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.
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