Proteins of Coagulation Pathways

这个由ARRA资助的修订后的两年续展项目延长了PI的基础和临床 凝血调节机制的研究及其转译临床研究 静脉血栓形成。血栓形成与抗凝剂和抗凝剂的失衡密切相关 促凝血机制。四个具体目标中有三个涉及小说的基础研究 调节凝血的血浆分子，而最后一个目标涉及翻译研究 寻找血栓形成的新生物标志物。对于目标1和目标2，我们假设 凝血酶的生成会受到少量单链血浆脂类的影响， 所谓的“可溶”脂类。基于表面等离子体共振(SPR)结合的研究 在凝血试验上，我们假设抗凝剂酰肉碱，包括 棕榈酰肉碱，通过与凝血因子Xa结合直接抑制凝血因子Xa。在预赛中 研究表明，棕榈酰肉碱与无GLA结构域的因子Xa结合并抑制其活性。 因此，这些初步数据和拟议的实验与一种新的 血浆脂类对凝血途径影响的范例。重组因子 来自不同物种的X/IX嵌合体和变异因子X分子将被用于 鉴定因子Xa上可能的脂结合结构域。对于目标2，我们假设 血浆磷脂转运蛋白(PLTP)可直接影响血浆凝固性 以及通过与凝血因子的新的直接相互作用产生凝血酶。弹簧 初步数据显示，PLTP与特定的凝血因子结合。对于目标3，我们将 测定凝血酶原酶的某些三维结构性质 磷脂膜上含有Xa、Va和凝血酶原的复合体。第一, 我们将引入半胱氨酸突变并制备荧光标记的因子Va。那我们 将使用福斯特共振能量转移(FRET)产生一组多点- 可用于生成并解释的点到点距离和点到面距离 凝血酶原酶复合体的FRET派生距离。对于目标4，基于 假设血浆抗凝剂少量丰度脂的失衡与 血栓形成风险，我们将使用已有的血栓形成冰冻血浆样本 患者和匹配的对照组确定特定的靶向血脂或两种血脂 结合血浆蛋白(PLTP或血清淀粉样蛋白A)是血栓形成的生物标志物。这 该项目将增加对血栓形成的病理生理学的了解，并可能改善 血栓形成的诊断和治疗。

This revised renewal two year ARRA-funded project extends the PI's basic and clinical research on regulation of blood coagulation and on translational clinical research of venous thrombosis. Thrombosis is strongly linked to an imbalance of anticoagulant and procoagulant mechanisms. Three of four specific aims involve basic studies of novel plasma molecules that regulate clotting while the last aim involves translational research to identify novel biomarkers for thrombosis. For aims 1 and 2, we hypothesize that thrombin generation can be influenced by minor abundance single chain plasma lipids, so-called "soluble" lipids. Based on Surface Plasmon Resonance (SPR) binding studies and on clotting assays, we hypothesize that anticoagulant acyl carnitines, including palmitoyl carnitine, directly inhibit coagulation factor Xa by binding to Xa. In preliminary studies, palmitoyl carnitine binds to Gla-domainless-factor Xa and inhibits its activity. These preliminary data and the proposed experimentation thus relate to a novel paradigm for the effects of plasma lipids on coagulation pathways. Recombinant factor X/IX chimeras and variant factor X molecules from various species will be used to identify putative lipid binding domains on factor Xa. For aim 2, we hypothesize that plasma phospholipid transfer protein (PLTP) can directly influence plasma coagulability and thrombin generation by novel direct interactions with clotting factors. SPR preliminary data show that PLTP binds to specific clotting factors. For aim 3, we will determine some of the three dimensional structural properties of the prothrombinase complex comprising factors Xa, Va and prothrombin on phospholipid membranes. First, we will introduce Cys mutations and prepare fluorescently labeled factor Va. Then we will use Forster Resonance Energy Transfer (FRET) to generate a set of multiple point- to-point and point-to-plane distances that can be used to generate and then interpret FRET-derived distances for the prothrombinase complex. For aim 4, based on the hypothesis that imbalances of plasma anticoagulant minor abundance lipids are linked to thrombosis risk, we will use already available frozen plasma samples from thrombosis patients and matched controls to determine if certain targeted plasma lipids or two lipid binding plasma proteins (PLTP or serum amyloid A) are biomarkers for thrombosis. This project will increase insights into the pathophysiology of thrombosis and may improve diagnosis and treatment of thrombosis.