DETERMINATION OF THE STRUCTURE OF 2 NOVEL ECHISTATIN VARIANTS AND COMPARISON OF THE ABILITY OF ECHISTATIN VARIANTS TO INHIBIT AGGREGATION OF PLATELETS FROM DIFFERENT SPECIES

Two new variants of short disintegrins were purified from the venom of Echis carinatus leakeyi and named echistatin beta and gamma. These pr oteins were found to be about 85% similar in amino acid sequence to ec histatin alpha which has been well studied. The disulphide pattern of echistatin gamma appeared to be identical with that of echistatin alph a. They all contain the adhesive recognition sequence Arg-Gly-Asp (RGD ) but inhibit the aggregation of platelets from human and other mammal s with different potencies. Echistatin beta and alpha are far more eff ective on platelets from humans and guinea pigs than those from rabbit s and rats whereas echistatin gamma is less discriminating of the plat elets of the species tested. This species-dependent platelet sensitivi ty to echistatin beta and gamma could be attributed to the variations in residues 15, 21, 22 and 27, which are close to or within the RGD lo op, rather than to the C-terminal variations after residue 46. Taking advantage of the presence of methionine residues Banking both sides of the ARGDDM motif in echistatin gamma, we deleted this hexapeptide by CNBr cleavage to produce des-(23-28)-echistatin gamma. The modified pr otein showed c.d. and fluorescent spectra grossly similar to the intac t echistatin but its antiplatelet potency decreased more than 200-fold . We thus propose that a favourable conformation of the RGD region is responsible mainly for the high-affinity binding of echistatin to the platelet glycoprotein IIb-IIIa as shown previously for the binding of medium-size disintegrin.