Search

The effect of zinc ions on kinetic and equilibrium steps that may contribute to the activation and subsequent autoactivation of human blood coagulation factor XII in the presence of 500-kDa dextran sulfate was studied. To analyze the results, the expression of the overall autoactivation constant that had been established from the model presented in a previous study was used. Comparison of the kinetics obtained at different levels of zinc, which included amounts lower than the residual concentration introduced by NaCl in the incubation mixture, suggested that the addition of Zn2+ up to 5 microM lowered the mean number of sites available for the binding of factor XII to the surface from 220 to 172 and increased the rate of the first-order activation of factor XII by one order of magnitude, from (1.6 +/- 0.4) x 10(-4) s(-1) to (8.0 +/- 0.4) x 10(-4) s(-1) in the presence of 550 nM dextran sulfate. Neither the factor XII/surface dissociation constant (1 microM), the apparent catalytic constant, nor the apparent Michaelis-Menten constant associated with the postulated multi-stage kinetic sequence were affected by the presence of zinc. Most experimental trends induced by the presence of zinc could be successfully interpreted by using the model, thus reinforcing its reliability under different conditions.

The autoactivation kinetics of purified factor XII (FXII) in the presence of dextran sulfate of 500000 Da was reexamined assuming the existence of two preceding activation steps. Kinetics were numerically simulated by using rate and equilibrium constants related to surface-bound species. Relevant feature parameters related to the polymer (number of binding sites and concentration, dissociation constant of FXII from the surface) and the zymogen (concentration. Michaelis-Menten constant of the autoactivation reaction, catalytic rate constant) were accordingly introduced in the mechanisms. Depending on the rate-limiting step i.e. whether the polymer or FXII predominates, numerical simulation analysis led to obtain for the observed autoactivation rate constant (kobs) two explicit expressions which included the contributing variables. One of the two proposed models was in good accordance with the experimental data obtained in this study and with others published previously. We were able to estimate the mean number of the FXII-activating sites supported by the polymer chains (220) and the equilibrium dissociation constant of FXII from the surface (1 microM). Further treatment led us to determine surface-concentration-independent constants (K(m) = 2510 nM and kcat = 0.01 s-1), as well as the rate constant (k1 = 1.6 x 10(-4) s-1) of the postulated first-order activation rate aimed at explaining the formation of the first trace amounts of FXIIa via an intramolecular mechanism. Overall, the treatment applied to the dextran sulfate case offers a quantitative tool by which data determined in the presence of other activating materials can be rationalized.