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: 6433736
• 负责人: HAROLD Ross ROBERTS
• 金额: $ 7.93万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6433736
• 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.

该提案的总体目标是将功能意义分配给凝血因子IX的特定结构区域。这些研究是在该项目赠款的最后一个周期所做工作的结果。使用相同的技术，成功地确定了内皮细胞因子IX受体的胶原蛋白IV型，我们已经确定了嵌合因子IX分子，不结合到血小板因子IX结合位点。我们假设，即使因子IX在其他方面是完全活跃的，不与血小板结合的因子IX在生理环境中也是无效的。我们建议通过在血友病犬和血友病B小鼠品系的体外研究和体内研究中使用该嵌合体进行研究，以了解血小板因子IX结合位点的作用，血友病犬和血友病B小鼠品系是在本项目资助的最后一个周期中开发的。我们还计划研究将因子IX中的Arg 338突变为Leu的生理后果。该突变使因子IXa活性增加至少3倍。该残基由CG突变热点编码，但在该位点尚未报道突变。这是特别值得注意的，因为因子IX中的16个热点占所有报告突变的40%。我们假设突变已经被报道，因为该位点的突变不会引起血友病，而是血栓形成。我们将在血友病B犬和小鼠中检验这一假设。我们提出的研究，以了解参与底物识别和切割的因子IX的残基。在本基金的最后一个周期中，我们确定了在缺乏因子VIIIa的情况下，猪因子IXa对人因子X的活性高于野生型因子IXa。我们将使用人-猪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抑制剂蛋白酶连接蛋白II与因子IXa的相互作用，以确定参与因子IXa的扩展结合位点的残基。最后，我们计划使用来自因子VIII的肽来定义因子VIIIa的结合位点，我们已经证明该肽与因子IXa结合并抑制其活性。我们将交联该肽与因子IXa，并分离与其结合的因子IXa的肽。总的来说，这些研究将使我们能够定义特定的结构区域的因子IX参与了一些重要的生理功能。