Complement and thrombosis in HIT

Duke University, Durham NC

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Abstract

ABSTRACT Heparin-induced thrombocytopenia (HIT) is a thrombotic disorder caused by ultra-large immune complexes (ULICs) composed of IgG antibodies (Abs) to platelet factor 4 (PF4) and heparin (H). Even with prompt recognition, discontinuation of heparin, and institution of alternative anticoagulants, thrombotic risk remains high and persists for weeks. Studies from our last funding cycle suggest that therapy targeting complement may improve outcomes. We showed that HIT ULICs potently activate the classical pathway of complement, elicit complement-dependent binding of ICs to neutrophils (PMNs) and monocytes, and promote Fcγ receptor (FcγR)- dependent PMN degranulation and monocyte tissue factor (TF) expression. Importantly, we saw striking differences in complement-dependent responses to HIT ULICs formed in the presence and absence of heparin. With heparin, HIT Abs bind to circulating PF4/H complexes forming soluble ULICs that bind to PMNs/monocytes in a complement-dependent manner to promote leukocyte-driven cytokine release and monocyte TF expression. Without heparin, HIT Abs bind to cell-surface GAGs on endothelial cells (ECs) and platelets, rather than white cells, forming cell-bound ICs that drive complement-mediated platelet adhesion and EC procoagulant activity. Based on these observations, we will test the hypothesis that complement responses to HIT ULICs are impacted by heparin itself and involve distinct cellular targets, activation pathways and effector mechanisms though the following specific aims: 1) Cell injury by soluble HIT ULICs requires complement and Fcγ receptors. We show the importance of the proximal complement pathway (C1/C3) for binding of soluble HIT ULICs to PMNs and monocytes via complement receptors (CRs). We hypothesize that binding of soluble HIT ULICs to CR1/CR3 initiates cell signaling and cytoskeletal reorganization, FcγR clustering, membrane scramblase and monocyte TF expression. 2) Cell-bound HIT ICs promote complement-dependent platelet adhesion and direct endothelial injury. We show terminal complement pathway mediated platelet adhesion to injured EC and upregulation of EC procoagulant activity by cell-bound ICs. We hypothesize that cell-bound HIT Abs initiate sublytic C5aR-mediated membrane injury leading to further upregulation of C5aRs, release of high molecular weight von Willebrand Factor multimers, and C5aR-dependent TF expression. 3) Complement activation as a biomarker for pathogenic HIT antibodies. Our studies show a strong correlation of complement activation with platelet-activating effects of HIT ULICs. We will test the hypothesis that complement activation identifies a subset of pathogenic HIT Abs by characterizing the biologic properties of HIT IgG Abs that do/do not activate complement and form soluble and cell-bound ULICs, develop complement activation as a biomarker assay, and examine complement’s role in the therapeutic efficacy of intravenous immunoglobulin. Together, these proposed studies of complement in HIT will advance our understanding of its underlying pathology, should improve diagnostic testing and position complement inhibitors as adjunctive therapy for this prothrombotic disorder.