Thrombin has dual trypsin-like and chymotrypsin-like specificity

BACKGROUND:The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question as to whether thrombin is the physiological activator of protein C across vertebrates. OBJECTIVE:Establish if thrombin can cleave at Trp residues. METHODS:The activity of thrombin wild-type and mutant D189S was tested with a library of chromogenic substrates and toward protein C wild-type and mutants carrying substitutions at the site of cleavage. RESULTS:Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable to that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond. CONCLUSIONS:The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader than currently assumed.