Contribution of PAR-1, PAR-4 and GPIbα in intracellular signaling leading to the cleavage of the β3 cytoplasmic domain during thrombin-induced platelet aggregation

Summary

Integrin αIIbβ3 plays a pivotal role in platelet aggregation by binding to fibrinogen. The β3 cytoplasmic domain of αIIbβ3 interacts with cytoskeletal and signaling proteins and is cleaved by µ-calpain, a calcium regulated cysteine protease. In the present study, we have investigated in more detail the cleavage of the β3 cytoplasmic domain during platelet aggregation induced by thrombin, TRAP-1 and TRAP-4. Our data show that β3 is cleaved in all three cases. The time course of β3 cleavage and the amount of cleaved β3 depends on the way platelets are activated and on the complete activation of µ-calpain, with a maximum of 90% of cleaved β3 obtained when thrombin is used. Furthermore, our results also show that the cleaved αIIbβ3 is mainly distributed in the Triton soluble fraction, indicating its inability to bind to the cytoskeleton. Interestingly, in the absence of GPIbα or following inhibition of thrombin binding to GPIbα, there is a reduction in the thrombin-induced calcium flux, β3 cleavage and µ-calpain activation. These results suggest that cleavage of the β3 cytoplasmic domain by µ-calpain might be an important step regulating the link between the cytoskeleton and αIIbβ3 during platelet aggregation, and that GPIbα could function as a cofactor for the complete activation of platelets by thrombin.

Keywords

Integrin αIIbβ3 - β3 cytoplasmic domain - PAR - GPIbα

• References

• 1 Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987; 238: 491-7.
• 2 Bennett VGJS. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest 1979; 64: 1393-1401.
• 3 Phillips DR, Charo IF, Parise LV. et al. The platelet membrane glycoprotein IIb-IIIa complex. Blood 1988; 71: 831-43.
• 4 Phillips DR, Jennings LK, Edwards HH. Identification of membrane proteins mediating the interaction of human platelets. J Cell Biol 1980; 86: 77-86.
• 5 Fox JE, Boyles JK, Berndt MC. et al. Identification of a membrane skeleton in platelets. J Cell Biol 1988; 106: 1525-38.
• 6 Fox JE, Lipfert L, Clark EA. et al. On the role of the platelet membrane skeleton in mediating signal transduction. Association of GP IIb-IIIa, pp60c-src, pp62c-yes, and the p21ras GTPase-activating protein with the membrane skeleton. J Biol Chem 1993; 268: 25973-84.
• 7 Hotchin NA, Hall A. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J Cell Biol 1995; 131: 1857-65.
• 8 Fox JE, Shattil SJ, Kinlough-Rathbone RL. et al. The platelet cytoskeleton stabilizes the interaction between alphaIIbbeta3 and its ligand and induces selective movements of ligand- occupied integrin. J Biol Chem 1996; 271: 7004-11.
• 9 Knezevic I, Leisner TM, Lam SC. Direct binding of the platelet integrin alphaIIbbeta3 (GPIIb-IIIa) to talin. Evidence that interaction is mediated through the cytoplasmic domains of both alphaIIb and beta3. J Biol Chem 1996; 271: 16416-21.
• 10 Otey C, Griffiths W, Burridge K. Characterization of monoclonal antibodies to chicken gizzard talin. Hybridoma 1990; 09: 57-62.
• 11 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: 808-11.
• 12 O’Toole TE, Ylanne J, Culley BM. Regulation of integrin affinity states through an NPXY motif in the beta subunit cytoplasmic domain. J Biol Chem 1995; 270: 8553-8.
• 13 Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelet paradigm. Blood 1998; 91: 2645-57.
• 14 Beckerle MC, Burridge K, DeMartino GN. et al. Colocalization of calcium-dependent protease II and one of its substrates at sites of cell adhesion. Cell 1987; 51: 569-77.
• 15 Fox JE. Regulation of platelet function by the cytoskeleton. Adv Exp Med Biol 1993; 344: 175-85.
• 16 Fox JE, Taylor RG, Taffarel M. et al. Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIIa. J Cell Biol 1993; 120: 1501-7.
• 17 Fox JE, Goll DE, Reynolds CC. et al. Identification of two proteins (actinbinding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent protease during platelet aggregation. J Biol Chem 1985; 260: 1060-6.
• 18 Gache Y, Landon F, Touitou H. et al. Susceptibility of platelet alpha-actinin to a Ca2+-activated neutral protease. Biochem Biophys Res Commun 1984; 124: 877-81.
• 19 Patel Y, Kakkar VV, Authi KS. Calpaininduced down-regulation of protein kinase C inhibits dense-granule secretion in human platelets. Inhibition of platelet aggregation or calpain activity preserves protein kinase C and restores full secretion. Biochim Biophys Acta 1994; 1224: 480-8.
• 20 Frangioni JV, Oda A, Smith M. et al. Calpaincatalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets. Embo J 1993; 12: 4843-56.
• 21 Oda A, Druker BJ, Ariyoshi H. et al. pp60src is an endogenous substrate for calpain in human blood platelets. J Biol Chem 1993; 268: 12603-8.
• 22 Banno Y, Asano T, Nozawa Y. Proteolytic modification of membrane-associated phospholipase C-beta by mucalpain enhances its activation by G-protein beta gamma subunits in human platelets. FEBS Lett 1994; 340: 185-8.
• 23 Cooray P, Yuan Y, Schoenwaelder SM. et al. Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain. Biochem J 1996; 318: 41-7.
• 24 Schoenwaelder SM, Yuan Y, Cooray P. et al. Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin alphaIIbbeta3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots. J Biol Chem 1997; 272: 1694-1702.
• 25 Du X, Saido TC, Tsubuki S. et al. Calpain cleavage of the cytoplasmic domain of the integrin beta 3 subunit. J Biol Chem 1995; 270: 26146-51.
• 26 Kouns WC, Hadvary P, Haering P. et al. Conformational modulation of purified glycoprotein (GP) IIb-IIIa allows proteolytic generation of active fragments from either active or inactive GPIIb-IIIa. J Biol Chem 1992; 267: 18844-51.
• 27 Meyer S, Kresbach G, Haring P. et al. Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells. Incorporation of GP Ib alpha into the cell surface membrane. J Biol Chem 1993; 268: 20555-62.
• 28 Fox JE, Reynolds CC, Boyles JK. Studying the platelet cytoskeleton in Triton X-100 lysates. Methods Enzymol 1992; 215: 42-58.
• 29 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-50.
• 30 Lopez JA. The platelet glycoprotein Ib-IX complex. Blood Coagul Fibrinolysis 1994; 05: 97-119.
• 31 Ward CM, Vinogradov DV, Andrews RK. et al. Characterization of mocarhagin, a cobra venom metalloproteinase from Naja mocambique mocambique, and related proteins from other Elapidae venoms. Toxicon 1996; 34: 1203-6.
• 32 Dubois C, Steiner B, Kieffer N. et al. Thrombin binding to GPIbalpha induces platelet aggregation and fibrin clot retraction supported by resting alphaIIbbeta3 interaction with polymerized fibrin. Thromb Haemost 2003; 89: 853-65.
• 33 Malik MN, Ramaswamy S, Tuzio H. et al. Micromolar Ca2+ requiring protease from human platelets: purification, partial characterization and effect on the cytoskeletal proteins. Life Sci 1987; 40: 593-604.
• 34 Calderwood DA, Zent R, Grant R. et al. The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation. J Biol Chem 1999; 274: 28071-4.
• 35 Lyman S, Gilmore A, Burridge K. et al. Integrinmediated activation of focal adhesion kinase is independent of focal adhesion formation or integrin activation. Studies with activated and inhibitory beta3 cytoplasmic domain mutants. J Biol Chem 1997; 272: 22538-47.
• 36 Goldmann WH. Kinetic determination of focal adhesion protein formation. Biochem Biophys Res Commun 2000; 271: 553-7.
• 37 Reddy KB, Gascard P, Price MG. et al. Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells. J Biol Chem 1998; 273: 35039-47.
• 38 Calderwood DA, Yan B, de Pereda JM. et al. The phosphotyrosine binding-like domain of talin activates integrins. J Biol Chem 2002; 277: 21749-58.
• 39 Yan B, Calderwood DA, Yaspan B. et al. Calpain cleavage promotes talin binding to the beta 3 integrin cytoplasmic domain. J Biol Chem 2001; 276: 28164-70.
• 40 Bialkowska K, Kulkarni S, Du X. et al. Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active. J Cell Biol 2000; 151: 685-96.
• 41 Law DA, Nannizzi-Alaimo L, Phillips DR. Outside-in integrin signal transduction. Alpha IIb beta 3-(GP IIb IIIa) tyrosine phosphorylation induced by platelet aggregation. J Biol Chem 1996; 271: 10811-5.
• 42 Cowan KJ, Law DA, Phillips DR. Identification of shc as the primary protein binding to the tyrosine- phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin platelet signaling. J Biol Chem 2000; 275: 36423-9.
• 43 Jenkins AL, Nannizzi-Alaimo L, Silver D. et al. Tyrosine phosphorylation of the beta3 cytoplasmic domain mediates integrin-cytoskeletal interactions. J Biol Chem 1998; 273: 13878-85.
• 44 De Candia E, Hall SW, Rutella S. et al. Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets. J Biol Chem 2001; 276: 4692-8.
• 45 Adam F, Verbeuren TJ, Fauchère J-L. et al. Thrombin-induced platelet PAR4 activation: role of glycoprotein Ib and ADP. J Thromb Haemost 2003; 01: 798-804.