Charge reversal at the P3′ position in protein C optimally enhances thrombin affinity and activation rate

We have examined the properties of several human protein C (HPC) derivatives with substitutions for acidic residues near the thrombin cleavage site, including changing the P3′ Asp to Asn (D172N), Gly (D172G), Ala (D172A), or Lys (D172K). The rate of thrombin‐catalyzed activation of D172N, D172G, and D172A was increased 4‐9‐fold compared to wildtype HPC, primarily due to a reduction in the inhibitory effect of calcium and a resulting increase in affinity for free a‐thrombin. There was no significant increase in activation rate or affinity with these 3 derivatives in the absence of calcium, confirming that P3′ Asp affects calcium dependency in the native protein C molecule. With charge reversal at P3′ (D172K), there was a 30‐fold increase in activation rate in the presence of calcium, but unlike the other derivatives, there was a substantial effect (5‐fold) on the activation rate and affinity for free α‐thrombin in the absence of calcium. Thus, protein C affinity for thrombin appears to be influenced by a combination of calcium‐dependent and ‐independent effects of the acidic P3′ residue.