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 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 KD of 46 nM versus 4.3 nM with phospholipid membranes. The Km 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-L-serine-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 VIIIa-factor IXa enzyme complex.
Gilbert
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Department of Medicine, Brockton-West Roxbury VA Medical Center, Massachusetts 02132, USA. GILBERT_MD,GARY_E.+@brockton.va.gov
Arena
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D000818:Animals; D001921:Brain; D002417:Cattle; D004789:Enzyme Activation; D015949:Factor IXa / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D010718:Phosphatidylserines / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D008081:Liposomes; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D015944:Factor VIIIa; D015949:Factor IXa; D013917:Thrombin