Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Jul 15;269(2):465–473. doi: 10.1042/bj2690465

Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors.

W R Bishop 1, J August 1, J M Petrin 1, J K Pai 1
PMCID: PMC1131600  PMID: 2386487

Abstract

Stimulation of platelets with thrombin leads to rapid degradation of inositol phospholipids, generation of diacylglycerol (DAG) and subsequent activation of protein kinase C (PKC). Previous studies indicated that prior activation of PKC with phorbol myristate acetate (PMA) desensitizes platelets to thrombin stimulation, as indicated by a decreased production of inositol phosphates and decreased Ca2+ mobilization. This suggests that PKC activation generates negative-feedback signals, which limit the phosphoinositide response. To test this hypothesis further, we examined the effects of PKC activators and inhibitors on thrombin-stimulated DAG mass formation in platelets. Pretreatment with PMA abolishes thrombin-stimulated DAG formation (50% inhibition at 60 nM). Pretreatment of platelets with the PKC inhibitors K252a or staurosporine potentiates DAG production in response to thrombin (3-4-fold) when using concentrations required to inhibit platelet PKC (1-10 microM). K252a does not inhibit phosphorylation of endogenous DAG or phosphorylation of a cell-permeant DAG in unstimulated platelets, indicating that DAG over-production is not due to inhibition of DAG kinase. Sphingosine, a PKC inhibitor with a different mechanism of action, also potentiates DAG formation in response to thrombin. Several lines of evidence indicate that DAG formation under the conditions employed occurs predominantly by phosphoinositide (and not phosphatidylcholine) hydrolysis: (1) PMA alone does not elicit DAG formation, but inhibits agonist-stimulated DAG formation; (2) thrombin-stimulated DAG formation is inhibited by neomycin (1-10 mM) but not by the phosphatidate phosphohydrolase inhibitor propranolol; and (3) no metabolism of radiolabelled phosphatidylcholine was observed upon stimulation by thrombin or PMA. These data provide strong support for a role of PKC in limiting the extent of platelet phosphoinositide hydrolysis.

Full text

PDF
465

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abdel-Latif A. A., Smith J. P. Effects of DL-propranolol on the synthesis of glycerolipids by rabbit iris muscle. Biochem Pharmacol. 1976 Aug 1;25(15):1697–1704. doi: 10.1016/0006-2952(76)90401-9. [DOI] [PubMed] [Google Scholar]
  2. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  3. Banno Y., Yada Y., Nozawa Y. Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets. J Biol Chem. 1988 Aug 15;263(23):11459–11465. [PubMed] [Google Scholar]
  4. Bell R. L., Kennerly D. A., Stanford N., Majerus P. W. Diglyceride lipase: a pathway for arachidonate release from human platelets. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3238–3241. doi: 10.1073/pnas.76.7.3238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bennett C. F., Balcarek J. M., Varrichio A., Crooke S. T. Molecular cloning and complete amino-acid sequence of form-I phosphoinositide-specific phospholipase C. Nature. 1988 Jul 21;334(6179):268–270. doi: 10.1038/334268a0. [DOI] [PubMed] [Google Scholar]
  6. Bennett C. F., Crooke S. T. Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo. J Biol Chem. 1987 Oct 5;262(28):13789–13797. [PubMed] [Google Scholar]
  7. Besterman J. M., Duronio V., Cuatrecasas P. Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6785–6789. doi: 10.1073/pnas.83.18.6785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Billah M. M., Eckel S., Mullmann T. J., Egan R. W., Siegel M. I. Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in signal transduction. J Biol Chem. 1989 Oct 15;264(29):17069–17077. [PubMed] [Google Scholar]
  9. Billah M. M., Lapetina E. G. Rapid decrease of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets. J Biol Chem. 1982 Nov 10;257(21):12705–12708. [PubMed] [Google Scholar]
  10. Billah M. M., Pai J. K., Mullmann T. J., Egan R. W., Siegel M. I. Regulation of phospholipase D in HL-60 granulocytes. Activation by phorbol esters, diglyceride, and calcium ionophore via protein kinase- independent mechanisms. J Biol Chem. 1989 May 25;264(15):9069–9076. [PubMed] [Google Scholar]
  11. Bishop W. R., Bell R. M. Attenuation of sn-1,2-diacylglycerol second messengers. Metabolism of exogenous diacylglycerols by human platelets. J Biol Chem. 1986 Sep 25;261(27):12513–12519. [PubMed] [Google Scholar]
  12. Bishop W. R., Bell R. M. Functions of diacylglycerol in glycerolipid metabolism, signal transduction and cellular transformation. Oncogene Res. 1988 Feb;2(3):205–218. [PubMed] [Google Scholar]
  13. Bishop W. R., Ganong B. R., Bell R. M. Attenuation of sn-1,2-diacylglycerol second messengers by diacylglycerol kinase. Inhibition by diacylglycerol analogs in vitro and in human platelets. J Biol Chem. 1986 May 25;261(15):6993–7000. [PubMed] [Google Scholar]
  14. Blackmore P. F., Exton J. H. Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate. J Biol Chem. 1986 Aug 25;261(24):11056–11063. [PubMed] [Google Scholar]
  15. Bocckino S. B., Blackmore P. F., Wilson P. B., Exton J. H. Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism. J Biol Chem. 1987 Nov 5;262(31):15309–15315. [PubMed] [Google Scholar]
  16. Broekman M. J., Ward J. W., Marcus A. J. Fatty acid composition of phosphatidylinositol and phosphatidic acid in stimulated platelets. Persistence of arachidonyl-stearyl structure. J Biol Chem. 1981 Aug 25;256(16):8271–8274. [PubMed] [Google Scholar]
  17. Carlson K. E., Brass L. F., Manning D. R. Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than Gi in human platelets. J Biol Chem. 1989 Aug 5;264(22):13298–13305. [PubMed] [Google Scholar]
  18. Crouch M. F., Lapetina E. G. A role for Gi in control of thrombin receptor-phospholipase C coupling in human platelets. J Biol Chem. 1988 Mar 5;263(7):3363–3371. [PubMed] [Google Scholar]
  19. Crouch M. F., Lapetina E. G. Dual mechanisms of platelet hormone receptor desensitization. Differential importance between agonists of protein kinase C-dependent and -independent pathways. J Biol Chem. 1989 Jan 5;264(1):584–588. [PubMed] [Google Scholar]
  20. Daniel L. W., Waite M., Wykle R. L. A novel mechanism of diglyceride formation. 12-O-tetradecanoylphorbol-13-acetate stimulates the cyclic breakdown and resynthesis of phosphatidylcholine. J Biol Chem. 1986 Jul 15;261(20):9128–9132. [PubMed] [Google Scholar]
  21. Eichberg J., Gates J., Hauser G. The mechanism of modification by propranolol of the metabolism of phosphatidyl-CMP (CDP-diacylglycerol) and other lipids in the rat pineal gland. Biochim Biophys Acta. 1979 Apr 27;573(1):90–106. doi: 10.1016/0005-2760(79)90176-0. [DOI] [PubMed] [Google Scholar]
  22. Hakata H., Kambayashi J., Kosaki G. Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine platelets. J Biochem. 1982 Sep;92(3):929–935. doi: 10.1093/oxfordjournals.jbchem.a134008. [DOI] [PubMed] [Google Scholar]
  23. Hannun Y. A., Greenberg C. S., Bell R. M. Sphingosine inhibition of agonist-dependent secretion and activation of human platelets implies that protein kinase C is a necessary and common event of the signal transduction pathways. J Biol Chem. 1987 Oct 5;262(28):13620–13626. [PubMed] [Google Scholar]
  24. Hannun Y. A., Loomis C. R., Merrill A. H., Jr, Bell R. M. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J Biol Chem. 1986 Sep 25;261(27):12604–12609. [PubMed] [Google Scholar]
  25. Irving H. R., Exton J. H. Phosphatidylcholine breakdown in rat liver plasma membranes. Roles of guanine nucleotides and P2-purinergic agonists. J Biol Chem. 1987 Mar 15;262(8):3440–3443. [PubMed] [Google Scholar]
  26. Kase H., Iwahashi K., Nakanishi S., Matsuda Y., Yamada K., Takahashi M., Murakata C., Sato A., Kaneko M. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Commun. 1987 Jan 30;142(2):436–440. doi: 10.1016/0006-291x(87)90293-2. [DOI] [PubMed] [Google Scholar]
  27. Kikuchi A., Ikeda K., Kozawa O., Takai Y. Modes of inhibitory action of protein kinase C in the chemotactic peptide-induced formation of inositol phosphates in differentiated human leukemic (HL-60) cells. J Biol Chem. 1987 May 15;262(14):6766–6770. [PubMed] [Google Scholar]
  28. King W. G., Rittenhouse S. E. Inhibition of protein kinase C by staurosporine promotes elevated accumulations of inositol trisphosphates and tetrakisphosphate in human platelets exposed to thrombin. J Biol Chem. 1989 Apr 15;264(11):6070–6074. [PubMed] [Google Scholar]
  29. Kolesnick R. N. 1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells. J Biol Chem. 1987 Dec 15;262(35):16759–16762. [PubMed] [Google Scholar]
  30. Kolesnick R. N., Paley A. E. 1,2-Diacylglycerols and phorbol esters stimulate phosphatidylcholine metabolism in GH3 pituitary cells. Evidence for separate mechanisms of action. J Biol Chem. 1987 Jul 5;262(19):9204–9210. [PubMed] [Google Scholar]
  31. Lambeth J. D., Burnham D. N., Tyagi S. R. Sphinganine effects on chemoattractant-induced diacylglycerol generation, calcium fluxes, superoxide production, and on cell viability in the human neutrophil. Delivery of sphinganine with bovine serum albumin minimizes cytotoxicity without affecting inhibition of the respiratory burst. J Biol Chem. 1988 Mar 15;263(8):3818–3822. [PubMed] [Google Scholar]
  32. Lipsky J. J., Lietman P. S. Aminoglycoside inhibition of a renal phosphatidylinositol phospholipase C. J Pharmacol Exp Ther. 1982 Feb;220(2):287–292. [PubMed] [Google Scholar]
  33. MacDonald M. L., Mack K. F., Richardson C. N., Glomset J. A. Regulation of diacylglycerol kinase reaction in Swiss 3T3 cells. Increased phosphorylation of endogenous diacylglycerol and decreased phosphorylation of didecanoylglycerol in response to platelet-derived growth factor. J Biol Chem. 1988 Jan 25;263(3):1575–1583. [PubMed] [Google Scholar]
  34. MacDonald M. L., Mack K. F., Williams B. W., King W. C., Glomset J. A. A membrane-bound diacylglycerol kinase that selectively phosphorylates arachidonoyl-diacylglycerol. Distinction from cytosolic diacylglycerol kinase and comparison with the membrane-bound enzyme from Escherichia coli. J Biol Chem. 1988 Jan 25;263(3):1584–1592. [PubMed] [Google Scholar]
  35. Majerus P. W., Connolly T. M., Deckmyn H., Ross T. S., Bross T. E., Ishii H., Bansal V. S., Wilson D. B. The metabolism of phosphoinositide-derived messenger molecules. Science. 1986 Dec 19;234(4783):1519–1526. doi: 10.1126/science.3024320. [DOI] [PubMed] [Google Scholar]
  36. Muir J. G., Murray A. W. Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C. J Cell Physiol. 1987 Mar;130(3):382–391. doi: 10.1002/jcp.1041300311. [DOI] [PubMed] [Google Scholar]
  37. Muir J. G., Murray A. W. Phosphatidylcholine synthesis in platelets is stimulated by diacylglycerol but not by phorbol ester. Biochem Int. 1988 Jun;16(6):1145–1151. [PubMed] [Google Scholar]
  38. Orellana S., Solski P. A., Brown J. H. Guanosine 5'-O-(thiotriphosphate)-dependent inositol trisphosphate formation in membranes is inhibited by phorbol ester and protein kinase C. J Biol Chem. 1987 Feb 5;262(4):1638–1643. [PubMed] [Google Scholar]
  39. Pai J. K., Siegel M. I., Egan R. W., Billah M. M. Activation of phospholipase D by chemotactic peptide in HL-60 granulocytes. Biochem Biophys Res Commun. 1988 Jan 15;150(1):355–364. doi: 10.1016/0006-291x(88)90528-1. [DOI] [PubMed] [Google Scholar]
  40. Pai J. K., Siegel M. I., Egan R. W., Billah M. M. Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes. J Biol Chem. 1988 Sep 5;263(25):12472–12477. [PubMed] [Google Scholar]
  41. Paris S., Magnaldo I., Pouysségur J. Homologous desensitization of thrombin-induced phosphoinositide breakdown in hamster lung fibroblasts. J Biol Chem. 1988 Aug 15;263(23):11250–11256. [PubMed] [Google Scholar]
  42. Pittet D., Krause K. H., Wollheim C. B., Bruzzone R., Lew D. P. Nonselective inhibition of neutrophil functions by sphinganine. J Biol Chem. 1987 Jul 25;262(21):10072–10076. [PubMed] [Google Scholar]
  43. Preiss J., Loomis C. R., Bishop W. R., Stein R., Niedel J. E., Bell R. M. Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J Biol Chem. 1986 Jul 5;261(19):8597–8600. [PubMed] [Google Scholar]
  44. Rhee S. G., Suh P. G., Ryu S. H., Lee S. Y. Studies of inositol phospholipid-specific phospholipase C. Science. 1989 May 5;244(4904):546–550. doi: 10.1126/science.2541501. [DOI] [PubMed] [Google Scholar]
  45. Rider L. G., Dougherty R. W., Niedel J. E. Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils. J Immunol. 1988 Jan 1;140(1):200–207. [PubMed] [Google Scholar]
  46. Rittenhouse S. E., Sasson J. P. Mass changes in myoinositol trisphosphate in human platelets stimulated by thrombin. Inhibitory effects of phorbol ester. J Biol Chem. 1985 Jul 25;260(15):8657–8660. [PubMed] [Google Scholar]
  47. Rubin R. Phosphatidylethanol formation in human platelets: evidence for thrombin-induced activation of phospholipase D. Biochem Biophys Res Commun. 1988 Nov 15;156(3):1090–1096. doi: 10.1016/s0006-291x(88)80744-7. [DOI] [PubMed] [Google Scholar]
  48. Ryu S. H., Cho K. S., Lee K. Y., Suh P. G., Rhee S. G. Purification and characterization of two immunologically distinct phosphoinositide-specific phospholipases C from bovine brain. J Biol Chem. 1987 Sep 15;262(26):12511–12518. [PubMed] [Google Scholar]
  49. Siess W., Siegel F. L., Lapetina E. G. 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 Sep 25;258(18):11236–11242. [PubMed] [Google Scholar]
  50. Slivka S. R., Insel P. A. Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells. Evidence that bradykinin mediates noninterdependent activation of phospholipases A2 and C. J Biol Chem. 1988 Oct 15;263(29):14640–14647. [PubMed] [Google Scholar]
  51. Slivka S. R., Meier K. E., Insel P. A. Alpha 1-adrenergic receptors promote phosphatidylcholine hydrolysis in MDCK-D1 cells. A mechanism for rapid activation of protein kinase C. J Biol Chem. 1988 Sep 5;263(25):12242–12246. [PubMed] [Google Scholar]
  52. Smith C. D., Uhing R. J., Snyderman R. Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters. J Biol Chem. 1987 May 5;262(13):6121–6127. [PubMed] [Google Scholar]
  53. Takamura H., Narita H., Park H. J., 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 Feb 15;262(5):2262–2269. [PubMed] [Google Scholar]
  54. Takuwa N., Takuwa Y., Rasmussen H. A tumour promoter, 12-O-tetradecanoylphorbol 13-acetate, increases cellular 1,2-diacylglycerol content through a mechanism other than phosphoinositide hydrolysis in Swiss-mouse 3T3 fibroblasts. Biochem J. 1987 May 1;243(3):647–653. doi: 10.1042/bj2430647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
  56. Tohmatsu T., Nakashima S., Hattori H., Suganuma A., Nozawa Y. A role of diacylglycerol kinase in stimulus-secretion coupling of human platelets. Dissociation of serotonin secretion from Ca2+ mobilization. Thromb Res. 1987 Jul 1;47(1):25–35. doi: 10.1016/0049-3848(87)90237-4. [DOI] [PubMed] [Google Scholar]
  57. Tysnes O. B., Steen V. M., Holmsen H. Neomycin inhibits platelet functions and inositol phospholipid metabolism upon stimulation with thrombin, but not with ionomycin or 12-O-tetradecanoyl-phorbol 13-acetate. Eur J Biochem. 1988 Oct 15;177(1):219–223. doi: 10.1111/j.1432-1033.1988.tb14365.x. [DOI] [PubMed] [Google Scholar]
  58. Watson S. P., Lapetina E. G. 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 U S A. 1985 May;82(9):2623–2626. doi: 10.1073/pnas.82.9.2623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Watson S. P., McNally J., Shipman L. J., Godfrey P. P. The action of the protein kinase C inhibitor, staurosporine, on human platelets. Evidence against a regulatory role for protein kinase C in the formation of inositol trisphosphate by thrombin. Biochem J. 1988 Jan 15;249(2):345–350. doi: 10.1042/bj2490345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wilson D. B., Neufeld E. J., Majerus P. W. Phosphoinositide interconversion in thrombin-stimulated human platelets. J Biol Chem. 1985 Jan 25;260(2):1046–1051. [PubMed] [Google Scholar]
  61. Yamada K., Iwahashi K., Kase H. K252a, a new inhibitor of protein kinase C, concomitantly inhibits 40K protein phosphorylation and serotonin secretion in a phorbol ester-stimulated platelets. Biochem Biophys Res Commun. 1987 Apr 14;144(1):35–40. doi: 10.1016/s0006-291x(87)80471-0. [DOI] [PubMed] [Google Scholar]
  62. Zavoico G. B., Halenda S. P., Sha'afi R. I., Feinstein M. B. Phorbol myristate acetate inhibits thrombin-stimulated Ca2+ mobilization and phosphatidylinositol 4,5-bisphosphate hydrolysis in human platelets. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3859–3862. doi: 10.1073/pnas.82.11.3859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. de Chaffoy de Courcelles D. C., Roevens P., Van Belle H. R 59 022, a diacylglycerol kinase inhibitor. Its effect on diacylglycerol and thrombin-induced C kinase activation in the intact platelet. J Biol Chem. 1985 Dec 15;260(29):15762–15770. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES