Antiphospholipid syndrome (APS) is characterized by increased risk of vascular thrombosis, involving the venous, arterial, and placental circulatory systems. The pathogenic mechanisms for antiphospholipid antibody (aPL)–induced thrombosis are incompletely understood. Passive transfer of IgG from aPL-positive sera (IgG-APS) has been found to induce fetal loss, thrombosis, and endothelial cell activation in mice, suggesting a direct pathogenic role of aPL (1–3). Complement activation is a necessary intermediary event in the pathogenesis of fetal loss associated with aPL in this model (4, 5).
It is well established that activated complement fragments themselves have the capacity to bind and activate inflammatory and endothelial cells, as well as induce a prothrombotic phenotype either directly through C5b–9 (membrane attack complex [MAC]) or through C5a receptor (C5aR)–mediated effects (6, 7). Endothelial cells can release tissue factor in response to C5a activation (8). Inflammatory cells, when triggered by complement proteolytic products C5a and C3a, respond with the production of selected procoagulant activities, thereby initiating the coagulation pathways. MAC has also been associated with thrombosis. Studies performed in rats showed that CD59, an inhibitor of C5b–9 assembly and insertion, serves a protective role in a rat model of thrombotic microangiopathy, demonstrating that C5b–9 plays a critical role in the pathogenesis of thrombosis (9).
In previous studies, we demonstrated that the complement C3 convertase inhibitor, Crry, inhibited IgG-APS–induced thrombosis, suggesting that complement activation is required in IgG-APS–induced thrombophilia (4). Moreover, Girardi and coworkers proposed that heparin, the current standard treatment in patients with APS, prevents obstetric complications by blocking activation of complement, as opposed to preventing placental thrombosis (10). We therefore tested the hypothesis that complement activation mediates endothelial cell activation and the thrombogenic effects of IgG-APS.
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- MATERIALS AND METHODS
Using a model of surgically induced thrombus formation, we demonstrated that complement activation plays an important role in thrombosis induced by aPL in mice. Specifically, we identified C3 and C5 as the critical intermediaries linking pathogenic aPL to WBC adhesion and development of thrombosis. Our conclusions are based on the prevention of thrombophilia observed in C3−/− and C5−/− mice and the protective effects of anti-C5 mAb. In previous studies from our group, the findings suggested that C3 activation is required for aPL-induced thrombosis. We demonstrated that Crry-Ig, an inhibitor of C3 convertase, blocks thrombosis initiated by aPL (4). Subsequently, Girardi et al showed that complement activation, specifically C5a–C5aR interaction, is required for aPL-induced pregnancy loss and suggested that C5a promotes neutrophil infiltration of decidual tissue (5). Evidence that neutrophils activated by C5a release procoagulant substances and that monocytes activated by C5a release tissue factor suggests that infiltrating leukocytes stimulated by complement split products can initiate placental infarction and ultimately cause fetal death (8).
In the current study, we have extended our findings by demonstrating that complement is required for aPL-mediated thrombosis and for increased leukocyte adhesion to endothelium. In the absence of C3 or C5, we observed neither enhanced leukocyte adherence nor increased thrombosis associated with aPL treatment. Furthermore, we found that anti-C5 mAb prevented aPL-mediated thrombosis, emphasizing the role of C5 (either C5a, C5aR, or C5b–9) in induction of thrombophilia. C5a binding to endothelial cells results in increased expression of P-selectin and markedly increases neutrophil adhesion (14), and binding of C5b to target surfaces initiates assembly of the MAC that triggers proinflammatory signaling pathways and induces a prothrombotic phenotype in vascular tissue (6). Observations that blockade of C5aR prevents thrombus formation and leukocyte accumulation in a rat model of antibody-mediated thrombotic glomerulonephritis underscore the linkage between complement activation and thrombophilia (15).
Thrombosis in APS is sporadic and may occur in any vein or artery of the body. In this study, we used a mouse model of thrombosis induced by a standardized pinch injury in the femoral vein to define the mediators of thrombophilia associated with aPL (2, 3). Recently, other investigators have demonstrated enhancement of thrombosis by aPL in an experimental model of photochemically induced vascular injury in hamsters (16). Patients with APS often have aPL for prolonged periods of time without clinical manifestations, and thrombosis occurs after a triggering event such as an infection, immobilization, or surgery. Therefore, our experimental model of injury-induced thrombosis, although artificial, simulates a “second hit” that triggers thrombotic episodes in susceptible patients and mimics sporadic clotting as observed clinically in APS.
We recognize that there are differences between the human and the murine complement systems. However, the anti-C5 mAb (BB5.1) used in these studies has been shown to effectively block C5 activation in vitro and in vivo in mice and in humans (13, 17, 18). Independent of the initiator of the complement cascade, this mAb prevents C5 activation and thus prevents the generation of the potent proinflammatory factors, C5a and C5b–9. Anti-C5 mAb precipitates the 2 chains of C5 from normal mouse serum and inhibits C5-dependent hemolysis in a functional complement test. It has been shown to prevent aPL-induced pregnancy loss, in which thrombosis plays an important role (5).
Anti-C5 biologic therapy has been extensively investigated in several other animal models of complement-mediated diseases, including collagen-induced arthritis and lupus-like autoimmune disease in (NZB/NZW)F1 mice (19, 20). Eculizumab (5G1.1), the humanized anti-C5 mAb, is considered a potential treatment for several chronic inflammatory diseases, including rheumatoid arthritis and nephritis, and phase II trials have been initiated for these indications. Furthermore, eculizumab has been shown to prevent C5 activation in humans and to have beneficial effects in patients with paroxysmal nocturnal hemoglobinuria; specifically, it reduces intravascular hemolysis, hemoglobinuria, and the need for transfusion in these patients, providing a proof-of-concept that blockade of complement activation is feasible and tolerable in patients with chronic disease (18).
We propose that pathogenic aPL, in addition to their direct effects on platelet and endothelial cell targets, induce complement activation, and thus generate complement split products that attract inflammatory cells and initiate thrombosis and tissue injury. Our finding that blockade of C5 is effective in preventing thrombosis in a mouse model of APS has important therapeutic implications. Blockade of complement activation may be a valuable target for interventions that prevent, arrest, or modify the thrombogenic effects of aPL.