Effect of thrombomodulin on the molecular recognition and early catalytic events in thrombin-protein C interaction

A viscosity perturbation method allowed to compute the second order rate constant, k+1, for the formation of thrombin-Protein C complex, both in the absence and presence of thrombomodulin (TM) at pH 8.00 and 37 degrees C. In the absence of TM the second order rate constant was found equal to 7.9 +/- 0.6 x 10(3) M-1 sec-1, whereas it was enhanced to 9.9 +/- 0.4 x 10(4) M-1 s-1 by a saturating (100 nM) TM concentration. Addition of 5 mM Ca++ to solution containing 100 nM TM induced a further increase of k+1 value up to 7.3 +/- 0.5 x 10(5) M-1 s-1. Moreover, it was demonstrated that the thrombin-PC complex undergoes the acylation reaction more rapidly than it dissociates to form free thrombin and substrate (stickiness ratio = 2.4 +/- 0.9). This tendency is even favored when thrombin is bound to TM both in the absence and presence of Ca++ (stickiness ratio = 9 +/- 6 in the absence of Ca++ and 16 +/- 10 in the presence of Ca++). Altogether these results demonstrate that TM is able to positively affect both the molecular encounter and the kinetics of the early catalytic events of the thrombin-Protein C interaction.