A complementary role for tetraspanin superfamily member CD151 and ADP purinergic P2Y₁₂ receptor in platelets

Summary

P2Y₁₂ receptor is required for sustained activation of integrin α_llbβ₃, irreversible platelet aggregation and thrombus stabilisation. Tetraspanin superfamily member CD151 associates with integrin aNbp3 and plays critical roles in regulation of thrombus growth and stability in vivo. The possible functional relationship between P2Y₁₂ and CD151 in a molecular cluster in platelets may affect thrombus formation. Hence our aim was to investigate the physical and functional requirements for this association in platelets. Our investigations reveal a specific and constitutive association between CD151 and P2Y₁₂ receptor in human platelets shown by immunoprecipitation/western blot studies and by flow cytometry. Specifically, the prominent association involves CD151 with P2Y₁₂ oligomers, and to a lesser extent P2Y₁₂ monomers. This association is not altered by platelet aggregation induced by different agonists. There is also a distinct complex of tetraspanin CD151 with ADP purinergic receptor P2Y₁₂ but not P2Y₁. P2Y₁₂ oligomer interaction with CD151 is selective as compared to other tetraspanins. To investigate the functional relationship between these receptors in platelets we used wild-type or CD151 knockout (KO) mice treated with either PBS or 50 mg/kg clopidogrel. CD151 KO mice treated with clopidogrel exhibited synergy in delayed kinetics of clot retraction, in PAR-4 and collagen-mediated platelet aggregation, platelet spreading on fibrinogen and without restricting cAMP inhibition. Clopidogrel treated CD151 KO arterioles showed smaller and less stable thrombi with increased tendency to embolise ex vivo and in vivo. These studies demonstrate a complementary role between CD151 and P2Y₁₂ receptor in platelets in regulating thrombus growth and stability.

Keywords

Platelets - CD151 - mice - clopidogrel - purinergic P2Y12

• References

• 1 Gachet C. The platelet P2 receptors as molecular targets for old and new antiplatelet drugs. Pharmacol Therap 2005; 108: 180-192.
• 2 Rivera J, Lozano ML, Navarro-Núñez L. et al. Platelet receptors and signalling in the dynamics of thrombus formation. Haematologica 2009; 94: 700-711.
• 3 Hollopeter G, Jantzen HM, Vincent D. et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001; 409: 202-207.
• 4 von Kügelgen I. Pharmacological profiles of cloned mammalian P2Y-receptor subtypes. Pharmacol Therap 2006; 110: 415-432.
• 5 Dorsam RT, Kunapuli SP. Central role of the P2Y12 receptor in platelet activation. J Clin Invest 2004; 113: 340-345.
• 6 Kahner BN, Shankar H, Murugappan S. et al. Nucleotide receptor signalling in platelets. J Thromb Haemost 2006; 4: 2317-2326.
• 7 André 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 Cattaneo M, Gachet C. ADP receptors and clinical bleeding disorders. Arterioscl Thromb Vasc Biol 1999; 19: 2281-2285.
• 9 Humbert M, Nurden P, Bihour C. et al. 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. Arterioscl Thromb Vasc Biol 1996; 16: 1532-1543.
• 10 Hemler ME. Specific tetraspanin functions. J Cell Biol 2001; 155: 1103-1108.
• 11 Goschnick MW, Jackson DE. Tetraspanins-structural and signalling scaffolds that regulate platelet function. Mini Rev Med Chem 2007; 7: 1248-1254.
• 12 Stipp CS. Laminin-binding integrins and their tetraspanin partners as potential antimetastatic targets. Exp Rev Mol Med 2010; 12: 0.
• 13 Haining EJ, Yang J, Tomlinson MG. Tetraspanin microdomains: fine-tuning platelet function. Biochem Soc Transact 2011; 39: 518-523.
• 14 Sincock PM, Fitter S, Parton RG. et al. PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function. J Cell Sci 1999; 112: 833-844.
• 15 Lau LM, Wee JL, Wright MD. et al. The tetraspanin superfamily member CD151 regulates outside-in integrin aIIbp3 signalling and platelet function. Blood 2004; 104: 2368-2375.
• 16 Orlowski E, Chand R, Yip J. et al. A platelet tetraspanin superfamily member, CD151, is required for regulation of thrombus growth and stability in vivo. J Thromb Haemost 2009; 7: 2074-2084.
• 17 Wee JL, Jackson DE. The Ig-ITIM superfamily member PECAM-1 regulates the “outside-in” signalling properties of integrin αIIbβ3 in platelets. Blood 2005; 106: 3816-3823.
• 18 Wright MD, Geary SM, Fitter S. et al. Characterization of mice lacking the tetraspanin superfamily member CD151. Mol Cell Biol 2004; 24: 5978-5988.
• 19 Högberg C, Svensson H, Gustafsson R. et al. The reversible oral P2Y12 antagonist AZD6140 inhibits ADP-induced contractions in murine and human vasculature. Int J Cardiol 2010; 142: 187-192.
• 20 Jackson DE, Ward CM, Wang R. et al. The Protein-tyrosine Phosphatase SHP-2 Binds Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1) and Forms a Distinct Signalling Complex during Platelet Aggregation. J Biol Chem 1997; 272: 6986-6993.
• 21 Goschnick MW, Lau LM, Wee JL. et al. Impaired outside-in integrin aIIb|33 signalling and thrombus stability in TSSC6-deficient mice. Blood 2006; 108: 1911-1918.
• 22 Wong C, Liu Y, Yip J. et al. CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo. Blood 2009; 113: 1818-1828.
• 23 Walsh TG, Harper MT, Poole AW. SDF-1a is a novel autocrine activator of platelets operating through its receptor CXCR4. Cell Signal 2015; 27: 37-46.
• 24 Azam M, Andrabi SS, Sahr KE. et al. Disruption of the Mouse µ-Calpain Gene Reveals an Essential Role in Platelet Function. Mol Cell Biol 2001; 21: 2213-2220.
• 25 Little KD, Hemler ME, Stipp CS. Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells: central role of CD81 in facilitating GPR56-G±q/11 association. Mol Biol Cell 2004; 15: 2375-2387.
• 26 Alshahrani MM, Yang E, Yip J. et al. CEACAM2 negatively regulates hemi (ITAM-bearing) GPVI and CLEC-2 pathways and thrombus growth in vitro and in vivo. Blood 2014; 124: 2431-2441.
• 27 Bianco F, Fumagalli M, Pravettoni E. et al. Pathophysiological roles of extracellular nucleotides in glial cells: differential expression of purinergic receptors in resting and activated microglia. Brain Res Rev 2005; 48: 144-156.
• 28 Kaji K, Oda S, Shikano T. et al. The gamete fusion process is defective in eggs of Cd9-deficient mice. Nature Gen 2000; 24: 279-282.
• 29 Le Naour F, Rubinstein E, Jasmin C. et al. Severely reduced female fertility in CD9-deficient mice. Science 2000; 287: 319-321.
• 30 Miyado K, Yamada G, Yamada S. et al. Requirement of CD9 on the egg plasma membrane for fertilization. Science 2000; 287: 321-324.
• 31 Deng J, Dekruyff RH, Freeman GJ. et al. Critical role of CD81 in cognate T-B cell interactions leading to Th2 responses. Int Immunol 2002; 14: 513-523.
• 32 Gachet C. P2Y12 receptors in platelets and other hematopoietic and non-hematopoietic cells. Purin Signal 2012; 8: 609-619.
• 33 Burkhart JM, Vaudel M, Gambaryan S. et al. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012; 120: e73-82.
• 34 Jantzen HM, Milstone DS, Gousset L. et al. Impaired activation of murine platelets lacking G alpha(i2). J Clin Invest 2001; 108: 477-483.
• 35 Nishiuchi R, Sanzen N, Nada S. et al. Potentiation of the ligand-binding activity of integrin ±3²1 via association with tetraspanin CD151. Proc Natl Acad Sci USA 2005; 102: 1939-1944.