Molecular Biology Of Human Coagulation Factor V

凝血酶原复合体产生的凝血酶在心肌梗死、深静脉血栓形成、肺栓塞、脑卒中等常见临床疾病的发病机制中起着重要作用。凝血酶原复合体由酶因子Xa、辅因子Va和磷脂膜表面组成。Va因子与血小板膜的相互作用需要活化的血小板或内皮细胞表面表达磷脂酰丝氨酸（PS）。该项目的长期目标是利用综合的分子、结构和生物物理方法来了解Va因子与生物膜的相互作用。一个膜结合位点定位于因子vc2结构域，并阐明了该结构域的两种晶型结构。这些结构提出了一个Va因子与磷脂膜相互作用的工作模型，包括：(1)暴露的疏水残基浸泡在极性膜核中；(2)与PS头基团的立体定向相互作用；(3)碱性侧链与带负电荷的膜磷酸基团的良好静电接触。这个模型的几个方面已经得到了验证。两个色氨酸的吲哚侧链位于C2结构域内的移动溶剂暴露环上，插入到膜双分子层中，是高亲和膜结合所必需的。位于同源因子V C1结构域中类似位置的酪氨酸和亮氨酸残基在与含有低浓度PS的膜结合中发挥作用。本研究的目的是验证和完善我们的因子Va与生物膜关联的工作模型。本提案的具体目的是确定因子V C1和C2结构域内的PS特异性口袋和膜结合位点，并确定它们对磷脂囊泡和细胞膜上凝血酶原组装的贡献。结合位点将使用重组因子Va突变体、重组轻链结构域、单链抗体和可溶性磷脂类似物来确定。结合相互作用将采用定量荧光结合测定和功能凝血酶原测定表征。我们假设因子Va和含有PS的膜之间的复杂相互作用允许对凝血酶原复合物进行精细调节。提出的实验可能会导致抗血栓治疗的新靶点。

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.