Tityus Discrepans Scorpion Venom Activates Platelets Through GPVI and a Novel Src-Dependent Signaling Pathway
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Platelets, May 2011; 22(3): 165–172 Copyright ß 2011 Informa UK Ltd. ISSN: 0953-7104 print/1369-1635 online DOI: 10.3109/09537104.2010.544343 ORIGINAL ARTICLE Tityus discrepans scorpion venom activates platelets through GPVI and a novel Src-dependent signaling pathway JOSMARY BRAZO´ N1,2, CRAIG E. HUGHES2, JUN MORI2, CARLOS SEVCIK1, GINA D’SUZE1, & STEVE P. WATSON2 1Laboratorio de Neurofarmacologı´a Celular, Centro de Biofı´sica y Bioquı´mica. Instituto Venezolano de Investigaciones Cientı´ficas (IVIC), Apartado 20632, Caracas, 1020-A, Venezuela and 2Centre for Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK Abstract In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin IIb 3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR -chain, Syk, and PLC 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR -chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway. Keywords: Scorpion venom, collagen, GPVI, integrin, tyrosine kinases, platelet For personal use only. Introduction There are several reports of the effect of scorpion venom on platelets. Charybdotoxin is a toxin in Platelets are critical elements for the maintenance of Leiurus quinquestriatus venom that inhibits the pro- haemostasis. They express a diverse range of surface coagulant response of human platelets through Ca2þ receptors, which induce activation through a variety - of mechanisms including regulation of heterotrimeric activated potassium channels [5–7]. A bioactive G proteins and tyrosine kinases. polypeptide from Buthus martensii Karsh venom has been reported to inhibit rabbit and rat platelet Platelets Downloaded from informahealthcare.com by University of Birmingham on 03/01/12 One of the most thoroughly characterized path- ways of platelet activation is through the immuno- aggregation by thrombin or ADP but the underlying globulin receptor GPVI, which is a receptor for mechanism is not known [8]. There is also a report subendothelial collagen and laminin, and a variety of describing partial aggregation of dog platelets by snake venom toxins [1, 2]. GPVI forms a complex Centruroides sculpturatus venom, although the molec- with the FcR -chain in the membrane. Clustering ular basis of this is also not known [9]. of GPVI leads to phosphorylation of two conserved The Venezuelan scorpion Tityus discrepans induces tyrosines in the immunoreceptor tyrosine based a potentially fatal systemic response consisting of activation motif (ITAM) of the FcR -chain. This hypertension or hypotension, tachycardia, tachypnea, leads to recruitment and activation of the tyrosine hypothermia, leucocytosis, sialorrhoea, myocarditis, kinase Syk and initiation of a downstream pancreatitis and respiratory distress syndrome [10]. signaling cascade that culminates in activation of Scorpionism victims develop fibrin deposits in the PLC 2 [3, 4]. alveoli, prolongation or shortening in prothrombin Correspondence: Dr Josmary Brazo´n, Laboratorio de Neurofarmacologı´a Celular, Centro de Biofı´sica y Bioquı´mica. Instituto Venezolano de Investigaciones Cientı´ficas (IVIC). Apartado 20632. Caracas, 1020-A, Venezuela. Tel: 58-212-504-1479. Fax: 58-212-504-1093. E-mail: [email protected]; [email protected] (received 8 October 2010; revised 15 November 2010; accepted 29 November 2010) 166 J. Brazo´n et al. and partial thromboplastin times [11–13]. breedings were used as controls. Mouse blood was Fibrinolytic and anti-fibrinolytic components have drawn from CO2 asphyxiated mice by portal vein been identified in the venom [14]. In the present puncture and taken into 100 mL of ACD. Platelets study, we demonstrate platelet activation by T. were separated as described above for human plate- discrepans venom (TdV) revealing a GPVI-activating lets and resuspended in Tyrode´s buffer at a concen- component as well as evidence for a second novel tration of 2  108 platelets/mL. tyrosine kinase-dependent mechanism of activation. Platelet aggregation Materials and methods Platelet aggregation was monitored by light trans- Animals and venom mittance using a Born aggregometer (Chronolog, Havertown, PA, USA). Mouse or washed human Tityus discrepans scorpions were collected in the areas platelets were incubated at 37C with continuous surrounding Caracas, Venezuela. Adult scorpions stirring at 1200 rpm for 5 min before treatment with (100) were kept alive in a laboratory with food and Integrilin (9 mM) or 4-amino-5-(4-chlorophenyl)- water ad libitum. Venom was extracted from them 7-(dimethylethyl)pyrazolo[3,4,d]pyrimidine (PP2) once a month. The animals were immobilized with (20 mM) for 1 min prior to agonist stimulation. CO2 before venom extraction by electrical stimula- tion. The venom was dissolved in double distilled water and centrifuged at 15000 g for 15 min at 4C Protein tyrosine phosphorylation to remove insoluble material. The protein concen- Platelets were treated with Integrilin (9 mM) or PP2 tration in the supernatant was estimated by spectro- (20 M) for one minute before stimulation by colla- photometry as previously described [15]. This m yielded approximately 0.5–1 mg of protein at a gen (10 mg/mL) or TdV (100 mg/mL). Platelets were concentration of 70 mg/mL per extraction. The lysed with equal volumes of ice-cold 2 lysis buffer venom was lyophilized and stored at À80C until (2% Triton X-100, 2% dodecyl maltoside, 4 mM required. 4-(2-aminoethyl)benzenesulfonyl fluoride), 20 mg/ mL aprotinin, 20 mg/mL leupeptin, 2 mg/mL pepsta- Washed human platelets tin, 10 mM sodium orthovanadate, pH 7.5). The insoluble material was removed by centrifugation For personal use only. Venous blood was drawn on the day of the exper- and the sample was used to determine tyrosine iment from healthy, drug-free volunteers and mixed phosphorylation in whole cell lysates and following 1:9 (v/v) with 4% sterile trisodium citrate and 1:9 immunoprecipitation. For the latter, lysates were (v/v) acid citrate dextrose (Acid Citrate Dextrose pre-cleared with either protein A or protein G (ACD): 120 mM sodium citrate, 110 mM glucose, Sepharose beads for 30 min at 4C and mixed with 80 mM citric acid) and transferred to 5 mL polypro- either 2 mL anti-Syk or 2 mL anti-PLC 2 polyclonal pylene tubes, 75  12 mm, for centrifugation at 200 g antibodies (kindly provided by J.B. Bolen, DNAX, for 20 min at room temperature. Platelet rich plasma California, USA) at a dilution of 1/500. The mixture (upper layer) was gently pipetted into a 50 mL was rotated for 2 h at 4C followed by washing with polypropylene tube using a plastic pipette followed three changes of lysis buffer. Immunoprecipitated Platelets Downloaded from informahealthcare.com by University of Birmingham on 03/01/12 by addition of 10 mg/ml prostacyclin to prevent proteins were then eluted off the beads by addition of platelet activation. The tubes were mixed gently by 2 Laemmli reducing sample buffer and boiling. inversion and immediately centrifuged at 1000 g for Whole cell lysate samples were also boiled following 10 min. The platelet number was measured using a addition of an equal volume of 2 Laemmli reducing Coulter Z2 analyser. The washed platelets were sample buffer. Samples were separated by SDS- resuspended in Tyrode’s buffer (134 mM NaCl, PAGE on 10% BisTris gels (Invitrogen, Paisley, UK) 2.9 mM KCl, 0.34 mM Na2HPO4 Á 12H2O, 12 mM and transferred to polyvinylidene difluoride mem- NaHCO3, 20 mM HEPES, 1 mM MgCl2) contain- brane. Phosphotyrosine was detected by western  8 ing 5 mM glucose at a concentration of 4 10 blotting using 4G10 primary antibody (Upsate platelets/ml for western blotting or 2  108 platelets/ Biotechnology, Milton Keynes, UK), HRP-conju- ml for aggregation and secretion. gated secondary antibody (Amersham Biosciences, Buckinghamshire, UK), enhanced chemilumines- Mouse platelets cence reagents (Perkin Elmer, Boston, MA, USA) FcR -chain [3] and PLC 2 [16] deficient mice were and Hyperfilm (Amersham Biosciences). For immu- bred either as homozygotes or heterozygotes, respec- noprecipitated lysates, the membranes were tively, on a C57Bl/6 background. Wild type mice on subsequently stripped and reprobed with anti-Syk the same background or littermates from the PLC 2 or anti-PLC 2 antibodies. Tityus discrepans venom and platelets aggregation 167 counted by trypan blue exclusion, and samples were divided for luciferase assay (2  106 cells/mL) and flow cytometry (5  105 cells/sample) as previ- ously described [17]. Phorbol 12-myristate 13- acetate (50 ng/mL, PMA) plus 1 mM ionomycin were used as positive control. Luciferase activity was measured with a Centro LB 960 microplate luminometer (Berthold Technologies, Bad Wildbad, Germany). Data is expressed as the n-fold increase over basal. Receptor expression was confirmed by flow cytometry as described [17]. Venom purification Whole venom (100 mg resuspended in 1 mL of 20 mM of ammonium acetate, pH 4.7) was fraction- ated by gel filtration chromatography on a Sephadex G-50 column (1  200 cm). The venom components were eluted with 20 mM of ammonium acetate, pH 4.7, at 0.25 mL/min flow rate with detection at 280 nm.