Molecular Recognition in Factor VIIa Induced Coagulation

因子 VIIa 诱导凝血中的分子识别

• 批准号: 6852031
• 负责人: S Paul Bajaj
• 金额: $ 19.39万
• 依托单位: ORTHOPAEDIC HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6852031
• 关键词: X ray crystallography; activation product; active sites; binding sites; blood coagulation; calcium ion; chemical binding; coagulation factor VII; coagulation factor X; endopeptidases; enzyme activity; enzyme inhibitors; epidermal growth factor; molecular dynamics; molecular shape; mutant; protease inhibitor; protein purification; protein structure; recombinant proteins; sodium ion; thromboplastin; zinc; zymogens

This application is based upon the conviction that activation of factor X by the tissue factor/factor VIIa (TF/VIIa) complex represents a key step in the initiation of coagulation by the extrinsic pathway. Further, factor VIIa has virtually no enzymatic activity prior to its complexation with TF. Tissue factor pathway inhibitor (TFPI) contains two functional Kunitz domains (K1 and K2) and it first inhibits factor Xa via its K2 domain. The resulting Xa TFPI complex then inhibits TF/VIIa via its K1 domain; the light chain of factor Xa also appears to be important for this interaction. Detailed molecular bases for these interactions are only partially understood. In the first two specific aims, we will define the structural basis for the K2 domain to inhibit factor Xa and for the K1 domain to inhibit factor VIIa. By molecular modeling, we have identified the putative residues to be mutated, and the expression systems for factor VII, factor X and TFPI are well established in the applicant's laboratory. Further, we hypothesize that EGF1 domain contained in the light chain of factor Xa plays an important role in binding of Xa TFPI to TF/VIIa. This will be evaluated in this proposal. In Specific Aim 3, we will define the linkage between TF binding, Na+ binding, Ca2+ binding, and Zn2+ binding in the protease domain, as well as occupancy of the S1 site in the TF induced development of catalytic efficiency in factor VIIa. In Specific Aim 4, we will crystallographically determine the structure of zymogen VII complexed with soluble TF (sTF), variously active-site inhibited factor VIIa/sTF (e.g., occupancy of S1 site, S1 and S3/S4 sites, S1, S2 and S3/S4 sites, and isolated K1 domain bound to VIIa), and of Gla domainless Xa-K2 complex. We have crystals of zymogen VII/sTF and several variously active-site inhibited VIIa/sTF complexes (including reversible inhibitors). We have collected x-ray intensity data on several of these crystals and are in the processes of refining some of the structures. Since our crystals are obtained in Ca2+/Mg2+/Zn2+-containing solution, we have located and are refining these sites in factor VIIa as well. The crystallographic studies will complement the biochemical studies proposed in the first three specific aims. Our integrated approach is expected to provide new valuable information regarding the assembly of the extrinsic coagulation complex and its regulation by TFPI. The knowledge gained from such studies is essential for developing new generations of antithrombotics.

该应用是基于以下信念：通过组织因子/因子VIIA（TF/VIIA）复合物激活因子X，这代表了外部途径启动凝血的关键步骤。 此外，VIIA在与TF络合之前几乎没有酶促活性。 组织因子途径抑制剂（TFPI）包含两个功能性kunitz结构域（K1和K2），首先通过其K2结构域抑制因子XA。 然后，由此产生的XA TFPI复合物通过其K1域抑制TF/VIIA。 Xa因子的轻链对于这种相互作用似乎也很重要。 这些相互作用的详细分子碱基仅部分理解。 在前两个特定目标中，我们将定义K2结构域抑制因子XA和K1域抑制VIIA的结构基础。 通过分子建模，我们已经确定了要突变的假定残基，并且在申请人的实验室中，因子VII，因子X和TFPI的表达系统已很好地确定。 此外，我们假设在XA的轻链中包含的EGF1结构域在XA TFPI与TF/VIIA结合中起重要作用。 这将在此提案中进行评估。 在特定的目标3中，我们将定义蛋白酶结构域中TF结合，Na+结合，Ca2+结合和Zn2+结合之间的联系，以及在TF诱导的VIIA催化效率的发展中S1位点的占用。 在特定目标4中，我们将在晶体学上确定与可溶性TF（STF）复合的Zymogen VII的结构，各种活动位置抑制因子VIIA/STF（例如，S1位点的占用率为S1和S3/S4位点，S1和S1和S1，S2，S2，S2和S3/S4位点，以及隔离的K1 domain domain to Viia和viia和gaia和gla and and gla and gla and comply和compliain。 我们有扎伊斯原VII/STF的晶体和几种抑制VIIA/STF复合物（包括可逆抑制剂）的多种活性位点。 我们已经在这些晶体中收集了X射线强度数据，并且正在精炼某些结构。 由于我们的晶体是在Ca2+/Mg2+/Zn2+溶液中获得的，因此我们在VIIA因子中也将这些位点进行了完善。 晶体学研究将补充前三个特定目的中提出的生化研究。 我们的综合方法有望提供有关外部凝血综合体及其对TFPI的调节的新有价值的信息。 从这些研究中获得的知识对于发展新一代的抗肉眼学至关重要。