Complement and Thrombosis in HIT

摘要 肝素诱导的血小板减少症（HIT）是一种由超大免疫引起的严重血栓性疾病 复合物 (ULIC) 含有针对多价抗原的 IgG 抗体，该多价抗原由血小板因子 4 (PF4) 和 肝素 (H)。 HIT 患者面临死亡、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 中血栓形成的病理学，这将导致有针对性的新型非 抗凝干预作为当代治疗的补充。我们发布的试点数据表明 补体途径的激活填补了这一空白。具体来说，我们展示了 HIT ULIC：1）交互和绑定 补体成分和冯维勒布兰德因子 (vWF) 多聚体，2) 生成可溶性补体 通过经典途径的成分，3) 将 C3 沉积在中性粒细胞、单核细胞和内皮细胞 (EC) 上，4) 触发补体依赖性中性粒细胞脱颗粒、单核细胞组织因子 (TF) 和促凝血活性 C5 上游，5) 即使在 DTI 存在的情况下也能激活补体，6) 促进补体沉积 在 HIT 小鼠血栓形成模型中。基于这些发现，我们假设补体激活是通过 HIT ULICs 通过表面表达介导的直接 EC 损伤促进 HIT 中的血栓前状态 补体受体 (CR) 并通过促进与中性粒细胞上的 Fc-RIIA 的协同作用来放大信号传导 和单核细胞。我们将实现以下具体目标：1）检查 HIT ULIC-补体相互作用并 补体激活对 EC 的影响 我们将检验补体掺入扩大的假设 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）检查 HIT ULIC、补体和单核细胞/中性粒细胞 FcR 的协同相互作用。我们将检验假设 含有补体的 ULICS 通过促进与细胞 CR 的协同作用来放大 Fc-RIIA 信号传导 中性粒细胞和单核细胞。我们将检查 ULIC 成分对细胞活化的影响，识别 CR 参与结合 HIT ULIC，研究可溶性和细胞补体调节机制，并表征 有或没有 HIT 的血清阳性患者的补体激活。 3) 检查补体抑制作为 HIT 中血栓形成的治疗策略。我们将使用微流体测定和小鼠血栓形成模型来 检验 HIT ULIC 激活经典补体途径促进大血管的假设 激活的 EC 释放 vWF 并增强细胞促凝血活性，从而形成血栓。我们 将检查近端和末端补体途径抑制作为降低 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 广泛介导血栓形成，并为补体抑制提供详细的机制途径 HIT 治疗的基本原理、有效且非抗凝依赖性策略。

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.