Specific contribution of different phospholipid surfaces to the activation of prothrombin by the fully assembled prothrombinase.
This paper addresses, in thermodynamic and kinetic terms, the reasons for the acidic lipid specificity of the human prothrombinase complex. We obtained, from the measured lipid titrations of the initial rates of prothrombin activation, the empirical binding constants for prothrombinase assembly on different membranes. These favored assembly on phosphatidylserine (PS)- as opposed to phosphatidylglycerol (PG)-containing membranes. In addition, we have used full time courses of prothrombin activation, in conjunction with a calculation of the equilibrium distribution of factor Xa between four enzymatic forms, to obtain the intrinsic kinetic constants of the prothrombinase assembled on PS- or PG-containing membranes. The resulting values of kcat, Km, and kcat/Km increased as acidic lipid content increased, and kcat/Km reached a plateau at 12 mol % PS and 50 mol % PG. Using the measured assembly and kinetic constants, the observed shapes of the phospholipid titration curves of human prothrombin activation were interpreted. We conclude that the difference in activity of prothrombinase assembled on PS- versus PG-containing membranes results both from the different binding properties of factors Xa and Va to these surfaces and from the different intrinsic activities of the prothrombinase when assembled on different membranes.
Pei
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Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260.
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D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D004789:Enzyme Activation; D015951:Factor Xa / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D010715:Phosphatidylglycerols / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology*; D011516:Prothrombin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013499:Surface Properties; D013816:Thermodynamics; D013925:Thromboplastin / Q000378:metabolism*
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D010743:Phospholipids; C019162:dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D011516:Prothrombin; D013925:Thromboplastin; D001120:Arginine; D015951:Factor Xa