Coagulation Factor XII Recruitment and Activation During Thrombus Formation

项目总结 与出血增加无关的抗血栓药物将是一个变革性的进步 凝血障碍的治疗。抑制凝血因子XII(FXII)已成为一种治疗方法 可将止血与血栓形成“脱钩”以在不出血的情况下实现抗血栓效果的策略 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 在临床前模型中，FXII缺乏与出血素质无关，而FXII被阻断或缺失 始终如一地预防血栓形成。然而，尽管它具有潜在的临床重要性，但其机制 体内潜在的血小板依赖的FXII激活尚不清楚。使用基于质谱学的 蛋白质组学筛选，我们已经确定整合素αIIbβ3可能是FXII的血小板受体。这些结果产生了 随后进行了一些额外的研究，重复地证明了FXII-αIIbβ3的相互作用和 将整合素结合区定位于FXII重链的Kd区。我们的中心假设 FxII酶原与血小板整合素αIIbβ3结合增强FxII活性是必要和充分的吗 用于FXII介导的凝血。在本提案的目标1中，我们将绘制FXII KD负责的区域(S) 用于使用重组产生的KD片段通过竞争抑制试验与αIIbβ3结合。我们 还将评估FXII KD在小鼠血栓形成模型中的作用，以确定KD的丢失是否可以 防止体内血栓形成。在Aim 2中，我们将与我们的合作者密切合作，进行精确度 FxII-αIIbβ3复合体的结构研究在目标3中，我们将重点介绍FXII结合的机制 到αIIb，β3导致FxII的激活和下游凝血。拟议的工作将提供重要的新的 对FXII在血小板表面募集、激活和增殖的分子机制的认识 并为开发基于抑制FxII-αIIbβ3复合体的新疗法的努力提供信息。

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.