Mutagenesis studies toward understanding the mechanism of the cofactor function of thrombomodulin.

Thrombomodulin (TM) is as essential cofactor in protein C activation by thrombin. To investigate the cofactor effect of TM on the P3-P3' binding specificity of thrombin, we prepared a Gla-domainless protein C (GDPC) and an antithrombin (AT) mutant in which the P3-P3' residues of both molecules were replaced with the corresponding residues of the factor Xa cleavage site in prethrombin-2. TM is known to interact with GDPC, but not AT in the complex. Thrombin did not react with either mutant in the absence of a cofactor. While the thrombin-TM complex also did not react with the AT mutant, it activated the GDPC mutant with a normal k(cat), but an approximately 4-fold impaired K(m) value. Further studies revealed that the active-site directed inhibitor p-aminobenzamidine acts as a competitive inhibitor of both wild-type and GDPC mutant in reaction with the thrombin-TM complex. These results suggest that the interaction of the P3-P3' residues of GDPC with the active-site pocket of the thrombin-TM complex makes a dominant contribution to the binding specificity of the reaction. Moreover, the observation that the GDPC mutant, but not the AT mutant, functions as an effective substrate for the thrombin-TM complex suggests that GDPC interaction with the thrombin-TM complex may be associated with the alteration of the conformation of the P3-P3' residues of the substrate.