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: 10424806
• 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/10424806
• 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; Research Personnel; Risk; Role; Signal Transduction; Site; Solid; Specificity; Surface; Surface Plasmon Resonance; System; Testing; Therapeutic; Thrombosis; Treatment Efficacy; Wild Type Mouse; Work; base; 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; 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 所有目前可用的抗血栓药物都有出血的风险， 许多患者出现毁灭性的并发症，可以迅速逆转治疗的益处。 鉴于这一现实，凝血因子XII（FXII）已成为一种有前途的新药靶点， 可能会改变抗血栓治疗。临床前FXII的阻断或缺失 动物模型一直被证明对血栓形成有保护作用，但严重的血栓形成是由动物模型引起的。 先天性FXII缺乏与出血素质无关。因此，抑制FXII 可能代表了长期寻求的将止血与血栓形成“分离”的方法， 抗血栓疗效好，无出血并发症。然而，尽管其潜在的临床 重要的是，对血小板依赖性FXII激活的潜在机制知之甚少 in vivo.使用质谱蛋白质组学筛选，我们已经确定整合素αIIbβ3为 FXII的假定血小板受体。这些结果已被一些后续行动， 其他研究可重现地证明了FXII-整联蛋白αIIbβ3相互作用。我们的中央 假设是FXII酶原表现出与血小板表面上整联蛋白αIIbβ3的特异性结合， 这增强了其蛋白水解活性，并且对于依赖于FXII的 凝血在本提案的目标1中，我们将绘制负责结合的FXII区域， 整合素αIIbβ3通过竞争性抑制试验，使用重组产生的 FXII.我们还将利用表面等离子体共振来评估FXII的特异性， 整合素αIIbβ3相互作用并获得结合动力学。在目标2中，我们将探讨整合素的作用， 使用整合素αIIbβ 3包被的珠粒和来自野生型血小板的FXII活化中的αIIbβ3结合。 型和整合素β3（ITGB 3）缺失小鼠。拟议的工作将提供重要的新见解 FXII在血小板上募集、活化和增殖的分子机制 表面和通知的努力，开发新的治疗的基础上FXII抑制。