PHOSPHOLIPID BINDING STRUCTURES OF FACTOR VIII

这一建议试图为因子VIII与含有磷脂酰丝氨酸(PS)的膜的高亲和力和特异性结合作用提供结构上的解释。这项提议是出于三个方面的考虑。首先，在细胞膜上暴露合适的含PS的部位可能是止血的限速步骤。其次，因子VIII的高亲和力、特异性的膜结合，包括静电和疏水相互作用，本质上是有趣的，对结构的理解可能作为其他膜结合作用的原型。第三，因子的PS结合肽似乎具有生化重要性，它还与因子IVa或因子X以及von Willebrand因子结合。关于磷脂和膜的性质，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的相互作用部分除包括磷酸-L-丝氨酸外，还包括sn-2酰链，可能还包括酰基-甘油键。3)因子VIII的高亲和力结合取决于因子VIII的PS结合基序附近的侧膜压力的降低。我们假设因子IXa活性的增强是由于因子VIII的多肽与因子IXa或因子X的GLA结构域结合所致。该提议的目的是i)确定影响因子VIII结合的膜结构和性质。2)表征fVIII2303-23与因子IXa和/或因子X、与磷脂酰丝氨酸、以及与von Willebrand因子的相互作用。研究旨在确定fV2303-23对因子IXA的动力学效应和增强活性的机制。我们将确定PS与FvIII2303-23对接的三维结构，并确定与PS、因子IXa和von Willebrand因子结合所必需的氨基酸。3)C2结构域的结构与磷脂、VWF和因子IXa的结合有关。我们将使用多维核磁共振技术解决因子VIII的C2结构域的三维结构。我们将进行定点突变以确认与PS、因子IXa和von Willebrand因子相互作用的氨基酸。

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.