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

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Abstract
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.