and chemokines, and release of procoagulant and proinflammatory microparticles . Annexin A2, a cell-surface receptor for plasminogen and plasminogen activator, mediates the binding of β2GPI to cells [20,25]. However, annexin A2 does not have a transmembrane domain; therefore, other co-receptors must be involved in cellular activation. A recent study suggested that anti-β2GPI antibodies induce signaling through a multiprotein complex that includes annexin A2, calreticulin, nucleolin, and Toll-like receptor 4 (TLR4) , with TLR4 inducing activation of a TLR4/myeloid differentiation factor 88 (MyD88)-dependent pathway culminating in NF-κB activation [26,27▪▪]. The involvement of p38 mitogen activated protein kinase in endothelial activation has also been demonstrated . In addition to annexin A2, other potential receptors such as apoER2  and TLR2 may also play a role in endothelial activation .
β2GPI co-localizes with annexin 2 and TLR4 on the lipid rafts of monocytes and anti-β2GPI antibodies stimulate monocyte tissue factor expression . Antiphospholipid antibody-induced tissue factor expression in monocytes occurs through phosphorylation of MEK-1/ERK proteins, and the p38 mitogen activated protein kinase-dependent nuclear translocation and activation of NF-κB/Rel proteins . Tissue factor is the main initiator of the extrinsic coagulation pathway and is likely a key mediator of APS-related thrombosis.
Recombinant dimerized β2GPI and preformed, immobilized complexes of β2GPI and anti-β2GPI monoclonal antibodies induce platelet adhesion to collagen-coated surfaces in a glycoprotein IB and apoER2-dependent manner [33,34]. Yet, despite evidence for platelet activation in patients with APS, specific binding of monomeric β2GPI to unactivated platelets has not been convincingly demonstrated.
Anticoagulant and fibrinolytic systems
Antiphospholipid antibodies promote thrombosis via interference with the anticoagulant activity of protein C, protein S , annexin V , and antithrombin . Antiphospholipid antibodies inhibit the activation and activity of protein C, both in the fluid phase and on cell surfaces, and in doing so may enhance thrombin generation . Anti-prothrombin antibodies from APS patients have lupus anticoagulant activity, inhibit the inactivation of thrombin by antithrombin, induce tissue factor expression, and display prothrombotic properties in vivo. Antiphospholipid antibodies may also inhibit the interactions of antithrombin with anticoagulant glycosaminoglycans on cell surfaces .
Elevated levels of coagulation factor XI have been identified as a risk factor for thrombosis in the general population. APS patients have higher levels of the active free thiol form of factor XI than age and sex-matched controls . The free thiol form of β2GPI is formed by the action of thioredoxin-I and protein disulfide isomerase. Whether aPL and antiβ2GPI antibodies have direct effects on protein disulfide isomerase is unknown.
Annexin A5 binds to phosphatidylserine on cell surfaces and forms a shield that inhibits the formation of coagulation complexes. Anti-β2GPI antibodies complexed with β2GPI can disrupt this shield, exposing procoagulant phosphatidylserine and promoting thrombosis . Hydroxychloroquine has been reported to protect the annexin V shield and has shown efficacy against aPL-mediated thrombosis in a murine model [42,43].
β2GPI may have intrinsic fibrinolytic activity, and several studies have suggested that aPL may inhibit fibrinolysis through interactions with tissue plasminogen activator and/or plasminogen [18,44].
Activation of the complement system has been implicated in the development of thrombosis and fetal loss in APS . Activated complement fragments bind to and activate cells through the C5b-9 membrane attack complex or C5a-receptor-mediated effects. Complement activation by aPL may generate the potent inflammatory mediator C5a, which recruits neutrophils and monocytes and leads to exposure of tissue factor by endothelial cells and neutrophils . Mice deficient in C3, C4, C5, or C5a receptor are protected from fetal loss induced by aPL IgG . Complement deficiency is also protective against thrombosis in some murine models . However, although elevated levels of complement split products have been identified in patients with APS, these have not been demonstrated to correlate with thrombosis . Several recent case reports document the successful use of eculizumab (humanized anti-C5a monoclonal antibody) in patients with CAPS and APS complicating renal transplantation [50▪▪].
Origin of antiphospholipid
Loss of immune tolerance is thought to be responsible for the origin of pathogenic aPL, which appears to be predominantly antigen driven. Bacterial and viral infections have been implicated in the development of aPL and shown to induce pathogenic antibodies against β2GPI. Antiphospholipid antibodies develop in mice immunized with a cytomegalovirus-derived peptide , and a recent study demonstrated that protein H of Streptococcus pyogenes can bind β2GPI and expose neoepitopes that induce production of anti-β2GPI antibodies . Oxidation of β2GPI by reactive oxygen and nitrosative species also increase in its immunogenicity .
Rauch et al. demonstrated that TLR4 is involved in inducing a break in immune tolerance and production of aPL. Dysregulation of other TLRs including TLR7, TLR8, and TLR9 may also contribute to the development of aPL . Hydroxychloroquine inhibits TLR7 and is associated with reduced persistence of aPL in patients with systemic lupus erythematosus (SLE) [56▪▪].
Anticoagulation with heparin followed by long-term anticoagulation with a vitamin K antagonist is the mainstay of therapy for thrombotic APS. However, a significant proportion of patients have recurrent thrombosis despite antithrombotic therapy . Vitamin K antagonists are also problematic because of food and drug interactions, bleeding complications, and need for frequent monitoring. Furthermore, aPL interact differently with different thromboplastin reagents affecting monitoring of the prothrombin time and international normalized ratio .
The oral direct thrombin inhibitors (dabigatran) and direct factor Xa inhibitors (rivaroxaban and apixaban) overcome some of these disadvantages – they are fixed dose, do not need routine monitoring, and have few drug or food interactions. However, they are irreversible and there is limited experience in patients with APS. The Rivaroxaban in AntiPhospholipid Syndrome (RAPS) trial is an ongoing, open-label, prospective, noninferiority randomized trial evaluating the efficacy of rivaroxaban in patients with thrombotic APS. Oral direct inhibitors should be considered in APS patients with a first or recurrent venous thromboembolis (VTE) only when there is vitamin K antagonist intolerance or poor anticoagulant control. There are no data to recommend their use in APS patients with recurrent VTE occurring on therapeutic anticoagulation or in APS-related arterial thrombosis [59▪].
Advances in the understanding of pathogenic mechanisms involved in APS have led to the identification of new therapeutic approaches. These include inhibition of cellular activation and intracellular signaling pathways, antiplatelet agents, and immunomodulatory therapies (Table 2).
Statins display anti-inflammatory properties and inhibit cellular activation by aPL. Fluvastatin prevents the expression of adhesion molecules and tissue factor by aPL-treated endothelial cells in vitro. Simvastatin and pravastatin also reduced fetal loss in a mouse model . In a recent prospective study, Erkan et al.[62▪] demonstrated that fluvastatin treatment reduced the levels of biomarkers of inflammation and thrombosis in patients with aPL. Modulation of aPL effects on vascular cells by statins could be a valuable approach in the management of APS. At this time, however, statins are not recommended in patients with APS in the absence of hyperlipidemia [59▪]. Clinical studies are needed to evaluate their role in adjuvant therapy and for primary thromboprophylaxis in aPL-positive patients.
Hydroxychloroquine is an established treatment for SLE due to its anti-inflammatory and immunomodulatory effects. Hydroxychloroquine has been reported to protect against arterial and venous thrombosis in SLE patients with and without aPL . This is likely mediated by a decrease in lupus activity and cardiovascular risk factors as well as modulation of aPL effects. Hydroxychloroquine inhibits platelet aggregation and arachidonic acid release from stimulated platelets. It also inhibits binding of anti-β2GPI antibodies to purified phospholipid membranes. Recent studies by Rand and colleagues have shown that hydroxychloroquine protects the annexin A5 shield on endothelium and placental syncytiotrophoblast from disruption by aPL and preserves anticoagulant activity . Observational studies have shown that hydroxychloroquine decreased aPL titers and lowered the odds of having persistently positive aPL [56▪▪].
Hydroxychloroquine is currently recommended for all aPL-positive patients with SLE [59▪]. There are no strong data to support the use of hydroxychloroquine in patients with aPL without systemic autoimmune diseases. A recently published prospective trial comparing oral anticoagulation (fluindione) with or without hydroxychloroquine in primary APS patients reported that there were six (30%) VTE events in the monotherapy group versus zero events in the hydroxychloroquine group at 6 and 36 months [65▪]. A multicenter, randomized control trial (NCT 01784523) to determine the efficacy of hydroxychloroquine for primary thrombosis prevention in patients with aPL without systemic autoimmune diseases is currently underway.
Complement is implicated in the pathogenesis of thrombosis and fetal loss in APS via generation of C5a. Anti-C5a monoclonal antibodies and C5a-receptor antagonist peptides have demonstrated efficacy in attenuating thrombosis and preventing fetal loss in murine models of APS. Clinical studies of complement inhibition in APS are limited to case reports describing the successful use of eculizumab in patients with refractory CAPS and APS-related postrenal transplant thrombotic microangiopathy [50▪▪]. An ongoing phase II study (NCT01029587) is evaluating the efficacy of eculizumab to prevent recurrent CAPS after kidney transplantation in patients with a prior history of CAPS. Eculizumab carries a risk of infection with encapsulated organisms and patients should be immunized against meningococcus before starting treatment [59▪].
Defibrotide is an adenosine receptor antagonist with antithrombotic, profibrinolytic, and anti-inflammatory effects on vascular endothelial cells that also blocks neutrophil tissue factor expression. It is been successfully used in hepatic veno-occlusive disease and multiorgan failure after stem cell transplant. Defibrotide could potentially decrease endothelial activation in APS and has been used for the treatment of refractory CAPS .
B cells contribute to APS pathogenesis through the production of pathogenic aPL. Rituximab (anti-CD20 chimeric monoclonal antibody) induces B-cell depletion and has been used with success in some patients with refractory CAPS . The Rituximab in AntiPhospholipid Syndrome (RITAPS) trial, a prospective, open-label, phase II trial of rituximab in primary APS patients, reported that rituximab is effective in controlling some noncriteria manifestations of APS such as thrombocytopenia, hemolytic anemia, skin ulcers, and nephropathy [68▪▪].
Two studies have shown a benefit of B-cell modulatory therapies in murine models of APS. The first focused on co-stimulatory blockage with cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4-Ig) and demonstrated that CTLA4-Ig could not treat APS but was able to prevent B-cell activation and aPL production if administered prior to aPL antibody development . The second approach targeted B cell activating factor (BAFF), a tumor necrosis factor (TNF)-like cytokine that supports B-cell survival and differentiation. In contrast to co-stimulatory blockade, BAFF antagonists prevented APS onset and prolonged survival. Based on these studies, B-cell modulation shows some promise as a therapeutic approach in APS . Abatacept, a CTLA4 blocker, and belimumab, a BAFF antagonist, are approved for use in rheumatoid arthritis and SLE, respectively, but have not been used in patients with APS.
Inhibition of intracellular signaling pathways
As described above, several intracellular signaling pathways are involved in aPL-mediated pathogenic effects. Several of these pathways pose potential therapeutic targets. For example, NF-κB inhibition inhibits aPL-induced cellular activation and thrombosis , and TLR4 inhibition decreases tissue factor, MyD88, and TNF expression in in-vitro and in-vivo studies [72▪].
Lifelong anticoagulation remains the mainstay of therapy for thrombotic APS. The management of asymptomatic patients with aPL is still controversial, as the pathogenesis of this disorder is still not well understood or supported by a unifying hypothesis. Innovative therapeutic approaches such as immune modulation, complement inhibition, and inhibiting intracellular signaling pathways have shown promising results in preclinical studies. Further mechanistic and clinical studies are needed to develop and implement improved therapies for this devastating illness.
This work was supported by a Bridge Grant from the American Society of Hematology (to K.R.M.) and NIH Grant P50 HL081011 (K.R.M. project leader).
Conflicts of interest
The authors have no conflicts of interest to disclose.
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62▪. Erkan D, Willis R, Murthy VL, et al. A prospective open-label pilot study of fluvastatin on proinflammatory and prothrombotic biomarkers in antiphospholipid antibody positive patients. Ann Rheum Dis. 2013; 73(6):1176–1180.
This prospective clinical study demonstrated that proinflammatory and prothrombotic biomarkers, which are upregulated in persistently aPL-positive patients, can be reversibly reduced by fluvastatin.
63. Petri M. Use of hydroxychloroquine to prevent thrombosis in systemic lupus erythematosus and in antiphospholipid antibody-positive patients. Curr Rheumatol Rep. 2011; 13:77–80.
64. Wu XX, Guller S, Rand JH. Hydroxychloroquine reduces binding of antiphospholipid antibodies to syncytiotrophoblasts and restores annexin A5 expression. Am J Obstet Gynecol. 2011; 205:e7–e14.
65▪. Schmidt-Tanguy A, Voswinkel J, Henrion D, et al. Antithrombotic effects of hydroxychloroquine in primary antiphospholipid syndrome patients. J Thromb Haemost. 2013; 11:1927–1929.
This is the first prospective study investigating the use of hydroxychloroquine for secondary venous thrombosis prophylaxis in primary APS.
66. Espinosa G, Berman H, Cervera R. Management of refractory cases of catastrophic antiphospholipid syndrome. Autoimmun Rev. 2011; 10:664–668.
67. Berman H, Rodriguez-Pinto I, Cervera R, et al. Rituximab use in the catastrophic antiphospholipid syndrome: descriptive analysis of the CAPS registry patients receiving rituximab. Autoimmun Rev. 2012; 12:1085–1090.
68▪▪. Erkan D, Vega J, Ramón G, et al. A pilot open-label phase II trial of rituximab for noncriteria manifestations of antiphospholipid syndrome. Arthritis Rheum. 2013; 65:464–471.
This phase II pilot study showed that rituximab is well tolerated and effective in treating some noncriteria manifestations of APS.
69. Akkerman A, Huang W, Wang X, et al. CTLA4Ig prevents initiation but not evolution of antiphospholipid syndrome in NZW/BXSB mice. Autoimmunity. 2004; 37:445–451.
70. Kahn P, Ramanujman M, Bethunaickan R, et al. Prevention of murine antiphospholipid syndrome by BAFF blockade. Arthritis Rheum. 2008; 58:2824–2834.
71. Montiel-Manzano G, Romay-Penabad Z, Papalardo de Martinez E, et al. In vivo effects of an inhibitor of nuclear factor kappa B on thrombogenic properties of antiphospholipid antibodies. Ann N Y Acad Sci. 2007; 1108:540–553.
72▪. Xie H, Zhou H, Wang H, et al. Antiβ(2)GPI/β(2)GPI induced TF and TNF-α expression in monocytes involving both TLR4/MyD88 and TLR4/TRIF signaling pathways. Mol Immunol. 2013; 53:246–254.
This study showed that antiβ(2)GPI/β(2)GPI complex induced tissue factor and TNF-α expression involves TLR4/MyD88 and TLR4/TRIF signaling pathways, and this can be blocked by inhibiting TLR4-mediated signal transduction.