Thromb Haemost 1998; 80(03): 463-468
DOI: 10.1055/s-0037-1615230
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Abnormal Tyrosine Phosphorylation Linked to a Defective Interaction between ADP and Its Receptor on Platelets

Sylviane Lévy-Toledano
1   From the INSERM U348, IFR Circulation Lariboisière, Hôpital Lariboisière, Paris
,
Jacques Maclouf
1   From the INSERM U348, IFR Circulation Lariboisière, Hôpital Lariboisière, Paris
,
Jean-Philippe Rosa
1   From the INSERM U348, IFR Circulation Lariboisière, Hôpital Lariboisière, Paris
,
Carole Gallet
1   From the INSERM U348, IFR Circulation Lariboisière, Hôpital Lariboisière, Paris
,
Géraldine Vallès*
,
Paquita Nurden*
,
Alan T. Nurden*
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Publikationsverlauf

Received 28. Oktober 1997

Accepted after resubmission 13. Mai 1998

Publikationsdatum:
08. Dezember 2017 (online)

Summary

ADP, a primary stimulus of platelets, binds to one or more populations of receptors on the platelet surface. These receptors are linked to discrete activation pathways. Both G proteins and tyrosine kinases have been implicated in the cellular responses to this agonist. We have studied a patient with a congenital abnormality of ADP-induced platelet aggregation in an effort to gain information on the signalling pathways used by ADP. Immunoblotting with a broadly reactive rabbit antibody recognizing the GTP-binding domain of G protein α-subunits, and with rabbit antibodies specific for Giαl-3, and Gα12 all showed normal reactivity when tested against the patient‘s platelets. The phosphorylation of proteins was studied using an anti-phosphotyrosine MoAb (4G10) and platelets stimulated in a platelet aggregometer with ADP, a thromboxane A2 mimetic (IBOP), TRAP-14-mer peptide and α-thrombin. With normal platelets, a time-dependent phosphorylation of several bands in the 60 to 130 kDa mol. wt. range was observed with all agonists. For the patient, minimal aggregation and little or no phosphorylation of proteins of 80-85 kDa (cortactin), 100-105 kDa and 125-130 kDa were seen in response to ADP. The aggregation and phosphorylation responses were slightly modified in the presence of low doses of thrombin but were normal with high doses. Aggregation and tyrosine phosphorylation were virtually absent with IBOP, a finding reproduced when normal platelets were incubated with IBOP and the CP/CPK ADP scavenging system, thereby underlining the role of ADP in the response to IBOP. Our results show that the ADP receptor pathway deficient in the patient is linked to a selective tyrosine phosphorylation response.

* UMR 5533 CNRS, Hôpital Cardiologique, Pessac, France


 
  • References

  • 1 Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-9.
  • 2 Mustard JF, Packham MA, Kinlough-Rathbone RL, Perry DW, Regoeczi E. Fibrinogen and ADP-induced platelet aggregation. Blood 1978; 52: 453-66.
  • 3 Du X, Ginsberg MH. Integrin αIIbβ3 and platelet function. Thromb Haemostas 1997; 78: 96-100.
  • 4 Mills DCB. ADP receptors on platelets. Thromb Haemost 1996; 76: 835-56.
  • 5 Gachet C, Hechler B, Léon C, Vial C, Leray C, Ohlmann P, Cazenave J-P. Activation of ADP receptors and platelet function. Thromb Haemost 1997; 7: 271-5.
  • 6 Léon C, Hechler B, Vial C, Leray C, Cazenave J-P, Gachet C. The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells. FEBS Letts 1997; 403: 26-30.
  • 7 Daniel JL, Dangelmaier C, Jin J, Ashby B, Smith B, Kunapuli SP. Molecular basis for ADP-induced platelet activation. Evidence for three distinct ADP receptors on human platelets. J Biol Chem 1998; 273: 2024-9.
  • 8 Jin J, Daniel JL, Kunapuli SP. Molecular basis for ADP-induced platelet activation. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem 1998; 273: 2030-4.
  • 9 Cattaneo MB, Lecchi A, Randi AM, McGregor JL, Mannucci PM. Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate. Blood 1992; 80: 2787-96.
  • 10 Nurden P, Savi P, Heilmann E, Bihour C, Herbert JM, Maffrand JP, Nurden AT. An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Evidence for more than one pathway of GP IIb-IIIa complex activation. J Clin Invest 1995; 95: 1612-22.
  • 11 Gachet C, Cattaneo M, Ohlmann P, Hechler B, Lecchi A, Chevalier J, Cassel D, Mannucci PM, Cazenave J-P. Purinoreceptors on blood platelets: Further pharmacological and clinical evidence to suggest the presence of two receptors. Br J Haematol 1995; 91: 434-44.
  • 12 Humbert M, Nurden P, Bihour C, Pasquet J-M, Winckler J, Heilmann E, Savi P, Herbert J-M, Kunicki TJ, Nurden AT. Ultrastructural studies of platelet aggregates from human subjects receiving clopidogrel and from a patient with an inherited defect of an ADP-dependent pathway of platelet activation. Arterioscler Thromb Vasc Biol 1996; 15: 1532-43.
  • 13 Brass LF, Hoxie JA, Manning DR. Signalling through G proteins-coupled receptors during platelet activation. Thromb Haemost 1993; 70: 217-23.
  • 14 Clark EA, Brugge JS. Tyrosine phosphorylation in platelets. Potential roles in intracellular signal transduction. Trends Cardiovasc Med 1993; 3: 218-27.
  • 15 Lévy-Toledano S, Gallet C, Nadal F, Bryckaert M, Maclouf J, Rosa J-P. Phosphorylation and dephosphorylation mechanisms in platelet function: a tightly regulated balance. Thromb Haemost 1997; 78: 226-33.
  • 16 Kovacsovics TJ, Bachelot C, Toker H, Vlahos CJ, Duckworth B, Cantley L, Hartwig JH. Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. J Biol Chem 1995; 270: 11358-66.
  • 17 Ohlmann P, Laugwitz KL, Spicher K, Nurnberg K, Schultz G, Cazenave J-P, Gachet C. The human platelet ADP receptor activates Giα2 proteins. Biochem J 1995; 312: 775-9.
  • 18 Macfarlane DE, Mills DCB. 2-methylthioadenosine(β-32P)diphosphate, an agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets. J Clin Invest 1983; 71: 420-8.
  • 19 Yukawa M, Yokota R, Eberhardt RT, von Adrian L, Ware JA. Differential desensitization of thromboxane A2 receptor subtypes. Circ Res 1997; 80: 551-6.
  • 20 Rosa J-P, Artçanuthurry V, Grelac F, Maclouf J, Caen J-P, Lévy-Toledano S. Reassessment of protein tyrosine phosphorylation in thrombasthenic platelets: Evidence that phosphorylation of cortactin and a 64-kD protein is dependent on thrombin activation and integrin αIIbβ3. Blood 1997; 89: 4385-92.
  • 21 Blanco A, Habib A, Levy-Toledano S, Maclouf J. Involvement of tyrosine kinases in the induction of cyclo-oxygenase-2 in human endothelial cells. Biochem J 1995; 312: 419-23.
  • 22 Pasquet J-M, Dachary-Prigent J, Nurden AT. Calcium influx is a determining factor of calpain activation and microparticle formation in platelets. Eur J Biochem 1996; 239: 647-54.
  • 23 Golden A, Brugge JS. Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets. Proc Natl Acad Sci USA 1989; 86: 901-5.
  • 24 Rao AK, Willis J, Holmsen H. A major role of ADP in thromboxane transfer experiments : studies in patients with platelet secretion defects. J Lab Clin Med 1984; 104: 116-26.
  • 25 Mackenzie AB, Mahaut-Smith MP, Sage SO. Activation of receptor-operated channels via P2X1 not P2T purinoreceptors in human platelets. J Biol Chem 1996; 27 (01) 2879-81.
  • 26 Gachet C, Cazenave J-P, Ohlmann P, Hilf G, Wieland T, Jakobs KH. ADP receptor-induced activation of guanine nucleotide-binding proteins in human platelet membranes. Eur J Biochem 1992; 207: 259-63.
  • 27 Gabbeta J, Yang X, Kowalska MA, Sun L, Dhanasekaran N, Rao AK. Platelet signal transduction defect with Gα subunit dysfunction and diminished Gαq in a patient with abnormal platelet responses. Proc Natl Acad Sci USA 1997; 94: 8750-5.
  • 28 Ferrel JE, Martin GS. Tyrosine-specific protein phosphorylation is regulated by glycoprotein IIb-IIIa in platelets. Proc Natl Acad Sci USA 1989; 86: 2234-9.
  • 29 Golden A, Brugge JS, Shattil SJ. Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins. J Cell Biol 1990; 111: 3117-25.
  • 30 Cattaneo M, Canciani MT, Lecchi A, Kinlough-Rathbone RL, Packham MA, Mannucci PM, Mustard JF. Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates. Blood 1990; 75: 1081-6.
  • 31 Kinlough-Rathbone RL, Reimers HJ, Mustard JF, Packham MA. Sodium arachidonate can induce platelet shape change and aggregation which are independent of the release reaction. Science, 1976; 192: 1011-2.
  • 32 Charo IF, Feinman RD, Detwiler TC, Smith JB, Ingerman CM, Silver MJ. Prostaglandin endoperoxides and thromboxane A2 can induce platelet aggregation in the absence of secretion. Nature, 1977; 269: 66-9.
  • 33 Savi P, Dol F, Herbert JM. ADP-dependence of platelet activation induced by a thrombin receptor agonist. Nouv Rev Fr Hematol 1993; 35: 115-9.
  • 34 Savi P, Herbert J-M. ADP receptors and ADP-selective antiaggregating agents. Medicinal Research Reviews 1996; 16: 159-79.
  • 35 Savi P, Laplace MC, Herbert JM. Evidence for the existence of two different ADP-binding sites on rat platelets. Thromb Res 1994; 76: 157-69.
  • 36 Savi P, Artçanuthurry V, Bornia J, Grelac F, Maclouf J, Lévy-Toledano S, Herbert JM. Effect of clopidogrel treatment on ADP-induced phosphorylations in rat platelets. Brit J Haematol 1997; 97: 185-91.