Platelets are required for enhanced activation of the endothelium and fibrinogen in a mouse thrombosis model of APS

Abstract

• The anti-b2GP1 autoantibody/ b2GP1 complex binds to the platelet thrombus, amplifying platelet activation.• Platelets are required for enhanced activation of the endothelium and fibrin generation by the anti-b2GP1 autoantibody/b2GP1 complex.Antiphospholipid syndrome (APS) is defined by thrombosis, fetal loss, and the presence of antiphospholipid antibodies, including anti-b2-glycoprotein-1 autoantibodies (antib2GP1) that have a direct role in the pathogenesis of thrombosis in vivo. The cellular targets of the anti-b2GP1 autoantibody/b2GP1 complex in vivo were studied using a laserinduced thrombosis model of APS in a live mouse and human anti-b2GP1 autoantibodies affinity-purified from APS patients. Cell binding of fluorescently labeled b2GP1 and antib2GP1 autoantibodies revealed their colocalization on the platelet thrombus but not the endothelium. Anti-b2GP1 autoantibodies enhanced platelet activation, monitored by calcium mobilization, and endothelial activation, monitored by intercellular adhesion molecule-1 expression. When eptifibatide was infused to block platelet thrombus formation, enhanced fibrin generation and endothelial cell activation were eliminated. Thus, the anti-b2GP1 autoantibody/b2GP1 complex binds to the thrombus, enhancing platelet activation, and platelet secretion leads to enhanced endothelium activation and fibrin generation. These results lead to a paradigm shift away from the concept that binding of the anti-b2GP1 autoantibody/b2GP1 complex activates both endothelial cells and platelets toward one in which activation of platelets in response to anti-b2GP1 autoantibody/b2GP1 complex binding leads to subsequent enhanced endothelium activation and fibrin generation. (Blood. 2014;124(4):611-622) IntroductionAntiphospholipid syndrome (APS) is characterized by venous or arterial thrombosis and/or pregnancy morbidity and is associated with circulating antiphospholipid (aPL) autoantibodies.1-3 These antibodies, including anti-b2-glycoprotein-1 (anti-b2GP1) autoantibodies, recognize plasma proteins that bind to anionic phospholipids, among which b2GP1 is the major target. 4 Antibodies directed against b2GPI 5,6 are associated with thrombotic events in APS. Antib2GP1 autoantibodies from patients with APS and thrombosis enhance arterial thrombus formation after injury in a mouse model of APS, 7 with dramatic increases in platelet thrombus size and fibrin generation.The mechanisms leading to thrombosis in APS are unresolved. In vitro and in vivo studies using animal models demonstrated that aPL antibodies interact with endothelial cells and monocytes to increase tissue factor expression and complement activation and proinflammatory cytokines. 8,9 In vitro, platelet activation occurs after the binding of complexes of anti-b2GP1 antibodies and dimerized b2GP1 to GPIba and ApoER2. [10][11][12] Furthermore, APS patients exhibit markers of platelet activation. 13 The conventional understanding is that the antib2GP1/b2GP1 complex binds to receptors on both the endothelial cell and the platelet, lea...

Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome

“…Quantitative results are presented for the median fluorescence associated with platelets and fibrin from multiple thrombi in each group ( Figure 1B-C Although the defect in platelet accumulation was expected on the basis of the absence of platelet dense granules, thus confirming previous studies demonstrating diminished platelet thrombi in ruby-eye mice in response to vessel wall injury, 22 the defect in fibrin generation was unanticipated. The absence of fibrin generation cannot be explained by the absence of platelet accumulation because we have previously shown normal fibrin formation in Par4 2/2 mice in whom platelets cannot be thrombin activated 23 ; in WT mice treated with eptifibatide, an inhibitor that blocks platelet accumulation 15,24 ; and in chimeric mice lacking platelet b3 integrins. 17 In contrast, there is no fibrin generation in b3 2/2 mice lacking both platelet aIIbb3 and endothelial aVb3.…”

Activated signature of antiphospholipid syndrome neutrophils reveals potential therapeutic target

“…Finally, we felt that our recently described animal model was particularly amenable to assessing the role of neutrophil adhesion in antiphospholipid antibody-mediated thrombosis (21). Notably, the majority of models used to study APS in mice have relied on explicit vessel wall damage such as femoral vein pinch injury (13,54), laser injury to the cremaster microcirculation (55,56), and ferric chloride application (57). The flow restriction model used by our While the PSGL-1 -/-mice used here are global KOs, we added specificity to the model with adoptive transfer experiments.…”

β2-Glycoprotein I/HLA class II complexes are novel autoantigens in antiphospholipid syndrome

“…55,56 Conversely, a recent study suggested that activation of platelets by aPL antibodies is associated with thrombosis formation. 57 In these ways, aPL antibodies seem to be a major contributor to the pathogenesis of APS. [6][7][8][9] HLA class II expression is induced on endothelial cells after exposure to cytokines such as IFN-g and TNF-a.…”