Complement and Thrombosis in HIT

摘要 肝素诱导的血小板减少症（HIT）是一种由超大型免疫球蛋白引起的严重血栓性疾病， 含有抗多价抗原的IgG抗体的复合物（ULIC），所述多价抗原由血小板因子4（PF 4）和 肝素（H）。HIT患者有死亡、截肢、复发性血栓栓塞和出血的风险， 接受最大耐受剂量的Xa因子或直接凝血酶抑制剂（DTI）。因此，有一个未满足的 需要更深入地了解HIT中血栓形成的病理生物学，这将导致靶向新的非血栓性疾病。 抗凝干预，以补充当代治疗。我们公布的数据和试点数据表明， 补体途径的激活填补了这一空白。具体来说，我们表明，HIT ULIC：1）相互作用和绑定 补体成分和血管性血友病因子（vWF）多聚体，2）产生可溶性补体 3）C3在中性粒细胞、单核细胞和内皮细胞（EC）上存款， 触发补体依赖性中性粒细胞脱粒、单核细胞组织因子（TF）和促凝血活性 C5上游，5）即使在存在DTI的情况下也激活补体，和6）促进补体沉积 在HIT的鼠血栓形成模型中。基于这些发现，我们假设补体激活是由 HIT ULIC通过表面表达的ULIC介导的直接EC损伤促进HIT的血栓前状态。 补体受体（CR），并通过促进与中性粒细胞上的Fc γ RIIA的协同性来放大信号传导 和单核细胞。我们将解决以下具体目标：1）检查HIT ULIC-补体相互作用， 补体激活对内皮细胞的影响我们将检验补体掺入增加内皮细胞的假设。 并稳定组装的ULIC，损害补体调节功能，并促进EC损伤，导致 释放vWF多聚体，进一步放大ULIC形成和补体激活。2)审查 HIT ULIC、补体和单核细胞/中性粒细胞Fc β R的协同相互作用。我们将检验这个假设 含有补体的ULICS通过促进与细胞CRs的协同性来放大Fc γ RIIA信号传导， 中性粒细胞和单核细胞。我们将研究ULIC组合物对细胞活化的影响，鉴定CRs， 参与结合HIT ULIC，研究可溶性和细胞补体调节机制，并表征 在有和没有HIT的血清阳性患者中补体激活。3)检查补体抑制， HIT中血栓形成的治疗策略。我们将使用微流体分析和小鼠血栓形成模型， 检验HIT ULIC激活经典补体途径促进大血管 通过从活化的EC释放vWF和放大细胞促凝血活性来抑制血栓形成。我们 将检查近端和末端补体途径抑制的有效性，作为降低 治疗HIT所需的抗血栓治疗强度。总之，这些研究将为IC提供新的见解- 广泛介导的血栓形成，并提供了补体抑制的详细机制途径， 合理性、有效性和非抗凝剂依赖性治疗HIT的策略。

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.