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Study of breaking down Fibrin Amyloid Clots

Counteracting effect of glycyrrhizin on the hemostatic
abnormalities induced by Bothrops jararaca snake venom
1Mariane Assafim, 1Marcos S. Ferreira, 1Fla´ via S. Frattani, 2Jorge A. Guimara˜ es,
1Robson Q. Monteiro & *,1Russolina B. Zingali
1Instituto de Bioquı´mica Me´dica, Programa de Biologia Estrutural, Centro de Cieˆncias da Sau´ de, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil and 2Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
1 Envenomation by the snake Bothrops jararaca is typically associated with hemostatic
abnormalities including pro- and anticoagulant disturbances. Glycyrrhizin (GL) is a plant-derived
thrombin inhibitor that also exhibits in vivo antithrombotic properties. Here, we evaluated the ability
of GL to counteract the hemostatic abnormalities promoted by B. jararaca venom.
2 GL inhibited the human fibrinogen clotting (IC50¼B1.0 mg ml1; 1.2mM), H-D-phenylalanyl-
L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride hydrolysis (IC50¼B0.4 mg ml1; 0.47mM) and
platelet aggregation (IC50¼B0.28mg ml1; 0.33mM) induced by B. jararaca venom, in vitro.
3 The in vivo effect of GL was tested in rats using a model of venous thrombosis in which
intravenous (i.v.) administration of B. jararaca venom (100 mgkg1) produced in all animals a
thrombus with a mean weight of 10.671.7 mg.
4 Prior administration of GL (180mg kg1) or antibothropic serum (27 ml kg1) inhibited thrombus
formation by 86 and 67%, respectively. Remarkably, co-administration of ineffective doses of GL and
antibothropic serum markedly decreased thrombus weight, suggesting a synergistic effect.
5 Co-administration of GL with antibothropic serum abolished venom-induced bleeding. Ex vivo
clotting times showed that rat plasma was non-clotting after i.v. administration of B. jararaca venom.
Treatment with GL, antibothropic serum or both before venom administration efficiently prevented
this abnormality.
6 Altogether, we demonstrate here that GL prevents both in vitro and in vivo venom-induced changes
in hemostasis, suggesting a potential antiophidic activity.
British Journal of Pharmacology (2006) 148, 807–813. doi:10.1038/sj.bjp.0706786;
published online 5 June 2006
Keywords: Glycyrrhizin; B. jararaca venom; venous thrombosis; antibothropic serum
Abbreviations: APTT, activated partial thromboplastin time; GL, glycyrrhizin; PT, prothrombin time; S-2238, H-Dphenylalanyl-
L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride
In Brazil, snakebite accidents represent an important public
health problem, with the genus Bothrops (including the
species B. jararaca, B. moojeni, B. erythromelas and B. atrox)
being responsible for more than 90% of the registered cases
(Cardoso, 1990). A number of proteins from bothropic
venoms interfere with the hemostatic system and have been
characterized in detail (Markland, 1998; Castro et al., 2004)
as procoagulant, anticoagulant or fibrinolytic factors (Marsh,
1994). In addition, several components may alter platelet
function displaying either pro- or antiaggregating properties
(Markland, 1998). The signs and symptoms presented by
patients include local (pain, swelling, ecchymosis and necrosis)
and systemic (blood incoagulability, hemorrhage) manifestations
(Kamiguti et al., 1986; Maruyama et al., 1990).
Envenomation by these snakes generally results in strong
coagulopathy with persistent bleeding owing to fibrinogen
degradation as well as consumption of blood coagulation
factors (Maruyama et al., 1990; Kamiguti et al., 1991). On the
other hand, massive blood clotting activation may cause
thrombosis in small vessels (Thomas et al., 1995). Therefore,
severe cases of envenomotion may lead to permanent tissue
loss, disability or amputation.
The effective therapeutic treatment for ophidian accidents
nowadays is serotherapy (Heard et al., 1999). Nevertheless,
alternative medication has been proposed. The use of heparin
was first proposed in the late 40s (Ahuja et al., 1946). Although
it is a well-known anticoagulant, heparin does not neutralize
the thrombin-like activity of bothropic venoms and does
not prevent the defibrinogenating syndrome induced by these
venoms (Nahas et al., 1975). In fact, structural differences
between thrombin and venom-derived thrombin-like enzymes
impair the inhibitory action of heparin–antithrombin complex
as well as other coagulation inhibitors such as hirudin (Castro
et al., 2004).
A number of studies have also reported the use of plants
(or their extracts) known in popular medicine for treatment
of snakebite (Mors et al., 1989; 2000; Mors, 1991; Martz, 1992;
Houghton & Osibogun, 1993; Houghton & Skari, 1994).
Among these studies, some focus on the inhibition of snake
*Author for correspondence at: Instituto de Bioquı´mica Me´dica,
Universidade Federal do Rio de Janeiro, Av Trompovski s/no, CCS
Bloco H2sala 04, Cidade Universitaria, 21941-590 Rio de Janeiro, RJ,
Brazil. E-mail:
British Journal of Pharmacology (2006) 148, 807–813 & 2006 Nature Publishing Group All rights reserved 0007–1188/06 $30.00
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