In Vivo Antithrombotic Effect of Triflavin, an Arg-Gly-Asp Containing Peptide on Platelet Plug Formation in Mesenteric Microvessels of Mice

Summary

Triflavin, an Arg-Gly-Asp-containing snake venom peptide, inhibits platelet aggregation through the blockade of fibrinogen binding to the activated platelets. In this study, platelet thrombus formation was induced by irradiation of the mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. Electron microscopy reveals moderately damaged endothelial cells, as well as aggregates consisting almost exclusively of platelets with pseudopod formation, and degranulated appearance. Triflavin (10-20 µg/g) significantly prolonged the lag period of inducing platelet plug formation in mesenteric venules when it was intravenously infused. Triflavin (20 µg/g) prolonged the occlusion time about 2-fold (from control 112 ± 23 to 240 ± 47 s). Furthermore, PGE, briefly prolonged the occlusion time about 1.5-fold (from 105 ± 21 to 168 ± 20 s) when it was given by continuous infusion (40 µg/kg/min). On the other hand, triflavin was also effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at dose of 2-4 µg/g. Heparin (1.5 U/g) and indomethacin (200 µg/g) had no significant effect in prolonging the occlusion time or in reducing ADP-induced pulmonary embolism in mice. Therefore, triflavin is an effective antithrombotic agent in preventing the thromboembolism in these two in vivo models.

• References

• 1 Gent M, Blakely JA, Easton JD, Ellis DJ, Hachinsky VC, Harbison JW, Panak E, Roberts RS, Sicarella J. Turpie AGG and the CATS Group. The Canadian American Ticlopidine Study (CATS) in thromboembolic stroke. Lancet 1989; 2: 1215-20
• 2 Hass WK, Easton JD, Adams HP, Pryse-Philips W, Molony BA, Anderson S, Kamm B. A randomized trial comparing ticlopidine hydrochloride with aspirin for the prevention of stroke in high-risk patients. N Engl J Med 1989; 321: 501-7
• 3 Seegers WH, Ouyang C. Snake venoms and blood coagulation. In: Snake Venoms, Handbook of Experimental Pharmacology Lee CY. ed Berlin -Heidelberg - New York: Springer Verlag; 1979. 52 pp 684-750
• 4 Ouyang C, Teng CM, Huang TF. Characterization of the purified principles of Formosan snake venoms which affect blood coagulation and platelet aggregation. J Formosan Med Assoc 1982; 81: 781-790
• 5 Ouyang C, Teng CM, Huang TF. Characterization of snake venom principles affecting blood coagulation and platelet aggregation. J Asia Pacific J Pharmacol 1987; 2: 169-176
• 6 Boffa BC, Boffa GA. Correlations between the enzymatic and factors active on blood coagulation and platelet aggregation from the venom of Vipera aspis. Biochim Biophys Acta 1974; 354: 275-290
• 7 Ouyang C, Huang TF. Inhibition of platelet aggregation by 5’-nucleotidase purified from Trimeresurus gramineus snake venom. Toxicon 1983; 21: 491-501
• 8 Ouyang C, Huang TF. Platelet aggregation inhibitors from Agkistorodon acutus snake venom. Toxicon 1986; 24: 1099-1106
• 9 Ouyang C, Huang TF. Platelet aggregation inhibitor from Trimeresurus gramineus snake venom. Biochim Biophys Acta 1983; 757: 332-341
• 10 Huang TF, Holt JC, Lukasiewcz H, Niewiarowski S. Trigramin, a low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb/IIIa complex. J Biol Chem 1987; 262: 16157-16163
• 11 Huang TF, Holt JC, Kirby EP, Niewiarowski S. Trigramin: primary structure and its inhibition of von Willebrand factor binding to glycoprotein IIb/IIIa complex on human platelets. Biochemistry 1989; 28: 661-666
• 12 Coller BS, Pearschke El, Scudder LE, Sullivan CA. A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thromboasthenic-like state in normal platelets and binds to glycoprotein IIb/IIIa. J Clin Invest 1983; 72: 325-338
• 13 Huang TF, Wang WJ, Teng CM, Ouyang C. Mechanism of action of the antiplatelet peptide, arietin, from Bids arietans venom. Biochim Biophys Acta 1991; 1074: 144-150
• 14 Huang TF, Liu CZ, Ouyang C, Teng CM. Halysin, an antiplatelet Arg-Gly-Asp-containing snake venom peptide, as fibrinogen receptor antagonist. Biochem Pharmacol 1991; 42: 1209-1219
• 15 Gan ZR, Gould RJ, Jacobs JW, Friedman PA, Polokoff MA. Echistatin, a potent platelet aggregation inhibitor from the venom of the viper, Echis ca-rinatus. J Biol Chem 1989; 263: 19827-19832
• 16 Chao BH, Jakubowski JA, Savage B, Chow EP, Marzec LM, Harker LA, Maraganore JM. Agkistrodon piscivorus piscivorus platelet aggregation inhibitor: a potent inhibitor of platelet activation. Proc Natl Acad Sci USA 1989; 86: 8050-8054
• 17 Shebuski RJ, Ramjit DR, Bencen GH, Polokoff MA. Characterization and platelet inhibitory activity of bitistatin, a potent arginine-glycine-aspartic acid-containing peptide from the venom of the viper Bids arietans. J Biol Chem 1990; 264: 21550-21556
• 18 Williams JA, Rucinski B, Holt JC, Niewarowski S. Elegantin and albo-labrin purified peptides from viper venoms; homologies with the RGDS domain of fibrinogen and von Willebrand factor. Biochim Biophys Acta 1990; 1039: 81-89
• 19 Gould RJ, Polokoff MA, Friedman PA, Huang TF, Holt JC, Cook JJ, Nie-wiarowski S. Disintegrins: a family of integrin inhibitory proteins from viper venom. Proc Soc Exp Biol Med 1990; 195: 169-171
• 20 Sheu JR, Lin CH, Chung JL, Teng CM, Huang TF. Triflavin, an Arg-Gly-Asp containing antiplatelet peptide inhibits cell-substratum adhesion and melanoma cell-induced lung colonization. Jpn J Cancer Res 1992; 83: 885-193
• 21 Sheu JR, Lin CH, Chung JL, Teng CM, Huang TF. Triflavin, an Arg-Gly-Asp containing snake venom peptide, inhibits aggregation of human platelets induced by human hepatoma cell lines. Thromb Res 1992; 66: 679-691
• 22 Sheu JR, Lin CH, Peng HC, Teng CM, Huang TF. Triflavin, an Arg-Gly-Asp-containing peptide, inhibits tumor cell-induced platelet aggregation. Jpn J Cancer Res 1993; 84: 1062-1071
• 23 Sheu JR, Lin CH, Huang TF. Triflavin, an antiplatelet peptide inhibits tumor cell-extracellular matrix adhesion via an RGD-dependent mechanism. J Lab Clin Med 1994; 123: 256-263
• 24 Huang TF, Sheu JR, Teng CM. A potent antiplatelet peptide, triflavin, from Trimeresurus flavoviridis snake venom. Biochem J 1991; 277: 351-357
• 25 Huang TF, Sheu JR, Teng CM. Mechanism of action of a potent antiplatelet peptide, triflavin from Trimeresurus flavoviridis snake venom. Thromb Haemost 1991; 66: 489-493
• 26 Huang TF, Sheu JR, Teng CM, Chen SW, Liu CS. Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein Ilb/IIIa complex. J Biochem 1991; 109: 328-334
• 27 Sheu JR, Teng CM, Huang TF. Triflavin, an RGD-containing antiplatelet peptide, binds to GPIIIa of ADP-stimulated platelets. Biochem Biophys Res Commun 1992; 189: 1236-1212
• 28 Rosenblum WI, EL-Sabban F. Platelet aggregation in the cerebral microcirculation: Effect of aspirin and other agents. Circ Res 1977; 40: 320-328
• 29 Sato M, Ohshima N. Platelet thrombus induced in vivo by filtered light and fluorescent dye in mesenteric microvessels of the rat. Thromb Res 1984; 35: 319-334
• 30 Sato M, Ohshima N. Hemodynamic at stenoses formed by growing platelet thrombi in mesenteric microvasculature of rat. Microvasc Res 1986; 31: 66-76
• 31 Nordoy A, Chandler AB. Platelet thrombosis induced by adenosine diphosphate in the rat. Scand J Haematol 1964; 1: 16-25
• 32 Nowak J, Wennmalm A. Influence of indomethacin and of prostaglandin E, on total and regional blood flow in man. Acta Physiol Scand 1978; 102: 484-491
• 33 Vigdahl RL, Marquis NR, Tavormina PA. Platelet aggregation. Biochem Biophys Res Commun 1969; 37: 409-415
• 34 Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibrinogen can be duplicated by small synthetic fragments of the molecule. Nature 1984; 309: 30-33
• 35 Doolittle RF. Fibrinogen and fibrin. Ann Rev Biochem 1984; 53: 195-227
• 36 Titani K, Takio K, Ericsson LH, Wade RD, Ashida K, Walsh SA, Chopek MW, Sadler JE, Fujikawa IA. Amino acid sequence of human von Willebrand factor. Biochemistry 1981; 25: 3171-3184
• 37 Hovig T, McKenzie FN, Arfors KE. Measure of the platelet response to laser induced microvascular injury; ultrastructural studies. Thromb Diath Haemorrh 1974; 32: 695-703
• 38 Callahan AB, Lutz BR, Fulton GP, Degelman J. Smooth muscle and thrombus thresholds to unipolar stimulation of small blood vessels. Angiology 1960; 11: 35-39
• 39 Beviglia L, Poggi A, Rossi C, McLane MA, Calabrese R, Scanziani E, Cook JJ, Niewiarowski S. Mouse antithrombotic assay. Inhibition of platelet thromboembolism by disintegrins. Thrombos Res 1993; 71: 301-315
• 40 Musial J, Niewiarowski S, Rucinski B, Stewart GJ, Cook JJ, Edmunds LH. Inhibition of platelet adhesion to surfaces of extracorporeal circuits by disintegrins RGD-containing peptides from viper venoms. Circulation 1990; 82: 261-273