ACTIVATION OF THE FACTOR VIIIA-FACTOR IXA ENZYME COMPLEX OF BLOOD-COAGULATION BY MEMBRANES CONTAINING PHOSPHATIDYL-L-SERINE

Factor IXa, a serine protease of blood coagulation, functions at least 100,000 times more efficiently when bound to factor VIIIa on a phosph olipid membrane than when free in solution. We have utilized the catal ytic activity of the factor VIIIa-factor IXa complex to report the eff ect of phospholipid membranes on binding of factor IXa to factor VIIIa and on enzymatic cleavage of the product. The apparent affinity of fa ctor IXa for factor VIIIa was 10-fold lower in the absence of phosphol ipid membranes with a K-D of 46 nM versus 4.3 nM with phospholipid mem branes. 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, phosph atidylethanolamine, and phosphatidylcholine at a ratio of 4:20:76. The largest effect of phosphatidyl-L-serine-containing membranes on the f actor VIIIa-factor IXa complex was the accelerated rate of peptide bon d cleavage, with the k(cat) increased by 1,500-fold from 0.022 to 33 m in(-1). Membranes in which phosphatidyl-L-serine was replaced by phosp hatidyl-D-serine, phosphatidic acid, or phosphatidylglycerol were at l east 10-fold less effective for enhancing the k(cat). Thus, while memb ranes containing phosphatidyl-L-serine enhance condensation of the enz yme with its cofactor and substrate, their largest effect is activatio n of the assembled factor VIIIa-factor IXa enzyme complex.