Mechanisms Involved in the Antiplatelet Activity of Staphylococcus aureus Lipoteichoic Acid in Human Platelets

Summary

In this study, Gram-positive Staphylococcus aureus lipoteichoic acid (LTA) dose-dependently (0.1-1.0 µg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LTA also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+² mobilization in human platelets stimulated by collagen. LTA (0.5 and 1.0 µg/ml) also significantly inhibited thromboxane A₂ formation stimulated by collagen in human platelets. Moreover, LTA (0.1-1.0 µg/ml) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. Rapid phosphorylation of a platelet protein of Mr. 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (30 nM). This phosphorylation was markedly inhibited by LTA (0.5 and 1.0 µg/ml) within a 10-min incubation period.

These results indicate that the antiplatelet activity of LTA may be involved in the following pathways: LTA’s effects may initially be due to induction of conformational changes in the platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A₂ formation, thereby leading to inhibition of both intracellular Ca+² mobilization and phosphorylation of P47 protein. Therefore, LTA-mediated alteration of platelet function may contribute to bleeding diathesis in Gram-positive septicemic and endotoxemic patients.

Key words

Gram-positive bacteria - lipoteichoic acid - platelet aggregation - membrane fluidity - protein kinase C

• References

• 1 Bave AE. The multiple organ or system failure syndrome. In: Pathophysiology of shock, sepsis and organ failure. Schlag G, Redl H. ed. 1993: 1004-18 Berlin: Springer Verlag.;
• 2 Bone RC. Gram-positive organisms and sepsis. Arch Intern Med 1994; 154: 26-34.
• 3 Bone RC. The pathogenesis of sepsis. Annal Intern Med 1991; 115: 457-69.
• 4 Wicken AJ, Knox KW. Lipoteichoic acids: new class of bacterial antigen. Science 1975; 187: 1161-7.
• 5 Lonchampt MO, Auguet M, Delaflotte S, Goulin-Schulz J, Chabrier PE, Braquet P. Lipoteichoic acid: a new inducer of nitric oxide synthase. J Cardiovasc Pharmacol 1992; 20: s145-7.
• 6 Kengatharan M, De Kimpe SJ, Thiemermann C, Vane JR. Characterization of the mechanism of enhanced nitrite formation by lipoteichoic acid (from Saureus) in cultured J 774.2 macrophage cells. Br J Pharmacol 1995; 115: 9P.
• 7 Neame PB, Kelton JG, Walker IR, Stewart IO, Nossel HL, Hirsh J. Thrombocytopenia in septicemia: the role of disseminated intravascular coagulation. Blood 1980; 56: 88-92.
• 8 Matera C, Falzarano C, Berrino L, Rossi F. Effects of tetanus toxin Salmonella typhimurium porin, and bacterial lipopolysaccharide on platelet aggregation. J Med 1992; 23: 327-38.
• 9 Isogai E, Kitagawa H, Isogai H, Matsuzawa T, Shimizu T, Yanagihara Y, Kitami K. Effect of leptospiral lipopolysaccharide on rabbit platelets. Int J Med Microbiol 1992; 271: 186-96.
• 10 Mueller-Eckhardt CH, Luscher EF. Immune reactions of human blood platelets. IV. Investigators on the problem of an immunologically induced effect of endotoxin on human platelets. Thromb Diath Haemorrh 1968; 20: 336-44.
• 11 Saba HI, Saba SR, Morelli G, Hartmann RG. Endotoxin-mediated inhibition of human platelet aggregation. Thromb Res 1984; 34: 19-33.
• 12 Beachey EH, Chiang TM, Ofek I, Kang AH. Interaction of lipoteichoic acid of group A. Streptococci with human platelets. Infect Immun 1977; 16: 649-54.
• 13 Huang TF, Sheu JR, Teng CM. A potent antiplatelet peptide, triflavin, from Trimeresurus flavoviridis snake venom. Biochem J 1991; 277: 351-7.
• 14 Born GVR, Cross MJ. The aggregation of blood platelets. J Physiol 1963; 168: 178-95.
• 15 Sheu JR, Teng CM, Huang TF. Triflavin, an RGD-containing antiplatelet peptide, binds to GP IIIa of ADP-stimulated platelets. Biochem Biophys Res Commun 1992; 189: 1236-42.
• 16 Sheu JR, Lin CH, Peng CH, Huang TF. Triflavin, an Arg-Gly-Asp-containing peptide, inhibits the adhesion of tumor cells to matrix protein via binding to multiple integrin receptors expressed on human hepatoma cells. Proc Soc Exp Biol Med 1996; 213: 71-9.
• 17 Lowry DH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-75.
• 18 Neylon CB, Summers RJ. Stimulation of α₁-adrenoceptor in rat kidney mediates increased inositol phospholipid hydrolysis. Br J Pharmacol 1987; 91: 367-76.
• 19 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ induces with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-50.
• 20 Kitagawa S, Shinohara T, Kametani F. Effects of alcohols on ADP-induced aggregation and membrane fluidity of gel-filtered bovine blood platelets. J Membrane Biol 1984; 79: 97-102.
• 21 Wroblewski F, Ladue JS. Lactic dehydrogenase activity in blood. Proc Soc Exp Biol Med 1955; 90: 210-5.
• 22 Grabarek J, Raychowdhury M, Ravid K, Kent KC, Newman PJ, Ware JA. Identification and functional characterization of protein kinase C isozymes in platelets and HEL cells. J Biol Chem 1992; 267: 10011-7.
• 23 Laemmli K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
• 24 Sheu JR, Hung WC, Wu CH, Ma MC, Kan YC, Lin CH, Lin MS, Luk HN, Yen MH. Reduction in lipopolysaccharide-induced thrombocytopenia by triflavin in a rat model of septicemia. Circulation 1999; 99: 3056-62.
• 25 Broekman MJ, Ward JW, Marcus AJ. Phospholipid metabolism in phosphatidyl inositol, phosphatic acid and lysophospholipids. J Clin Invest 1980; 66: 275-83.
• 26 Siess W, Lapetina EG. Platelet aggregation induced by alpha 2-adrenoceptor and protein kinase C activation A novel synergism. Biochem J 1989; 263: 377-85.
• 27 Kengatharan KM, De Kimpe SJ, Thiemermann C. Role of nitric oxide in the circulatory failure and organ injury in a rodent model of Gram-positive shock. Br J Pharmacol 1996; 119: 1411-21.
• 28 Charo IF, Feinman RD, Detwiler TC. Inhibition of platelet secretion by an antagonist of intracellular calcium. Biochem Biophys Res Commun 1976; 72: 1462-7.
• 29 Kirk CJ, Creba JA, Downes CP, Michell RH. Hormone-stimulated metabolism of inositol lipids and its relationship to hepatic receptor function. Biochem Soc Transact 1981; 09: 377-9.
• 30 Berridge MJ. Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem J 1983; 212: 249-58.
• 31 Hornby EJ. Evidence that prostaglandin endoperoxides can induce platelet aggregation in the absence of thromboxane A2 production. Biochem Pharmacol 1982; 31: 1158-60.
• 32 McKean ML, Smith JB, Silver WJ. Formation of lysophosphatidylcholine in human platelets in response to thrombin. J Biol Chem 1981; 256: 1522-4.
• 33 Kraft AS, Andersion WB. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature (London) 1983; 301: 621-3.
• 34 Murohara T, Parkinson SJ, Waldman SA, Lefer AM. Inhibition of nitric oxide biosynthesis promotes P-selectin expression in platelets. Role of protein kinase C. Arterioscler Thromb Vas Biol 1995; 15: 2068-75.
• 35 Bhargava M, Kumari S, Bhargava SK. Impairment of platelet function in neonatal septicemia. Indian J Med Res 1978; 68: 964-69.