Abstract
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.
Highlights
From the Department of Biochemistry and Biophysics, C B 7260, The Universityof North Carolina, Chapel Hill, North Carolina 27599-7260
Not all negatively charged phospholipidmembranes furnish catalytic surfaces that are effective for prothrombin activation
We conclude that the that the phospholipidsurfacemay alterin a lipid-specific difference in activity of prothrombinase assembled on fashion at least one componentf the catalytic
Summary
Phospholipid Dependence of the Rate of Prothrombin Acti- and peak heights of lipid titration curves shown inFig. 2 were vation-From time courses such asshown, the initial reflective of three key parameters:thekineticconstants velocity of prothrombin activation was determined asa func- (mainly kcat)for activation of prothrombin by the assembled tion of both composition andconcentration of membrane prothrombinase, the Kd for factor Va binding to membranes, vesicles containing acidic phospholipid. Containing membranes norfor membranes containing different surface concentrations of acidic phospholipid For these reasons, we have adjusted the Kd for factor Xa binding to Va.PL to obtain a best least-squares fit of calculated initial rates (see Appendix) to our own lipid titration experiments.
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