Proteins of Coagulation Pathways

凝血途径的蛋白质

• 批准号: 7748029
• 负责人: JOHN H GRIFFIN
• 金额: $ 56.86万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7748029
• 关键词: 3-Dimensional; Affect; Anticoagulants; Arteries; Basic Science; Binding; Binding Proteins; Binding Sites; Biological Assay; Biological Markers; Blood; Blood Clot; Blood Coagulation Factor; Blood coagulation; Carnitine; Chimera organism; Clinical; Clinical Research; Coagulation Process; Complex; Data; Diagnosis; Disease; Dyslipidemias; Elements; Energy Transfer; Factor Va; Factor X; Factor Xa; Fatty acid glycerol esters; Freezing; Functional disorder; Funding; Generations; Glucosylceramides; Goals; Hand; Knowledge; Label; Leg; Life; Link; Lipid Binding; Lipids; Lung; Maps; Membrane; Methods; Minor; Molecular; Mutation; Myocardial Infarction; Pathway interactions; Patients; Phospholipid Transfer Proteins; Phospholipids; Plasma; Plasma Proteins; Property; Protein Binding; Proteins; Prothrombin; Recombinants; Registries; Regulation; Risk; Sampling; Serum amyloid A protein; Solutions; Sphingosine; Study Subject; Surface; Surface Plasmon Resonance; Testing; Thrombin; Thrombophilia; Thromboplastin; Thrombosis; Translating; Translational Research; Variant; Veins; Venous; Venous Thrombosis; base; bench to bedside; clinically relevant; improved; innovation; insight; lipid transfer protein; mutant; novel; plasma phospholipid transfer protein; prothrombinase complex

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.

这个修订后的续期两年 ARRA 资助的项目扩展了 PI 的基础和临床 凝血调节研究及转化临床研究 静脉血栓形成。血栓形成与抗凝剂和抗凝剂失衡密切相关 促凝血机制。四个具体目标中的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转化研究 识别血栓形成的新生物标志物。对于目标 1 和 2，我们假设 凝血酶的产生可能受到少量单链血浆脂质的影响， 所谓的“可溶性”脂质。基于表面等离子共振 (SPR) 结合研究 在凝血试验中，我们假设抗凝酰基肉碱，包括 棕榈酰肉碱，通过与 Xa 结合直接抑制凝血因子 Xa。在初步 研究发现，棕榈酰肉碱与 Gla 无结构域因子 Xa 结合并抑制其活性。 因此，这些初步数据和拟议的实验涉及一种新颖的 血浆脂质对凝血途径影响的范例。重组因子 来自不同物种的 X/IX 嵌合体和变体 X 因子分子将用于 鉴定 Xa 因子上假定的脂质结合域。对于目标 2，我们假设 血浆磷脂转移蛋白（PLTP）可直接影响血浆凝固性 以及通过与凝血因子直接相互作用产生凝血酶。表面等离子体共振 初步数据表明 PLTP 与特定的凝血因子结合。对于目标 3，我们将 确定凝血酶原酶的一些三维结构特性 磷脂膜上包含因子 Xa、Va 和凝血酶原的复合物。第一的， 我们将引入Cys突变并制备荧光标记的Va因子。然后我们 将使用福斯特共振能量转移（FRET）来生成一组多点- 可用于生成然后解释的点到平面距离 凝血酶原复合物的 FRET 距离。对于目标 4，基于 假设血浆抗凝剂少量丰度脂质的不平衡与 血栓形成风险，我们将使用现有的血栓形成冰冻血浆样本 患者和匹配的对照，以确定是否某些目标血浆脂质或两种脂质 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病理生理学的了解，并可能改善 血栓形成的诊断和治疗。