Mutation of Glu-80–>Lys results in a protein C mutant that no longer requires Ca2+ for rapid activation by the thrombin-thrombomodulin complex.

Abstract

Binding Ca2+ to a high affinity site in protein C and Gla-domainless protein C (protein C lacking residues 1-44) results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. Protein C modeling studies suggested the single high affinity Ca2+ binding-site might be present in a loop in the protease domain and involve Glu-70 and -80 (chymotrypsin numbering system). This loop, which is a known Ca(2+)-binding site in trypsin, is also conserved in other coagulation proteases, including factors VII, IX,and X. In thrombin, which does not bind Ca2+, Glu-70 is replaced by Lys, creating an internal salt bridge with Glu-80. We constructed and expressed a Gla-domainless protein C mutant in which Glu-80 is replaced with Lys. The activation of the resultant mutant is accelerated by thrombomodulin in a Ca(2+)-independent fashion. Unlike wild type Gla-domainless protein C, Ca2+ no longer inhibits activation of the mutant by free thrombin, and Ca2+ stimulation of chromogenic activity is also absent. The characteristic Ca(2+)-dependent quenching of Gla-domainless protein C intrinsic fluorescence is also absent in the mutant. We conclude that the high affinity Ca(2+)-binding site in protein C critical for zymogen activation involves Glu-80. The Glu-80 to Lys mutation probably results in a salt bridge with Glu-70 that stabilizes protein C zymogen in a conformation similar, if not identical, to the Ca(2+)-stabilized conformation favorable for rapid activation by the thrombin-thrombomodulin complex.