Inhibitory effect of fucoidan on the activities of crotaline snake venom myotoxic phospholipases A 2 Yamileth Angulo a,b,* , Bruno Lomonte a a Instituto Clodomiro Picado, Facultad de Microbiologı ´a, Universidad de Costa Rica, San Jose ´, Costa Rica b Departamento de Bioquı ´mica, Escuela de Medicina, Universidad de Costa Rica, San Jose ´, Costa Rica Received 25 June 2003; accepted 24 July 2003 Abstract Myotoxic phospholipases A 2 account for most of the muscle necrosis that results from envenenomation by crotaline snakes. In this study, we investigated the protective effect of fucoidan, a natural sulfated polysaccharide obtained from the brown seaweed Fucus vesiculosus, against the cytotoxic and myotoxic activities of a group of phospholipase A 2 myotoxins from crotaline snake venoms: Bothrops asper myotoxins I, II, III, and IV, Cerrophidion godmani myotoxins I and II, Atropoides nummifer myotoxins I and II, and Bothriechis schlegelii myotoxin I. All of the toxins tested were efficiently inhibited by fucoidan, in both their cytotoxic and myotoxic effects. The basis for this inhibition appears to be the rapid formation of complexes between fucoidan and myotoxins, as evidenced by turbidimetric analysis. The possible binding site of fucoidan on the myotoxins was investigated using short synthetic peptides that represent the membrane-damaging region (residues 115–129) for three of these toxins. Fucoidan clearly inhibited the cytolytic activity of the peptides, indicating its ability to interact with the C-terminal myotoxic region of these phospholipases A 2 . Fucoidan significantly inhibited muscle damage in mice, when administered locally, immediately after experimental envenomation with crude venom from B. asper . These results encourage further studies of sulfated fucans as compounds of potential use to improve the treatment of envenomations by crotaline snakes. # 2003 Elsevier Inc. All rights reserved. Keywords: Fucoidan; Myotoxin; Phospholipase A 2 ; Snake venom; Inhibition; Polysaccharide 1. Introduction Phospholipases A 2 (PLA 2 ; EC 3.1.1.4) are ubiquitous enzymes that catalyze the hydrolysis of the sn-2 position of glycerophospholipids, leading to production of free fatty acids and lysophospholipids [1]. Although all PLA 2 s cat- alyze essentially the same reaction, their biologic activities vary considerably. In snake venoms, PLA 2 s have evolved into potent toxins with diverse specific activities such as neurotoxicity, myotoxicity, stimulation or inhibition of platelet aggregation, anticoagulant, hypotensive, cardio- toxic, edema-inducing [2], and bactericidal [3] effects. Crotaline snakes (family Viperidae, subfamily Crotali- nae) are widely distributed in America [4], comprising a large number of species which are responsible for the majority of snakebite envenomations in this continent [5]. Myotoxic PLA 2 s have been described in the venoms of crotaline species from the genera Bothrops, Agkistrodon, Porthidium, Trimeresurus, Atropoides, Crotalus, Bothrie- chis, Calloselasma, and Cerrophidion [6–10], as compo- nents that have a prominent role in the induction of skeletal muscle necrosis. These PLA 2 s are structurally classified into group IIA [11], belonging either to the enzymatically- active (Asp49-type) or enzymatically-inactive (Lys49-type) subgroups. The clinical relevance of myonecrosis in snakebite envenomations has motivated the search for nonimmune neutralizing molecules against myotoxic PLA 2 s, including natural inhibitors of animal and plant origins [12–16], that could complement conventional antivenom therapy. Among the different types of inhibitors studied, some anionic polysaccharides such as heparin have been shown to neutralize myotoxic PLA 2 s isolated from Bothrops jararacussu [17] and B. asper [18,19] venoms. On this basis, we decided to investigate if fucoidan, a complex sulfated polysaccharide extracted from the brown seaweed Fucus vesiculosus, could inhibit the toxic activities of a group of myotoxic PLA 2 s from crotaline venoms. Biochemical Pharmacology 66 (2003) 1993–2000 0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-2952(03)00579-3 * Corresponding author. Tel.: þ506-229-0344; fax: þ506-292-0485. E-mail address: yangulo@cariari.ucr.ac.cr (Y. Angulo). Abbreviations: PLA 2 , phospholipase A 2 ; CK, creatine kinase; LDH, lactic dehydrogenase.