CA2+ DEPENDENCE OF THE INTERACTIONS BETWEEN PROTEIN-C, THROMBIN, AND THE ELASTASE FRAGMENT OF THROMBOMODULIN - ANALYSIS BY ULTRACENTRIFUGATION

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3-degrees-C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (K(d) < 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (K(d) almost-equal-to 4-mu-M in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (K(d) almost-equal-to 1-mu-M) and what appears to be a 1:2 thrombin-protein C complex. The K(d) for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca2+-independent 1:1 complex (K(d) almost-equal-to 80-mu-M). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent K(d) almost-equal-to 1-mu-M. Nearly identical binding to elTM-thrombin was observed for activated protein C and protein C, suggesting that there is little discrimination between substrate and product. This was confirmed kinetically. Activated protein C is a potent inhibitor of its own formation both by thrombin alone (K(i) almost-equal-to 0.5-mu-M) and by thrombin bound to elTM (K(i) almost-equal-to 4-mu-M). Bovine protein C lacking the 41 amino acid gamma-carboxyglutamic acid containing peptide (Gla domain) exhibited virtually no interaction with either elTM or elTM-thrombin, whereas the Gla domain competed effectively and specifically with protein C in these interactions. Finally, binding of the Gla domain to elTM-thrombin was observed. These results indicate that the Gla domain may be involved directly in the binding of protein C to both thrombin and elTM.