COMPARATIVE STUDY ON THE EFFECTS OF HEMISCORPIUS LEPTURUS, HOTTENTOTTA SAULCYI AND MESOBUTHUS EUPEUS SCORPIONS ON BLOOD HEMOSTASIS IN RATS

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1 Biochem. Cell. Arch. Vol. 11, No. 1, pp , 2011 ISSN COMPARATIVE STUDY ON THE EFFECTS OF HEMISCORPIUS LEPTURUS, HOTTENTOTTA SAULCYI AND MESOBUTHUS EUPEUS SCORPIONS ON BLOOD HEMOSTASIS IN RATS M. Razi Jalali 1, S. R. Fatemi 2 and T. Shirali 3 ¹Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran. ²Department of Basic Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran. ³Graduated of Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran. jalali_m@scu.ac.ir, mrazijalali@yahoo.com (Accepted 15 February 2011) ABSTRACT Scorpion venom can induce significant alterations in blood parameters. Several publications have reported on the effects of scorpion envenomation on blood coagulation. The aim of this research was to compare the effects of H. lepturus, M. eupeus and H. saulcyi scorpion venoms on blood coagulation system in rats. For this aim 96 Wistar rats were divided randomly into 4 equal groups (A, B, C and D). Group A, received normal saline intraperitonealy as control group. Group B, C and D received H. lepturus (3mg/kg), M. eupeus (1.5mg/kg) and H. saulcyi (1mg/kg) venom ip, respectively. Blood samples were taken by heart puncture with sodium citrate 3.6%, at 1, 3, 24 and 48 hours after envenomation. Samples were used for evaluation of Prothrombin time (PT), Activated partial thromboplastin time (APTT), fibrinogen level and platelet count. Statistical analysis was done by SPSS_ 16, Anova test for evaluation of the data. Results showed that PT and APTT in different times after envenomatin were increased significantly in groups B and C compare to control group. The maximum alterations in these tests were seen at 3 hours after envenomatin in both groups. Plasma fibrinogen also was increased in group B and C at 1 and 3 hours after envenomatin. After 24 and 48 hours plasma fibrinogen was gradually reduced to the level of control group. On the other hand the plasma fibrinogen significantly increased after envenomation with H. saulcyi at different times. Platelet count was decreased significantly at 3 and 24 hours after envenomatin in groups B and C. However, Platelet count was increased gradually. Based on the results of this study it seems that the effects of H. lepturus and M. eupeus on blood hemostasis were similar to each other. In addition it seems that H. saulcyi has no significant effects on blood hemostasis and the diagnosis of the effective mechanism on hemostatic system needs more research. Key words : Scorpion, venom, coagulation test, rat. INTRODUCTION Scorpion sting is an acute life-threatening, time-limiting medical emergency more commonly seen in villagers. Among the different species of scorpions in Iran, Hemiscorpius lepturus, Mesobuthus eupeus and Hottentota saulcyi are of medical importance. Investigations showed that following these scorpion sting, victims have presented severe pain, hyperemia, edema, thirst, dry mouth, hypotension, nausea, hypertension, elevated bronchial secretion, difficulty in breathing, tachycardia and cyanosis (Ozkan et al, 2008). Changes in blood coagulation profiles and presence of acute disseminated intravascular coagulation (DIC) were observed in animals and man after scorpion venom injection (Murthy et al, 1988). Direct effect of toxins on neurons could contribute to seizures and encephalopathy in some children. However, hemiplegia and other neurological lesions have been attributed to fibrin deposition resulting from DIC. These were confirmed in autopsy studies of human scorpion sting victims. Acute rise in blood pressure due to sympathetic stimulation, rupture of un-protected perforating arteries, intracerebral hemorrhage and cerebral infarction due to DIC are possibly related to CNS manifestations. Some species of scorpion (Tityus discrepans) cause abundant micro thrombi in rabbit lungs. It is suggested that these clotting alterations are fundamental to produce acute lung injury and increased alveolocapillary membrane permeability (D Suze et al, 1999). Venom MATERIALS AND METHODS Hemiscorpius lepturus, Hottentotta saulcyi and Mesobuthus eupeus venom were obtained by electric shock at the scorpion telson in the department of venomous animals, Razi Vaccine and Serum Research Institute of Ahvaz. It was lyophilized and stored at 4 C until use. Experimental protocol Ninety six Wistar rats with an average weight of 200g were used in this study. All animals were housed under

2 126 Razi Jalali et al conditions of controlled light (12-hour-light 12-hour-dark cycle), temperature (24 ± 1 C) and humidity (55 ± 5%), with standard diet and water available ad libitum. All animals were kept in compliance based on the Guide for Care and Use of Laboratory Animals. Rats were divided randomly into 4 equal groups as below: Group A: Received normal saline intraperitonealy as control group. Group B: Received Hemiscorpius lepturus scorpion venom (3mg/kg) Intraperitonealy. Group C: Received Mesobothus eupeus scorpion venom (1.5mg/kg) Intraperitonealy. Group D: Received Hottentota saulcyi scorpion venom (1mg/kg) Intraperitonealy. Blood samples were taken by heart puncture with sodium citrate 3.6%, at 1, 3, 24 and 48 hours after envenomation. Plasmas were used for evaluation of Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), fibrinogen level and platelet count. Prothrombin time Prothrombin time was assessed on citrated plasma by means of a standard kit. The PT kit was based on the assay principle that the addition of an adequately calcified amount of tissue factor (factor III) to citrated plasma activates factor VII, which induced the formation of a stable plug. The assay procedure was performed by placing 200 µl of tissue factor (PT reagent) in a test tube preheated to 37 C and subsequently adding 100 µl of citrated plasma. Upon the addition of test plasma, a stopwatch was started, and the clotting time was measured. The time in seconds from plasma-reagent mixing to visual clot formation was defined as the PT Activated partial thromboplastin Activated partial thromboplastin time was determined from citrated plasma by means of a standard kit. The APTT kit was based on the addition of a platelet substitute (phospholipids and ellagic acid as a soluble activator) and calcium chloride, which induced the formation of a stable plug. The assay procedure was performed by placing 100 µl of citrated plasma and 100 µl of APTT reagent (preheated to 37 C) in a test tube preheated to 37 C, followed by an additional incubation for 3 min at 37 C, and then adding 100 µl of calcium chloride that had been preheated to 37 C. Upon the addition of calcium chloride, a stopwatch was started and the clotting time was measured. The time in seconds from calcium chloride addition to visual clot formation was defined as the APTT Fibrinogen determination Fibrinogen determination was performed using citrated plasma by means of a standard kit. The standard kit for the quantitative determination of fibrinogen was based on the addition of a relatively large amount of thrombin to diluted citrated plasma, ensuring that the clotting time depended on only the fibrinogen contained in the sample. The assay procedure consisted of placing 200 µl of diluted plasma (diluted 1:10 by the combination of 100 µl of plasma µl of buffer) in a test tube preheated to 37 C, incubating for an additional 2 min at 37 C, and then adding 100 µl of the fibrinogen reagent. Upon the addition of fibrinogen reagent, a stopwatch was started, and the clotting time was measured. The time (seconds) until clot formation was automatically converted into mg/dl by the automated mechanical endpoint coagulation instrument RESULTS AND DISCUSSION Animal venoms are known for causing coagulopathy disorders (Brazon et al, 2008; Han et al, 2008; Marino et al, 2009; Oliveira-Carvalho et al, 2008; Valdez- Cruz et al, 2004). The best anticoagulants or hemorrhagic components studied are those from venomous snakes (Kini 2005; Kini and Evans, 1987). Several of these specific components are well characterized and grouped into families of proteins such as proteinases and metalloproteinases, C-type lectins, desintegrins and phospholipases. They usually affect: procoagulation, fibrinogen clotting, fibrinolysis, platelet-activation, anticoagulation, and might cause thrombotic or haemorrhagic manifestations (Kini and Evans, 1989). Some scorpion venoms also cause coagulopathy; however the number and type of components thus far characterized is small. There are reports indicating that the venom from the scorpions Hottentotta judaicus, Heterometrus spinnifer, Parabuthus transvaalicus, Androctonus australis, Scorpio maurus palmatus, Leiurus quinquestriatus habraeus and Pandinus imperator species, delay the clotting time of plasma. In particular the venom of P. imperator and P. transvaalicus venoms delay the clotting time by 2.5 and 2.3 fold respectively; whereas the other venoms delay the clotting around times (Tan and Ponnudurai, 1992). Venom from P. imperator and P. transvaalicus compared with snake venoms are 3 4 times less anticoagulant. The venom from the scorpion Buthus tamulus, in general causes coagulopathy, and it has been shown to cause disseminated intravascular coagulopathy

3 Effects of scorpion on blood of rats 127 Table1 : Changes of Mean± SE of coagulation test in different times in control group. Table 2 : Changes of Mean± SE of coagulation test in different times in group B (Hemiscorpius lepturus). PT ( sec) 10.6± ± ± ±2.1 APTT( sec) 29.7± ± ± ±3.1 Fibrinogen mg/dl 316±23 321±21 311±29 321±22 Platelet k/ul 222±23 228±24 236±27 234±34 Table3 : Changes of Mean± SE of coagulation test in different times in group C. (Mesobothus eupeus). PT ( sec) 15.1± ± ± ±2.2 bcd acd ab ab APTT( sec) 39.4± ± ± ±3.8 Fibrinogen 397±21 326± ± ±3.8 mg/dl bcd acd ab ab Platelet k/ul 201±14 175±12 152±19 191±18 PT( sec) 16.1± ± ± ±2.7 bc ad ad bc APTT( sec) 37.4± ± ± ±4.5 bcd acd abd abc Fibrinogen 321± ±31 307±16 315±18 mg/dl b acd b b Platelet k/ul 217±19 187±14 199±19 221±16 bc ad ad bc Table 4 : Changes of Mean± SE of coagulation test in different times in group D (Hottentota saulcyi) PT ( sec) 11.7± ± ± ±2.4 APTT( sec) 3.4± ± ± ±3.6 Fibrinogen 324±21 385±31 362±26 341±32 mg/dl bcdbcd acd abd abc Platelet k/ul 224±21 227±24 216±19 229±18 (DIC), (Radhakrishna Murthy et al, 1988). Injection of this scorpion venom intravenously (i.v.) at sub lethal dose into dogs and rabbits causes alteration in the coagulation mechanism (Gajalakshmi, 1982). Human accidents due to sting by the scorpion Palamneus gravimanus activates coagulation, probably mediated by a venom component acting on Factor X; it also has an inhibitory effect over thrombin (Hamilton et al, 1974). It has been demonstrated that high concentration of T. discrepans venom in human plasma fraction, increases the severity of the envenoming symptoms, by modification of the partial thromboplastin time (PTT) and prothrombin time (PT), rising the cytokines levels and increasing amylasemia and glycemia (D Suze et al, 2003). Brazon et al (2008) reported separation of T. discrepans venom into six sub-fractions by gel filtration on Protein-Pack 125 and assayed the effects on PTT and PT. Fraction 1 contains high molecular weight proteins, which decreases the PTT time in a dose dependent manner. Fractions 2 and 6 contain peptides that prolong the PTT and PT time. The latter are probably peptides like serpin that inhibit serine-protease activities. Results of this study showed that PT and APTT in different times after envenomatin were increased significantly in groups B (H. lepturus) and C (M. eupeus) compare to control group. The maximum alterations in these tests were seen at 3 hours after envenomation in both groups. Plasma fibrinogen also was increased in group B and C at 1 and 3 hours after envenomatin. After 24 and 48 hours plasma fibrinogen was gradually reduced to the level of control group. On the other hand the plasma fibrinogen significantly increased after envenomation with H. saulcyi at different times. Platelet count was decreased significantly at 3 and 24 hours after envenomatin in groups B and C. However, Platelet count was increased gradually. Patel et al (1992) showed that blood clotting time was significantly increased by venom injection (0.5 mg/ kg, IV) in rabbits at 0.5 and 6 hours interval (P < 0.05). This increase in blood clotting time was not significant at the interval of 24 and 48 hrs. Increased blood clotting time and hemorrhages observed in this study are correlated with the published observations that venom has anticoagulant property, which may possibly be due to calcium lack. It has been suggested that the venom is likely to produce chelation with ionic calcium and to

4 128 Razi Jalali et al change capillary permeability (Ismail et al, 1973). In other study on the effects of different doses of scorpion (Odontobuthus doriae) venom on hematological values in dog showed that Sedimentation rate, coagulation time, bleeding time and partial thromboplastin time significantly increased. Neutrophilic leukocytosis in addition to eosinopenia, thrombocytopenia and monocytopenia were also observed (p<0.05). Results indicate that hematological changes in the dogs led to a reactional leukocytosis and coagulopathies. The effect of toxin on liver led to coagulation. This suggests the probability of the existence of hepatotoxic factor(s) in the venom of this type of scorpion (Shorijeh et al, 2003). Kochar et al (2002) reported a case of scorpion sting that presented with right hemiparesis and deranged level of consciousness. Prolonged bleeding time and clotting time, decreased platelet counts, prolongation of PT and APTT with positive fibrinogen degradation products (FDP) D-dimer and CT scan findings of multiple cerebral hemorrhagic infacts indicating the presence of disseminated intravascular coagulation (DIC) as a cause of hemiparesis. Patient was managed with fresh blood transfusion and conventional treatment with favorable outcome. Disseminated intravascular coagulation occurred in dogs given scorpion venom subcutaneously in doses of 3 mg/kg (Bothotus tumulus) body weight. Treatment with heparin reversed the coagulation abnormality of the syndrome and 10 out of 12 dogs survived. Necropsy findings in human patients stung by scorpions suggest that this syndrome also occurs in man (Sita devi et al, 1970). In other study venom from the scorpion (Heterometrus fulvipes) was injected in sublethal doses and haematological studies were observed before and after venom administration. Distrubances in coagulation was not significant, but they may account for the cardiovascular changes frequently seen in scorpion envenomation. Present study clearly reveals that the coagulation abnormalities are not responsible for other clinical changes like myocardial infractions, cardiovascular changes of scorpion sting (Vanja et al, 1996). Emam et al (2008) in evaluation the effects of H. lepturus scorpion venom on hematological changes showed that quick diagnosis and treatment of scorpion stinging is very important factor for decreasing the mortality in sorpion envenomation. According to the results of this research and compare to other studies, it has been shown that hemostatic system can be affected by scorpion venom as a target organ. Based on the results of this study it seems that the effects of H. lepturus and M. eupeus on blood hemostasis were similar to each other. 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