Complement and Thrombosis in HIT

摘要 肝素诱导的血小板减少症(HIT)是由超长免疫引起的一种严重的血栓性疾病 含有抗血小板第4因子(PF4)和多价抗原的抗体的复合体(ULIC) 肝素(H)。HIT患者有死亡、截肢、复发血栓栓塞症和出血的风险 接受最大耐受量的凝血因子Xa或直接凝血酶抑制剂(DTI)。因此，有一个未得到满足的 需要更深入地了解HIT中血栓形成的病理生物学，这将导致有针对性的新的非 抗凝干预，以补充现代治疗。我们公布的和试点的数据表明 补体途径的激活填补了这一缺口。具体地说，我们展示了HIT ULIC：1)交互和绑定 补体成分和von Willebrand因子(VWF)多聚体，2)产生可溶性补体 通过经典途径的成分，3)在中性粒细胞、单核细胞和内皮细胞(ECs)上沉积C3，4) 触发补体依赖型中性粒细胞脱颗粒、单核细胞组织因子和促凝血活性 C5的上游，5)即使在DTI存在的情况下也激活补体，以及6)促进补体沉积 在HIT的小鼠血栓模型中。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内皮损伤参与HIT的血栓前状态 补体受体(CRs)和通过促进与FcRIIA在中性粒细胞上的协同作用来放大信号 和单核细胞。我们将解决以下具体目标：1)研究HIT-ULIC-补语相互作用和 补体激活对内皮细胞的影响我们将检验补体掺入扩大的假说 稳定组装的ULICs，损害补体调节功能，促进EC损伤，导致 释放vWF多聚体，进一步放大ULIC的形成和补体激活。2)审查 HIT ULICs、补体和单核/中性粒细胞FcR的协同作用我们将检验这一假说 含有补体的ULICS通过促进与细胞内CRs的协同作用而放大FcRIIA信号 中性粒细胞和单核细胞。我们将研究ULIC组成对细胞激活的影响，确定CRS 参与结合HIT ULICs，研究可溶性和细胞补体调节机制，并表征 血清阳性患者有无HIT时补体激活。3)将补体抑制作为 HIT血栓形成的治疗策略。我们将使用微流控分析和小鼠血栓形成模型 验证HIT ULICs激活经典补体途径促进大血管生成的假说 通过从激活的内皮细胞释放vWF和放大细胞促凝血活性而形成血栓。我们 将研究近端和末端补体途径抑制的有效性，作为降低 治疗HIT需要抗血栓治疗的强度。总之，这些研究将为IC提供新的见解- 广泛地介导血栓形成，并为补体抑制提供了一条详细的机制途径 治疗HIT的合理、有效和非抗凝剂依赖的策略。

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.