BOTHROALTERNIN, A THROMBIN INHIBITOR FROM THE VENOM OF BOTHROPS ALTERNATUS

Bothroalternin (MW 27 kDa), a new member of the family of C-type lecti ns is a thrombin inhibitor which was purified from pooled B. alternatu s venom by affinity chromatography on PPACK-thrombin-Sepharose, follow ed by size exclusion and reverse-phase on HPLC columns. Material retai ned on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregatio n of rabbit platelets induced by alpha-thrombin (IC50 = 28 mu g/ mi). A single band of similar to 27 kDa was recognized in Western-blot assa ys using polyclonal antibodies raised against bothrojaracin, a thrombi n inhibitor purified from B. jararaca venom (Zingali et al., 1993). Th e immunological similarity of this fraction to bothrojaracin was confi rmed by ELISA and competitive ELISA, Further purification by size excl usion and reverse-phase on HPLC, produced a single homogenous peak cal led bothroalternin. This protein was highly homologous to bothrojaraci n (95% in its N-terminal sequence-for residues to 25) but displaying l ower inhibitory effect on thrombin induced platelet aggregation (IC50 = 0.19 mu g/ml) compared to bothrojaracin (IC50 = 0.06) Altogether, bo throalternin is a new thrombin inhibitor isolated from Bothrops altern atus venom and has been characterized as a bothrojaracin-like protein. (C) 1998 Elsevier Science Ltd. All rights reserved.