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 wild-type HPC, primarily due to a reduction in the inhibitory effect of calcium and a resulting increase in affinity for free alpha-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 alpha-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.
Richardson
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MA
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285-0434.
Gerlitz
B
B
Grinnell
B W
BW
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D004563:Electrochemistry; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011486:Protein C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology
D011486:Protein C; D013917:Thrombin; D002118:Calcium