Physiologic and Pharmacologic Regulation of Factor IXa

因子 IXa 的生理和药理学调节

• 批准号: 6906901
• 负责人: JOHN Patrick SHEEHAN
• 金额: $ 31.95万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6906901
• 关键词: binding sites; coagulation factor IX; coagulation factor VIII; combinatorial chemistry; drug design /synthesis /production; drug discovery /isolation; enzyme activity; enzyme complex; fluorescent dye /probe; gene mutation; hemophilia B; heparin; high throughput technology; ionophores; laboratory mouse; low density lipoprotein receptor; mucopolysaccharides; protein binding; protein purification; recombinant proteins; small molecule; surface plasmon resonance; thrombin; thromboembolism

DESCRIPTION (provided by applicant): The intrinsic tenase complex (ITC) is rate-limiting for thrombin generation, and animal models suggest that therapeutic targeting of this complex reduces bleeding risk. Our central hypothesis is that the heparin-binding exosite of factor IXa is critical to regulation of intrinsic tenase activity, thrombin generation, and response to injury. This hypothesis is based on our demonstration that heparin inhibits the ITC by disrupting the interaction of factor VIlla with this exosite. The rationale for these studies is that compounds utilizing this mechanism will represent effective antithrombotic agents with reduced bleeding risk. The specific aims of this proposal are to: 1) determine the extent to which the factor VIlla and heparin-binding sites overlap on factor IXa, 2) demonstrate the role of the heparin binding exosite in the ex vivo and in vivo regulation of factor IXa activity, and 3) identify novel compounds that specifically target this exosite. Recombinant factor IX mutants will be expressed, purified, and characterized with respect to factor IXa-heparin affinity by surface plasmon resonance and factor IXa-factor VIlla affinity by functional binding assays. The mutant proteins will be critical reagents to analyze the contribution of this exosite to ex vivo coagulant activity, half-life, and thrombin generation in the absence and presence of heparins. Likewise, the effect of these mutations on plasma clearance, cellular degradation by the LDL receptor-related protein (LRP), response to venous endothelial injury, and efficacy of heparins will be examined in the hemophilia B mouse. Finally, glycosaminoglycans and small molecule chemical libraries will undergo high throughput screening for compounds that disrupt the factor IXa-heparin interaction, and these compounds will be characterized with regard to ITC inhibition. These studies will provide a detailed molecular understanding of how heparin disrupts critical protease interactions with the factor VIlla A2 domain, demonstrate the critical role of the factor IXa heparin-binding exosite for thrombin generation, in vivo clearance of factor IX(a) and response to injury, and the therapeutic efficacy of heparins. Finally, these studies will provide proof of principle for this exosite as a critical therapeutic target, and identify lead compounds for a novel class of antithrombotics.

描述（由申请人提供）：内源性tenase复合物（ITC）是凝血酶生成的限速因子，动物模型表明该复合物的治疗靶向可降低出血风险。我们的中心假设是，IXa因子的肝素结合外位点对调节内源性tenase活性、凝血酶生成和对损伤的反应至关重要。这一假设是基于我们的证明，肝素抑制ITC通过破坏因子VIIa与这个外部位点的相互作用。这些研究的基本原理是，利用这种机制的化合物将代表具有降低出血风险的有效抗血栓形成剂。该提议的具体目的是：1）确定因子VIIa和肝素结合位点在因子IXa上重叠的程度，2）证明肝素结合外位点在因子IXa活性的离体和体内调节中的作用，和3）鉴定特异性靶向该外位点的新化合物。将表达、纯化重组因子IX突变体，并通过表面等离子体共振表征因子IXa-肝素亲和力，通过功能结合测定表征因子IXa-因子VIIa亲和力。突变蛋白将是关键的试剂来分析这种外位点的贡献离体凝血活性，半衰期，和凝血酶的产生在肝素的存在和不存在。同样，将在血友病B小鼠中检查这些突变对血浆清除率、LDL受体相关蛋白（LRP）的细胞降解、对静脉内皮损伤的反应和肝素疗效的影响。最后，将对糖胺聚糖和小分子化学文库进行高通量筛选，以寻找破坏因子IXa-肝素相互作用的化合物，并将对这些化合物的ITC抑制进行表征。这些研究将提供肝素如何破坏与因子VIIa A2结构域的关键蛋白酶相互作用的详细分子理解，证明因子IXa肝素结合外位点对于凝血酶生成、因子IX（a）的体内清除和对损伤的反应以及肝素的治疗功效的关键作用。最后，这些研究将为这种外部位作为关键治疗靶点提供原理证明，并确定一类新型抗血栓药物的先导化合物。