Kallikrein-Like Activity Of Crotalase, A Snake Venom Enzyme Which Clots Fibrinogen

During amino acid sequence determination of crotalase, a thrombin-like enzyme from Crotalus adamanteus (eastern diamondback rattlesnake) venom, we found that in addition to the expected structural homology with bovine thrombin, there was even greater homology with porcine pancreatic kallikrein. In exploring further the similarity between crotalase and kallikrein, several completely unexpected and interesting observations were made. First, crotalase was rapidly and specifically inhibited by the tripeptide, affinity-labelling chloromethyl ketone inhibitor Pro-Phe-Arg-CH₂Cl, which is known to be a specific inhibitor of urinary kallikrein. Further, crotalase exhibits significant activity not only with the thrombin chromogenic substrate S-2238 (H-D-Phe- pipecolyl-Arg-p-nitroanilide) and the general serine protease substrate S-2160 (N-benzoyl-Fhe-Val-Arg-p-nitroanilide) but also with the plasma kallikrein substrate S-2302 (H-D- Pro-Phe-Arg-p-nitroanilide) and the glandular kallikrein substrate S-2266 (H-D-Val-Leu-Arg-p-nitroanilide). Additionally, SDS polyacrylamide gel electrophoresis reveals that crotalase cleaves the plasma kallikrein susceptible bonds in human high molecular weight kiniogen (HMWK) producing intermediates with procoagulant activity. Analyses for bradykinin release from HMWK are presently in progress. Interestingly, one of the normal activities of plasma kallikrein, the activation of human plasminogen, was not one of the activities possessed by crotalase.

In summary, it would appear that crotalase has significant kallikrein-like activity. Whether this will prove to be of importance in the ongoing clinical application of fibrinogen clotting snake venom enzymes, such as ancrod and batroxobin, remains to be shown.