Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the β subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin α and β subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the α subunit and computer models of the α and β subunits. The sequence of α echicetin is highly similar to the α and β chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated α or β subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor–ristocetin or α-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin β subunit might have been due to partial reduction of the protein.

Several studies have shown that Asp-49 is the residue that controls calcium binding in, and so plays a critical role in the calcium-mediated activation of, low- M r group I-III phospholipases A 2 (PLA 2 s). The present paper provides experimental evidence that Asp-49 is not an absolute prerequisite for the enzymic activity of PLA 2 s, and that proteins with amino acid(s) other than Asp at position 49 can exhibit significant phospholipase activity. The purification, complete amino acid sequence and characterization of ecarpholin S, a PLA 2 from Echis carinatus sochureki (saw-scaled viper) venom, is described. This single-chain, 122-amino-acid, basic (pI 7.9) protein is a group II PLA 2 . Although Asp-49 is replaced by Ser and Tyr-28 by Phe (both of these positions being involved in the Ca 2+ -binding site of PLA 2 s), the lipolysis of soybean phosphatidylcholine and egg yolk in the presence of 10 mM CaCl 2 was 1.5 times and 2.9 times greater respectively with ecarpholin S than with recombinant human group II PLA 2 . The Ca 2+ -dependencies of the enzymic activities of ecarpholin S and rPLA 2 were found to be similar. Ecarpholin S added to washed platelets induced aggregation; the presence of Ca 2+ was a prerequisite for this platelet-aggregating effect. Computer modelling of the Ca 2+ -binding site of Ser-49 PLA 2 compared with the Asp-49 and Lys-49 forms, for which crystallographic data exist, shows that the Ca 2+ -binding site is sterically blocked by Lys-49 but not by Ser-49; in the latter, the Ser hydroxy group may replace the Asp carboxylate in stabilization of Ca 2+ binding. Sequence comparisons of ecarpholin S and other low- M r PLA 2 s predicts the presence of a Ser-49 group in the protein family of low- M r PLA 2 s that is distinct from the Asp-49 and Lys-49 groups.