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PMID: 169298 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Thrombin-induced protein phosphorylation in human platelets.

The Journal of clinical investigation ·Vol. 56 ·No. 4 ·1975-10-00 ·Pages 924-36

Lyons RM, Stanford N, Majerus PW

Abstract

Intact human platelets loaded with 32PO4 contain multiple phosphorylated proteins. Thrombin treatment of intact 32PO4-loaded platelets results in a 2-6-fold increase in phosphorylation of a platelet protein (designated "peak 7" protein) of approximately 40,000 mol wt as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by gel filtration on Sephadex G-150. A similar increase in phosphorylation was observed in a platelet protein (designated "peak 9" protein) of approximately 20,000 mol wt. The time for half-maximal phosphorylation of peak 7 and peak 9 protein was 10-14 s. The concentration of thrombin at half-maximal phosphorylation was 0.25 U/ml for both proteins. Prior incubation of platelets with dibutyryl cyclic adenosine 3',5'-monophosphate or prostaglandin E1 inhibited thrombin-induced peak 7 and peak 9 protein phosphorylation. The erythroagglutinating phytohemagglutinin of Phaseolus vulgaris, a non-proteolytic release-inducing agent, induced peak 7 and peak 9 protein phosphorylation. Thus, the characteristics of peak 7 and peak 9 protein phosphorylation are similar to those of the platelet release reaction, suggesting that the phosphorylation of these proteins may play a role in the platelet release reaction. When platelet sonicates or the supernatant fraction from platelet sonicates were incubated with [gamma-32P]ATP there was phosphorylation of both peak 7 and peak 9 proteins. This phosphorylation was unaffected by either added thrombin or adenosine 3',5'-cyclic monophosphate (cAMP) despite the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. Thus, the thrombin-dependent phosphorylation depends upon intact platelets. When the supernatant fraction from platelet sonicates was fractionated by histone-Sepharose affinity chromatography, two distinct protein kinase enzymes were resolved, one a cAMP-dependent holoenzyme and the other a cAMP-independent enzyme. The isolated cAMP-dependent enzyme fraction catalyzed the cAMP-(but not thrombin-) stimulated phosphorylation of a protein that co-electrophoresed with peak 7 protein.

MeSH Terms
Animals Blood Platelets/drug effects,enzymology,metabolism Chemical Fractionation Chromatography, Affinity Chromatography, Gel Cyclic AMP/metabolism,pharmacology Electrophoresis, Polyacrylamide Gel Humans Molecular Weight Muscle Proteins/metabolism Phosphoproteins/biosynthesis,blood Protein Kinases/metabolism Rabbits Sepharose Stimulation, Chemical Thrombin/administration & dosage,pharmacology Time Factors
Chemicals
Muscle Proteins Phosphoproteins Sepharose Cyclic AMP Protein Kinases Thrombin
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Lyons R M
Stanford N
Majerus P W
References (45)
45 references, click to expand
  1. Phosphorylation of endogenous protein of rat brain by cyclic adenosine 3',5'-monophosphate-dependent protein kinase.
    J Biol Chem. 1971 Dec 25;246(24):7731-9 PMID: 4332564
  2. Isolation and properties of a thrombin-sensitive protein of human platelets.
    J Biol Chem. 1972 May 10;247(9):2723-31 PMID: 4260214
  3. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  4. Binding of thrombin to human platelets.
    Nature. 1974 Feb 1;247(5439):306-7 PMID: 4818365
  5. An assay for adenosine 3',5'-cyclic monophosphate based on the association of the nucleotide with a partially purified binding protein.
    Biochemistry. 1970 Oct 13;9(21):4223-9 PMID: 4318805
  6. Separation of regulatory and catalytic subunits of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase(s) of rabbit skeletal muscle.
    Biochem Biophys Res Commun. 1971 Jan 22;42(2):187-94 PMID: 4322813
  7. Regulation of adenosine 3',5'-monophosphate-dependent protein kinase. I. Preliminary characterization of the adipose tissue enzyme in crude extracts.
    J Biol Chem. 1973 Mar 10;248(5):1813-21 PMID: 4348550
  8. Phosvitin kinase from brain: activation by ions and subcellular distribution.
    Biochem J. 1964 Oct;93(1):84-91 PMID: 4284465
  9. The effects of thrombin on adenyl cyclase activity and a membrane protein from human platelets.
    J Clin Invest. 1972 Jan;51(1):81-8 PMID: 4331802
  10. MOLECULAR EXCLUSION AND RESTRICTED DIFFUSION PROCESSES IN MOLECULAR-SIEVE CHROMATOGRAPHY.
    Biochemistry. 1964 May;3:723-30 PMID: 14193644
  11. Regulation of phosphorylation of a specific protein in toad-bladder membrane by antidiuretic hormone and cyclic AMP, and its possible relationship to membrane permeability changes.
    Proc Natl Acad Sci U S A. 1973 Mar;70(3):880-4 PMID: 4351809
  12. Phosphoproteins.
    Adv Protein Chem. 1974;28:1-210 PMID: 4275513
  13. Quantification of human platelet inositides and the influence of ionic environment on their incorporation of orthophosphate-32P.
    J Clin Invest. 1971 Apr;50(4):762-72 PMID: 4323126
  14. Nuclear protein kinases. Evidence for their heterogeneity, tissue specificity, substrate specificities, and differential responses to cyclic adenosine 3':5'-monophosphate.
    J Biol Chem. 1974 Feb 10;249(3):750-60 PMID: 4359766
  15. Activation by adenosine 3':5'-monophosphate of a membrane-bound phosphoprotein phosphatase from toad bladder.
    Proc Natl Acad Sci U S A. 1973 Jun;70(6):1831-5 PMID: 4352657
  16. Krebs EG: Purification and characterization of a protein inhibitor of adenosine 3',5'-monophosphate-dependent protein kinases.
    J Biol Chem. 1971 Apr 10;246(7):1977-85 PMID: 4324557
  17. The phosphorylase b to a converting enzyme of rabbit skeletal muscle.
    Biochim Biophys Acta. 1956 Apr;20(1):150-7 PMID: 13315361
  18. Phosphorylation of casein. Role of the golgi apparatus.
    J Biol Chem. 1972 Dec 25;247(24):8193-4 PMID: 4629742
  19. The binding of thrombin to the surface of human platelets.
    J Biol Chem. 1974 Apr 25;249(8):2646-51 PMID: 4856653
  20. Aldosterone-induced increase in protein phosphatase activity of toad bladder.
    Proc Natl Acad Sci U S A. 1974 Oct;71(10):3869-73 PMID: 4372594
  21. Human platelet myosin. I. Purification by a rapid method applicable to other nonmuscle cells.
    Anal Biochem. 1974 Jul;60(1):258-66 PMID: 4277511
  22. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis.
    J Biol Chem. 1969 Aug 25;244(16):4406-12 PMID: 5806584
  23. Studies on the adenosine 3',5'-monophosphate-dependent protein kinases of rabbit skeletal muscle.
    J Biol Chem. 1972 Dec 10;247(23):7790-8 PMID: 4344233
  24. Phosphoprotein kinases associated with rat liver chromatin.
    Biochem Biophys Res Commun. 1971 Jan 8;42(1):103-10 PMID: 5546345
  25. Cyclic AMP and histone phosphorylation.
    Ann N Y Acad Sci. 1971 Dec 30;185:166-80 PMID: 4330489
  26. Role of cyclic AMP in platelet function.
    Adv Cyclic Nucleotide Res. 1972;1:231-47 PMID: 4353170
  27. Phosphorylation of human platelet myosin.
    Proc Natl Acad Sci U S A. 1973 Nov;70(11):3115-9 PMID: 4522294
  28. The binding of phytohemagglutinins to human platelet plasma membranes.
    J Biol Chem. 1972 Jul 10;247(13):4253-7 PMID: 4113818
  29. Inhibition of platelet energy production and release reaction by PGE1, theophylline and cAMP.
    Biochem Biophys Res Commun. 1970 Oct 9;41(1):128-34 PMID: 4318850
  30. Purification and properties of rabbit skeletal muscle adenosine 3',5'-monophosphate-dependent protein kinases.
    J Biol Chem. 1971 Apr 10;246(7):1986-95 PMID: 4324558
  31. INHIBITION OF CHYMOTRYPSIN ACTIVITY IN CRYSTALLINE TRYPSIN PREPARATIONS.
    J Biol Chem. 1964 Jun;239:1799-803 PMID: 14213354
  32. Cyclic nucleotide-dependent protein kinases. V. Preparation and properties of adenosine 3',5'-monophosphate-dependent protein kinase from various bovine tissues.
    Biochim Biophys Acta. 1970 Jul 15;212(1):79-91 PMID: 4322697
  33. Kinetics of the thrombin-induced release of calcium (II) by platelets.
    Biochemistry. 1973 Jan 16;12(2):282-9 PMID: 4683003
  34. Cyclic adenosine 3',5'-monophosphate dependent protein kinase of human platelets: membrane phosphorylation and regulation of platelet function.
    Ser Haematol. 1973;6(3):351-66 PMID: 4360128
  35. Cell cycle variation in cyclic adenosine 3',5'-monophosphate-dependent inhibition of protein kinase from Physarum polycephalum.
    Biochem Biophys Res Commun. 1972 Oct 17;49(2):414-9 PMID: 4344888
  36. Chemical characterization of the phosphoprotein phosvitin.
    J Biol Chem. 1965 Oct;240(10):3892-8 PMID: 5891575
  37. Protein measurement with the Folin phenol reagent.
    J Biol Chem. 1951 Nov;193(1):265-75 PMID: 14907713
  38. Cyclic nucleotide-dependent protein kinases. 3. Purification and properties of adenosine 3',5'-monophosphate-dependent protein kinase from bovine brain.
    J Biol Chem. 1969 Dec 10;244(23):6395-402 PMID: 4311198
  39. Isolation of human platelets and platelet surface membranes.
    Methods Enzymol. 1974;31:149-55 PMID: 4213668
  40. The endometrial adenosine cyclic 3':5'-monophosphate-dependent protein kinase. Distribution, subunit structure, and kinetics of adenosine cyclic 3':5'-monophosphate binding.
    J Biol Chem. 1973 May 25;248(10):3593-600 PMID: 4349870
  41. A simple apparatus for vertical flat-sheet polyacrylamide gel electrophoresis.
    Anal Biochem. 1968 Mar;22(3):374-81 PMID: 5672484
  42. Induction of the platelet release reaction by phytohemagglutinin.
    J Clin Invest. 1974 Jan;53(1):211-8 PMID: 4357613
  43. Kinetics of the thrombin-induced release of adenosine triphosphate by platelets. Comparison with release of calcium.
    Biochemistry. 1973 Jun 19;12(13):2462-8 PMID: 4709942
  44. Hydrolysis of cyclic guanosine and adenosine 3',5'-monophosphates by rat and bovine tissues.
    J Biol Chem. 1970 Nov 10;245(21):5649-55 PMID: 4319563
  45. Conformational states of the subunit of Escherichia coli alkaline phosphatase.
    Biochemistry. 1967 Nov;6(11):3552-9 PMID: 4864145
Article Info
Journal
The Journal of clinical investigation
Abbr.
J Clin Invest
ISSN
0021-9738
DOI
10.1172/JCI108172
Published
1975-10-00
Pages
924-36
Language
English
Region
United States
NLM ID
7802877
PMCID
PMC301948
Subset
IM
External Links
PubMed source DOI: 10.1172/JCI108172 PMC Full Text View journal details
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