β-arrestin-1 participates in thrombosis and regulates integrin α_IIbβ₃ signalling without affecting P2Y receptors desensitisation and function

 

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Summary

β-arrestin-1 (β-arr1) and β-arrestin-2 (β-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of β-arr1 or β-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in β-arr1^−/− and β-arr2^−/− platelets. In addition, deficiency in β-arr1 or β-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between β-arr1 and β-arr2 may explain these unchanged platelet responses. Interestingly, β-arr1^−/− but not β-arr2^−/− mice were protected against laser- and FeCl₃-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in β-arr1^−/− and β-arr2^−/− mice, suggesting no defect in haemostasis. β-arr1^−/− platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin α_IIbβ₃. β-arr1 appeared to be located downstream of Src family kinases and to regulate α_IIbβ₃ signalling by increasing Akt phosphorylation. Overall, this study supports a role for β-arr1 in promoting thrombus formation, in part through its participation in α_IIbβ₃ signalling, and no role of β-arr1 and β-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.

• References

• 1 DeWire SM, Ahn S, Lefkowitz RJ. et al. Beta-arrestins and cell signaling. Ann Rev Physiol 2007; 69: 483-510.
  CrossrefPubMedGoogle Scholar
• 2 Gachet C. P2 receptors, platelet function and pharmacological implications. Thromb Haemost 2008; 99: 466-472.
  Article in Thieme ConnectPubMedGoogle Scholar
• 3 Baurand A, Eckly A, Bari N. et al. Desensitization of the platelet aggregation response to ADP: differential down-regulation of the P2Y1 and P2cyc receptors. Thromb Haemost 2000; 84: 484-491.
  Article in Thieme ConnectPubMedGoogle Scholar
• 4 Baurand A, Eckly A, Hechler B. et al. Differential regulation and relocalization of the platelet P2Y receptors after activation: a way to avoid loss of hemostatic properties?. Mol Pharmacol 2005; 67: 721-733.
  PubMedGoogle Scholar
• 5 Hardy AR, Conley PB, Luo J. et al. P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms. Blood 2005; 105: 3552-3560.
  CrossrefPubMedGoogle Scholar
• 6 Hoffmann C, Ziegler N, Reiner S. et al. Agonist-selective, receptor-specific interaction of human P2Y receptors with beta-arrestin-1 and –2. J Biol Chem 2008; 283: 30933-30941.
  CrossrefPubMedGoogle Scholar
• 7 Reiner S, Ziegler N, Leon C. et al. beta-Arrestin-2 interaction and internalization of the human P2Y1 receptor are dependent on C-terminal phosphorylation sites. Mol Pharmacol 2009; 76: 1162-1171.
  CrossrefPubMedGoogle Scholar
• 8 Mundell SJ, Luo J, Benovic JL. et al. Distinct clathrin-coated pits sort different G proteincoupled receptor cargo. Traffic 2006; 07: 1420-1431.
  CrossrefPubMedGoogle Scholar
• 9 Watson SP, Auger JM, McCarty OJ. et al. GPVI and integrin alphaIIb beta3 signaling in platelets. J Thromb Haemost 2005; 03: 1752-1762.
  CrossrefPubMedGoogle Scholar
• 10 Woulfe DS. Akt signaling in platelets and thrombosis. Expert Rev Hematol 2010; 03: 81-91.
  CrossrefPubMedGoogle Scholar
• 11 Banfic H, Downes CP, Rittenhouse SE. Biphasic activation of PKBalpha/Akt in platelets. Evidence for stimulation both by phosphatidylinositol 3,4-bisphosphate, produced via a novel pathway, and by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998; 273: 11630-11637.
  CrossrefPubMedGoogle Scholar
• 12 Valiyaveettil M, Feng W, Mahabaleshwar G. et al. Phosphorylation of platelet alphaIIbbeta3 is crucial for arterial thrombosis in vivo and microparticule generation. Abstracts from American Heart Association Scientific Sessions Orlando 2007 Nov 4–7; Circulation 2007; 116: II_75 Abstract 450.
  PubMedGoogle Scholar
• 13 Law DA, DeGuzman FR, Heiser P. et al. Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function. Nature 1999; 401: 808-811.
  CrossrefPubMedGoogle Scholar
• 14 Goschnick MW, Lau LM, Wee JL. et al. Impaired „outside-in“ integrin alphaIIbbeta3 signaling and thrombus stability in TSSC6-deficient mice. Blood 2006; 108: 1911-1918.
  CrossrefPubMedGoogle Scholar
• 15 Takizawa H, Nishimura S, Takayama N. et al. Lnk regulates integrin alphaIIbbeta3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo. J Clin Invest 2010; 120: 179-190.
  CrossrefPubMedGoogle Scholar
• 16 Li D, D'Angelo L, Chavez M. et al. Arrestin-2 differentially regulates PAR4 and ADP receptor signaling in platelets. J Biol Chem 2011; 286: 3805-3814.
  CrossrefPubMedGoogle Scholar
• 17 Cazenave JP, Ohlmann P, Cassel D. et al. Preparation of washed platelet suspensions from human and rodent blood. Methods Mol Biol 2004; 272: 13-28.
  PubMedGoogle Scholar
• 18 Conner DA, Mathier MA, Mortensen RM. et al. beta-Arrestin1 knockout mice appear normal but demonstrate altered cardiac responses to beta-adrenergic stimulation. Circulation Res 1997; 81: 1021-1026.
  CrossrefPubMedGoogle Scholar
• 19 Bohn LM, Lefkowitz RJ, Gainetdinov RR. et al. Enhanced morphine analgesia in mice lacking beta-arrestin 2. Science 1999; 286: 2495-2498.
  CrossrefPubMedGoogle Scholar
• 20 Schaff M, Receveur N, Bourdon C. et al. Novel function of tenascin-C, a matrix protein relevant to atherosclerosis, in platelet recruitment and activation under flow. Arterioscl Thromb Vasc Biol 2011; 31: 117-124.
  CrossrefPubMedGoogle Scholar
• 21 Barragan P, Paganelli F, Camoin-Jau L. et al. Validation of a novel ELISA-based VASP whole blood assay to measure P2Y12-ADP receptor activity. Thromb Haemost 2010; 104: 410-411.
  Article in Thieme ConnectPubMedGoogle Scholar
• 22 Boulay-Moine D, Bourguet N, Miet S. et al. Evaluation of platelet agonists and antagonists by a novel ELISA-based VASP assay and compatibility with whole blood samples from several animal species. Poster presentation at the XXIII Congress of the International Society on Thrombosis and Haemostasis Kyoto. 2011 Jul 23–28; Poster P-MO-050.
  PubMedGoogle Scholar
• 23 Mangin P, Yuan Y, Goncalves I. et al. Signaling role for phospholipase C gamma 2 in platelet glycoprotein Ib alpha calcium flux and cytoskeletal reorganization. Involvement of a pathway distinct from FcR gamma chain and Fc gamma RIIA. J Biol Chem 2003; 278: 32880-32891.
  CrossrefPubMedGoogle Scholar
• 24 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.
  PubMedGoogle Scholar
• 25 Mangin P, David T, Lavaud V. et al. Identification of a novel 14–3–3zeta binding site within the cytoplasmic tail of platelet glycoprotein Ibalpha. Blood 2004; 104: 420-427.
  CrossrefPubMedGoogle Scholar
• 26 Hechler B, Nonne C, Eckly A. et al. Arterial thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization. J Thromb Haemost 2010; 08: 173-184.
  CrossrefPubMedGoogle Scholar
• 27 Eckly A, Hechler B, Freund M. et al. Mechanisms underlying FeCl(3) -induced arterial thrombosis. J Thromb Haemost 2011; 09: 779-789.
  CrossrefPubMedGoogle Scholar
• 28 Eckly A, Gendrault JL, Hechler B. et al. Differential involvement of the P2Y1 and P2YT receptors in the morphological changes of platelet aggregation. Thromb Haemost 2001; 85: 694-701.
  Article in Thieme ConnectPubMedGoogle Scholar
• 29 Goel R, Phillips-Mason PJ, Raben DM. et al. alpha-Thrombin induces rapid and sustained Akt phosphorylation by beta-arrestin1-dependent and -independent mechanisms, and only the sustained Akt phosphorylation is essential for G1 phase progression. J Biol Chem 2002; 277: 18640-1868.
  CrossrefPubMedGoogle Scholar
• 30 Kohout TA, Lin FS, Perry SJ. et al. beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking. Proc Natl Acad Sci USA 2001; 98: 1601-1606.
  PubMedGoogle Scholar
• 31 Nisar S, Daly ME, Federici AB. et al. An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets. Blood 2011; 118: 5641-5651.
  CrossrefPubMedGoogle Scholar
• 32 Chen J, De S, Damron DS. et al. Impaired platelet responses to thrombin and collagen in AKT-1-deficient mice. Blood 2004; 104: 1703-1710.
  CrossrefPubMedGoogle Scholar
• 33 Gong H, Shen B, Flevaris P. et al. G protein subunit Galpha13 binds to integrin al-phaIIbbeta3 and mediates integrin “outside-in“ signaling. Science 2010; 327: 340-343.
  CrossrefPubMedGoogle Scholar
• 34 Akbar H, Shang X, Perveen R. et al. Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation. PLoS One 2011; 06: e22117.
  CrossrefPubMedGoogle Scholar

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