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: 6564781
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564781
• 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赋予凝血因子 IX 的特定结构区域功能意义。这些研究是该计划项目资助的最后一个周期所做工作的产物。使用成功鉴定内皮细胞因子 IX 受体为 IV 型胶原的相同技术，我们鉴定了一种不与血小板因子 IX 结合位点结合的嵌合因子 IX 分子。我们假设，即使因子 IX 在其他方面完全活跃，不与血小板结合的因子 IX 在生理环境中也将无效。我们建议通过在血友病狗和本项目最后一个周期开发的血友病 B 小鼠品系的体外研究和体内研究中使用该嵌合体来了解血小板因子 IX 结合位点的作用，在血友病狗和本项目资助的最后一个周期开发的血友病 B 小鼠品系中进行研究。我们还计划检查将因子 IX 中的 Arg338 突变为 Leu 的生理后果。该突变使 IXa 因子活性增加至少 3 倍。该残基由 CG 突变热点编码，但尚未报道该位点发生突变。这是特别值得注意的，因为 IX 因子中的 16 个热点占所有报告突变的 40%。我们假设已经报道了突变，因为该位点的突变不会导致血友病，而是会导致血栓形成。我们将在 B 型血友病狗和小鼠身上检验这一假设。我们建议进行研究以了解参与底物识别和切割的因子 IX 残基。在本次资助的最后一个周期中，我们确定，在缺乏因子 VIIIa 的情况下，猪因子 IXa 对人因子 X 的活性比野生型因子 IXa 更高。我们将使用人-猪嵌合体和点突变来分离导致这种较高活性的残基。此外，我们还将检查 Kunitz 抑制剂蛋白酶 nexin II 与因子 IXa 的相互作用，以确定参与因子 IXa 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，我们已证明该肽可以与因子 IXa 结合并抑制其活性。我们将该肽与因子 IXa 交联，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涉及许多重要生理功能的因子 IX 的特定结构区域。