Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a ␤-hairpin turn, and also by net positive charge and specific interactions with phospho-L-serine. , and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipidlimiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced ϳ20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced ϳ5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipidbinding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.Factor VIII is a phosphatidyl-L-serine (PS) 1 binding cofactor (1, 2) for the vitamin K-dependent serine protease, factor IXa, that also binds to PS-containing membranes (3, 4). The membrane-bound factor VIIIa-factor IXa complex cleaves the zymogen, factor X, to factor Xa, which is then responsible for catalyzing prothrombin activation (5). The importance of this enzyme complex is illustrated by hemophilia, a disease in which a deficiency of either factor VIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. Factor IXa gains more than 100,000-fold greater efficiency in activating factor X by assembling with factor VIIIa on a PS-containing membrane than when free in solution (6). We have recently found that the predominant effect of PS-containing membranes on the factor VIIIa-factor IXa complex is to increase the k cat by more than 1000-fold (7). These membranes also increase the affinity of factor IXa for factor VIIIa and for factor X. The central importance of the membrane binding function of factor VIII motivates studies to define the membrane-binding motif.Factor VIII, with M r 280,000, is homologous to another procoagulant protein, factor V, in amino acid sequence (8 -10) and in function, as a membrane-bound enzyme cofactor (5, 11-13). The proteins share a repeating domain structure of A1-A2-B-A3-C1-C2 in which the A domains are homologous with ceruloplasmin, the B domain is unique to each protein, and the C domains are homologous with discoidin I, a phospholipid-binding lectin (14), and with a murine milk fat globule membrane ...

Factor IXa, a serine protease of blood coagulation, functions at least 100,000 times more efficiently when bound to factor VIIIa on a phospholipid membrane than when free in solution. We have utilized the catalytic activity of the factor VIIIa-factor IXa complex to report the effect of phospholipid membranes on binding of factor IXa to factor VIIIa and on enzymatic cleavage of the product. The apparent affinity of factor IXa for factor VIIIa was 10-fold lower in the absence of phospholipid membranes with a K D of 46 nM versus 4.3 nM with phospholipid membranes. The K m for activation of factor X by the factor VIIIa-factor IXa complex was 1700 nM in solution, 70-fold higher than the value of 28 nM when bound to membranes containing phosphatidyl-L-serine, phosphatidylethanolamine, and phosphatidylcholine at a ratio of 4:20:76. The largest effect of phosphatidyl-Lserine-containing membranes on the factor VIIIa-factor IXa complex was the accelerated rate of peptide bond cleavage, with the k cat increased by 1,500-fold from 0.022 to 33 min ؊1. Membranes in which phosphatidyl-L-serine was replaced by phosphatidyl-D-serine, phosphatidic acid, or phosphatidylglycerol were at least 10-fold less effective for enhancing the k cat . Thus, while membranes containing phosphatidyl-L-serine enhance condensation of the enzyme with its cofactor and substrate, their largest effect is activation of the assembled factor VIIIafactor IXa enzyme complex.

Synthetic membranes of phosphatidylcholine require inclusion of at least 5% phosphatidylserine (Ptd-L-Ser) to form binding sites for factor VIII. The relatively high requirement for Ptd-L-Ser suggests that stimulated platelets may contain another membrane constituent that enhances expression of factor VIII-binding sites. We report that phosphatidylethanolamine (PE), which is exposed in concert with Ptd-L-Ser in the course of platelet stimulation, induces high affinity binding sites for factor VIII on synthetic membranes containing 1-15% Ptd-L-Ser. The affinity of factor VIII for binding sites on membranes of Ptd-L-Ser/PE/phosphatidylcholine in a 4:20:76 ratio was 10.2 +/- 3.5 nM with 180 +/- 33 phospholipid molecules/site. PE did not induce binding sites on membranes of 4% Ptd-D-Ser, indicating that the induced binding sites require the correct stereochemistry of Ptd-L-Ser as well as PE. Egg PE and dimyristoyl-PE were equivalent for inducing factor VIII-binding sites, indicating that hexagonal phase-inducing properties of PE are not important. We conclude that PE induces high affinity factor VIII-binding sites on membranes with physiologic mole fractions of Ptd-L-Ser, possibly including those of stimulated platelets.