Physiologic and Pharmacologic Regulation of Factor IXa

因子 IXa 的生理和药理学调节

• 批准号: 7179277
• 负责人: JOHN Patrick SHEEHAN
• 金额: $ 28.36万
• 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 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7179277
• 关键词: Affect; Affinity; Animal Model; Anticoagulants; Antithrombins; Binding; Binding Sites; Biological Assay; Blood Vessels; Blood coagulation; Cell Line; Chronic; Class; Coagulants; Coagulation Process; Complex; Development; Disease; Elderly; Electrostatics; Endopeptidases; Exhibits; Factor IX; Factor IXa; Factor X; Fibrinolytic Agents; Generations; Glycosaminoglycans; Half-Life; Helix (Snails); Hemophilia B; Hemorrhage; Heparin; Heparin Binding; In Vitro; Incidence; Injury; LDL-Receptor Related Proteins; Label; Lead; Modeling; Molecular; Molecular Target; Mus; Mutation; Nature; Oligosaccharides; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Physiological; Plasma; Preparation; Protease Domain; Proteins; Rate; Reagent; Recombinants; Recurrence; Regulation; Research Personnel; Risk; Risk Factors; Role; Site; Small Molecule Chemical Library; Surface Plasmon Resonance; Therapeutic; Thrombin; Thrombosis; Treatment Efficacy; United States; Venous; Vitamin K; Work; base; ear helix; factor IXa-factor VIIIa; fluorophore; high throughput screening; in vivo; mutant; novel; response; response to injury; therapeutic target; uptake

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）：固有的心脏复合物（ITC）是凝血酶产生的速率限制，动物模型表明该复合物的治疗靶向降低了出血风险。我们的核心假设是，IXA因子的肝素结合外源对于调节内在的十酶活性，凝血酶产生和对损伤的反应至关重要。该假设基于我们的证明，即肝素通过破坏因子别墅与该外部的相互作用来抑制ITC。这些研究的理由是，利用这种机制的化合物将代表有效的抗血栓形成剂，其出血风险降低。该提案的具体目的是：1）确定因子和肝素结合位点在因子IXA上重叠的程度，2）证明肝素结合外源在离体和体内调节因子IXA活性中的作用，以及3）确定特定靶向该exosite的新颖化合物。重组因子IX突变体将通过表面等离子体的共振和IXA-FACTOR因子与IXA-FACTOR VICATOR VILLA亲和通过功能结合测定法表达，纯化和表征有关IXA-Heparin亲和力的特征。突变蛋白将是关键试剂，以分析该外部对在不存在和存在肝素的情况下对离体凝结活性，半衰期和凝血酶产生的贡献。同样，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以及肝素的疗效将在血友病B小鼠中检查。最后，糖胺聚糖和小分子化学文库将对破坏IXA-肝素相互作用的化合物进行高吞吐量筛选，并且这些化合物将在ITC抑制方面表征。这些研究将对肝素如何破坏与因子A2域的关键蛋白酶相互作用提供详细的分子理解，并证明了IXA IXA肝素结合因子对凝血酶产生的关键作用，体内IX（A）的体内清除以及对损伤的反应以及肝素的治疗效率。最后，这些研究将为该外部作为关键治疗靶点提供原理证明，并确定新型抗血栓形成类的铅化合物。