Molecular Biology Of Human Coagulation Factor V

描述（由申请方提供）：凝血酶原酶复合物产生的凝血酶在常见临床疾病（如心肌梗死、深静脉血栓形成、肺栓塞和卒中）的发病机制中起重要作用。凝血酶原酶复合物由酶因子Xa、辅因子Va和磷脂膜表面组成。因子Va与血小板膜的相互作用需要磷脂酰丝氨酸（PS）在活化的血小板或内皮细胞表面上的表达。该项目的长期目标是使用综合的分子，结构和生物物理方法来了解因子Va与生物膜的相互作用。一个膜结合位点被定位到因子V C2结构域和该结构域的两种晶体形式的结构已被阐明。这些结构表明了因子Va与磷脂膜相互作用的工作模型，包括：（1）暴露的疏水残基浸入非极性膜核心;（2）与PS头基的立体特异性相互作用和（3）碱性侧链与带负电荷的膜磷酸基团的有利静电接触。该模型的几个方面已经得到验证。位于C2结构域内的移动的溶剂暴露环上的两个色氨酸的吲哚侧链插入膜双层中，并且是高亲和力膜结合所需的。酪氨酸和亮氨酸残基位于一个类似的位置内的同源因子V C1结构域中发挥作用，结合到膜含有低浓度的PS。本研究的目的是验证和完善我们的工作模型的关联因子Va与生物膜。本提案的具体目的是定义PS特异性口袋（S）和膜结合位点内的因子V C1和C2域，并确定其对凝血酶原酶组装磷脂囊泡和细胞膜的贡献。将使用重组因子Va突变体、重组轻链结构域、scFv抗体和可溶性磷脂类似物来定义结合位点。将使用定量荧光结合试验和功能性凝血酶原酶试验表征结合相互作用。我们推测，复杂的相互作用之间的因子Va和PS包含膜允许凝血酶原酶复合物的精细调节。拟议的实验可能会导致新的抗血栓治疗的目标。

DESCRIPTION (provided by applicant): Thrombin generated by the prothrombinase complex contributes significantly to the pathogenesis of common clinical disorders such as myocardial infarction, deep vein thrombosis, pulmonary embolism, and stroke. The prothrombinase complex consists of the enzyme factor Xa, the cofactor factor Va and a phospholipid membrane surface. The interaction of factor Va with platelet membranes requires expression of phosphatidylserine (PS) on the surface of activated platelets or endothelial cells. The long-term goal of this project is to use integrated molecular, structural and biophysical approaches to understand the interaction of factor Va with biological membranes. A membrane binding site was localized to the factor V C2 domain and the structures of two crystal forms of this domain have been elucidated. These structures have suggested a working model for the interaction of factor Va with phospholipid membranes that includes:(1) immersion of exposed hydrophobic residues in the apolar membrane core; (2) stereospecific interactions with PS head groups and (3) favorable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups. Several aspects of this model have been validated. The indole side chains of two tryptophans located on a mobile solvent exposed loop within the C2 domain insert into the membrane bilayer and are required for high affinity membrane binding. Tyrosine and leucine residues located in an analogous location within the homologous factor V C1 domain play a role in binding to membranes containing low concentrations of PS. The goals of the present study are to validate and refine our working model for the association of factor Va with biological membranes. The specific aims of the present proposal are to define the PS specificity pocket(s) and membrane binding sites within the factor V C1 and C2 domains and to determine their contribution to prothrombinase assembly on both phospholipid vesicles and cellular membranes. Binding sites will be defined using recombinant factor Va mutants, recombinant light chain domains, scFv antibodies and soluble phospholipid analogues. Binding interactions will be characterized using quantitative fluorescence binding assays and functional prothrombinase assays. We hypothesize that complex interactions between factor Va and PS containing membranes allow for the fine regulation of the prothrombinase complex. The proposed experiments could lead to novel targets for anti-thrombotic therapy.