Physiologic and Pharmacologic Regulation of Factor IXa

描述（由申请人提供）：内在张力酶复合物（ITC）是凝血酶生成的限速物，动物模型表明该复合物的治疗靶向性可降低出血风险。我们的中心假设是，IXa因子的肝素结合外源对调节内在紧张酶活性、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，肝素通过破坏因子VIlla与外源物的相互作用来抑制ITC。这些研究的基本原理是，利用这一机制的化合物将代表有效的抗血栓药物，降低出血风险。该提案的具体目标是：1)确定因子VIlla和肝素结合位点在因子IXa上重叠的程度，2)证明肝素结合外源位点在因子IXa活性的体外和体内调节中的作用，以及3)鉴定特异性靶向该外源位点的新化合物。重组因子IX突变体将通过表面等离子体共振和功能结合试验表达、纯化和表征因子ixa -肝素亲和力和因子ixa -因子VIlla亲和力。突变蛋白将是分析这种外源对体外凝血活性、半衰期和肝素缺失或存在时凝血酶生成的贡献的关键试剂。同样，这些突变对血浆清除率、低密度脂蛋白受体相关蛋白（LRP）的细胞降解、对静脉内皮损伤的反应以及肝素的功效的影响将在血友病B小鼠中进行研究。最后，糖胺聚糖和小分子化学文库将进行高通量筛选，以寻找破坏ixa -肝素相互作用因子的化合物，并对这些化合物进行ITC抑制。这些研究将为肝素如何破坏关键蛋白酶与VIlla A2结构域的相互作用提供详细的分子理解，证明IXa肝素结合外源性因子在凝血酶生成、IX因子(a)的体内清除和对损伤的反应以及肝素的治疗效果方面的关键作用。最后，这些研究将为这种外来物作为关键治疗靶点提供原理证明，并鉴定出一类新型抗血栓药物的先导化合物。

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.