Enzymatic inactivation of human antithrombin III. Limited proteolysis of the inhibitor by snake venom proteinases in the presence of heparin.

Incubation of human plasma antithrombin III with Crotalid, Viperid and Colubrid snake venoms resulted in the enzymatic inactivation of the inhibitor, as evidenced by a gradual loss of inhibitory activity against trypsin and thrombin. This indicates that proteinases which selectively inactivate antithrombin III are widespread among the families of poisonous snakes. The inactivation was due to metalloproteinases present in the venoms, since the reaction could be terminated by the addition of EDTA. Elapid venoms were tested and shown to be devoid of activity on antithrombin III. Preincubation of the anthrombin III with heparin accelerated the reaction, and less venom was required to achieve total inactivation. Several venoms had very little effect on antithrombin III in the absence of heparin, but inactivated the inhibitor completely within 2 h when heparin was present. Optimal rates of inactivation were observed with antithrombin III: heparin ratios of approx. 3 : 1. When heparin was present in excess, the inactivation was retarded. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the antithrombin III/venom proteinase reaction mixtures indicated that intact antithrombin III (63 000 daltons) was converted to an inactive form (57 500 daltons) by limited proteolysis. No complex formation between antithrombin III and venom proteinases was detectable. The inactivating cleavage occurred within a disulfide loop of the antithrombin III molecule, since the lower molecular weight species was detected only under reducing conditions.