In vitro and in vivo anti-coagulant activity and toxicological studies of marine sulfated glycosaminoglycans Fatma Krichen a , Zohra Ghlissi b , Ikram Ben Amor c , Nadhem Sayari a , Rim Kallel d , Jalel Gargouri c , Zouheir Sahnoun b , Tahia Boudawara d , Ali Bougatef a, * a Laboratoire d’Amélioration des Plantes et Valorisation des Agro-Ressources, ENIS, Université de Sfax, 3038 Tunisia b Unité de Recherche Pharmacologie et Toxicologie des Xénobiotiques, Faculté de Médecine de Sfax, Sfax, 3029, Tunisia c Centre Régional de Transfusion Sanguine de Sfax, Route El-Ain Km 0.5, CP 3003 Sfax, Tunisia d Laboratoire Anatomie Pathologie, CHU Habib Bourguiba, Sfax 3029, Tunisia A R T I C L E I N F O Article history: Received 9 June 2016 Received in revised form 18 September 2016 Accepted 1 November 2016 Keywords: Anticoagulant activity Fish skins Sulfated glycosaminoglycans Toxicological studies A B S T R A C T The present study aimed to characterize and evaluate the in vitro and in vivo anticoagulant activity of sulfated glycosaminoglycans from the skins of smooth hound (SHSG) and grey triggerfish (GTSG). The analysis of SHSG and GTSG with acetate cellulose electrophoresis in Zn-acetate revealed the presence of hyaluronic acid (HA), chondroitin sulfate (CS) and dermatan sulfate (DS). Both glycosaminoglycans were evaluated for their in vitro anticoagulant activities using activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. SHSG and GTSG and calciparin were tested as in vivo anticoagulants by subcutaneous (s.c) injection to adult female Wistar rats in a concentration of 75 mg/kg of body weight. The administration of SHSG, GTSG and calciparin to rats induced a significant decrease of platelet rates compared to the control. The aPTT assay of SHSG and GTSG was prolonged 1.3 and 1.23-fold respectively compared with the control. Toxicity studies were performed to investigate whether or not SHSG and GTSG can cause pathological changes in the liver, proteins and DNA. The concentration and catalytic activity of liver oxidative stress markers and enzymes, respectively, as well as the observed hepatic morphological changes indicated that calciparin induced hepatic toxicity and oxidative damage in the liver. The higher activity and lower toxicity of SHSG and GTSG recommended these compounds as a better drug candidate than calciparin. ã 2016 Elsevier GmbH. All rights reserved. 1. Introduction The physiological process involving blood coagulation and its interruption is the second steps of haemostasis. The imbalance in this process is the origin of many ischemic and hemorrhagic diseases. Heparin is far used as an anticoagulant and antithrombotic drug, mainly by binding to antithrombin (AT) and heparin cofactor II (HCII), accelerating the rate at which these protease inhibitors form complexes with enzymes implied in blood clotting, particularly – factor Xa and thrombin, inactivate them (Casu, 1985; Bourin et al., 1993; Mourão and Pereira, 1999). Recently, the low molecular weight heparin became an alternative for prophylaxis of venous thromboembolism because of its longer half-life in blood circulation system, higher bioavailability and lower variability in coagulation response. The big advantage is the possibility of administration this drug subcutaneously at fixed doses without need for diagnostic laboratory monitoring (Paw- laczyk et al., 2011). Although, the clinical use of heparin has limitations since its anticoagulant effect is unpredictable, resulting in risk of bleeding (Warkentin, 2006). Heparin suffers other disadvantages including the problem of heparin-induced throm- bocytopenia, poor bioavailability, and risk of animal diseases which can possibly affect human health such as Mad Cow disease. So there is still a clinical need for new parental anticoagulants that are effective and safe when used in conjunction with either fibrinolytic therapy or antiplatelet agents in patients with cardiovascular diseases (Hirsh, 2001). Therefore, the replacement of heparin by natural or chemical sulfated polysaccharides may have benefits due to health (Mourão and Pereira, 1999; Maas et al., * Corresponding author. E-mail address: ali.bougatef79@gmail.com (A. Bougatef). http://dx.doi.org/10.1016/j.etp.2016.11.002 0940-2993/ã 2016 Elsevier GmbH. All rights reserved. Experimental and Toxicologic Pathology xxx (2016) xxx–xxx G Model ETP 50983 No. of Pages 9 Please cite this article in press as: F. Krichen, et al., In vitro and in vivo anti-coagulant activity and toxicological studies of marine sulfated glycosaminoglycans, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.11.002 Contents lists available at ScienceDirect Experimental and Toxicologic Pathology journal homepage: www.else vie r.de /et p