Inhibition of Prothrombinase at Macroscopic Lipid Membranes: Competition between Antithrombin and

ABSTRACT: 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,, near the prothrombin converting surface using earlier developed transport theory [Billy, D., et al. (1995) J. Biol. Chem. 270, 1029-10341. It appears that the inhibition rate is proportional to the fraction of enzyme, K,/(Km-I- C,), not occupied by substrate. The value of K, of prothrombinase estimated from the dependence of the inhibition rate on the prothrombin concentration (K, = 2-3 nM) is in excellent agreement with the value estimated from the substrate conversion rate (K, = 3 nM). Therefore inhibition of prothrombinase by antithrombin and antithrombin-heparin complexes is fully competitive with th