Inherited dysfunctional platelet P2Y12 receptor mutations associated with bleeding disorders

Anna Lecchi; Eti A. Femia; Silvia Paoletta; Arnaud Dupuis; Philippe Ohlmann; Christian Gachet; Kenneth A. Jacobson; Katharina Machura; Gian M. Podda; Barbara Zieger; Marco Cattaneo

doi:10.5482/HAMO-16-03-0010

Hämostaseologie, Table of Contents

Hamostaseologie 2016; 36(04): 279-283
DOI: 10.5482/HAMO-16-03-0010

Original article

Schattauer GmbH

Inherited dysfunctional platelet P2Y₁₂ receptor mutations associated with bleeding disorders

Angeborene dysfunktionale thrombozytäre P2Y₁₂-Rezeptormutationen mit assoziierten Blutungs symptomen

Authors

Anna Lecchi

¹Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy
Eti A. Femia

²Divisione di Medicina 3, ASST Santi Paolo e Carlo, Dipartimento di Scienze della Salute - Università degli Studi di Milano, Milan, Italy
Silvia Paoletta

³Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
Arnaud Dupuis

⁴Unite Mixte de Recherche_S949, Inserm, Université de Strasbourg, Etablissement Français du Sang-Alsace-Lorraine-Champagne-Ardennes, Strasbourg, France
Philippe Ohlmann

⁴Unite Mixte de Recherche_S949, Inserm, Université de Strasbourg, Etablissement Français du Sang-Alsace-Lorraine-Champagne-Ardennes, Strasbourg, France
Christian Gachet

⁴Unite Mixte de Recherche_S949, Inserm, Université de Strasbourg, Etablissement Français du Sang-Alsace-Lorraine-Champagne-Ardennes, Strasbourg, France
Kenneth A. Jacobson

³Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
Katharina Machura

⁵Department of Pediatrics and Adolescent Medicine, Division of Pediatric Hematology and Oncology, Medical Center – University of Freiburg, Faculty of Medicine, Germany
Gian M. Podda

²Divisione di Medicina 3, ASST Santi Paolo e Carlo, Dipartimento di Scienze della Salute - Università degli Studi di Milano, Milan, Italy
Barbara Zieger

⁵Department of Pediatrics and Adolescent Medicine, Division of Pediatric Hematology and Oncology, Medical Center – University of Freiburg, Faculty of Medicine, Germany
Marco Cattaneo

¹Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy

Abstract

Summary

The platelet adenosine 5’-diphosphate (ADP) receptor P2Y₁₂ (P2Y₁₂R) plays a critical role in platelet aggregation. The present report illustrates an update of dysfunctional platelet P2Y₁₂R mutations diagnosed with congenital lifelong bleeding problems. Described patients with heterozygous or homozygous substitution in the P2Y₁₂R gene and qualitative abnormalities of the platelet P2Y₁₂R are summarized. Recently, a further dysfunctional variant of P2Y₁₂R has been identified in two brothers who presented with a lifelong severe bleeding disorder. During in vitro aggregation studies, the patient´s platelets show a markedly reduced and rapid reversible ADP-promoted aggregation. A homozygous c.561T>A substitution that changes the codon for His187 to Gln (p.His187Gln) in the P2Y₁₂R gene has been identified. This mutation causes no change in receptor expression but decreases the affinity of the ligand for the receptor, even at high concentrations. Structure modelling studies indicated that the p.His187Gln mutation, located in the fifth transmembrane spanning domain (TM5), impairs conformational changes of the receptor. Structural integrity of the TM5 region is necessary for agonist and antagonist binding and for correct receptor function.

Zusammenfassung

Der thrombozytäre Adenosin-5‘-diphosphat(ADP)-Rezeptor P2Y₁₂ (P2Y₁₂R) spielt eine entscheidende Rolle bei der Thrombozytenaggregation. Im Folgenden soll ein kurzes Update über dysfunktionale thrombozytäre P2Y₁₂R-Mutationen, welche im Zusammenhang mit angeborenen, lebenslang bestehenden Blutungsproblemen diagnostiziert wurden, gegeben werden. Patienten mit bereits beschriebenen homo- und heterozygoten Substitutionen im P2Y₁₂ -Gen sowie die daraus folgenden qualitativen Abnormitäten des thrombozytären P2Y₁₂R werden hier zusammengefasst. Zudem wurde kürzlich bei einem Brüderpaar mit einer langjährigen schweren Blutungsstörung eine weitere dysfunktionale Variante des P2Y₁₂R identifiziert. In in vitro- Aggregationsstudien zeigen die Thrombozyten dieser beiden Patienten eine erheblich reduzierte und schnell reversible ADP-induzierte Aggregation. Hierbei wurde eine homozygote c.561T>A Substitution, die das Kodon für His187 zu Gln (p.His187Gln) im P2Y₁₂R-Gen verändert, nachgewiesen. Die Mutation verursacht dabei keine Änderung der Rezeptorexpression, erhöht aber selbst bei einer hohen Ligandenkonzentration die Affinität des Liganden zum Rezeptor. „Strukturmodelling“-Studien zeigen, dass die in der fünften Transmembrandomäne (TM5) lokalisierte p.His187Gln Mutation die Konformationsänderung des Rezeptors beeinträchtigt, was darauf hindeutet, dass die strukturelle Integrität der TM5 Region für die Bin-dung von Agonisten als auch von Antagonisten notwendig ist und damit zu einer normalen Rezeptorfunktion beiträgt.

Keywords

Platelets - mutation - P2Y₁₂ receptor

Schlüsselwörter

Thrombozyten - Mutation - P2Y₁₂-Rezeptor

Full Text

References

References
1 Cattaneo M. The platelet P2Y₁₂ receptor for adenosine diphosphate: congenital and drug-induced defects. Blood 2011; 117: 2102-2112.
2 Hollopeter G, Jantzen HM, Vincent D. et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001; 409: 202-207.
3 Fabre JE, 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.
4 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.
5 Savoia A, Kunishima S, De Rocco D. et al. Spectrum of the mutations in Bernard-Soulier syndrome. Human Mutation 2014; 35: 1033-1045.
6 Foster CJ, Prosser DM, Agans JM. et al. Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine anti-thrombotic drugs. J Clin Invest 2001; 107: 1591-1598.
7 Andre P, Delaney SM, LaRocca T. et al. P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries. J Clin Invest 2003; 112: 398-406.
8 Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 1998; 94: 657-666.
9 Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 1998; 95: 8070-8074.
10 Cattaneo M, Gachet C. ADP receptors and clinical bleeding disorders. Arterioscler Thromb Vasc Biol 1999; 19: 2281-2285.
11 Nicholas RA. Insights into platelet P2Y12 receptor activation. Blood 2015; 125: 893-895.
12 Kauffenstein G, Hechler B, Cazenave JP, Gachet C. Adenine triphosphate nucleotides are antagonists at the P2Y receptor. J Thromb Haemost 2004; 2: 1980-1988.
13 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.
14 Gachet C. ADP receptors of platelets and their inhibition. Thromb Haemost 2001; 86: 222-232.
15 Dorsam RT, Kunapuli SP. Central role of the P2Y12 receptor in platelet activation. J Clin Invest 2004; 113: 340-345.
16 Podda G, Femia EA, Pugliano M, Cattaneo M. Congenital defects of platelet function. Platelets 2012; 23: 552-563.
17 Cattaneo M, Lecchi A, Randi AM. et al. Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate. Blood 1992; 80: 2787-2796.
18 Nurden P, Savi P, Heilmann E, Bihour C. 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.
19 Cattaneo M, Lecchi A, Lombardi R. et al. 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 舘primary secretion defect’ are heterozygous for a defect of P2CYC receptors. Arterioscler Thromb Vasc Biol 2000; 20: E101-E106.
20 Cattaneo M. New P2Y₁₂ inhibitors. Circulation 2010; 121: 171-179.
21 Zighetti ML, Carpani G, Sinigaglia E, Cattaneo M. Usefulness of a flow cytometric analysis of intra-platelet vasodilator-stimulated phosphoprotein phosphorylation for the detection of patients with genetic defects of the platelet P2Y₁₂ receptor for ADP. J Thromb Haemost 2010; 8: 2332-2334.
22 Cattaneo M, Zighetti ML, Lombardi R. et al. Molecular bases of defective signal transduction in the platelet P2Y12 receptor of a patient with congenital bleeding. Proc Natl Acad Sci USA 2003; 100: 1978-1983.
23 Remijn JA, MJ IJ, Strunk AL, Abbes AP. et al. Novel molecular defect in the platelet ADP receptor P2Y12 of a patient with haemorrhagic diathesis. Clin Chem Lab Med 2007; 45: 187-189.
24 Daly ME, Dawood BB, Lester WA. et al. Identification and characterization of a novel P2Y 12 variant in a patient diagnosed with type 1 von Willebrand disease in the European MCMDM-1VWD study. Blood 2009; 113: 4110-4113.
25 Oral presentations. J Thromb Haemost 2009; 7: 1-316.
26 Patel YM, Lordkipanidze M, Lowe GC. et al. A novel mutation in the P2Y₁₂ receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding. J Thromb Haemost 2014; 12: 716-725.
27 Lecchi A, Razzari C, Paoletta S. et al. Identification of a new dysfunctional platelet P2Y12 receptor variant associated with bleeding diathesis. Blood 2015; 125: 1006-1013.
28 El-Tayeb A, Griessmeier KJ, Muller CE. Synthesis and preliminary evaluation of [³H]PSB-0413, a selective antagonist radioligand for platelet P2Y₁₂ receptors. Bioorg Med Chem Lett 2005; 15: 5450-5452.
29 Ohlmann P, Lecchi A, El-Tayeb A. et al. The platelet P2Y₁₂ receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [H]PSB-0413. Purinergic Signal 2013; 9: 59-66.
30 Saposnik B, Binard S, Fenneteau O. et al. Mutation spectrum and genotype-phenotype correlations in a large French cohort of MYH9-related disorders. Mol Genet Genomic Med 2014; 2: 297-312.
31 Zhang J, Zhang K, Gao ZG. et al. Agonist-bound structure of the human P2Y₁₂ receptor. Nature 2014; 509: 119-122.
32 Zhang K, Zhang J, Gao ZG. et al. Structure of the human P2Y₁₂ receptor in complex with an anti-thrombotic drug. Nature 2014; 509: 115-118.