lonomycin-stimulated Arachidonic Acid Release in Human Platelets: a Role for Protein Kinase C and Tyrosine Phosphorylation

Philip G Hargreaves; Susanne Jenner; Janet E Merritt; Stewart O Sage; Richard W Farndale

doi:10.1055/s-0038-1650563

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1996; 76(02): 248-252
DOI: 10.1055/s-0038-1650563

Original Article

Schattauer GmbH Stuttgart

lonomycin-stimulated Arachidonic Acid Release in Human Platelets: a Role for Protein Kinase C and Tyrosine Phosphorylation

Philip G Hargreaves

¹The Department of Biochemistry, Cambridge, UK

,

Susanne Jenner

²Physiological Laboratory, University of Cambridge, Cambridge, UK

,

Janet E Merritt

³Roche Research Centre, Welwyn Garden City, Herts, UK

,

Stewart O Sage

²Physiological Laboratory, University of Cambridge, Cambridge, UK

,

Richard W Farndale

¹The Department of Biochemistry, Cambridge, UK

› Author Affiliations

Abstract

PDF Download

Summary

Collagen (10-90 Μg/ml) and ionomycin (1 ΜM; a calcium iono-phore) each evoked rises in intracellular free calcium, protein kinase C activity and arachidonic acid release in human platelets, and as previously demonstrated for collagen, ionomycin (1 p,M) stimulated protein tyrosine phosphorylation. However, at lower concentrations (60 and 250 nM) ionomycin selectively mobilised calcium. Ro31-8220 (a selective inhibitor of protein kinase C) inhibited (by 50%) ionomycin-stimulated arachidonic acid release. Genistein (an inhibitor of protein tyrosine kinases) also reduced by 50% ionomycin-stimulated arachidonic acid release. In combination, genistein and Ro31-8220 abolished ionomycin-stimulated arachidonic acid release. These findings show 1) that a rise in calcium is not sufficient, and 2) the activation of both protein kinase C and protein tyrosine phosphorylation is necessary, for full ionomycin-stimulated arachidonic acid release in human platelets.

PDF (364 kb)

References

References
1 Purdon AD, Patelumas D, Smith JB. Evidence for the release of arachidonic acid through the selective action of phospholipase A₂ in thrombin-stimulated human platelets. Biochim Biophys Acta 1987; 920: 205-214
2 Nozawa Y, Nakashima S, Nagata K. Phospholipid-mediated signalling in receptor activation of human platelets. Biochim Biophys Acta 1991; 1082: 219-238
3 Turini ME, Holub BJ. Eicosanoid/thromboxane A₂-independent and -dependent generation of lysoplasmenylethanolamine via phospholipase A2 in collagen-stimulated human platelets. Biochem J 1993; 289: 641-646
4 Brass LF, Hoxie JA, Manning DR. Signaling through G proteins and G protein-coupled receptors during platelet activation. Thromb Haemost 1993; 70: 217-223
5 Knezevic I, Borg C, Le Breton GC. Identification of G_q as one of the G-pro-teins which copurify with human platelet thromboxane A₂/prostaglandin H₂ receptors. J Biol Chem 1993; 268: 26011-26017
6 Kramer RM, Jakubowski JA, Deykin D. Hydrolysis of l-alkyl-2-arachi-donoyl-sn-glycero-3-phosphocholine, a common precursor of platelet-activating factor and eicosanoids, by human platelet phospholipase A₂ . Biochim Biophys Acta 1988; 959: 269-279
7 Smith JB, Selak MA, Dangelmaier C, Daniel JL. Cytosolic calcium as a second messenger for collagen-induced platelet responses. Biochem J 1992; 288: 925-929
8 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: 869-877
9 Halenda SP, Banga H, Zavoico GB, Lau L-F, Feinstein MB. Synergistic release of arachidonic acid from platelets by activation of protein kinase C and CA²⁺ ionophores. Evidence for the role of protein phosphorylation in the activation of phospholipase A₂ and independence from the Na⁺/H⁺ exchanger. Biochemistry (USA) 1989; 28: 7356-7363
10 Murphy CT, Elmore M, Kellie S, Westwick J. Comparison of the role of protein kinase C in platelet functional responses induced by three different mechanisms, PAF, ionomycin and arachidonic acid. Biochim Biophys Acta 1991; 1133: 46-54
11 Borsch-Haubold AG, Kramer RM, Watson SP. Cytosolic phospholipase A₂ is phosphorylated in collagen- and thrombin-stimulated human platelets independent of protein kinase C and mitogen-activated protein kinase. J Biol Chem 1995; 270: 25885-25892
12 Clark EA, Brugge JS. Tyrosine phosphorylation in platelets: potential roles in intracellular signal transduction. Trends Cardiovasc Med 1993; 3: 218-227
13 Ferrell JE, Martin GS. Platelet tyrosine-specific protein phosphorylation is regulated by thrombin. Mol Cell Biol 1988; 8: 3603-3610
14 Golden A, Brugge JS. Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets. Proc Natl Acad Sci (USA) 1989; 86: 901-905
15 Nakamura S, Yamamura H. Thrombin and collagen induce rapid phosphorylation of a common set of cellular proteins on tyrosine in human platelets. J Biol Chem 1989; 264: 7089-7091
16 Rendu F, Eldor A, Grelac F, Levy-Toledano S, Levitzki A. Tyrosine kinase blockers: new platelet activation inhibitors. Blood Coagulation and Fibrinolysis 1990; 1: 713-716
17 Salari H, Duronio V, Howard SL, Demos M, Jones K, Reany A, Hudson AT, Pelech SL. Erbstatin blocks platelet activating factor-induced protein-tyrosine phosphorylation, polyphosphoinositide hydrolysis, protein kinase C activation, serotonin secretion and aggregation of rabbit platelets. FEBS Lett 1990; 263: 104-108
18 Dillon AMR, Heath MF. Protein tyrosine phosphorylation in equine platelets: the effect of stimulation by thrombin and platelet-activating factor (PAF). Equine Vet J 1995; 27: 448-458
19 Hargreaves PG, Licking EF, Sargeant P, Barnes MJ, Famdale RW. The tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, inhibit the release of [³H] arachidonate from human platelets stimulated by thrombin or collagen. Thromb Haemost 1994; 72: 634-642
20 McNicol A, Robertson C, Gerrard JM. Vanadate activates platelets by enhancing arachidonic acid release. Blood 1993; 81: 2329-2338
21 Murthy M, Rao GH, Robinson P, Reddy S. Influx of extracellular calcium and agonist-coupling appear essential for the activation of thromboxane A₂-dependent phospholipase A₂ in human platelets. Prostaglandins Leuko-trienes and Essential Fatty Acids 1995; 53: 31-39
22 Sargeant P, Farndale RW, Sage SO. The tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and genistein reduce thrombin-evoked tyrosine phosphorylation and CA²⁺ entry in human platelets. FEBS Lett 1993; 315: 242-246
23 Kramer RM, Roberts EF, Manetta JV, Hyslop PA, Jakubowski JA. Thrombin-induced phosphorylation and activation of CA²⁺-sensitive cytosolic phospholipase A₂ in human platelets. J Biol Chem 1993; 268: 26796-26804
24 Glaser KB, Mobilo D, Chang JY, Senko N. Phospholipase A₂ enzymes: regulation and inhibiton. Trends in Pharmacol Sci 1993; 14: 92-98
25 Halenda SP, Rehm AG. Thrombin and C-kinase activators potentiate calcium-stimulated arachidonic acid release in human platelets. Biochem J 1987; 248: 471-475
26 Kramer RM, Roberts EF, Manetta JV, Sportsman JR, Jakubowski JA. CA²⁺-sensitive cytosolic phospholipase A₂ (cPLA₂) in human platelets. J Lipid Mediator 1993; 6: 209-216
27 Kramer RM, Roberts EF, Hyslop PA, Utterback BG, Hui KY, Jakubowski JA. Differential activation of cytosolic phospholipase A₂ (cPLA₂) by thrombin and thrombin receptor agonist peptide in human platelets. J Biol Chem 1995; 270: 14816-14823
28 Kramer RM, Roberts EF, Strifler BA, Johnstone EM. Thrombin induces activation of p38 MAP kinase in human platelets. J Biol Chem 1995; 270: 27395-27398
29 Saklatvala J, Rawlinson L, Waller RJ, Sarsfield S, Lee JC, Morton LF, Barnes MJ, Famdale RW. Role for p38 mitogen-activated protein kinase in platelet activation caused by collagen or a thromboxane analogue. J Biol Chem 1996; 27: 6586-6589