Inhibition of prothrombinase at macroscopic lipid membranes: Competition between antithrombin and prothrombin

Didier Billy, Han Speijer, Theo Lindhout, H. Coenraad Hemker, George M. Willems

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    The-kinetics of inhibition of prothrombinase during prothrombin conversion by antithrombin and antithrombin-heparin complexes was studied in a tubular flow reactor. Prothrombinase was assembled at a macroscopic phospholipid membrane, composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine, deposited on the inner wall of a glass capillary, by perfusion with a factor Xa-factor Va mixture. Measurement of thrombin production allowed estimation of the amount of prothrombinase present at the capillary wall. Perfusion with a mixture of prothrombin and antithrombin or antithrombin-heparin complexes caused a progressive decline of the prothrombinase activity. The rate of inactivation steeply decreased with increasing prothrombin concentrations, indicating competitive inhibition. Analysis of competitive inhibition data requires estimation of the time-dependent substrate concentration, C-o, near the prothrombin converting surface using earlier developed transport theory [Billy, D., et al. (1995) J. Biol. Chem. 270, 1029-1034]. It appears that the inhibition rate is proportional to the fraction of enzyme, K-m/(Km + C-o), not occupied by substrate. The value of K-m of prothrombinase estimated from the dependence of the inhibition rate on the prothrombin concentration (K-m = 2-3 nM) is in excellent agreement with the value estimated from the substrate conversion rate (K-m = 3 nM). Therefore inhibition of prothrombinase by antithrombin and antithrombin-heparin complexes is fully competitive with the substrate: prothrombin. Our results show that prothrombinase assembled on macroscopic lipid surfaces by virtue of its low K-m value is protected for inhibition due to highly effective competition of prothrombin with antithrombin for the active site of factor Xa.
    Original languageEnglish
    Pages (from-to)13699-13704
    Number of pages6
    Issue number41
    Publication statusPublished - 17 Oct 1995

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