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

血小板表面凝血因子 XII 招募和激活的机制

• 批准号: 10588194
• 负责人: Pavan Bendapudi
• 金额: $ 8.65万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10588194
• 关键词: Animal Model; Binding; Biological Assay; Blood Platelets; Cell Line; Clinical; Co-Immunoprecipitations; Coagulation Process; Competitive Binding; Complication; Development; Disease susceptibility; Drug Targeting; Enzyme Precursors; Event; Exhibits; Factor XII; Factor XII Deficiency; Factor XIIa; Fibrinolytic Agents; Flow Cytometry; Generations; Hemorrhage; Hemostatic function; Human; ITGB3 gene; Integrin Binding; Integrins; Kinetics; Knockout Mice; Length; Maps; Mass Spectrum Analysis; Membrane Proteins; Molecular; Molecular Conformation; Pathway interactions; Patients; Phase; Physiological; Plasma; Play; Pre-Clinical Model; Process; Property; Proteomics; RGD (sequence); Reagent; Recombinants; Reproducibility; Research Personnel; Risk; Role; Signal Transduction; Site; Solid; Specificity; Surface; Surface Plasmon Resonance; System; Testing; Therapeutic; Thrombosis; Treatment Efficacy; Wild Type Mouse; Work; experimental study; factor A; follow-up; in vivo; insight; interest; member; new therapeutic target; novel; novel strategies; novel therapeutics; pre-clinical; reaction rate; receptor; recruit; thrombotic; treatment strategy; vascular injury

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.

项目摘要 目前所有可用的抗血栓药物都有出血风险，通常 许多患者的毁灭性并发症可以迅速扭转治疗的益处。 鉴于这一现实，凝血因子XII（FXII）已成为一个有希望的新药物目标 可能会改变抗血栓疗法。临床前的封锁或删除FXII 动物模型始终被证明受到保护免受血栓形成，但严重 先天性FXII缺乏与出血性透性无关。因此，抑制FXII 可以代表长期以来从血栓形成“解矛”止血并实现的手段 抗血栓性效率而没有出血并发症。但是，它的潜在临床 重要的是，关于血小板依赖性FXII激活的机制知之甚少 体内。使用质谱法蛋白质组学筛选，我们已经确定整联蛋白αIIBβ3为 推定的FXII血小板受体。这些结果已得到了许多 可重复证明FXII-IntegrinαIIBβ3相互作用的其他研究。我们的中心 假设是FXII Zymogen在血小板表面上表现出与整联蛋白αIIBβ3的特异性结合， 这增强了其蛋白水解活性，并且足以依赖FXII 凝血。在本提案的目标1中，我们将绘制负责约束的FXII区域 通过使用重组生成的片段，通过竞争性抑制测定法进行整合蛋白αIIBβ3 fxii。我们还将利用表面等离子体共振来评估FXII的特异性 整联蛋白αIIBβ3相互作用并获得结合动力学。在AIM 2中，我们将探索整合素的作用 使用整合素αIIBβ3包涂的珠和血小板从野生 - 类型和整联蛋白β3（ITGB3）-Null小鼠。拟议的工作将提供重要的新见解 进入血小板的FXII募集，激活和传播的分子机制 表面并为基于FXII抑制而开发新疗法的努力。