PHOSPHOLIPID BINDING STRUCTURES OF FACTOR VIII

该提案旨在开发一种结构解释的高亲和力，特异性结合相互作用的因子VIII与磷脂酰丝氨酸（PS）的含膜。该建议是出于三个考虑。首先，细胞膜上合适的含PS位点的暴露可能是止血的限速步骤。第二，高亲和力，特异性膜结合因子VIII，涉及静电和疏水相互作用，是内在的利益和结构的理解可以作为其他膜结合相互作用的原型。第三，因子VIII的PS结合肽似乎具有生物化学重要性，也结合因子Iva或因子X和血管性血友病因子。关于磷脂和膜性质，我们假设：I）大部分结合能来自因子VIII与单个PS分子的相互作用。2)PS的相互作用部分除了磷酸-L-丝氨酸外，还包括sn-2酰基链和可能的酰基-甘油键。3)因子VIII的高亲和性结合取决于紧邻因子VIII的PS结合基序的侧膜压力的降低，我们假设因子IXa活性的增强是由于因子VIII肽与因子IXa或因子X的Gla结构域的结合.该提案的目的是I）确定影响因子VIII结合的膜结构和性质。2)表征fVIII 2303 -23与因子IXa和/或因子X、与磷脂酰丝氨酸和与血管性血友病因子的相互作用。本研究旨在确定fV 2303 -23对因子IXA的动力学效应和增强活性的机制。我们将确定与fvIII 2303 -23对接的PS的三维结构，并确定与PS，因子IXa和von Willebrand因子结合所必需的氨基酸。3)将C2结构域的结构与磷脂、vWf和因子IXa结合相关。使用多维NMR，我们将解决因子VIII的C2结构域的三维结构。我们将进行定点诱变，以确认与PS，因子IXa和von Willebrand因子相互作用的氨基酸的鉴定。

This proposal seeks to develop a structural explanation for the high affinity, specific binding interaction of factor VIII with phosphatidylserine (PS)- containing membranes. The proposal is motivated by three considerations. First, exposure of suitable PS-containing sites on cell membranes is probably a rate-limiting step in hemostasis. Second, the high affinity, specific membrane binding of factor VIII, involving both electrostatic and hydrophobic interactions, is intrinsically interest and a structural understanding may serve as a prototype for other membrane-binding interactions.. Third, a PS-binding peptide of factor VIII appears to have biochemical importance, binding also to factor Iva or factor X and to von Willebrand factor. With regard to phospholipid and membrane properties we have hypothesized that: I) The majority of the binding energy arises from interaction of factor VIII with a single PS molecule. 2) That the interactive moieties of PS include the sn-2 acyl chain, and possibly the acyl-glycerol linkage, in addition to phospho-L-serine. 3) That high affinity binding of factor VIII is contingent upon decreased lateral membrane pressure in the immediate vicinity of a PS -binding motif of factor VIII we hypothesize that enhancement of factor IXa activity results from binding of the factor VIII peptides to the Gla domain of factor IXa or factor X. The aims of the proposal are I) Identify membrane structures and properties that influence binding of factor VIII. 2) Characterize the interaction of fVIII2303-23 with factor IXa and/or factor X, with phosphatidylserine, and with von Willebrand factor. Studies are directed toward determining the kinetic effects of fV2303-23 on factor IXA and the mechanism of enhanced activity. We will determine the 3-dimensional structure of PS docking with fvIII2303-23 and identify amino acids that are necessary for binding to PS, to factor IXa, and to von Willebrand factor. 3) Correlate structure of the C2 domain with binding to phospholipid, vWf, and factor IXa. Using multi- dimensional NMR we will solve the 3-dimensional structure of the C2 domain of factor VIII. We will perform site-directed mutagenesis to confirm the identify of amino acids that interact with PS, factor IXa, and von Willebrand factor.