Specificity of activated human protein C.

Area of Science:

• Biochemistry
• Enzymology
• Proteolysis

Background:

• Activated human Protein C is a key serine protease in the coagulation cascade.
• Understanding its substrate specificity is crucial for developing targeted therapeutics and diagnostic tools.

Purpose of the Study:

• To elucidate the substrate specificity of activated human Protein C.
• To identify optimal peptide substrates and inhibitors for activated Protein C.
• To provide insights for developing assays for functional protein C and thrombomodulin.

Main Methods:

• Synthesis and use of peptide p-nitroanilide substrates.
• Synthesis and use of peptidylchloromethane inhibitors.
• Determination of second-order rate constants for enzyme-inhibitor/substrate interactions.

Main Results:

• Activated Protein C exhibited highest activity with substrates containing arginine at the P1 position.
• Optimal substrates and inhibitors featured an apolar residue at the P2 position, accommodating hydrophobic residues like phenylalanine and leucine.
• The enzyme preferred apolar D-amino acid residues at the P3 position.

Conclusions:

• Defined the P1, P2, and P3 residue preferences for activated human Protein C.
• Identified specific substrates and inhibitors suitable for assays of functional protein C and thrombomodulin.
• Highlighted differences in substrate accommodation compared to thrombin.