Phospholipase C from Clostridium Perfringens Induces Human Platelet Aggregation in Plasma

Summary

We studied the aggregating effect of different concentrations of phospholipase C (PLC) (extracted from Clostridium perfringens) on human platelet-rich plasma (PRP). PRP was preincubated with PLC for 3 min at 37° C and the platelet aggregation was followed for 10 min. The threshold aggregating concentration (TAG) of PLC was 3-4 U/ml.

We also studied the potentiation of PLC with other stimuli on platelet aggregation. Potentiating stimuli, such as arachidonic acid (AA), ADP. Platelet Activating Factor (PAF) and U-46619 (a stable analogue of cyclic endoperoxides) were all used at subthreshold concentrations. We also studied the possible inhibitory effect of aspirin, apyrase, TMQ, a prostaglandin endoper- oxide/thromboxane receptor antagonist and BN-52021, a PAF receptor antagonist. Only aspirin and apyrase were able to reduce aggregation induced by PLC alone and PLC + AA and PLC + ADP respectively. TMQ and BN-52021 were inactive. In ex vivo experiments oral aspirin (500 mg) partially inhibited platelet aggregation induced by PLC alone, PLC + AA and PLC + ADP 2 and 24 h after administration. Aspirin 20 mg for 7 days also reduced aggregation induced by PLC + AA.

Keywords

Phospholipase C - Human platelet aggregation - Aspirin

• References

• 1 Lapetina EG. IV, metabolism of inositides and the activation of platelets. Life Sci 1983; 32: 2069-2082
• 2 Lapetina EG, Siess W. The role of phospholipase C in platelet responses. Life Sci 1983; 33: 1011-1018
• 3 Rittenhouse SE. Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ADP. Biochem J 1984; 222: 103-110
• 4 Mahadevappa VG, Holub BJ. Diacylglycerol lipase pathway is a minor source of released arachidonic acid in thrombin-stimulated human platelets. Biochem Biophys Res Commun 1986; 134: 1327-1333
• 5 Berridge MJ. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J 1984; 220: 345-360
• 6 Nishizuka Y. Turnover of inositol phospholipids and signal transduction. Science 1984; 225: 1365-1370
• 7 Siess W. Inositol phospholipid metabolism and platelet function. Biochem Pharmacol 1986; 35: 3184-3187
• 8 Navran SS, Romstedt K, Chang J, Miller DD, Feller DR. Huzoor-Akbar. Human platelet activation by bacterial phospholipase C is mediated by phosphatidylinositol hydrolysis but not generation of phosphatidic acid: Inhibition by a selective inhibitor of phospholipase C Thromb Res 1984; 33: 499-510
• 9 Chap H, Douste-Blazy L. Reaction de liberation plaquettaire induite par la phospholipase C. Eur J Biochem 1974; 48: 351-355
• 10 Otnaess A-B, Holm T. The effect of phospholipase C on human blood platelets. J Clin Invest 1976; 57: 1419-1425
• 11 Sugahara T, Takahashi T, Yamaya S, Ohsaka A. In vitro aggregation of platelets induced by a-toxin (phospholipase C) of Clostridium perfringens. Jpn J Med Sci Biol 1976; 29: 255-263
• 12 Kaplan KL. Platelet granule proteins: Localization and secretion. In: Platelets in biology and pathology-2. Gordon JL. (ed) pp 77-98 Elsevier/North Holland; Amsterdam: 1981
• 13 Lauri D, Cerletti C, de Gaetano G. Amplification of primary response of human platelets to platelet-activating factor: Aspirin-sensitive and aspirin-insensitive pathways. J Lab Clin Med 1985; 105: 653-658
• 14 Cerletti C, Carriero MR, de Gaetano G. Platelet-aggregation response to single or paired aggregating stimuli after low-dose aspirin. N Engl J Med 1986; 314: 316-318
• 15 Di Minno G, Silver MJ, de Gaetano G. Prostaglandins as inhibitors of human platelet aggregation. Br J Haematol 1979; 43: 637-647
• 16 Di Minno G, Bertele V, Bianchi L, Barbieri B, Cerletti C, Dejana E, de Gaetano G, Silver MJ. Effects of an epoxymethano stable analogue of prostaglandin endoperoxides (U-46619) on human platelets. Thromb Haemostas 1981; 45: 103-106
• 17 Molnar J, Lorand L. Studies on apyrases. Arch Biochem Biophys 1961; 93: 353-363
• 18 Mukhopadhyay A, Navran SS, Amin HM, Abdel-Aziz SA, Chang J, Sober DJ, Miller DD, Feller D. Effect of trimetoquinol analogs for antagonism of endoperoxide/thromboxane A2-mediated responses in human platelets and rat aorta. J Pharmacol Exp Ther 1985; 232: 1-9
• 19 Touvay C, Etienne A, Braquet P. Inhibition of antigen-induced lung anaphylaxis in the guinea-pig by BN 52021 a new specific paf-acether receptor antagonist isolated from Ginkgo biloba. Agents Actions 1985; 17: 371-372
• 20 Colotta F, Bersani L, Lazzarin A, Poli G, Mantovani A. Rapid killing of actinomycin D-treated tumor cells by human monocytes. II. Cytotoxicity is independent of secretion of reactive oxygen intermediates and is suppressed by protease inhibitors J Immunol 1985; 134: 3524-3531
• 21 Patrignani P, Filabozzi P, Patrono C. Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J Clin Invest 1982; 69: 1366-1372
• 22 Cerletti C, Minoldo S, Bucchi F, Del Maschio A, de Gaetano G. Requirement of ADP for arachidonic acid-induced platelet aggregation: Studies with selective thromboxane-synthase inhibitors. Biochem Pharmacol 1986; 35: 1201-1203
• 23 Lauri D, Cerletti C, de Gaetano G. Contribution of ADP to the amplification of primary platelet aggregation by platelet activating factor (PAF): Modulatory role of aspirin. Agents Actions 1985; 17: 506-511
• 24 Schick PK, Yu BP. The role of platelet membrane phospholipids in the platelet release reaction. J Clin Invest 1974; 54: 1032-1039
• 25 Huzoor-Akbar NavranS S, Chang J, Miller DD, Feller DR. Investigation of the effects of phospholipase C on human platelets: Evidence that aggregation induced by phospholipase C is independent of prostaglandin generation, released ADP and is modulated by cyclic AMP. Thromb Res 1982; 27: 405-417
• 26 Kawahara Y, Takai Y, Minakuchi R, Sano K, Nishizuka Y. Phospholipid turnover as a possible transmembrane signal for protein phosphorylation during human platelet activation by thrombin. Biochem Biophys Res Commun 1980; 97: 309-17
• 27 Tapley PM, Murray AW. Platelet Ca²⁺-activated, phospholipid- dependent protein kinase: Evidence for proteolytic activation of the enzyme in cells treated with phospholipase C¹ . Biochem Biophys Res Commun 1984; 118: 835-841