Coagulation Factor XII Recruitment and Activation During Thrombus Formation

项目摘要 一种与出血增加无关的抗血栓药物将是一种变革性的进步， 凝血障碍的治疗。抑制凝血因子XII（FXII）已成为治疗 可以将止血与血栓形成“脱钩”以实现抗血栓形成疗效而不出血的策略 并发症对FXII作为治疗靶点的热情源于观察到严重的先天性 FXII缺乏与出血素质无关，而在临床前模型中阻断或缺失FXII 始终如一地防止血栓形成。然而，尽管其潜在的临床重要性， 体内潜在的血小板依赖性FXII活化仍不清楚。使用基于质谱的 通过蛋白质组学筛选，我们鉴定了整合素αIIbβ3为FXII的假定血小板受体。这些结果 随后进行了一些额外的研究，可重复地证明了FXII-αIIbβ3相互作用， 将整联蛋白结合区定位于FXII重链的kringle结构域（KD）。我们的核心假设 FXII酶原与血小板整合素αIIbβ3的结合增强了FXII的活化， 用于FXII介导的凝血。在本提案的目标1中，我们将绘制负责FXII KD的区域 使用重组产生的KD片段，通过竞争性抑制试验与αIIbβ3结合。我们 还将评估FXIIKD在小鼠血栓形成模型中的作用，以确定KD的丧失是否可以 防止体内血栓形成。在目标2中，我们将与合作者密切合作， FXII-αIIbβ3复合物的结构研究。在目标3中，我们将重点关注FXII结合的机制。 转化为αIIbβ3导致FXII活化和下游凝血。这项工作将提供重要的新 深入了解FXII在血小板表面的募集、活化和增殖的分子机制 并为开发基于FXII-αIIbβ3复合物抑制的新疗法提供信息。

PROJECT SUMMARY An antithrombotic medication not associated with increased hemorrhage would be a transformative advance in the treatment of coagulation disorders. Inhibiting coagulation factor XII (FXII) has emerged as a therapeutic strategy that could “decouple” hemostasis from thrombosis to achieve antithrombotic efficacy without bleeding complications. Enthusiasm for FXII as a therapeutic target stems from the observation that severe congenital FXII deficiency is not associated with a bleeding diathesis while blockade or deletion of FXII in preclinical models consistently protects against thrombosis. However, despite its potential clinical importance, the mechanisms underlying platelet-dependent FXII activation in vivo remain unclear. Using a mass spectrometry-based proteomic screen, we have identified integrin αIIbβ3 as the putative platelet receptor for FXII. These results have been followed up with a number of additional studies reproducibly demonstrating the FXII-αIIbβ3 interaction and localizing the integrin binding region to the kringle domain (KD) of the FXII heavy chain. Our central hypothesis is that binding of FXII zymogen to platelet integrin αIIbβ3 potentiates FXII activation and is necessary and sufficient for FXII-mediated coagulation. In Aim 1 of this proposal, we will map the region(s) of the FXII KD responsible for binding to αIIbβ3 via competitive inhibition assays using recombinantly-generated fragments of the KD. We will also evaluate the role of the FXII KD in a mouse model of thrombosis to determine if loss of the KD can prevent thrombus formation in vivo. In Aim 2, we will work closely with our collaborators to conduct precision structural studies of the FXII-αIIbβ3 complex. In Aim 3, we will focus on the mechanisms by which FXII binding to αIIbβ3 leads to activation of FXII and downstream coagulation. The proposed work will provide important new insights into the molecular mechanism of FXII recruitment, activation, and propagation at the platelet surface and inform efforts to develop novel therapeutics based on inhibition of the FXII-αIIbβ3 complex.