STRUCTURE FUNCTION STUDIES ON NORMAL AND MUTATED FACTOR IX

正常和突变因子 IX 的结构功能研究

• 批准号: 6302090
• 负责人: HAROLD Ross ROBERTS
• 金额: $ 29.23万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6302090
• 关键词: active sites; binding sites; chimeric proteins; coagulation factor IX; collagenase; crosslink; disease /disorder model; dogs; gamma carboxyglutamate; gene mutation; hemophilia B; hemostasis; human tissue; laboratory mouse; molecular dynamics; platelets; protease inhibitor; protein protein interaction; protein structure function; thrombosis

The overall aim of this proposal is to assign functional significance to specific structural regions of the coagulation factor IX. These studies have arisen as an outgrowth of work done in the last cycle of this program project grant. Using the same techniques that successfully identified the endothelial cell factor IX receptor as collagen type IV, we have identified a chimeric factor IX molecular that does not bind to the platelet factor IX binding site. We hypothesize that even if factor IX is otherwise fully active, factor IX that does not bind to platelets will be ineffective in a physiologic setting. We propose studies to understand the role of the platelet factor IX binding site by using this chimera in both in vitro studies and in vivo studies in hemophilic dogs and in a hemophilia B mouse strain developed in the last cycle of this program studies in hemophilic dogs and in hemophilia B mouse strain developed in the last cycle of this program project grant. We are also planning to examine the physiologic consequence of mutating Arg338 in factor IX to Leu. This mutation increases factor IXa activity at least 3 fold. This residue is coded for by a CG mutational hot spot, yet not mutations have been reported at this site. This is especially remarkable since 16 hot spots in factor IX account for 40% of all reported mutations. We hypothesize that mutations have been reported because mutation at that site do not cause hemophilia but rather are thrombogenic. We will test this hypothesis in hemophilia B dogs and mice. We are proposed studies to understand the residues of factor IX that are involved in substrate recognition and cleavage. In the last cycle of this grant, we established that, in the absence of factor VIIIa, porcine factor IXa has higher activity toward human factor X than does wild type factor IXa. We will isolate the residues responsible for this higher activity using a human-porcine chimera and point mutations. Also, we will examine the interactions of the Kunitz inhibitor protease nexin II with factor IXa to define residues involved in the extended binding site of factor IXa. Finally, we plan to define a binding site for factor VIIIa using a peptide from factor VIII that we have shown to bind to factor IXa and inhibit its activity. We will crosslink this peptide to factor IXa and isolate peptides of factor IXa to which it is bound. Overall, these studies will allow us to define specific structural regions of factor IX that are involved in a number of important physiologic functions.

该提案的总体目的是将功能意义分配给凝血因子IX的特定结构区域。这些研究已成为该计划项目资助的最后一个周期中完成的工作的产物。使用相同的技术，该技术成功地将内皮细胞因子IX受体识别为IV型胶原蛋白，我们已经鉴定出一种嵌合因子IX分子，该因子与血小板因子IX IX结合位点不结合。我们假设，即使因子IX是完全活跃的，与血小板不结合的因子IX在生理环境中也将无效。我们建议研究通过在体外研究中使用该嵌合体以及在该程序研究的最后一个狗的最后一个循环中，在该程序研究的最后一个b e菌菌株中，在该程序研究的最后一个计划项目赠款的最后一个周期中，在该程序研究的最后一个周期中，在该程序研究中开发的血小板IX IX结合位点的作用。我们还计划检查在leu第IX因子中突变Arg33的生理后果。该突变增加了IXA活性至少3倍。该残基由CG突变热点编码，但在该部位没有报告突变。这尤其引人注目，因为第IX因子中的16个热点占所有报告突变的40％。我们假设已经报告了突变，因为该部位的突变不会引起血友病，而是血栓形成。我们将在血友病B狗和小鼠中检验这一假设。我们提出的研究是为了了解与底物识别和裂解有关的因子IX的残基。在该赠款的最后一个周期中，我们确定，在没有因子VIIIA的情况下，猪IXA对人为因子X的活性高于野生型因子IXA。我们将使用人类孢子嵌合体和点突变分离负责这种较高活性的残基。此外，我们将研究Kunitz抑制剂蛋白酶Nexin II与IXA因子的相互作用，以定义与IXA因子扩展结合位点相关的残基。最后，我们计划使用来自VIII因子VIII的肽来定义一个因子VIIIA的结合位点，我们已证明该因子与因子结合并抑制其活性。我们将将此肽交联以IXA和分离肽的因子IXA的肽。总体而言，这些研究将使我们能够定义与许多重要生理功能有关的因子IX的特定结构区域。