Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Sep;80(17):5417–5420. doi: 10.1073/pnas.80.17.5417

Human platelets contain phospholipase C that hydrolyzes polyphosphoinositides.

S E Rittenhouse
PMCID: PMC384267  PMID: 6310576

Abstract

Stimulated human platelets are known to undergo marked and rapid changes in phosphoinositide metabolism consistent with the activation of phospholipase C. Such changes may promote a Ca2+ flux after platelets are exposed to agonists. I have examined this enzymatic activity by using disrupted platelets. When human platelets are sonicated and then incubated with phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) or phosphatidylinositol 4-monophosphate (PtdIns4P) in the presence of Ca2+ and deoxycholate, marked hydrolysis of these substrates occurs. Characterization of the hydrolysis products by anion exchange and thin-layer chromatography indicates that the bulk of this activity is enzymatic and attributable to phospholipase C. In the absence of Ca2+ or deoxycholate, only phosphomonoesterase activity is observed. I partially purified the soluble phospholipase C on DEAE-cellulose in order to minimize phosphomonoesterase activity. Fractions eluting at low salt concentrations contain the highest phospholipase C activity with respect to PtdIns4,5P2 and PtdIns4P and the lowest phosphomonoesterase activity. The enzyme(s) in these fractions is (are) maximally active in the presence of 0.1 mM Ca2+ and deoxycholate (1 mg/ml) and display(s) substrate affinities in the order PtdIns greater than PtdIns4P greater than PtdIns4,5P2 and maximum rates in the order PtdIns4P greater than PtdIns4,5P2 greater than PtdIns. This order of substrate preference appears to differ from that observed for physiologically stimulated cells. Possible reasons for such a discrepancy are discussed.

Full text

PDF
5417

Selected References

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

  1. Agranoff B. W., Murthy P., Seguin E. B. Thrombin-induced phosphodiesteratic cleavage of phosphatidylinositol bisphosphate in human platelets. J Biol Chem. 1983 Feb 25;258(4):2076–2078. [PubMed] [Google Scholar]
  2. Atherton R. S., Hawthorne J. N. The phosphoinositide inositolphosphohydrolase of guinea-pig intestinal mucosa. Eur J Biochem. 1968 Mar;4(1):68–75. doi: 10.1111/j.1432-1033.1968.tb00173.x. [DOI] [PubMed] [Google Scholar]
  3. Billah M. M., Lapetina E. G. Platelet-activating factor stimulates metabolism of phosphoinositides in horse platelets: possible relationship to Ca2+ mobilization during stimulation. Proc Natl Acad Sci U S A. 1983 Feb;80(4):965–968. doi: 10.1073/pnas.80.4.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Cohen P., Broekman M. J., Verkley A., Lisman J. W., Derksen A. Quantification of human platelet inositides and the influence of ionic environment on their incorporation of orthophosphate-32P. J Clin Invest. 1971 Apr;50(4):762–772. doi: 10.1172/JCI106547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DESJOBERT A., PETEK F. Chromatographie sur papier des esters phosphoriques de l'inositol; application a l'étude de la dégradation hydrolytique de l'inositolhexaphosphate. Bull Soc Chim Biol (Paris) 1956 Sep 4;38(5-6):871–883. [PubMed] [Google Scholar]
  7. Downes C. P., Michell R. H. The polyphosphoinositide phosphodiesterase of erythrocyte membranes. Biochem J. 1981 Jul 15;198(1):133–140. doi: 10.1042/bj1980133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fine J. B., Sprecher H. Unidimensional thin-layer chromatography of phospholipids on boric acid-impregnated plates. J Lipid Res. 1982 May;23(4):660–663. [PubMed] [Google Scholar]
  9. Gonzalez-Sastre F., Folch-Pi J. Thin-layer chromatography of the phosphoinositides. J Lipid Res. 1968 Jul;9(4):532–533. [PubMed] [Google Scholar]
  10. Hirasawa K., Irvine R. F., Dawson R. M. Proteolytic activation can produce a phosphatidylinositol phosphodiesterase highly sensitive to Ca2+. Biochem J. 1982 Sep 15;206(3):675–678. doi: 10.1042/bj2060675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hofmann S. L., Majerus P. W. Modulation of phosphatidylinositol-specific phospholipase C activity by phospholipid interactions, diglycerides, and calcium ions. J Biol Chem. 1982 Dec 10;257(23):14359–14364. [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Lloyd J. V., Mustard J. F. Changes in 32P-content of phosphatidic acid and the phosphoinositides of rabbit platelets during aggregation induced by collagen or thrombin. Br J Haematol. 1974 Feb;26(2):243–253. doi: 10.1111/j.1365-2141.1974.tb00469.x. [DOI] [PubMed] [Google Scholar]
  14. Michell R. H. Is phosphatidylinositol really out of the calcium gate? Nature. 1982 Apr 8;296(5857):492–493. doi: 10.1038/296492a0. [DOI] [PubMed] [Google Scholar]
  15. Palmer F. B. Chromatography of acidic phospholipids on immobilized neomycin. J Lipid Res. 1981 Nov;22(8):1296–1300. [PubMed] [Google Scholar]
  16. RAAFLAUB J. Applications of metal buffers and metal indicators in biochemistry. Methods Biochem Anal. 1956;3:301–325. doi: 10.1002/9780470110195.ch10. [DOI] [PubMed] [Google Scholar]
  17. Rittenhouse-Simmons S. Production of diglyceride from phosphatidylinositol in activated human platelets. J Clin Invest. 1979 Apr;63(4):580–587. doi: 10.1172/JCI109339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rittenhouse S. E., Allen C. L. Synergistic activation by collagen and 15-hydroxy-9 alpha,11 alpha-peroxidoprosta-5,13-dienoic acid (PGH2) of phosphatidylinositol metabolism and arachidonic acid release in human platelets. J Clin Invest. 1982 Dec;70(6):1216–1224. doi: 10.1172/JCI110720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rittenhouse S. E. Inositol lipid metabolism in the responses of stimulated platelets. Cell Calcium. 1982 Oct;3(4-5):311–322. doi: 10.1016/0143-4160(82)90019-7. [DOI] [PubMed] [Google Scholar]
  20. Rittenhouse S. E. Preparation of selectively labeled phosphatidylinositol and assay of phosphatidylinositol-specific phospholipase C. Methods Enzymol. 1982;86:3–11. doi: 10.1016/0076-6879(82)86161-2. [DOI] [PubMed] [Google Scholar]
  21. Schacht J. Purification of polyphosphoinositides by chromatography on immobilized neomycin. J Lipid Res. 1978 Nov;19(8):1063–1067. [PubMed] [Google Scholar]
  22. Schatzmann H. J. Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells. J Physiol. 1973 Dec;235(2):551–569. doi: 10.1113/jphysiol.1973.sp010403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shukla S. D., Hanahan D. J. AGEPC (platelet activating factor) induced stimulation of rabbit platelets: effects on phosphatidylinositol, di- and triphosphoinositides and phosphatidic acid metabolism. Biochem Biophys Res Commun. 1982 Jun 15;106(3):697–703. doi: 10.1016/0006-291x(82)91767-3. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES