Unsaturated phospholipid acyl chains are required to constitute membrane binding sites for factor VIII.
Membranes containing phosphatidyl-L-serine (PS) and phosphatidylethanolamine (PE) greatly enhance the function of the enzymatic cofactor factor VIII. The mechanisms of enhanced function involve condensation of enzyme (factor IXa), activated cofactor (factor VIIIa), and substrate (factor X) at a common location and, most dramatically, activation of the assembled enzyme-cofactor complex. We asked whether unsaturated phospholipid (PL) acyl chains are necessary to constitute factor VIII binding sites or to activate the factor VIIIa-factor IXa complex. We found 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 sites, and thrombin activation did not reduce the affinity for saturated membranes more than 2-fold so that the loss of functional activity is due to a requirement of the factor VIIIa-factor IXa complex for unsaturated acyl chains that exceeds the requirement for factor VIII binding 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 factor VIIIa-factor IXa activating activity. Lyso-PS did not support binding of factor VIII or function of the factor VIIIa-factor IXa 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 functional factor VIII binding sites on phospholipid membranes.
Gilbert
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Department of Medicine, Brockton-West Roxbury VA Medical Center, Boston, Massachusetts 02132, USA. ggilbert@massmed.org
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D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D015944:Factor VIIIa / Q000737:chemistry / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism
D005231:Fatty Acids, Unsaturated; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010743:Phospholipids; D015944:Factor VIIIa; D005169:Factor VIII; D002784:Cholesterol; D015951:Factor Xa