Platelet integrin αIIbβ3 possesses a Leu/Pro polymorphism at residue 33 (Leu33/HPA-1a or Pro33/HPA-1b). The Pro33 isoform has been suggested to exhibit prothrombotic features. αIIbβ3-expressing CHO (Chinese hamster ovary) cells on immobilized fibrinogen show activation of the MAP kinase family member ERK2, with an enhanced ERK2 activity in Pro33 cells compared to Leu33 cells. In our present work, we examined how the Leu/Pro polymorphism modulates the ERK2 activation stimulated by 2 differently triggered outside-in signalings. We either treated the CHO cells with Mn2+ or allowed them to adhere to fibrinogen. Moreover, we studied which signaling cascades are involved in ERK2 activation. In contrast to immobilized fibrinogen, Mn2+ did not significantly increase ERK2 activation. However, Mn2+ had a synergistic effect on ERK2 phosphorylation when combined with immobilized fibrinogen. Pro33 cells adherent to fibrinogen exhibited a significantly greater ERK2 activity than Leu33 cells in the presence of Mn2+, which peaked after 10 min of adhesion. Our data showed that Src family and rho kinases play a crucial role in the integrin αIIbβ3-dependent outside-in signaling to ERK2.

Keywords:
αIIbβ3, ERK signaling, Leu33, Pro33, Polymorphisms, Rho kinase, Src signaling
1.
Shattil SJ, Newman PJ: Integrins: dynamic scaffolds for adhesion and signaling in platelets. Blood 2004;104:1606-1615.
2.
Stegner D, Nieswandt B: Platelet receptor signaling in thrombus formation. J Mol Med (Berl) 2011;89:109-121.
3.
Varga-Szabo D, Pleines I, Nieswandt B: Cell adhesion mechanisms in platelets. Arterioscler Thromb Vasc Biol 2008;28:403-412.
4.
Michelson AD, Furman MI, Goldschmidt-Clermont P, Mascelli MA, Hendrix C, Coleman L, Hamlington J, Barnard MR, Kickler T, Christie DJ, Kundu S, Bray PF: Platelet GP IIIa PlA polymorphisms display different sensitivities to agonists. Circulation 2000;101:1013-1018.
5.
Feng D, Lindpaintner K, Larson MG, Rao VS, O'Donnell CJ, Lipinska I, Schmitz C, Sutherland PA, Silbershatz H, D'Agostino RB, Muller JE, Myers RH, Levy D, Tofler GH: Increased platelet aggregability associated with platelet GPIIIa PlA2 polymorphism: the Framingham Offspring Study. Arterioscler Thromb Vasc Biol 1999;19:1142-1147.
6.
Vijayan KV, Goldschmidt-Clermont PJ, Roos C, Bray PF: The PlA2 polymorphism of integrin beta3 enhances outside-in signaling and adhesive functions. J Clin Invest 2000;105:793-802.
7.
Loncar R, Stoldt V, Hellmig S, Zotz RB, Mihalj M, Scharf RE: HPA-1 polymorphism of αIIbβ3 modulates platelet adhesion onto immobilized fibrinogen in an in-vitro flow system. Thromb J 2007;5:2.
8.
Williams MS, Bray PF: Genetics of arterial prothrombotic risk states. Exp Biol Med (Maywood) 2001;226:409-419.
9.
Zotz RB, Winkelmann BR, Müller C, Boehm BO, März W, Scharf RE: Association of polymorphisms of platelet membrane integrins αIIbβ3 (HPA-1b/PlA2) and α2β1 (α2807TT) with premature myocardial infarction. J Thromb Haemost 2005;3:1522-1529.
10.
Litvinov RI, Nagaswami C, Vilaire G, Shuman H, Bennett JS, Weisel JW: Functional and structural correlations of individual αIIbβ3 molecules. Blood 2004;104:3979-3985.
11.
Petrich BG, Fogelstrand P, Partridge AW, Yousefi N, Ablooglu AJ, Shattil SJ, Ginsberg MH: The antithrombotic potential of selective blockade of talin-dependent integrin αIIbβ3 (platelet GPIIb-IIIa) activation. J Clin Invest 2007;117:2250-2259.
12.
Shattil SJ: Signaling through platelet integrin αIIbβ3: inside-out, outside-in, and sideways. Thromb Haemost 1999;82:318-325.
13.
Shattil SJ: Integrins and Src: dynamic duo of adhesion signaling. Trends Cell Biol 2005;15:399-403.
14.
Arias-Salgado EG, Lizano S, Sarkar S, Brugge JS, Ginsberg MH, Shattil SJ: Src kinase activation by direct interaction with the integrin beta cytoplasmic domain. Proc Natl Acad Sci USA 2003;100:13298-13302.
15.
Rosado JA, Sage SO: Phosphoinositides are required for store-mediated calcium entry in human platelets. J Biol Chem 2000;275:9110-9113.
16.
Zhu X, Assoian RK: Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. Mol Biol Cell 1995;6:273-282.
17.
Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA: Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol 1997;137:481-492.
18.
Vijayan KV, Liu Y, Dong JF, Bray PF: Enhanced activation of mitogen-activated protein kinase and myosin light chain kinase by the Pro33 polymorphism of integrin beta 3. J Biol Chem 2003;278:3860-3867.
19.
Kamm KE, Stull JT: Dedicated myosin light chain kinases with diverse cellular functions. J Biol Chem 2001;276:4527-4530.
20.
Vijayan KV, Bray PF: Molecular mechanisms of prothrombotic risk due to genetic variations in platelet genes: enhanced outside-in signaling through the Pro33 variant of integrin β3. Exp Biol Med (Maywood) 2006;231:505-513.
21.
Vijayan KV, Liu Y, Sun W, Ito M, Bray PF: The Pro33 isoform of integrin β3 enhances outside-in signaling in human platelets by regulating the activation of serine/threonine phosphatases. J Biol Chem 2005;280:21756-21762.
22.
Stoldt VR, Berendes S, Scharf RE: The HPA-1b (Pro33) variant integrin αIIbβ3 increases the resistance of adherent platelets and transfected CHO cells upon exposure to shear stress. 54th Annu Meet Soc Thromb Hemost, Nuremberg, 2010, A93.
23.
Buscà R, Pouyssegur J, Lenormand P: ERK1 and ERK2 map kinases: specific roles or functional redundancy? Front Cell Dev Biol 2016;4:53.
24.
Calvete JJ: Clues for understanding the structure and function of a prototypic human integrin: the platelet glycoprotein IIb/IIIa complex. Thromb Haemost 1994;72:1-15.
25.
Ruggeri ZM: Platelets in atherothrombosis. Nat Med 2002;8:1227-1234.
26.
Whalen AM, Galasinski SC, Shapiro PS, Nahreini TS, Ahn NG: Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase. Mol Cell Biol 1997;17:1947-1958.
27.
Jiang F, Jia Y, Cohen I: Fibronectin- and protein kinase C-mediated activation of ERK/MAPK are essential for proplateletlike formation. Blood 2002;99:3579-3584.
28.
Bazzoni G, Hemler ME: Are changes in integrin affinity and conformation overemphasized? Trends Biochem Sci 1998;23:30-34.
29.
Plow EF, Haas TA, Zhang L, Loftus J, Smith JW: Ligand binding to integrins. J Biol Chem 2000;275:21785-21788.
30.
Smith JW, Piotrowicz RS, Mathis D: A mechanism for divalent cation regulation of beta 3-integrins. J Biol Chem 1994;269:960-967.
31.
Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS, Lowell CA, Shattil SJ: Coordinate interactions of Csk, Src, and Syk kinases with αIIbβ3 initiate integrin signaling to the cytoskeleton. J Cell Biol 2002;157:265-275.
32.
Lee JW, Juliano R: Mitogenic signal transduction by integrin- and growth factor receptor-mediated pathways. Mol Cells 2004;17:188-202.
33.
Fincham VJ, James M, Frame MC, Winder SJ: Active ERK/MAP kinase is targeted to newly forming cell-matrix adhesions by integrin engagement and v-Src. EMBO J 2000;19:2911-2923.
34.
Tulasne D, Bori T, Watson SP: Regulation of RAS in human platelets. Evidence that activation of RAS is not sufficient to lead to ERK1-2 phosphorylation. Eur J Biochem 2002;269:1511-1517.
35.
Schoenwaelder SM, Hughan SC, Boniface K, Fernando S, Holdsworth M, Thompson PE, Salem HH, Jackson SP: RhoA sustains integrin αIIbβ3 adhesion contacts under high shear. J Biol Chem 2002;277:14738-14746.
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