Mechanisms of Coagulation Factor XII Recruitment and Activation at the Platelet Surface

项目总结 目前所有可用的抗血栓药物都有出血的风险，通常 许多患者出现毁灭性的并发症，可迅速逆转治疗的益处。 鉴于这一现实，凝血因子XII(FXII)已成为一种有希望的新药靶点 可能会改变抗血栓疗法。在临床前阶段阻断或删除FXII 动物模型一直被证明对血栓形成有保护作用，但严重 先天性FXII缺乏与出血素质无关。因此，抑制FXII 可以代表人们长期寻求的将止血与血栓形成“脱钩”的方法，并实现 抗血栓疗效好，无出血并发症。然而，尽管它具有潜在的临床应用价值 重要的是，对血小板依赖的FXII激活的机制知之甚少 在活体内。通过蛋白质组学筛选，我们鉴定整合素αIIbβ3为 推测为FXII的血小板受体。这些结果已经得到了一些后续行动 其他研究重复性地证明了FxII-整合素αIIbβ3的相互作用。我们的中央 假设FxII酶原与血小板表面的整合素αIIbβ3具有特异性结合， 这增强了它的蛋白分解活性，并且对FXII依赖是必要和充分的 凝结。在本提案的目标1中，我们将绘制FXII负责结合的区域(S) 使用重组产生的片段通过竞争抑制实验整合αIIbβ3 FXII.我们还将利用表面等离子共振来评估FXII- 整合素αIIbβ3相互作用并获得结合动力学。在目标2中，我们将探索整合素的作用 αIIbβ3结合使用整合素αIIbβ3包被的珠粒和来自野生动物的血小板激活FxII 类型和整合素β3(ITGB3)缺失的小鼠。拟议的工作将提供重要的新见解 FXII在血小板上募集、激活和增殖的分子机制 表面和信息努力开发基于FXII抑制的新疗法。

PROJECT SUMMARY All currently available antithrombotic medications carry the risk of hemorrhage, an often devastating complication in many patients that can rapidly reverse the benefits of therapy. Given this reality, coagulation factor XII (FXII) has emerged as a promising new drug target that could potentially transform antithrombotic therapy. Blockade or deletion of FXII in preclinical animal models has consistently been shown to be protective against thrombosis, yet severe congenital FXII deficiency is not associated with a bleeding diathesis. Therefore, inhibiting FXII could represent the long-sought means to “decouple” hemostasis from thrombosis and achieve antithrombotic efficacy without bleeding complications. However, despite its potential clinical importance, little is known about the mechanisms underlying platelet-dependent FXII activation in vivo. Using a mass spectrometry 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-integrin αIIbβ3 interaction. Our central hypothesis is that FXII zymogen exhibits specific binding to integrin αIIbβ3 on the platelet surface, which enhances its proteolytic activity and is necessary and sufficient for FXII-dependent coagulation. In Aim 1 of this proposal, we will map the region(s) of FXII responsible for binding to integrin αIIbβ3 via competitive inhibition assays using recombinantly-generated fragments of FXII. We will also utilize surface plasmon resonance to evaluate the specificity of the FXII- integrin αIIbβ3 interaction and obtain binding kinetics. In Aim 2, we will explore the role of integrin αIIbβ3 binding in the activation of FXII using integrin αIIbβ3-coated beads and platelets from wild- type and integrin β3 (ITGB3)-null mice. 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 FXII inhibition.