STRUCTURE FUNCTION STUDIES ON NORMAL AND MUTATED FACTOR IX

本提案的总体目标是为凝血因子IX的特定结构区域分配功能意义。这些研究是本项目最后一个周期所做工作的结果。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，我们已经鉴定出不与血小板因子IX结合位点结合的嵌合因子IX分子。我们假设，即使因子IX在其他方面是完全活跃的，因子IX不结合血小板将是无效的生理设置。我们建议研究血小板因子IX结合位点的作用，通过在血友病狗和血友病B小鼠株的体外和体内研究中使用这种嵌合体，在本项目最后一个周期中对血友病狗和血友病B小鼠株进行研究，在本项目资助的最后一个周期中进行研究。我们还计划研究将Arg338中的因子IX突变为Leu的生理后果，该突变使因子IXa活性至少增加3倍。该残基由一个CG突变热点编码，但在该位点尚未报道突变。这一点尤其值得注意，因为因子IX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引起血友病，而是引起血栓。我们将在B型血友病犬和小鼠中验证这一假设。我们建议研究了解参与底物识别和切割的因子IX的残基。在本拨款的最后一个周期中，我们确定，在缺乏viii型因子的情况下，猪型因子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出负责这种高活性的残基。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VIII的结合位点，我们已经证明该肽可以与因子IXa结合并抑制其活性。我们将这个肽与因子IXa交联，并分离与它结合的因子IXa的肽。总的来说，这些研究将使我们能够确定参与许多重要生理功能的因子IX的特定结构区域。

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.