Identification and Characterization of a Sodium Ion Binding Site on the Staphylocoagulase-Prothrombin Complex.

Sodium ion binding regulates allosterically the pro- and anti-coagulant functions of the central coagulation serine proteinase, thrombin (T). The conversion of the prothrombin (ProT) zymogen into T results in expression of the fibrinogen (Fbg) recognition site (exosite I) and may facilitate binding of a single Na⁺. Na⁺ binding stabilizes the “fast” form of T that mediates the efficient conversion of Fbg into fibrin. The “slow” form of T, lacking Na⁺, shows decreased Fbg substrate activity and slightly enhanced activity towards activation of protein C, which results in a net increase in the anticoagulant activity of T (Di Cera et al., 1995). Conversion of protein C to activated protein C results in cleavage and inactivation of factor Va and VIIIa, which inhibits reactions central to hemostasis. Staphylocoagulase (SC), a conformational activator of ProT, binds ProT with high affinity through proexosite I and imparts T-like activity to the zymogen-activator complex. To determine whether the Na⁺ binding site is present on the SC-(1-325)·ProT complex, kinetic studies in the presence and absence of Na⁺ were performed, using the non-interacting cation, choline to maintain constant ionic strength. Results indicate that indeed Na⁺ binds to the SC-(1-325)·ProT complex with a K_D of 17 ± 2 mM and causes an ~7-fold increase in the activity of SC-(1-325)·ProT complex toward a T-specific tripeptide chromogenic substrate. The chromogenic substrate activity of mouse ProT·SC-(1-325), unlike the human zymogen, was independent of Na⁺, consistent with the recent finding that mouse T lacks a functional Na⁺ binding site (Bush et al., 2006). Fbg clotting studies indicated that human T has 24-fold increased clotting activity in the presence of Na⁺, and the SC-(1-325)·T complex has 13-fold enhanced activity. Surprisingly, much unlike the results obtained from the chromogenic substrate studies, the Fbg clotting activity of the SC-(1-325)·ProT_human complex was less dependent on the presence of Na⁺, as indicated by a 4.1-fold increase in clotting activity for the complex in the presence of Na⁺. Further studies will be necessary to elucidate the functional significance of the Na⁺ binding site on the SC·ProT complex and its effect on Fbg recognition and cleavage.