CALCIUM INHIBITION OF THE ACTIVATION OF PROTEIN-C BY THROMBIN ROLE OF THE P3 AND P3' RESIDUES

Protein C, a precursor to a natural plasma anticoagulant, and the platelet thrombin receptor, involved in cell activation, both require proteolytic cleavage to be activated. In humans, the sequences adjacent to the scissile bond of protein C, DPR/LID and the thrombin receptor DPR/SFL are similar. Previous studies with Asp-->Gly mutants indicated that both the P3 and the P3' Asp residues make either peptides or protein C a poor substrate for free thrombin, but thrombin interaction with thrombomodulin overcomes these inhibitory interactions. Similar mechanisms are probably operative in the thrombin receptor. In rodents, the P3 Asp residue of the human thrombin receptor is replaced by Asn and in protein C, the P3' residue is Asn. To determine the functional significance of these Asp-->Asn substitutions, the Asp in the P3 or P3' position of human protein C was changed to Asn. The resultant mutants were still resistant to activation by thrombin, and still required Ca2+ for activation by thrombin-thrombomodulin complex. We conclude that, unlike activation of the Asp-->Gly mutants by thrombin, activation of the P3 and P3' Asp-->Asn mutants is still potently inhibited by physiological Ca2+. Furthermore, even though the charge has been deleted, thrombomodulin acceleration is retained.