• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Aug 1, 1998; 333(Pt 3): 591–599.
PMCID: PMC1219621

Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen.

Abstract

Phosphatidylserine exposure and microvesicle release give rise to procoagulant activity during platelet activation. We have previously shown that whereas the Ca2+ ionophore A23187 and 2,5-di-(t-butyl)-1, 4-benzohydroquinone, a Ca2+-ATPase inhibitor, induce phosphatidylserine exposure, only the former triggers microvesicle release. We now report that microvesicle formation with ionophore A23187 is specifically associated with mu-calpain activation, increased protein tyrosine phosphatase (PTP) activity and decreased tyrosine phosphorylation. The degree to which calpain and individual PTPs were activated in response to A23187 depended on the extent of bivalent cation chelation in the external medium. EGTA (2 mM) blocked or severely retarded their activation, and addition of extracellular Ca2+ in excess (2 mM) resulted in virtually immediate tyrosine dephosphorylation. Dephosphorylation was correlated with an increase in total PTP activity in platelet lysates. In platelets stimulated by a combination of thrombin and collagen, only the subpopulation undergoing microvesicle release and isolated by their binding to annexin-V-coated magnetic beads exhibited protein tyrosine dephosphorylation. Detection of PTP activity in an 'in-gel' assay showed the Ca2+-dependent appearance of active low-molecular-mass bands at 38, 36 and 27 kDa. Individual PTPs varied in their protease sensitivity to changes in intracellular Ca2+ levels. For example, PTP1B was a more sensitive substrate than SH2-domain-containing tyrosine phosphatase-1 for mu-calpain cleavage. Incubation of platelets with the PTP inhibitors, phenylarsine oxide and benzylphosphonic acid acetoxymethyl ester, led to increased tyrosine phosphorylation and the surface expression of aminophospholipids but little microvesicle formation. Furthermore, microvesicle release in response to ionophore A23187 was inhibited. We conclude that platelet microvesicle formation is associated with extensive protein tyrosine dephosphorylation.

Full Text

The Full Text of this article is available as a PDF (860K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood. 1997 Feb 15;89(4):1121–1132. [PubMed]
  • Bevers EM, Comfurius P, Zwaal RF. Platelet procoagulant activity: physiological significance and mechanisms of exposure. Blood Rev. 1991 Sep;5(3):146–154. [PubMed]
  • Comfurius P, Williamson P, Smeets EF, Schlegel RA, Bevers EM, Zwaal RF. Reconstitution of phospholipid scramblase activity from human blood platelets. Biochemistry. 1996 Jun 18;35(24):7631–7634. [PubMed]
  • Zhou Q, Zhao J, Stout JG, Luhm RA, Wiedmer T, Sims PJ. Molecular cloning of human plasma membrane phospholipid scramblase. A protein mediating transbilayer movement of plasma membrane phospholipids. J Biol Chem. 1997 Jul 18;272(29):18240–18244. [PubMed]
  • Dachary-Prigent J, Pasquet JM, Freyssinet JM, Nurden AT. Calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using Ca2+-ATPase inhibitors. Biochemistry. 1995 Sep 12;34(36):11625–11634. [PubMed]
  • Fox JE, Austin CD, Reynolds CC, Steffen PK. Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets. J Biol Chem. 1991 Jul 15;266(20):13289–13295. [PubMed]
  • Pasquet JM, Dachary-Prigent J, Nurden AT. Calcium influx is a determining factor of calpain activation and microparticle formation in platelets. Eur J Biochem. 1996 Aug 1;239(3):647–654. [PubMed]
  • Yano Y, Shiba E, Kambayashi J, Sakon M, Kawasaki T, Fujitani K, Kang J, Mori T. The effects of calpeptin (a calpain specific inhibitor) on agonist induced microparticle formation from the platelet plasma membrane. Thromb Res. 1993 Sep 1;71(5):385–396. [PubMed]
  • Clark EA, Shattil SJ, Brugge JS. Regulation of protein tyrosine kinases in platelets. Trends Biochem Sci. 1994 Nov;19(11):464–469. [PubMed]
  • Lipfert L, Haimovich B, Schaller MD, Cobb BS, Parsons JT, Brugge JS. Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets. J Cell Biol. 1992 Nov;119(4):905–912. [PMC free article] [PubMed]
  • Bachelot C, Cano E, Grelac F, Saleun S, Druker BJ, Levy-Toledano S, Fischer S, Rendu F. Functional implications of tyrosine protein phosphorylation in platelets. Simultaneous studies with different agonists and inhibitors. Biochem J. 1992 Jun 15;284(Pt 3):923–928. [PMC free article] [PubMed]
  • Jackson SP, Schoenwaelder SM, Yuan Y, Salem HH, Cooray P. Non-receptor protein tyrosine kinases and phosphatases in human platelets. Thromb Haemost. 1996 Nov;76(5):640–650. [PubMed]
  • Smilowitz HM, Aramli L, Xu D, Epstein PM. Phosphotyrosine phosphatase activity in human platelets. Life Sci. 1991;49(1):29–37. [PubMed]
  • Gu MX, York JD, Warshawsky I, Majerus PW. Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to cytoskeletal protein 4.1. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5867–5871. [PMC free article] [PubMed]
  • Frangioni JV, Oda A, Smith M, Salzman EW, Neel BG. Calpain-catalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets. EMBO J. 1993 Dec;12(12):4843–4856. [PMC free article] [PubMed]
  • Shen SH, Bastien L, Posner BI, Chrétien P. A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases. Nature. 1991 Aug 22;352(6337):736–739. [PubMed]
  • Li RY, Gaits F, Ragab A, Ragab-Thomas JM, Chap H. Tyrosine phosphorylation of an SH2-containing protein tyrosine phosphatase is coupled to platelet thrombin receptor via a pertussis toxin-sensitive heterotrimeric G-protein. EMBO J. 1995 Jun 1;14(11):2519–2526. [PMC free article] [PubMed]
  • Falet H, Ramos-Morales F, Bachelot C, Fischer S, Rendu F. Association of the protein tyrosine phosphatase PTP1C with the protein tyrosine kinase c-Src in human platelets. FEBS Lett. 1996 Apr 1;383(3):165–169. [PubMed]
  • Jackson DE, Ward CM, Wang R, Newman PJ. The protein-tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1 (PECAM-1) and forms a distinct signaling complex during platelet aggregation. Evidence for a mechanistic link between PECAM-1- and integrin-mediated cellular signaling. J Biol Chem. 1997 Mar 14;272(11):6986–6993. [PubMed]
  • Gu M, Majerus PW. The properties of the protein tyrosine phosphatase PTPMEG. J Biol Chem. 1996 Nov 1;271(44):27751–27759. [PubMed]
  • Burridge K, Nelson A. An in-gel assay for protein tyrosine phosphatase activity: detection of widespread distribution in cells and tissues. Anal Biochem. 1995 Nov 20;232(1):56–64. [PubMed]
  • Tonks NK, Neel BG. From form to function: signaling by protein tyrosine phosphatases. Cell. 1996 Nov 1;87(3):365–368. [PubMed]
  • Guinebault C, Payrastre B, Sultan C, Mauco G, Breton M, Levy-Toledano S, Plantavid M, Chap H. Tyrosine kinases and phosphoinositide metabolism in thrombin-stimulated human platelets. Biochem J. 1993 Jun 15;292(Pt 3):851–856. [PMC free article] [PubMed]
  • Rendu F, Eldor A, Grelac F, Bachelot C, Gazit A, Gilon C, Levy-Toledano S, Levitzki A. Inhibition of platelet activation by tyrosine kinase inhibitors. Biochem Pharmacol. 1992 Sep 1;44(5):881–888. [PubMed]
  • Dachary-Prigent J, Freyssinet JM, Pasquet JM, Carron JC, Nurden AT. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups. Blood. 1993 May 15;81(10):2554–2565. [PubMed]
  • Pasquet JM, Toti F, Nurden AT, Dachary-Prigent J. Procoagulant activity and active calpain in platelet-derived microparticles. Thromb Res. 1996 Jun 15;82(6):509–522. [PubMed]
  • Garcia-Morales P, Minami Y, Luong E, Klausner RD, Samelson LE. Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9255–9259. [PMC free article] [PubMed]
  • Zhang ZY, Van Etten RL. Purification and characterization of a low-molecular-weight acid phosphatase--a phosphotyrosyl-protein phosphatase from bovine heart. Arch Biochem Biophys. 1990 Oct;282(1):39–49. [PubMed]
  • Li RY, Ragab A, Gaits F, Ragab-Thomas JM, Chap H. Thrombin-induced redistribution of protein-tyrosine-phosphatases to the cytoskeletal complexes in human platelets. Cell Mol Biol (Noisy-le-grand) 1994 Jul;40(5):665–675. [PubMed]
  • Ariyoshi H, Oda A, Salzman EW. Participation of calpain in protein-tyrosine phosphorylation and dephosphorylation in human blood platelets. Arterioscler Thromb Vasc Biol. 1995 Apr;15(4):511–514. [PubMed]
  • Zwaal RF, Comfurius P, Bevers EM. Platelet procoagulant activity and microvesicle formation. Its putative role in hemostasis and thrombosis. Biochim Biophys Acta. 1992 Oct 13;1180(1):1–8. [PubMed]
  • Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995 Nov 1;182(5):1545–1556. [PMC free article] [PubMed]
  • Takayama H, Nakamura T, Yanagi S, Taniguchi T, Nakamura S, Yamamura H. Ionophore A23187-induced protein-tyrosine phosphorylation of human platelets: possible synergism between Ca2+ mobilization and protein kinase C activation. Biochem Biophys Res Commun. 1991 Jan 31;174(2):922–927. [PubMed]
  • Dachary-Prigent J, Pasquet JM, Nurden AT. Simultaneous detection of changes in cytoplasmic Ca(2+), aminophospholipid exposure and micro-vesiculation in activated platelets. Platelets. 1997;8(6):405–412. [PubMed]
  • Heemskerk JW, Vuist WM, Feijge MA, Reutelingsperger CP, Lindhout T. Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses. Blood. 1997 Oct 1;90(7):2615–2625. [PubMed]
  • Zhao Z, Shen SH, Fischer EH. Stimulation by phospholipids of a protein-tyrosine-phosphatase containing two src homology 2 domains. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4251–4255. [PMC free article] [PubMed]
  • Schoenwaelder SM, Kulkarni S, Salem HH, Imajoh-Ohmi S, Yamao-Harigaya W, Saido TC, Jackson SP. Distinct substrate specificities and functional roles for the 78- and 76-kDa forms of mu-calpain in human platelets. J Biol Chem. 1997 Oct 3;272(40):24876–24884. [PubMed]
  • Uchida T, Matozaki T, Noguchi T, Yamao T, Horita K, Suzuki T, Fujioka Y, Sakamoto C, Kasuga M. Insulin stimulates the phosphorylation of Tyr538 and the catalytic activity of PTP1C, a protein tyrosine phosphatase with Src homology-2 domains. J Biol Chem. 1994 Apr 22;269(16):12220–12228. [PubMed]
  • Wang J, Walsh CT. Mechanistic studies on full length and the catalytic domain of the tandem SH2 domain-containing protein tyrosine phosphatase: analysis of phosphoenzyme levels and Vmax stimulatory effects of glycerol and of a phosphotyrosyl peptide ligand. Biochemistry. 1997 Mar 11;36(10):2993–2999. [PubMed]
  • Swarup G, Cohen S, Garbers DL. Selective dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatases. J Biol Chem. 1981 Aug 10;256(15):8197–8201. [PubMed]
  • Brumell JH, Chan CK, Butler J, Borregaard N, Siminovitch KA, Grinstein S, Downey GP. Regulation of Src homology 2-containing tyrosine phosphatase 1 during activation of human neutrophils. Role of protein kinase C. J Biol Chem. 1997 Jan 10;272(2):875–882. [PubMed]
  • Artçanuthurry V, Grelac F, Maclouf J, Martin-Cramer E, Levy-Tolédano S. Serine/threonine dephosphorylation may be involved in tyrosine phosphorylation: a new mode of signal transduction in platelets. Semin Thromb Hemost. 1996;22(4):317–326. [PubMed]
  • Yano Y, Kambayashi J, Shiba E, Sakon M, Oiki E, Fukuda K, Kawasaki T, Mori T. The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane. Biochem J. 1994 Apr 1;299(Pt 1):303–308. [PMC free article] [PubMed]
  • Enouf J, Bobe R, Lacabaratz-Porret C, Bredoux R, Corvazier E, Kovacs T, Papp B. The platelet Ca2+ transport ATPase system. Platelets. 1997 Jan;8(1):5–14. [PubMed]
  • Fleming I, Fisslthaler B, Busse R. Interdependence of calcium signaling and protein tyrosine phosphorylation in human endothelial cells. J Biol Chem. 1996 May 3;271(18):11009–11015. [PubMed]
  • Stout JG, Bassé F, Luhm RA, Weiss HJ, Wiedmer T, Sims PJ. Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids. J Clin Invest. 1997 May 1;99(9):2232–2238. [PMC free article] [PubMed]
  • Dachary-Prigent J, Pasquet JM, Fressinaud E, Toti F, Freyssinet JM, Nurden AT. Aminophospholipid exposure, microvesiculation and abnormal protein tyrosine phosphorylation in the platelets of a patient with Scott syndrome: a study using physiologic agonists and local anaesthetics. Br J Haematol. 1997 Dec;99(4):959–967. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...