Evidence for a Role for Phospholipase C, but not Phospholipase A₂, in Platelet Activation in Response to Low Concentrations of Collagen

Summary

The release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 g/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen (1-10 g/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A₂, and p38^MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38^MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38^MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A₂ inhibitor, AACOCF₃, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38^MAPK phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Rö31-8220 inhibited collagen-induced aggregation, did not affect p38^MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase C 2 which was inhibited by pretreatment with U73122, unaffected by AACOCF₃ and enhanced by Rö31-8220. These results suggest that cytosolic phospholipase A₂ plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase C 2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.

Keywords

Platelets - collagen - arachidonic acid - phospholipase

• References

• 1 Sixma JJ, van Zanten GH, Huizinga EG, van der Plas RM, Verkley M, Wu YP, Gros P, de Groot PG. Platelet adhesion to collagen: an update. Thromb Haemost 1997; 78: 434-38.
• 2 Moroi M, Jung SM. Platelet receptors for collagen. Thromb Haemost 1997; 78: 439-44.
• 3 Watson SP. Collagen receptor signaling platelets and megakaryocytes. Thromb Haemost 1999; 82: 365-76.
• 4 Watson SP, Gibbins J. Collagen receptor signaling in platelets: extending the role of the ITAM. Immunol Today 1998; 19: 260-4.
• 5 Ezumi Y, Shindoh K, Tsuji M, Takayama H. Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets. J Exp Med 1998; 188: 267-76.
• 6 Briddon SJ, Watson SP. Evidence for the involvement of p59fyn and p53/56lyn in collagen receptor signalling in human platelets. Biochem J 1999; 338: 203-9.
• 7 Gibbins J, Asselin J, Farndale R, Barnes M, Law CL, Watson SP. Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets. J Biol Chem 1996; 271: 18095-9.
• 8 Gibbins JM, Okuma M, Farndale R, Barnes M, Watson SP. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain. FEBS Lett 1997; 413: 255-9.
• 9 Tsuji M, Ezumi Y, Arai M, Takayama H. A novel association of Fc receptor gamma-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets. J Biol Chem 1997; 272: 23528-31.
• 10 Yanaga F, Poole A, Asselin J, Blake R, Schieven GL, Clark EA, Law CL, Watson SP. Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the Fc gamma-IIA receptor. Biochem J 1995; 311: 471-8.
• 11 Ichinohe T, Takayama H, Ezumi Y, Arai M, Yamamoto N, Takahashi H, Okuma M. Collagen-stimulated activation of Syk but not c-Src is severely compromised in human platelets lacking membrane glycoprotein VI. J Biol Chem 1997; 272: 63-8.
• 12 Asselin J, Gibbins JM, Achison M, Lee YH, Morton LF, Farndale RW, Barnes MJ, Watson SP. A collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase C gamma2 in platelets independent of the integrin alpha2beta1. Blood 1997; 89: 1235-42.
• 13 Blake RA, Schieven GL, Watson SP. Collagen stimulates tyrosine phosphorylation of phospholipase C-gamma2 but not phospholipase C-gamma 1 in human platelets. FEBS Lett 1994; 353: 212-6.
• 14 Daniel JL, Dangelmaier C, Smith JB. Evidence for a role for tyrosine phosphorylation of phospholipase C gamma2 in collagen-induced platelet cytosolic calcium mobilization. Biochem J 1994; 302: 617-22.
• 15 Gibbins JM, Briddon S, Shutes A, van Vugt MJ, van de Winkel JG, Saito T, Watson SP. The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activitor of T cells (LAT) in platelets stimulated by collagen and convulxin. J Biol Chem 1998; 273: 34437-43.
• 16 Oda A, Ochs HD, Druker BJ, Ozaki K, Watanabe C, Handa M, Miyakawa Y, Ikeda Y. Collagen induces tyrosine phosphorylation of Wiskott-Aldrich syndrome protein in human platelets. Blood 1998; 92: 1852-8.
• 17 Quek LS, Bolen J, Watson SP. A role for Bruton’s tyrosine kinase (Btk) in platelet activation by collagen. Curr Biol 1998; 8: 1137-40.
• 18 Gross BS, Lee JR, Clements JL, Turner M, Tybulewicz VL, Findell PR, Koretzky GA, Watson SP. Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets. J Biol Chem 1999; 274: 5963-71.
• 19 Asazuma N, Wilde JI, Berlanga O, Leduc M, Leo A, Schweighoffer E, Tybulewicz V, Bon C, Liu SK, McGlade CJ, Schraven B, Watson SP. Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin. J Biol Chem 2000; 275: 33427-34.
• 20 Pasquet JM, Bobe R, Gross B, Gratacap MP, Tomlinson MG, Payrastre B, Watson SP. A collagen-related peptide regulates phospholipase Cgamma2 via phosphatidylinositol 3-kinase in human platelets. Biochem J 1999; 342: 171-7.
• 21 Pasquet JM, Quek L, Stevens C, Bobe R, Huber M, Duronio V, Krystal G, Watson SP. Phosphatidylinositol 3,4,5-trisphosphate regulates Ca(2+) entry via btk in platelets and megakaryocytes without increasing phospholipase C activity. EMBO J 2000; 19: 2793-802.
• 22 Keely PJ, Parise LV. The alpha2beta1 integrin is a necessary co-receptor for collagen-induced activation of Syk and the subsequent phosphorylation of phospholipase Cgamma2 in platelets. J Biol Chem 1996; 271: 26668-76.
• 23 Watson SP, Reep B, McConnell RT, Lapetina EG. Collagen stimulates [3H) inositol trisphosphate formation in indomethacin-treated human platelets. Biochem J 1985; 226: 831-7.
• 24 Smith JB, Selak MA, Dangelmaier C, Daniel JL. Cytosolic calcium as a second messenger for collagen-induced platelet responses. Biochem J 1992; 288: 925-9.
• 25 Börsch-Haubold AG, Kramer RM, Watson SP. Cytosolic phospholipase A 2 is phosphorylated in collagen- and thrombin-stimulated human platelets independent of protein kinase C and mitogen-activated protein kinase. J Biol Chem 1995; 270: 25885-92.
• 26 Börsch-Haubold AG, Kramer RM, Watson SP. Phosphorylation and activation of cytosolic phospholipase A₂ by 38-kDa mitogen-activated protein kinase in collagen-stimulated human platelets. Eur J Biochem 1997; 245: 751-9.
• 27 Narita H, Park HJ, Tanaka K, Matsuura T, Kito M. Insufficient mobilization of calcium by early breakdown of phosphatidylinositol 4,5-bisphosphate for aggregation of human platelets by collagen. J Biochem (Tokyo) 1985; 98: 1063-8.
• 28 Pollock WK, Rink TJ, Irvine RF. Liberation of [³H]-arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen. Biochem J 1986; 235: 869-77.
• 29 Kito M, Narita H, Takamura H, Park HJ, Matsuura T, Tanaka K. Dissociation of Ca²⁺ mobilization from breakdown of phosphatidylinositol 4,5-bisphosphate in activated human platelets. J Biochem (Tokyo) 1986; 99: 1277-80.
• 30 Seymour RA, Williams FM, Oxley A, Ward A, Fearns M, Brighan K, Rawlins MD, Jones PM. A comparative study of the effects of aspirin and paracetamol (acetaminophen) on platelet aggregation and bleeding time. Eur J Clin Pharmacol 1984; 26: 567-71.
• 31 Gerrard JM, Docherty JC, Israels SJ, Cheang MS, Bishop AJ, Kobrinsky NL, Schroeder ML, Israels ED. A reassessment of the bleeding time: association of age, hematocrit platelet function, von Willebrand factor, and bleeding time thromboxane B₂ with the length of the bleeding time. Clin Invest Med 1989; 12: 165-71.
• 32 McNicol A, Saxena SP, Becker AB, Brandes LJ, Gerrard JM. Further studies on the effects of the intracellular histamine antagonist DPPE on platelet function. Platelets 1991; 2: 215-21.
• 33 Scrutton MC. The platelet as a Ca²⁺-driven cell: Mechanisms which may modulate Ca²⁺-driven responses. Adv Exp Med Biol 1993; 344: 1-15.
• 34 Thomas LM, Holub BJ. BW755C or staurosporine inhibits collagen-stimulated phospho-inositide phosphorylation in platelets. Lipids 1991; 26: 689-95.
• 35 Saklatvala J, Rawlinson L, Waller RJ, Sarsfield S, Lee JC, Morton LF, Barnes MJ, Farndale RW. Role for p38 mitogen-activated protein kinase in platelet aggregation caused by collagen or a thromboxane analogue. J Biol Chem 1996; 271: 6586-9.
• 36 Barnes MJ, Knight CG, Farndale RW. The collagen-platelet interaction. Curr Opin Hematol 1998; 5: 314-20.
• 37 Karniguian A, Grelac F, Levy-Toledano S, Legrand YJ, Rendu F. Collagen-induced platelet activation mainly involves the protein kinase C pathway. Biochem J 1990; 268: 325-31.
• 38 McNicol A. Platelet preparation and estimation of functional responses. In: Platelets: A Practical Approach. Watson SP, Authi KS. eds. Oxford: Oxford University Press; 1996: 1-26.
• 39 Dole VP, Meinertz H. Microdetermination of long-chain fatty acids in plasma and tissue. J Biol Chem 1960; 235: 2595-9.
• 40 Püttmann M, Krug H, van Ochsenstein E, Kattermann R. Fast HPLC determination of serum free fatty acids in the picomole range. Clin Chem 1993; 39: 825-32.
• 41 Pampolina C, McNicol A. Anisomycin does not activate p38^MAPK in human platelets. Thromb Res 1999; 96: 27-35.
• 42 Bleasdale JE, Thakur NR, Gremban RS, Bundy GL, Fitzpatrick FA, Smith RJ, Bunting S. Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther 1990; 255: 756-68.
• 43 Lockhart LK, McNicol A. The phospholipase C inhibitor U73122 inhibits phorbol ester-induced platelet activation. J Pharmacol Exp Ther 1999; 289: 721-8.
• 44 McNicol A, Nickolaychuk BR. Inhibition of collagen-induced platelet activation by arachidonyl trifluoromethyl ketone. Biochem Pharmacol 1995; 50: 1795-802.
• 45 Rittenhouse SE, Allen CL. Synergistic activation by collagen and 15-hydroxy-9 alpha,11 alpha-peroxidoprosta-5,13-dienoic acid (PGH2) of phosphatidyl inositol metabolism and arachidonic acid release in human platelets. J Clin Invest 1982; 70: 1216-24.
• 46 Sano K, Takai Y, Yamanishi J, Nishizuka Y. A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions. J Biol Chem 1983; 258: 2010-3.
• 47 Vedelago HR, Mahadevappa VG. Mobilization of arachidonic acid in collagen-stimulated human platelets. Biochem J 1988; 256: 981-7.
• 48 Krishnamurthi S, Joseph S, Kakkar VV. Effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced arachidonate release and 5-hydroxytryptamine secretion in human platelets. Dependence of effects on agonist type and time of incubation with PMA. Biochim Biophys Acta 1987; 927: 429-36.
• 49 MacIntyre DE, McNicol A, Drummond AH. Tumour-promoting phorbol esters inhibit agonist-induced phosphatidate formation and Ca²⁺ flux in human platelets. FEBS Lett 1985; 180: 160-4.
• 50 Watson SP. Lapetina EG 1,2-Diacylglycerol and phorbol ester inhibit agonist-induced formation of inositol phosphates in human platelets: possible implications for negative feedback regulation of inositol phospholipid hydrolysis. Proc Natl Acad Sci USA 1985; 82: 2623-6.
• 51 Siess W, Siegel FL, Lapetina EG. Arachidonic acid stimulates the formation of 1,2-diacylglycerol and phosphatidic acid in human platelets. Degree of phospholipase C activation correlates with protein phosphorylation, platelet shape change, serotonin release, and aggregation. J Biol Chem 1983; 258: 11236-42.
• 52 Pollock WK, Armstrong RA, Brydon LJ, Jones RL, MacIntyre DE. Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium. Biochem J 1984; 219: 833-42.
• 53 Heemskerk JW, Farndale RW, Sage SO. Effects of U73122 and U73343 on human platelet calcium signalling and protein tyrosine phosphorylation. Biochim Biophys Acta 1997; 1355: 81-8.
• 54 Pulcinelli FM, Gresele P, Bonuglia M, Gazzaniga PP. Evidence for separate effects of U73122 on phospholipase C and calcium channels in human platelets. Biochem Pharmacol 1998; 56: 1481-4.
• 55 Börsch-Haubold AG, Ghomashchi F, Pasquet S, Goedert M, Cohen P, Gelb MH, Watson SP. Phosphorylation of cytosolic phospholipase A₂ in platelets is mediated by multiple stress-activated protein kinase pathways. Eur J Biochem 1999; 265: 195-203.
• 56 Kramer RM, Roberts EF, Um SL, Börsch-Haubold AG, Watson SP, Fisher MJ, Jakubowski JA. p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A₂ (cPLA₂) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA₂ . J Biol Chem 1996; 271: 27723-9.
• 57 Kramer RM, Roberts EF, Manetta JV, Sportsman JR, Jakubowski JA. Ca²⁺-sensitive cytosolic phospholipase A₂ (cPLA₂) in human platelets. J Lipid Mediat 1993; 6: 209-16.
• 58 Kramer RM, Roberts EF, Manetta JV, Hyslop PA, Jakubowski JA. Thrombin-induced phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A₂ in human platelets. J Biol Chem 1993; 268: 26796-804.
• 59 Börsch-Haubold AG, Bartoli F, Asselin J, Dudler T, Kramer RM, Apitz-Castro R, Watson SP, Gelb MH. Identification of the phosphorylation sites of cytosolic phospholipase A₂ in agonist-stimulated human platelets and HeLa cells. J Biol Chem 1998; 273: 4449-58.
• 60 Bartoli F, Lin HK, Ghomashchi F, Gelb MH, Jain MK, Apitz-Castro R. Tight binding inhibitors of 85-kDa phospholipase A₂ but not 14-kDa phospholipase A₂ inhibit release of free arachidonate in thrombin-stimulated human platelets. J Biol Chem 1994; 269: 15625-30.
• 61 Riendeau D, Guay J, Weech PK, Laliberte F, Yergey J, Li C, Desmarais S, Perrier H, Liu S, Nicoll-Griffith D, Street IP. Arachidonyltrifluoro methyl ketone, a potent inhibitor of 85-kDa phospholipase A₂, blocks production of arachidonate and 12-hydroxyeicosa-tetraenoic acid by calcium ionophore-challenged platelets. J Biol Chem 1994; 269: 15619-24.
• 62 Bell RL, Kennerly DA, Stanford N, Majerus PW. Diglyceride lipase: a pathway for arachidonate release from human platelets. Proc Natl Acad Sci USA 1979; 76: 3238-41.
• 63 Bell RL, Stanford N, Kennerly DA, Majerus PW. Diglyceride lipase: a pathway for arachidonate release from human platelets. Adv PG Tx Res 1980; 6: 219-24.
• 64 Rittenhouse-Simmons S. Indomethacin-induced accumulation of diglyce-ride in activated human platelets. The role of diglyceride lipase. J Biol Chem 1980; 255: 2259-62.
• 65 Takamura H, Narita H, Park HJ, Tanaka K, Matsuura T, Kito M. Differential hydrolysis of phospholipid molecular species during activation of human platelets with thrombin and collagen. J Biol Chem 1987; 262: 2262-9.
• 66 Moriyama T, Wada K, Oki M, Matsuura T, Kito M. The mechanism of arachidonic acid release in collagen-activated human platelets. Biosci Biotechnol Biochem 1994; 58: 93-8.
• 67 Chau LY, Tai HH. Monoglyceride and diglyceride lipases from human platelet microsomes. Biochim Biophys Acta 1988; 963: 436-44.
• 68 McNicol A, Nickolaychuk BR, Israels SJ. The effects of inhibitors on collagen-induced platelet activation. Blood 1995; 85: 2177.