Requirement of activation of complement C3 and C5 for antiphospholipid antibody–mediated thrombophilia




Antiphospholipid antibodies (aPL) have been shown to induce thrombosis, activate endothelial cells, and induce fetal loss. The pathogenesis of aPL-induced thrombosis, although not completely understood, may involve platelet and endothelial cell activation as well as procoagulant effects of aPL directly on clotting pathway components. Recent studies have shown that uncontrolled complement activation leads to fetal death in aPL-treated mice. In this study, we tested the hypothesis that aPL are responsible for activation of complement, thus generating split products that induce thrombosis.


To study thrombus dynamics and adhesion of leukocytes we used in vivo murine models of thrombosis and microcirculation, in which injections of aPL were used.


Mice deficient in complement components C3 and C5 were resistant to the enhanced thrombosis and endothelial cell activation that was induced by aPL. Furthermore, inhibition of C5 activation using anti-C5 monoclonal antibodies prevented thrombophilia induced by aPL.


These data show that complement activation mediates 2 important effectors of aPL, induction of thrombosis and activation of endothelial cells.

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.



C3-deficient mice were obtained from V. M. Holers (University of Colorado Health Sciences, Denver) (11). The mice were generated by intercrossing C3+/− mice at F1 during a backcross to C57BL/6 and then propagating C3−/− progeny. C5−/− mice (B10.D2-H2dH2-T18c Hco/o2Sn) and the C5+/+ background strain of mice (B10.D2-H2dH2-T18c Hco/oSnJ) were obtained from The Jackson Laboratories (Bar Harbor, ME). CD1 male mice (weight ∼30 grams) were obtained from Charles River Laboratories (Wilmington, DE). All animals were housed in the animal care (American Association of Laboratory Animal Care–approved) facilities of the Morehouse School of Medicine. Animals were handled by trained personnel according to Institutional Animal Care and Use Committee guidelines.

Preparation of IgG-APS.

Antiphospholipid antibodies from APS patients (IgG-APS) were affinity-purified using cardiolipin liposomes and protein G–Sepharose chromatography as previously described (2, 3). Human IgG from a non-autoimmune healthy individual (IgG-NHS) was purified by an identical method. The sterile-filtered IgG fractions were determined to be free of endotoxin contamination by the limulus amoebocyte lysate assay (E-Toxate; Sigma, St. Louis, MO). Protein concentration was determined using the method of Lowry (12). Levels of human anticardiolipin (aCL) and anti–β2-glycoprotein I antibodies were measured by standard enzyme-linked immunosorbent assay, performed as previously described (2, 3).

Analysis of thrombus dynamics and leukocyte adhesion in complement-deficient mice.

To investigate the role of C3 and C5 in thrombophilia induced by aPL, C3+/+ and C3−/− mice (n = 7–10 animals) and C5+/+ and C5−/− mice (n = 7–10 animals) were injected intraperitoneally with 500 μg of IgG-APS or with 500 μg of IgG-NHS twice, at time 0 and at 48 hours later. Surgical procedures to study thrombus dynamics and adhesion of leukocytes to the endothelium of postcapillary venules (number of adhering white blood cells [WBCs]) in the exposed cremaster muscle were performed 72 hours after the first IgG-APS or IgG-NHS injection, as described previously (2, 3). The mouse model of thrombosis formation used in this study has previously been described in detail (2, 3). Briefly, mice were anesthetized and the right femoral vein was exposed, resulting in a 0.5-cm segment of vein free for manipulation and observation. The vein was pinched with a pressure of 1,500 gm/mm2 to introduce a standardized injury that induced a clot. Clot formation and dissolution in the transilluminated vein were visualized with a microscope equipped with a closed-circuit video system (including a color monitor and a recorder). Thrombus size (expressed in μm2) was measured 1 minute after the pinch injury by freezing the digitized image and tracing the outer margin of the thrombus. Three-to-five thrombi were successfully induced in each animal, and mean values were computed.

Adhesion of leukocytes to endothelium in the cremaster muscle is an indication of endothelial cell activation (3). The number of leukocytes (WBCs) adhering within 5 different venules in the cremaster muscle was determined, and adhesion was defined as the presence of WBCs that remained stationary for at least 30 seconds (2, 3).

Analysis of thrombus dynamics using the anti-C5 monoclonal antibody (mAb).

Anti-C5 mAb have been shown to prevent C5 activation in vivo and in vitro and to protect mice from aPL-induced pregnancy loss (5, 13). To further investigate the role of C5 in thrombophilia induced by aPL, male CD1 mice (weight 25–30 grams; Charles River Laboratories) were injected intraperitoneally with 500 μg of IgG-APS or IgG-NHS (n = 10 mice per group) at time 0 and at 48 hours later. Half of the mice in each group received 1 mg of anti-C5 mAb (BB5.1) and half received 1 mg of murine IgG control at 30 minutes before each injection with IgG-APS or IgG-NHS. Surgical procedures to study thrombus dynamics were performed 72 hours after the first IgG-APS (or IgG-NHS) injection, as previously described (2, 3).

Statistical analysis.

An independent t-test was used to compare the antibody levels in different groups of mice. Student's unpaired t-test was used to compare the mean thrombus size and number of adhering WBCs between treated and control groups. P values of less than 0.05 were considered significant.


Protection of C3- and C5-deficient mice from IgG-APS–induced thrombophilia in vivo.

Consistent with our previous findings, IgG-APS significantly enhanced thrombus size (3.3-fold increase) in C3+/+ mice when compared with the effects of treatment with IgG-NHS in C3+/+ mice (2, 4). In C3−/− mice treated with IgG-APS, there was a significant reduction (P = 0.0002) in the size of injury-induced thrombi (67% decrease) when compared with the effects of IgG-APS in C3+/+ mice (Table 1). This reduction in thrombus size in C3−/− mice treated with IgG-APS was similar to that in C3−/− or C3+/+ mice treated with IgG-NHS (Table 1).

Table 1. Effects of IgG-APS on thrombus size and leukocyte adhesion to endothelial cells in C3−/− mice*
Mouse type, treatmentNo. of animalsThrombus size, μm2WBC adhesionaCL titer, GPL units
  • *

    Values are the mean ± SD. IgG-APS = IgG from antiphospholipid-positive sera; WBC = white blood cell (expressed as no. of leukocytes adhering within 5 different venules); aCL = anticardiolipin; GPL = IgG phospholipid; WT = wild-type; IgG-NHS = IgG from non-autoimmune healthy sera; C3−/− = deficient in C3.

  • Negative titer considered <10 GPL units.

 IgG-NHS9802 ± 39014.0 ± 5.0<10
 IgG-APS72,646 ± 98035.0 ± 12.078.0 ± 8.6
 IgG-NHS71,083 ± 4434.7 ± 0.9<10
 IgG-APS7873 ± 4254.7 ± 2.086.3 ± 10.4

In C5+/+ mice, IgG-APS induced a significant, 2.3-fold increase in thrombus size (P = 0.003) when compared with the effects of IgG-NHS in C5+/+ mice. The increase in thrombus size induced by IgG-APS was reduced by 54% (P = 0.011) in C5−/− mice (Table 2). The thrombus size in C5−/− mice treated with IgG-APS was not different from that in C5−/− or C5+/+ mice treated with IgG-NHS (Table 2).

Table 2. Effects of IgG-APS on thrombus size and leukocyte adhesion to endothelial cells in C5−/− mice*
Mouse type, treatmentNo. of animalsThrombus size, μm2WBC adhesionaCL titer, GPL units
  • *

    Values are the mean ± SD. See Table 1 for definitions.

  • Negative titer considered <10 GPL units.

 IgG-NHS7447 ± 1421.4 ± 0.4<10
 IgG-APS101,066 ± 1416.5 ± 2.5115.7 ± 74.2
 IgG-NHS7303 ± 2481.4 ± 0.4<10
 IgG-APS7493 ± 1491.7 ± 0.5102.3 ± 12.2

Protection of C3- and C5-deficient mice from IgG-APS–induced endothelial cell activation in vivo.

To determine whether complement is required for aPL induction of in vivo endothelial cell activation, we examined WBC adhesion to the endothelium of cremaster muscle. IgG-APS induced an increase in WBC adhesion in both C3+/+ and C5+/+ mice (2.5-fold [P = 0.01] and 4.6-fold [P = 0.0025], respectively) when compared with the effects of treatment with IgG-NHS (Tables 1 and 2). In contrast, adhesion of WBCs to cremaster endothelium was significantly reduced after treatment with IgG-APS in C3- and C5-deficient mice (reduction of 87% [P = 0.002] and 74% [P = 0.003], respectively) (Tables 1 and 2).

The titer of aCL antibodies in C3+/+ mice injected with IgG-APS was not different from the aCL titers in C3−/− mice treated with IgG-APS (mean ± SD 78 ± 8.6 IgG phospholipid [GPL] units versus 86.3 ± 10.4 GPL units). Similarly, the levels of aCL antibodies in C5+/+ mice treated with IgG-APS were not different from the aCL levels in C5−/− mice treated with IgG-APS (115.7 ± 74.2 GPL units versus 102.3 ± 12.2 GPL units). These findings exclude the possibility that the protective effects of IgG-APS in reducing proinflammatory and thrombogenic events in C3−/− and C5−/− mice were related to lower aCL titers in these mice.

Prevention of IgG-APS–induced thrombosis by anti-C5 mAb.

To confirm that C5 activation is required for the induction of thrombosis by aPL, mice treated with IgG-APS were injected with anti-C5 mAb to inhibit C5 cleavage. In control mice, IgG-APS caused an increase in thrombus size (mean ± SD 3,577 ± 1,129 μm2 with IgG-APS versus 712.0 ± 272 μm2 with IgG-NHS; P = 0.001) (Figure 1), whereas in mice treated with anti-C5 mAb and IgG-APS, the thrombus size was significantly smaller (838 ± 222 μm2; P = 0.005) when compared with mice treated with control IgG and IgG-APS (Figure 1). The mouse IgG used as the control for the anti-C5 mAb did not affect thrombus size in animals treated with IgG-APS or IgG-NHS. The aCL titer in IgG-APS–treated mice was 152.2 ± 14.8 GPL units and was not different from the aCL titer in mice treated with IgG-APS and anti-C5 mAb (135.3 ± 20.3 GPL units), thus excluding the possibility that the protective effects of anti-C5 mAb were due to a diminution in aCL titers because of interferences between IgG-APS and anti-C5 mAb.

Figure 1.

Effects of anti-C5 monoclonal antibody (mAb) on thrombosis induced by IgG from patients with antiphospholipid syndrome (IgG-APS). CD1 mice (5 per group) were injected with 1 mg of anti-C5 mAb or 1 mg of murine IgG control. After 30 minutes, the mice were injected with IgG-APS or with IgG from a non-autoimmune healthy individual (IgG-NHS) twice (at time 0 and 48 hours later). The size of induced thrombi (in μm2) was measured as described in Materials and Methods. Bars show the mean and SEM. ∗ = P = 0.001, IgG-APS control Ig versus IgG-NHS control Ig; ∗∗ = P = 0.005, IgG-APS anti-C5 mAb versus IgG-APS control Ig.


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.