MUTATION OF GLU-80 -] LYS RESULTS IN A PROTEIN-C MUTANT THAT NO LONGER REQUIRES CA2-THROMBOMODULIN COMPLEX( FOR RAPID ACTIVATION BY THE THROMBIN)

Binding Ca2+ to a high affinity site in protein C and Gla-domainless p rotein 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 studi es suggested the single high affinity Ca2+ binding-site might be prese nt in a loop in the protease domain and involve Glu-70 and -80 (chymot rypsin numbering system). This loop, which is a known Ca2+-binding sit e in trypsin, is also conserved in other coagulation proteases, includ ing 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. W e constructed and expressed a Gla-domainless protein C mutant in which Glu-80 is replaced with Lys. The activation of the resultant mutant i s accelerated by thrombomodulin in a Ca2+-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 a ctivity is also absent. The characteristic Ca2+-dependent quenching of Gla-domainless protein C intrinsic fluorescence is also absent in the mutant. We conclude that the high affinity Ca2+-binding site in prote in C critical for zymogen activation involves Glu-80. The Glu-80 to Ly s mutation probably results in a salt bridge with Glu-70 that stabiliz es protein C zymogen in a conformation similar, if not identical, to t he Ca2+-stabilized conformation favorable for rapid activation by the thrombin-thrombomodulin complex.