ACTIVATION OF FACTOR-VIII BY THROMBIN INCREASES ITS AFFINITY FOR BINDING TO SYNTHETIC PHOSPHOLIPID-MEMBRANES AND ACTIVATED PLATELETS

Membrane-bound thrombin-activated factor VIII (fVIIIa) functions as a cofactor for factor IXa in the factor Xase complex. We found that bind ing of heterotrimeric fVIIIa (A1.A2.A3-C1-C2) to synthetic vesicles wi th a physiologic content of 4% phosphatidylserine (PS), 76% phosphatid ylcholine, and 20% phosphatidylethanolamine occurs with a 10-fold high er affinity than that of factor VIII (fVIII), The increased affinity o f fVIIIa for PS-containing membranes resulted from the reduced rate of fVIIIa dissociation from the vesicles compared with that of fVIII, Si milar affinities of A3-C1-C2, A1.A2.A3-C1-C2, and A3-C1-C2.heavy chain for interaction with PS-containing membranes demonstrate that removal of the light chain (LCh) acidic region by thrombin is responsible for these increased affinities of fVIIIa and its derivatives. Similar kin etic parameters of fVIII and its LCh and C2 domain for binding to PS-c ontaining membranes and to activated platelets indicated that the C2 d omain is entirely responsible for the interaction of fVIII with membra nes. We conclude that the increased fVIIIa affinity for PS-containing membranes is a result of conformational change(s) within the C2 domain upon removal of the acidic region of the LCh, This conclusion is base d on the finding that binding of the monoclonal antibody ESH8 to the C 2 domain, which is known to prevent this conformational transition, re sulted in fVIIIa binding to PS/phosphatidylcholine/phosphatidylethanol amine vesicles (4/76/20) with a lower affinity similar to that of fVII I, In addition, stabilization of the low affinity binding conformation of the C2 domain of fVIIIa by this antibody led to an inhibition of t he fVIIIa activity in the factor X activation complex.