Interactions of human monoclonal and polyclonal antiphospholipid antibodies with serine proteases involved in hemostasis

Antiphospholipid antibodies (aPL) cause vascular thrombosis and/or pregnancy morbidity in the antiphospholipid syndrome (APS) (1). These clinical manifestations are triggered by the interaction of pathogenic aPL with various target cells, including monocytes, endothelial cells, and trophoblast cells, leading to the recruitment of cell surface receptors and subsequent perturbation of intracellular signaling pathways (2). These pathogenic aPL are generally IgG type (3, 4) and target a variety of antigens, including negative phospholipid, phospholipid binding proteins (particularly β2-glycoprotein I [β2GPI] and prothrombin), as well as other factors related to hemostasis, such as thrombin, protein C, activated protein C, protein S, plasmin, plasminogen, and tissue-type plasminogen activator (tPA) (5–13). In contrast, nonpathogenic aPL (found in 2–5% of healthy adults who lack features of the APS [14]) mostly bind directly to phospholipid (15). Thrombin, activated protein C, plasmin, and tPA, as well as activated factor VIIa (FVIIa), FIXa, FXa, and FXIIa, belong to the trypsin-like serine protease family of enzymes and are involved in the tight regulation of hemostasis (16). In previous studies, sera from between 13% and 54% of patients with the APS have been found to bind various different serine proteases (5, 8, 13). Furthermore, a panel of human monoclonal aPL produced from hybridomas displayed cross-reactivity with serine protease, binding to thrombin, activated protein C, plasmin, tPA, FIXa, and FXa (6–8, 17, 18). Overall, these serine proteases share ∼50% amino acid sequence similarity in their enzymatic domains but have greater homology at their catalytic sites. Given that several human monoclonal aPL have been found to inhibit the inactivation of procoagulant serine proteases and functional activities of anticoagulant/fibrinolytic serine proteases (7, 8, 13, 19), it has been suggested that some aPL may recognize the catalytic domain of serine proteases, leading to dysregulation of hemostasis and vascular thrombosis in the APS. To explore the interaction of aPL with target antigens in promoting thrombus formation, we have been studying a panel of recombinant human monoclonal IgG aPL, which differ from one another at points in their sequence precisely engineered by us. Studying this panel of IgG molecules has allowed us to investigate correlations between their sequences, binding, and biologic properties (20–23). These human monoclonal IgG aPL were all based on the human monoclonal IgG aPL IS4 (derived from a patient with APS), which binds β2GPI (24) and thrombin (8) and is thrombogenic in mice (25). Previously, we found that alterations in the pattern of somatic mutations in both the VH and VL regions of IS4 determined its ability to bind antigens relevant in the pathogenesis of the APS and to promote murine thrombogenesis (20–23). Interestingly, the in vivo thrombogenic effects of these monoclonal antibodies (mAb) were most closely predicted by their ability to bind thrombin, rather than phospholipid or β2GPI. Furthermore, mAb binding to thrombin followed a different pattern compared to the pattern observed with mAb binding to its zymogen prothrombin (21). Therefore, in the current study we used the same panel of mAb to examine whether binding to other serine proteases also parallels thrombogenicity in the mouse model, and whether the difference between binding to prothrombin and binding to thrombin is also seen with other zymogen/enzyme pairs, i.e., FIX and FIXa, or protein C and activated protein C. To assess the relevance of our findings obtained using monoclonal IgG aPL to polyclonal aPL found in vivo, we then tested serum samples and purified IgG samples from APS patients, systemic lupus erythematosus (SLE) patients without APS (subclassified according to positivity or negativity for aPL), and healthy controls. We investigated whether samples from those groups differed in the nature and avidity of their binding to serine proteases and ability to alter the functional activity of serine proteases.