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pubmed:abstractText |
A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-specific amidolytic chromogenic reaction. As thrombin accumulated initially at constant velocity, the simultaneous release from S-2238 of pNA conformed to an acceleration function. Adherence to the acceleration function of the temporally increasing A400 of pNA was evaluated after transforming the data into linear format which permitted linear regression analysis. High correlation coefficients, routinely greater than 0.99, verified linearity of thrombin production in individual assay mixtures. As prothrombin concentrations were varied, factor Xa exhibited Michaelis-Menten kinetics. Added choline produced a pattern of mixed-type inhibition. Replots of LB slopes and intercepts versus choline concentration gave apparent Ki and Ki' values (mM): 22 +/- 3 and 48 +/- 7 without factor Va, 25 +/- 4 and 41 +/- 4 with factor Va.
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