Physiologic and Pharmacologic Regulation of Factor IXa

描述(由申请人提供)：固有的张力酶复合体(ITC)对凝血酶的生成具有限速作用，动物模型表明，该复合体的治疗靶向降低了出血风险。我们的中心假设是，因子IXa的肝素结合外切体对于调节固有的张力酶活性、凝血酶的生成和对损伤的反应至关重要。这一假说是基于我们的论证，即肝素通过破坏因子Villa与这个外壁之间的相互作用来抑制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，并降低出血风险。这项建议的具体目的是：1)确定因子Villa和肝素结合位点在因子IXa上重叠的程度，2)展示肝素结合外切酶在体外和体内调节因子IXa活性的作用，以及3)识别专门针对该外切酶的新化合物。重组人凝血因子IX突变体将通过表面等离子共振法和功能结合实验分别表达、纯化和鉴定凝血因子IXa-肝素亲和力和凝血因子IXa-Villa亲和力。突变的蛋白质将是关键的试剂，以分析该外显子对体外凝血活性、半衰期和在没有和存在肝素的情况下凝血酶生成的贡献。同样，将在血友病B小鼠中检测这些突变对血浆清除、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疗效的影响。最后，糖胺聚糖和小分子化学库将对干扰因子IXa-肝素相互作用的化合物进行高通量筛选，并对这些化合物进行ITC抑制特性表征。这些研究将提供有关肝素如何破坏与凝血因子Villa A2结构域的关键蛋白酶相互作用的详细分子理解，展示凝血因子IXa肝素结合外切酶在凝血酶生成、体内清除凝血因子IX(A)和对损伤的反应以及肝素的治疗效果中的关键作用。最后，这些研究将为这种外植体作为关键的治疗靶点提供原理证据，并确定一类新的抗血栓药的先导化合物。

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.