RSS-Feed abonnieren
DOI: 10.1055/s-2005-869522
The P2 Receptors and Congenital Platelet Function Defects
Publikationsverlauf
Publikationsdatum:
26. April 2005 (online)
![](https://www.thieme-connect.de/media/sth/200502/lookinside/thumbnails/10.1055-s-2005-869522-1.jpg)
ABSTRACT
Platelets possess three P2 receptors: two (P2Y1 and P2Y12) are receptors for adenosine diphosphate (ADP), and one (P2X1) is a receptor for adenosine triphosphate (ATP). The P2Y1 receptor, which is coupled to Gq and phospholipase C-β, is responsible for mobilization of ionized calcium from internal stores and mediates the ADP-induced platelet shape change and initial wave of rapidly reversible aggregation. The other ADP receptor, P2Y12, is negatively coupled to adenylyl cyclase through Gi and mediates a progressive and sustained ADP-induced aggregation not preceded by shape change. In addition, this receptor plays an important role in the potentiation of platelet secretion induced by several platelet agonists. The combined action of P2Y1 and P2Y12 is necessary for the full platelet aggregation response to ADP. Four patients with severe deficiency of P2Y12 have been described so far. Sequence analysis of the P2Y12 locus of three of these patients revealed homozygous mutations that produced a frame shift mutation and premature truncation of the protein. The fourth patient had an allele with a frame shift mutation and a normal allele, which could be silenced by an additional, as yet unknown, mutation. More recently, we described a patient with a congenital bleeding disorder and a dysfunctional P2Y12. The patient is a compound heterozygote, in whom one allele contained a G to A transition resulting in an Arg256 to Gln codon substitution (R256Q) and the other allele contained a C to T transition resulting in an Arg265 to Trp codon substitution (R265W). The two substitutions are located in TM6 and EL3 of the receptor. Stable Chinese hamster ovaries (CHO) cell lines were established expressing either wild-type P2Y12 and P2Y12 R256Q or P2Y12 R265W. Neither mutation blocked the ability of the P2Y12 receptor to translocate to the CHO cell surface. ADP at all tested concentrations (0.1 to 10 μM) greatly inhibited the forskolin-induced increase of cyclic adenosine monophosphate (cAMP) in CHO cells transfected with wild-type P2Y12, whereas CHO cells transfected with either mutant protein were only partially inhibited by ADP. Thus, the molecular basis for the patient’s dysfunctional platelet phenotype is explained by missense mutations and the expression of a dysfunctional P2Y12 receptor. The localization of both mutations in TM6 and EL3 identifies this region of P2Y12 as a structurally and functionally critical region of the receptor.
KEYWORDS
Platelets - P2 receptors - congenital defects - dysfunctional platelets - P2Y12 defects
REFERENCES
- 1 Cattaneo M, Gachet C, Cazenave J P, Packham M A. Adenosine diphosphate (ADP) does not induce thromboxane A2 generation in human platelets. Blood. 2002; 99 3868-3869
- 2 Cattaneo M, Gachet C. ADP receptors and clinical bleeding disorders. Arterioscler Thromb Vasc Biol. 1999; 19 2281-2285
- 3 Jin J, Kunapuli S P. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci U S A. 1998; 95 8070-8074
- 4 Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev. 1998; 50 413-492
- 5 North R A. Families of ion channels with two hydrophobic segments. Curr Opin Cell Biol. 1996; 8 474-483
- 6 Brake A J, Wagenbach M J, Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature. 1994; 371 519-523
- 7 Valera S, Hussy N, Evans R J et al.. A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature. 1994; 371 516-519
- 8 Leon C, Hechler B, Vial C et al.. The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells. FEBS Lett. 1997; 403 26-30
- 9 Hollopeter G, Jantzen H M, Vincent D et al.. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature. 2001; 409 202-207
- 10 Zhang F L, Luo L, Gustafson E et al.. ADP is the cognate ligand for the orphan G protein-coupled receptor SP1999. J Biol Chem. 2001; 276 8608-8615
- 11 Sun B, Li J, Okahara K, Kambayashi J. P2X1 purinoceptor in human platelets. Molecular cloning and functional characterization after heterologous expression. J Biol Chem. 1998; 273 11544-11547
- 12 Clifford E E, Parker K, Humphreys B D, Kertesy S B, Dubyak G R. The P2X1 receptor, an adenosine triphosphate-gated cation channel, is expressed in human platelets but not in human blood leukocytes. Blood. 1998; 91 3172-3181
- 13 Vial C, Hechler B, Leon C, Cazenave J P, Gachet C. Presence of P2X1 purinoceptors in human platelets and megakaryoblastic cell lines. Thromb Haemost. 1997; 78 1500-1504
- 14 Scase T J, Heath M F, Allen J M, Sage S O, Evans R J. Identification of a P2X1 purinoceptor expressed on human platelets. Biochem Biophys Res Commun. 1998; 242 525-528
- 15 Mahaut-Smith M P, Ennion S J, Rolf M G, Evans R J. ADP is not an agonist at P2X(1) receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets. Br J Pharmacol. 2000; 131 108-114
- 16 Hechler B, Leon C, Vial C et al.. The P2Y1 receptor is necessary for adenosine 5′-diphosphate-induced platelet aggregation. Blood. 1998; 92 152-159
- 17 Savi P, Beauverger P, Labouret C et al.. Role of P2Y1 purinoceptor in ADP-induced platelet activation. FEBS Lett. 1998; 422 291-295
- 18 Jin J, Daniel J L, Kunapuli S P. Molecular basis for ADP-induced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem. 1998; 273 2030-2034
- 19 Jarvis G E, Humphries R G, Robertson M J, Leff P. ADP can induce aggregation of human platelets via both P2Y(1) and P(2T) receptors. Br J Pharmacol. 2000; 129 275-282
- 20 Geiger J, Honig-Liedl P, Schanzenbacher P, Walter U. Ligand specificity and ticlopidine effects distinguish three human platelet ADP receptors. Eur J Pharmacol. 1998; 351 235-246
- 21 Fagura M S, Dainty I A, McKay G D et al.. P2Y1-receptors in human platelets which are pharmacologically distinct from P2Y(ADP)-receptors. Br J Pharmacol. 1998; 124 157-164
- 22 Daniel J L, Dangelmaier C, Jin J et al.. Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets. J Biol Chem. 1998; 273 2024-2029
- 23 Jantzen H M, Gousset L, Bhaskar V et al.. Evidence for two distinct G-protein-coupled ADP receptors mediating platelet activation. Thromb Haemost. 1999; 81 111-117
- 24 Leon C, Hechler B, Freund M et al.. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice. J Clin Invest. 1999; 104 1731-1737
- 25 Fabre J E, Nguyen M, Latour A et al.. Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice. Nat Med. 1999; 5 1199-1202
- 26 Cattaneo M, Lecchi A, Randi A M, McGregor J L, Mannucci P M. Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate. Blood. 1992; 80 2787-2796
- 27 Leon C, Vial C, Gachet C et al.. The P2Y1 receptor is normal in a patient presenting a severe deficiency of ADP-induced platelet aggregation. Thromb Haemost. 1999; 81 775-781
- 28 Cattaneo M, Lecchi A, Lombardi R, Gachet C, Zighetti M L. Platelets from a patient heterozygous for the defect of P2CYC receptors for ADP have a secretion defect despite normal thromboxane A2 production and normal granule stores: further evidence that some cases of platelet ‘primary secretion defect’ are heterozygous for a defect of P2CYC receptors. Arterioscler Thromb Vasc Biol. 2000; 20 E101-E106
- 29 Cattaneo M, Lombardi R, Zighetti M L et al.. Deficiency of (33P)2MeS-ADP binding sites on platelets with secretion defect, normal granule stores and normal thromboxane A2 production. Evidence that ADP potentiates platelet secretion independently of the formation of large platelet aggregates and thromboxane A2 production. Thromb Haemost. 1997; 77 986-990
- 30 Trumel C, Payrastre B, Plantavid M et al.. A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase. Blood. 1999; 94 4156-4165
- 31 Cattaneo M, Canciani M T, Lecchi A et al.. Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates. Blood. 1990; 75 1081-1086
- 32 Savi P, Herbert J M. Pharmacology of ticlopidine and clopidogrel. Haematologica. 2000; 85 73-77
- 33 Humphries R G. Pharmacology of AR-C69931MX and related compounds: from pharmacological tools to clinical trials. Haematologica. 2000; 85 66-72
- 34 Rolf M G, Mahaut-Smith M P. Effects of enhanced P2X1 receptor Ca2+ influx on functional responses in human platelets. Thromb Haemost. 2002; 88 495-502
- 35 Oury C, Toth-Zsamboki E, Thys C et al.. The ATP-gated P2X1 ion channel acts as a positive regulator of platelet responses to collagen. Thromb Haemost. 2001; 86 1264-1271
- 36 Cattaneo M, Marchese P, Jacobson K A, Ruggeri Z M. The P2X1 receptor for ATP plays an essential role in platelet aggregation at high shear. Haematology J.. 2003; 4(suppl 2) 150 , (abst)
- 37 Hechler B, Lenain N, Marchese P et al.. A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo. J Exp Med. 2003; 198 661-667
- 38 Gachet C, Cattaneo M, Ohlmann P et al.. Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors. Br J Haematol. 1995; 91 434-444
- 39 Conley P B, Jurek M M, Vincent D, Lecchi A, Cattaneo M. Unique mutations in the P2Y12 locus of patients with previously described defects in ADP-dependent aggregation. Blood. 2001; 98 43b , (abst)
- 40 Nurden P, Savi P, Heilmann E et al.. An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function. J Clin Invest. 1995; 95 1612-1622
- 41 Cattaneo M, Zighetti M L, Lombardi R et al.. Phenotypic and genotypic characterization of a congenital bleeding disorder caused by a dysfunctional P2Y12 ADP receptor. Blood. 2001; 98 443a , (abst)
- 42 Moro S, Hoffmann C, Jacobson K A. Role of the extracellular loops of G protein-coupled receptors in ligand recognition: a molecular modeling study of the human P2Y1 receptor. Biochemistry. 1999; 38 3498-3507
- 43 Moro S, Guo D, Camaioni E et al.. Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites. J Med Chem. 1998; 41 1456-1466
- 44 Cattaneo M, Lecchi A. Patients with congenital abnormality of platelet aggregation induced by Ca(2+) ionophores may have a defect of the platelet P2Y(12) receptor for ADP. Br J Haematol. 2001; 115 485-487
- 45 Cattaneo M, Zighetti M L, Lombardi R, Mannucci P M. Role of ADP in platelet aggregation at high shear: studies in a patient with congenital defect of platelet responses to ADP. Br J Haematol. 1994; 88 826-829
- 46 Oury C, Lenaerts T, Peerlinck K, Vermylen J, Hoylaerts M F. Congenital deficiency of the phospholipase mC coupled platelet P2Y1 receptor leads to a mild bleeding disorder. Thromb Haemost. 1999; 85(suppl) 20 , (abst)
- 47 Oury C, Toth-Zsamboki E, Van Geet C et al.. A natural dominant negative P2X1 receptor due to deletion of a single amino acid residue. J Biol Chem. 2000; 275 22611-22614
Professor
Marco Cattaneo
Unit of Hematology and Thrombosis, Ospedale San Paolo, DMCO University of Milano
Via di Rudinì, 8, 20142 Milan, Italy
eMail: marco.cattaneo@unimi.it