Complement and Thrombosis in HIT

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ABSTRACT Heparin induced thrombocytopenia (HIT) is a severe thrombotic disorder initiated by ultralarge immune complexes (ULICs) containing IgG antibodies to a multivalent antigen composed of platelet factor 4 (PF4) and heparin (H). Patients with HIT are at risk for death, amputation, recurrent thromboembolism and bleeding while receiving maximally tolerated doses of factor Xa or direct thrombin inhibitors (DTIs). Thus, there is an unmet need for deeper insight into the pathobiology of thrombosis in HIT that will lead to targeted novel non- anticoagulant interventions to supplement contemporary therapy. Our published and pilot data demonstrate that activation of the complement pathway fulfills this gap. Specifically, we show that HIT ULICs: 1) interact and bind complement components and von Willebrand factor (vWF) multimers, 2) generate soluble complement components via the classical pathway, 3) deposit C3 on neutrophils, monocytes and endothelial cells (ECs), 4) trigger complement-dependent neutrophil degranulation, monocyte tissue factor (TF) and procoagulant activity upstream of C5, 5) activate complement even in the presence of DTIs, and 6) promote complement deposition in a murine thrombosis model of HIT. Based on these findings, we hypothesize that complement activation by HIT ULICs contributes to the prothrombotic state in HIT through direct EC injury mediated by surface expressed complement receptors (CRs) and by amplifying signaling by promoting cooperativity with FcRIIA on neutrophils and monocytes. We will address the following specific aims: 1) Examine HIT ULIC-complement interactions and effects of complement activation on ECs We will test the hypothesis that incorporation of complement enlarges and stabilizes assembled ULICs, impairs complement regulatory function, and promotes EC injury leading to release of vWF multimers that further amplify ULIC formation and complement activation. 2) Examine cooperative interactions of HIT ULICs, complement and monocyte/neutrophil FcR. We will test the hypothesis that complement-containing ULICS amplify FcRIIA signaling by promoting cooperativity with cellular CRs on neutrophils and monocytes. We will examine the effects of ULIC composition on cell activation, identify CRs involved in binding HIT ULICs, study soluble and cellular complement regulatory mechanisms, and characterize complement activation in seropositive patients with and without HIT. 3) Examine complement inhibition as a therapeutic strategy for thrombosis in HIT. We will use microfluidic assays and murine thrombosis models to test the hypothesis that activation of the classical complement pathway by HIT ULICs promotes macrovascular thrombosis through release of vWF from activated ECs and amplification of cellular procoagulant activity. We will examine the efficacy of proximal and terminal complement pathway inhibition as a strategy to lower the intensity of antithrombotic therapy needed to treat HIT. Together, these studies will provide new insights into IC- mediated thrombosis broadly and provide a detailed mechanistic pathway for complement inhibition as a rationale, potent and non anticoagulant-dependent strategy for the treatment of HIT.