Alpha 2-macroglobulin of rabbits inhibits the habutobin activity.

We have investigated whether alpha 2-macroglobulin (alpha 2M) of rabbits inhibits the activity of habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom. Rabbit alpha 2M was purified with ultracentrifugation, gel filtration on Sepharose 6B and ion exchange chromatography on DEAE-Sephacel. Inhibitory effects of rabbit alpha 2M on habutobin was determined by the fibrin forming activity, digestion of A alpha chain of fibrinogen, and the release of fibrinopeptide A from fibrinogen. As a results, purified alpha 2M showed a single band with high molecular weight, around 800,000 mol. wt by means of polyacrylamide gel electrophoresis using PhastSystem. Besides inhibiting amidolytic and caseinolytic activity of porcine trypsin, it has inhibited the activity of habutobin: that is, in the presence of rabbit alpha 2M, fibrin forming activity of habutobin was decreased and habutobin-induced digestion of A alpha chain was inhibited. In addition, rabbit alpha 2M reduced habutobin-induced release of fibrinopeptide A from rabbit fibrinogen.

Pharmacokinetics of habutobin in rabbits.

Following the administration of habutobin, the fibrinogen level in the circulating blood of the rabbits decreased. These results showed that the activity of habutobin was retained in vivo. The plasma level of habutobin was determined by a ELISA-double sandwich method. The pharmacokinetics of habutobin from Trimeresurus flavoviridis venom was studied in rabbits following i.v. administration of 50 micrograms kg-1 of habutobin. The time course of the plasma concentration of habutobin fitted a two-compartment open model. The half-life of the distribution phase was 4.43 +/- 1.28 min and that of the elimination phase was 50.42 +/- 7.89 min. The area under the plasma concentration-time curve (AUC) was 38.69 +/- 6.68 micrograms min ml-1. The total body clearance was 3.82 +/- 1.08 ml min-1. When the steady state was reached, the concentration ratio of habutobin in the tissue (Ct) to that in the plasma (Cc), Ct:Cc was 0.47:1. These findings suggest that relatively little habutobin tended to remain in the tissue.