UNSATURATED PHOSPHOLIPID ACYL CHAINS ARE REQUIRED TO CONSTITUTE MEMBRANE-BINDING SITES FOR FACTOR-VIII

Membranes containing phosphatidyl-L-serine (PS) and phosphatidylethano lamine (PE) greatly enhance the function of the enzymatic cofactor fac tor VIII. The mechanisms of enhanced function involve condensation of enzyme (factor Ma), activated cofactor (factor VIIIa), and substrate ( factor X) at a common location and, most dramatically, activation of t he assembled enzyme-cofactor complex. We asked whether unsaturated pho spholipid (PL) acyl chains are necessary to constitute factor VIII bin ding sites or to activate the factor VIIIa-factor IXa complex. We foun d that membranes composed of saturated, dimyristoyl phospholipids had 20-fold fewer factor VIII binding sites and that these sites supported less than 5% normal activity of the factor VIIIa-factor IXa complex. Thrombin-activated factor VIII bound to a similar number of membrane s ites, and thrombin activation did not reduce the affinity for saturate d membranes more than 2-fold so that the loss of functional activity i s due to a requirement of the factor VIIIa-factor Ma complex for unsat urated acyl chains that exceeds the requirement for factor VIII bindin g alone. Replacement of dimyristoyl-PS, -PE, or -PC individually with the corresponding unsaturated phospholipid restored 75%, 60%, and 15%, respectively, of factor VIII binding sites but less than 10% of facto r VIIIa-factor Ma activating activity. Lyso-PS did not support binding of factor VIII or function of the factor VIIIa-factor Ma complex even when PE and phosphatidylcholine contained unsaturated acyl chains. We conclude that the sn-2 acyl chain of PS and unsaturated phospholipid acyl chains are chemical requirements for constitution of fully functi onal factor VIII binding sites on phospholipid membranes.