Characterization of Ôbasparin A,Õ a prothrombin-activating metalloproteinase, from the venom of the snake Bothrops asper that inhibits platelet aggregation and induces defibrination and thrombosis Gilbert D. Lor  ıa, a,b Alexandra Rucavado, a Aura S. Kamiguti, c R. David G. Theakston, d Jay W. Fox, e Alberto Alape, a,f and Jos  e Mar  ıa Guti  errez a, * a Instituto Clodomiro Picado, Universidad de Costa Rica, San Jos e, Costa Rica b Departamento de Microbiolog  ıa e Inmunolog  ıa, Facultad de Microbiolog  ıa, Universidad de Costa Rica, San Jos e, Costa Rica c Department of Haematology, University of Liverpool, Royal Liverpool University Hospital, Liverpool, UK d Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, UK e Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA, USA f Departamento de Bioqu  ımica, Escuela de Medicina, Universidad de Costa Rica, San Jos e, Costa Rica Received 6 June 2003, and in revised form 17 July 2003 Abstract A prothrombin activator, named Ôbasparin A,Õ was isolated from the venom of the crotaline snake Bothrops asper, the species responsible for the majority of snakebite cases in Central America. It is an acidic (pI 5.4), 70 kDa, single chain P-III metallopro- teinase comprising, in addition to the metalloproteinase domain, disintegrin-like, and high-cysteine domains. Basparin A is a glycoprotein displaying immunological cross-reactivity with BaH1, a P-III hemorrhagic metalloproteinase isolated from the same venom. It activates prothrombin through the formation of meizothrombin, without requiring additional cofactors; it is, therefore, a class A snake venom prothrombin activator. In contrast with most venom metalloproteinases, it does not degrade components of the extracellular matrix. Apart from its clotting activity, basparin A inhibits collagen-dependent platelet aggregation in vitro, an effect that does not depend on proteolytic activity. Clotting activity on human plasma is not abrogated by the plasma proteinase inhibitors a 2 macroglobulin and murinoglobulin, whereas activity is completely inhibited by Costa Rican polyvalent (Crotalinae) anti-venom. Basparin A does not induce local tissue alterations, such as hemorrhage, myonecrosis, and edema, in mice. Moreover, it does not induce systemic hemorrhage, thrombocytopenia nor prolongation of the bleeding time following intravenous adminis- tration. At low doses, the only observed effect induced by basparin A, when injected intravenously or intramuscularly into mice, is defibrin(ogen)ation. At higher doses, intravenous administration resulted in sudden death due to numerous occluding thrombi in pulmonary vessels. Basparin A is likely to play an important role in the coagulopathy associated with B. asper envenoming. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Bothrops asper venom; Prothrombin activator; Metalloproteinase; Defibrination; Platelet aggregation inhibition Snake venoms possess a variety of components that affect hemostasis in many different ways. Some interfere with platelet function, whereas others act on clotting factors, either promoting or inhibiting coagulation [1,2]. Accordingly, clotting disorders constitute a common manifestation of snakebite envenomings, particularly in the case of species of the family Viperidae [3–6]. Procoagulant enzymes, such as thrombin-like serine proteinases and metalloproteinases displaying factor X- and prothrombin-activating effects, mediate coagu- lopathy associated with defibrin(ogen)ation and dis- seminated intravascular coagulation in envenomed patients [1,2]. Such clotting disorders may also con- tribute to the hemorrhagic syndrome characteristic of Archives of Biochemistry and Biophysics 418 (2003) 13–24 www.elsevier.com/locate/yabbi ABB * Corresponding author. Fax: +506-2920485. E-mail address: jgutierr@icp.ucr.ac.cr (J.M. Guti  errez). 0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0003-9861(03)00385-0