Regular Article Homocysteine decreases extracellular nucleotide hydrolysis in rat platelets Rafael Fernandes Zanin a , Luís Felipe Ingrassia Campesato a , Elizandra Braganhol a , Maria Rosa Chitolina Schetinger b , Angela Terezinha de Souza Wyse a , Ana Maria Oliveira Battastini a, ⁎ a Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, RS, Brazil abstract article info Article history: Received 6 May 2009 Received in revised from 11 August 2009 Accepted 23 September 2009 Available online 21 October 2009 Keywords: Platelet ADP ATP Homocysteine NTPDase Hyperhomocysteinemia is an independent risk factor for atherothrombotic disease. Platelets play an important role in cardiovascular disease and release pro-aggregates mediators when activated, such as ADP, a physiological agonist involved in normal hemostasis and thrombosis. NTPDases and 5'-nucleotidase are ecto-enzymes that hydrolyze ATP, ADP and AMP to adenosine playing an important role on blood flow and thrombogenesis by regulating ADP catabolism. The aim of the present study was evaluate extracellular adenine nucleotide hydrolysis of rat platelets exposed to homocysteine in vitro and in vivo. In vitro homocysteine (Hcy) in the concentration range of 20 to 500 μM caused a significant decrease on ATP (around 30%) and ADP (around 45%) hydrolysis, respectively, while AMP hydrolysis was not altered. Hcy was not able to inhibit the hydrolysis of ATP and ADP catalyzed by purified apyrase at the same concentrations tested in vitro on platelets, suggesting an indirect effect. The inhibitory effect of Hcy on platelets was prevented by antioxidants agents in vitro and in vivo. Furthermore homocysteine treatment increased platelet aggregation induced by ADP. Based on the results presented herein, we propose that inhibition of extracellular ATP and ADP hydrolysis caused by homocysteine was probably due oxidative stress, since antioxidants prevented such effects. These findings may contribute to an increase platelet response to ADP and consequence development of thrombotic risk attributed to hyperhomocysteinemia. © 2009 Elsevier Ltd. All rights reserved. Intoduction Homocysteine (Hcy) is a sulphur-containing amino acid that is closely related to the essential amino acid methionine and to cysteine [1]. Hcy is a metabolic intermediary in the transmethylation dependent of vitamin B12 and transsulfuration dependent of vitamin B6 [1,2]. In normal conditions, the plasma levels of Hcy in humans are low (5– 15 μM); however, genetic defects in the enzymes that metabolize Hcy or environmental factors markedly increase Hcy in the bloodstream [3]. Mild (15-25 μM) and intermediate (25-50 μM) hyperhomocysteinemia are mainly related to acquired factors whereas the severe hyperhomo- cysteinemia (> 100 μM) is associated to genetic defects in cystathionine beta synthase and methylenetetrahydrofolate reductase enzymes [3]. Elevated levels of plasma Hcy are recognized to be an independent risk factor for the development of atherosclerosis and thrombosis [4,5]. The possible mechanisms by which Hcy must be contributing to atherogenesis and thrombosis include increased smooth muscle cell proliferation, cytotoxicity, increased oxidative stress, stimulation of low- density lipoprotein oxidation, induction of endothelial dysfunction, enhanced coagulability and platelet activation [6,7]. In this context, recent studies have demonstrated involvement of Hcy actions linked to oxidative stress [8,9]. Besides that, some works have related Hcy effects on platelets function in vitro and in vivo [10–12]. Leoncini et al. [13] demonstrated that high plasmatic Hcy levels increased Ca 2+ levels and reduced nitric oxide formation, a potent antiaggregating agent of platelets. In addition, studies in vitro have shown that Hcy enhances thromboxane A 2 (TXA 2 ) levels [14] and TXA 2 biosynthesis in patients with homocystinuria [15]. Platelets are fundamental elements to the thrombogenesis process. Its activation leads to release of pro-aggregating mediators, such as ADP [16]. Even at micromolar concentration, ADP is able to induce platelet aggregation in vivo and its hydrolysis to adenosine, an inhibitor of platelet aggregation, is required to balance the hemostatic system [17,18]. Nucleotides exert their function through binding to purinergic receptors (P2), which comprises ionotropic receptors (P2X 1-7 , perme- able to Na + ,K + and Ca +2 ) or G-protein coupled receptors (eight subtypes, P2Y 1, 2, 4, 6, 11, 12, 13, 14 ) [19]. Platelets express P2Y 12 , P2Y 1 and P2X 1 , which are involved in the shape changes, aggregation, thrombox- ane A 2 generation, procoagulant activity, calcium influx, adhesion and thrombus formation [20]. The importance of adenine nucleotides in homeostasis and thrombosis is greatly correlated with the essential role of an enzymatic system that provides an adequate control of these signaling molecules in the extra- cellular medium. Members of several families of enzymes, known as ectonucleotidases, are able to hydrolyze extracellular nucleotides to their Thrombosis Research 125 (2010) e87–e92 ⁎ Corresponding author. Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600-anexo, 90035-003 Porto Alegre, RS, Brazil. Tel.: +55 51 33085554. E-mail address: abattastini@gmail.com (A.M. Oliveira Battastini). 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.09.020 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres