Determination of the structure of two novel echistatin variants and comparison of the ability of echistatin variants to inhibit aggregation of platelets from different species

Abstract

Two new variants of short disintegrins were purified from the venom of Echis carinatus leakeyi and named echistatin beta and gamma. These proteins were found to be about 85% similar in amino acid sequence to echistatin alpha which has been well studied. The disulphide pattern of echistatin gamma appeared to be identical with that of echistatin alpha. They all contain the adhesive recognition sequence Arg-Gly-Asp (RGD) but inhibit the aggregation of platelets from human and other mammals with different potencies. Echistatin beta and alpha are far more effective on platelets from humans and guinea pigs than those from rabbits and rats whereas echistatin gamma is less discriminating of the platelets of the species tested. This species-dependent platelet sensitivity 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 loop, rather than to the C-terminal variations after residue 46. Taking advantage of the presence of methionine residues flanking 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 protein showed c.d. and fluorescent spectra grossly similar to the intact 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.

Glycan structures and intrageneric variations of venom acidic phospholipases A2 from Tropidolaemus pitvipers

“…Some pitviper venoms contain several isoforms of acidic PLA 2 s [19–21], whereas some may express only one acidic PLA 2 . It is also known that platelets from different vertebrates may show different responses towards exogenous agents [23]. It has been suggested that dietary shifts may select genes that are effectively adapted to the new prey ecology [24,25].…”

Chloramine T‐induced structural and biochemical changes in echistatin

“…Echistatin was radiolabeled with 125 I either by lactoperoxidase (LP) or chloramine T (CT) method and its binding to purified α v β 3 was measured using a solid phase receptor binding assay as described in Section 2 [17, 26]. Incubation of purified α v β 3 (10 ng) with increasing concentrations of LP‐ or CT‐labeled echistatin for 3 h resulted in a saturable and specific binding (Fig.…”

“…Hence, we used an alternate approach. It is believed that the methionine residues are susceptible to oxidation during chloramine T treatment [26]. Echistatin contains a single methionine residue close to the RGD sequence [1–3].…”

Differential Expression and Geographic Variation of the Venom Phospholipases A2 of Calloselasma rhodostoma and Trimeresurus mucrosquamatus

“…The aggregation was initiated by the addition of 10 M ADP and followed for a period of 5 min. The IC 50 value of a PLA 2 , defined as the concentration to inhibit 50% of the aggregation of PRP caused by 10 M ADP, was determined from the dose-dependence curve (13).…”

“…6). It is known that the platelets from different mammals may have different susceptibilities toward antiplatelet agents (13). Thus, expression of the venom antiplatelet PLA 2 s possibly has been evolved to cope with the feeding ecology of the snakes (8,9,24).…”