Clotting of bovine fibrinogen. Kinetic analysis of the release of fibrinopeptides by thrombin and of the calcium uptake upon clotting at high fibrinogen concentrations

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Abstract
Kinetic data on the release of fibrinopeptides A and B from bovine fibrinogen by human thrombin were obtained at high fibrinogen concentrations, within the 0.8-8.8% range (0.227 .times. 10-4 to 2.60 .times. 10-4 M), at 25.degree.C, pH 7.26, 0.30 ionic strength, and 10-4 M free Ca2+ concentration. Release of fibrinopeptide A followed strictly first-order kinetics at all concentrations, in spite of the fact that the highest concentration was 26 times larger than the value of KM found in the literature. This behavior can be explained by inhibition of thrombin by the reaction products, with KI = KM. The equation describing the course of the reaction under these conditions can be rearranged into a linear relationship between 1/kobsd and substrate concentration. The slope of the line is equal to 1/kcat and the intercept to KM/kcat. The data points fell accurately on a straight line, and with the parameters of the latter, kcat and Km were calculated as (6.3 .+-. 0.11) .times. 10-10 M s-1 (unit of thrombin)-1 L-1 and (11.0 .+-. 3.0) .times. 10-6 M, respectively. These values agree well with those found in the literature. Release of fibrinopeptide B follows complex kinetics. Higgins et al. [Higgins, D. L., Lewis, S. D., and Shafer, J. A. (1983) J. Biol. Chem. 258, 9276-9282] suggested that it can be described as the result of two consecutive reactions, the first one being the release of fibrinopeptide A and the second one of fibrinopeptide B from those molecules that have already lost fibrinopeptide A in the previous step. An alternate model was also considered which allowed removal of fibrinopeptide B directly from the native molecule, running parallel to the consecutive reactions. These and several other mathematical models were tested by nonlinear least-squares fitting to our experimental data. Best fit was obtained with the model of two consecutive first-order reactions. Release of fibrinopeptide B from the native molecules, i.e., without previous removal of fibrinopeptide A, if at all possible, was nearly an order of magnitude slower. The rate constants for the release of fibrinopeptide B with the two consecutive reactions model were used as with fibrinopeptide A to obtain Kcat equal to (3.5 .+-. 0.2) .times. 10-10 M s-1 (unit of thrombin)-1 L-1 and KM equal to (6.0 .+-. 8.5) .times. 10-6 M. Data points for fibrinopeptide B were more scattered than those for fibrinopeptide A and hence the uncertainly of KM. Uptake of calcium associated with the clotting reaction followed the same mechanism as the release of fibrinopeptide B, and the rates of the two processes were also similar.