Thromb Haemost 1976; 35(02): 364-376
DOI: 10.1055/s-0038-1647930
Original Article
Schattauer GmbH

Metabolie Properties of Human Platelet Membranes

II. Thrombin-lnduced Phosphorylation of Membrane Lipids and Demonstration of Phosphorylating Enzymes in the Platelet Membrane[*]
Hermann Dietrich Kaulen
1   Medizinische Universitätsklinik Köln
,
Rudolf Gross
1   Medizinische Universitätsklinik Köln
› Author Affiliations
Further Information

Publication History

Received 21 April 1975

Accepted 18 November 1975

Publication Date:
02 July 2018 (online)

Summary

Washed human platelets have been labeled with either 32Pi or glycerol- 1-14C and the distribution of the label in the phospholipids determined. 32Pi was introduced primarily into polyphosphoinositides, i. e. di- and triphosphoinositide, whereas the label from glycerol which indicates de novo synthesis of lipid molecules did not appear in these phospholipids. In the course of thrombin-induced aggregation and release the phosphate incorporation into phos-phatic acid, di- and triphosphoinositide was rapidly stimulated in parallel to the platelet reaction. The incorporation of glycerol did not change under the same conditions.

It is concluded that phosphoinositides with rapid incorporation of phosphate groups are not as rapidly synthesized de novo and presumably form a separate phospholipid pool in the platelets. Only the phosphorylating reactions are stimulated by the thrombin aggregation. The necessary enzymes for these reactions, namely diglyceride kinase, phosphatidylinositol kinase, and phosphatidylinositol-phosphate kinase all can be shown to be associated with a well characterized platelet membrane fraction.

* Part I see this journal 30: 199 (1973)


 
  • References

  • 1 Agranoff B. W, Benjamin J. A, Hajra A. K. 1969; Biosynthesis of Phosphatidylinositol. Annals of the New York Academy of Sciences 165: 755.
  • 2 Aster R. H, Jandl J. H. 1964; Platelet Sequestration in Man. I. Methods. Journal of Clinical Investigation 43: 856.
  • 3 Brockerhoff H. 1963; Breakdown of phospholipid in mild alkaline hydrolysis. Journal of Lipid Research 4: 96.
  • 4 Brockerhoff H, Ballou C. E. 1962; Phosphate Incorporation in Brain Phosphoinositides. Journal of Biological Chemistry 237: 49.
  • 5 Buckley Th. J, Hawthorne J. N. 1972; Erythrocyte Membrane Polyphosphoinositide Metabolism and the Regulation of Calcium Binding. Journal of Biological Chemistry 247: 7218.
  • 6 Call II F. L, Williams W. J. 1970; Biosynthesis of Cytidine Diphosphate Diglyceride by Human Platelets. Journal of Clinical Investigations 49: 392.
  • 7 Call II F. L, Rubert M. 1973; Diglyceride kinase in Human Platelets. Journal of Lipid Research 14: 466.
  • 8 Cohen Ph, Derksen A. 1969; Comparison of phospholipid and fatty acid composition of human erythrocytes and platelets. British Journal of Haematology 17: 359.
  • 9 Cohen Ph, Broekman M. J, Verkley A, Lismann J. W. W, Derksen A. 1971; Quantification of Human Platelet Inositides and the Influence of the Ionic environment on their Incorporation of Orthophosphate - 32P. Journal of Clinical Investigations 50: 762.
  • 10 Deyktn D. 1973; Altered Lipid Metabolism in Human Platelets after primary aggregation. Journal of Clinical Investigations 52: 483.
  • 11 Deyktn D, Snyder D. 1973; Effect of epinephrine on platelet lipid metabolism. Journal of Laboratory and Clinical Medicine 82: 554.
  • 12 Ellis R. B, Galliard T, Hawthorne J. N. 1963; Phosphoinositides. 5. The Inositol Lipids of Ox Brain. Biochemical Journal 88: 125.
  • 13 Fisher D. B, Mueller G. C. 1971; Studies on the Mechanism by which Phytohämagglutinin rapidly stimulates Phospholipid Metabolism of Human Lymphocytes. Biochimica et Biophysica Acta 248: 434.
  • 14 Folch J. 1942; Brain Cephalin, a Mixture of Phosphatides. Journal of Biological Chemistry 146: 35.
  • 15 Folch J. 1949; Complete Fractionation of Brain Cephalin: Isolation from it of Phosphatidyl Serine, Phosphatidyl Ethanolamine, and Diphosphoinositide. Journal of Biological Chemistry 177: 497.
  • 16 Galliard T, Michell R. H, Hawthorne J. N. 1965; Incorporation of Phosphate into Diphosphoinositide by Subcellular Fractions from Liver. Biochemica et Biophysica Acta 106: 551.
  • 17 Glynn I. M, Chappell J. B. 1964; A simple method for the preparation of 32P-labeled adenosine triphosphate. Biochemical Journal 90: 147.
  • 18 Gonzalez-Sastre F, Folch-Pi J. 1968; Thin-Layer Chromatography of the Phosphoinositides. Journal of Lipid Research 9: 532.
  • 19 Grette K. 1962; Studies on the mechanism of thrombin catalyzed hemostatic reactions in blood platelets. Acta physioiogica Scandinavica 56 suppl. 195.
  • 20 Hajra A. K, Sequin E. B, Agranoff B. W. 1968; Rapid Labeling of Mitochondrial Lipid by labeled Orthophosphate and Adenosine Triphosphate. Journal of Biological Chemistry 243: 1609.
  • 21 Hendrickson St. H, Reinertsen J. L. 1969; Comparison of Metal-Binding Properties of trans-1,2-Cyclohexanediol Diphosphate and Deacylated Phosphoinositides. Biochemistry 8: 4855.
  • 22 Hoktn L. E. 1969; Functional Acticity in Glands and Synaptic Tissue and the Turnover of Phosphatidylinositol. Annals of the New York Academy of Sciences 165: 695.
  • 23 Hokin L. E, Hokin M. R. 1964; The Incorporation of 32P from Adenosine Triphosphate into Polyphosphoinositides and Phosphatide Acid in Erythrocyte Membranes. Biochimica et Biophysica Acta 84: 563.
  • 24 Holmsen H, Day H. J, Stormorken H. 1969 The Blood Platelet Release Reaction. Scandinavian Journal of Haematology. Suppl. 8.
  • 25 Kai M, Hawthorne J. N. 1969; Physiological significance of polyphosphoinositides in brain. Annals of the New York Academy of Sciences 165: 761.
  • 26 Kai M, Hawthorne J. N. 1966; Incorporation of Injected 32P-Phosphate into the Phosphoinositides of Subcellular Fractions from Young Rat Brain. Biochemical Journal 98: 62.
  • 27 Kai M, White G. L, Hawthorne J. N. 1966; The Phosphatidylinositol Kinase of Rat Brain. Biochemical Journal 101: 328.
  • 28 Karnovsky M. L, Wallach D. F. H. 1961; The Metabolic Basis of Phagocytosis. III. Incorporation of inorganic Phosphate into various Classes of Phosphatides during phagocytosis. Journal of Biological Chemistry 236: 1895.
  • 29 Kaulen H. D, Gross R. 1971; The Differentiation of Acid Phosphatases of Human Blood Platelets. Thrombosis et Diathesis Haemorrhagica 26: 353.
  • 30 Kaulen H. D. 1972; Separation of Phosphatidylserine and -inositol by One-dimensional Thin-layer Chromatography of Lipid Extracts. Analytical Biochemistry 45: 664.
  • 31 Kaulen H. D, Gross R. 1972; Demonstration of Adenosin-3’,5’-Monophosphate Dependent Protein Phosphokinase in Human Platelets. Klinische Wochenschrift 50: 1114.
  • 32 Kaulen H. D, Gross R. 1973; Metabolic Properties of Human Platelet Membranes. I. Characterization of Platelet Membranes prepared by Sucrose and Ficoll Density Gradients. Thrombosis et Diathesis Haemorrhagica 30: 199.
  • 33 Lloyd J. V, Nishizawa E. E, Haldar J, Mustard J. F. 1972; Changes in 32P-Labelling of Platelet Phospholipids in Response to ADP. British Journal of Haematology 23: 571.
  • 34 Lloyd J. V, Nishizawa E. E, Joist J. H, Mustard J. F. 1973; a Effect of ADP-Induced Aggregation on 32PO4 Incorporation into Phosphatidic Acid and the Phosphoinositides of Rabbit Platelets. British Journal of Haematology 24: 589.
  • 35 Lloyd J. V, Nishizawa E. E, Mustard J. F. 1973; b Effect of ADP-induced Shape Change on Incorporation of 32P into Platelet Phosphatidic Acid and Mono-, Di-, and Triphosphatidyl Inositol. British Journal of Haematology 25: 77.
  • 36 Lloyd J. V, Mustard J. F. 1974; Changes in the32P-Content of Phosphatidic Acid and the Phosphoinositides of Rabbit Platelets during Aggregation induced by Collagen or Thrombin. British Journal of Haematology 26: 243.
  • 37 Larrabee M. G, Leicht W. S. 1965; Metabolism of phosphatidylinositol and other lipids in active neurons of sympathetic ganglia and other peripheral nervous tissues. The Site of the Inositide effect. Journal of Neurochemistry 12: 1.
  • 38 Lewis N, Majerus Ph. W. 1969; Lipid Metabolism in Human Platelets. II. De-novo phospholipid synthesis and the effect of thrombin on the pattern of synthesis. Journal of Clinical Investigation 48: 2114.
  • 39 Lucas Ch. T, Call II F. L, Williams W. J. 1970; The Biosynthesis of Phosphatidyl Inositol in Human Platelets. Journal of Clinical Investigations 49: 1949.
  • 40 Mazuzawa Y, Osawa T, Inoue K, Nojima S. 1973; Effects of various mitogens on the phospholipid metabolism of human peripheral lymphocytes. Biochimica et Biophysica Acta 326: 339.
  • 41 Mustard J. F, Perry D. W, Ardlie N. G, Packham M. A. 1972; Preparation of Suspensions of Washed Platelets from Humans. British Journal of Haematology 22: 193.
  • 42 Paulus H, Kennedy E. P. 1960; The enzymatic synthesis of Inositol Monophosphatide. Journal of Biological Chemistry 235: 1303.
  • 43 Paysant M, Bursztein C, Maupin B, Polonovski J. 1962; Métabolisme phospholipidique du sang. IV. Incorporation in vitro du 32P dans les phospholipides plaquettaires du sang humain. Bulletin de la Société de chimie biologique 44: 477.
  • 44 Phillips D. R, Agin P. P. 1974; Thrombin substrates and the proteolytic site of thrombin action on human platelet plasma membranes. Biochimica et Biophysica Acta 352: 218.
  • 45 Santiago-Calvo E, Mule S, Redman C. M, Hokin M. R, Hokin L. E. 1964; The chromatographic separation of polyphosphoinositides and studies on their turnover in various tissues. Biochimica et Biophysica Acta 84: 550.
  • 46 Sastry P. S, Hokin L. E. 1966; Studies on the Role of Phospholipids in Phagocytosis. Journal of Biological Chemistry 241: 3354.
  • 47 Schneider R. P, Kirschner L. B. 1970; Di- and Triphosphoinositide Metabolism in swine erythrocyte membranes. Biochemica et Biophysica Acta 202: 283.
  • 48 Wells M. A, Dittmer J. C. 1965; The Quantitative Extraction and Analysis of Brain Phosphoinositides. Biochemistry 4: 2459.
  • 49 Wells M. A, Dittmer J. C. 1966; A Microanalytical Technique for the Quantitative Determination of Twenty-four Classes of Brain Lipids. Biochemistry 5: 3405.
  • 50 Westermann M. P, Jensen W. N. 1962; The in vitro Incorporation of Radiophosphorus into the Phosphatides of Normal Human Platelets. Blood 20: 796.
  • 51 White G. L, Schellhase H. U, Hawthorne J. N. 1974; Phosphoinositide metabolism in rat superior cervical ganglion, vagus and phrenic nerve: effects of electric stimulation and various blocking agents. Journal of Neurochemistry 22: 149.