Immune versus thrombotic stimulation of platelets differentially regulates signalling pathways, intracellular protein-protein interactions, and α-granule release

Summary

In addition to haemostasis, platelets mediate inflammation and clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve cytoskeletal rearrangements and release of granular content. Stimulation of the immune Toll-like receptor 2 (TLR2) on the platelet surface, activates phosphoinositide-3-kinase (PI3K) and causes platelet activation and plateletdependent thrombosis. It remains unknown if platelet activation by immune versus thrombotic pathways leads to the differential regulation of signal transduction, protein-protein interactions, and α-granule release, and the physiological relevance of these potential differences. We investigated these processes after immune versus thrombotic platelet stimulation. We examined selected signalling pathways and found that phosphorylation kinetics of Akt, ERK1/2 and p38 differed dramatically between agonists. Next, we investigated platelet protein-protein interactions by mass spectrometry (MS)-based proteomics specifically targeting cytosolic factor XIIIa (FXIIIa) because of its function as a cytoskeleton-crosslinking protein whose binding partners have limited characterisation. Four FXIIIa-binding proteins were identified, two of which are novel interactions: FXIIIa-focal adhesion kinase (FAK) and FXIIIa-gelsolin. The binding of FAK to FXIIIa was found to be altered differentially by immune versus thrombotic stimulation. Lastly, we studied the effect of thrombin versus Pam₃CSK₄ stimulation on α-granule release and observed differential release patterns for selected granule proteins and decreased fibrin clot formation compared with thrombin. The inhibition of PI3K caused a decrease in protein release after Pam₃CSK₄ ^-but not after thrombin-stimulation. In summary, stimulation of platelets by either thrombotic or immune receptors leads to markedly different signalling responses and granular protein release consistent with differential contribution to coagulation and thrombosis.

Keywords

Toll-like receptor 2 - thrombin - signal transduction - mass spectrometry-based proteomics - factor XIIIA

• References

• 1 Beutler B. Innate immunity: an overview. Mol Immunol 2004; 40: 845-859.
• 2 Akira S. Toll-like receptor signalling. J Biol Chem 2003; 278: 38105-38108.
• 3 Shiraki R, Inoue N, Kawasaki S. et al. Expression of Toll-like receptors on human platelets. Thromb Res 2004; 113: 379-385.
• 4 Cognasse F, Hamzeh H, Chavarin P. et al. Evidence of Toll-like receptor molecules on human platelets. Immunol Cell Biol 2005; 83: 196-198.
• 5 Weyrich AS, Zimmerman GA. Platelets: signalling cells in the immune continuum. Trends Immunol 2004; 25: 489-495.
• 6 Wetzler LM. The role of Toll-like receptor 2 in microbial disease and immunity. Vaccine 2003; 21: S2/55-S2/60.
• 7 Berg M, Offermanns S, Seifert R. et al. Synthetic lipopeptide Pam3CysSer(Lys)4 is an effective activator of human platelets. Am J Physiol 1994; 266: C1684-1691.
• 8 Blair P, Rex S, Vitseva O. et al. Stimulation of Tolllike receptor 2 in human platelets induces a thromboinflammatory response through activation of phosphoinositide 3-kinase. Circ Res. 2009 in press..
• 9 Brass LF, Stalker TJ, Zhu L. et al. Signal transduction during platelet plug formation. In: Platelets. 2nd ed. New York: Elsevier Science; 2007: 319-346.
• 10 Freedman JE, Loscalzo J, Barnard MR. et al. Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest 1997; 100: 350-356.
• 11 Achyuthan KE, Dobson JV, Greenberg CS. Gly-Pro-Arg-Pro modifies the glutamine residues in the alpha-and gamma-chains of fibrinogen: inhibition of transglutaminase cross-linking. Biochim Biophys Acta 1986; 872: 261-268.
• 12 Garcia BA, Smalley DM, Cho H. et al. The platelet microparticle proteome. J Proteome Res 2005; 04: 1516-1521.
• 13 Coppinger JA, Cagney G, Toomey S. et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 2004; 103: 2096-2104.
• 14 Bizios R, Lai L, Fenton 2nd JW, Malik AB. Thrombin-induced thromboxane generation by neutrophils and lymphocytes: dependence on enzymic site. J Cell Physiol 1987; 132: 359-362.
• 15 Yoon HY, Hwang SH, Lee EY. et al. Effects of ADP on different inhibitory properties of brain glutamate dehydrogenase isoproteins by perphenazine. Biochimie 2001; 83: 907-913.
• 16 Nadal-Wollbold F, Pawlowski M, Levy-Toledano S. et al. Platelet ERK2 activation by thrombin is dependent on calcium and conventional protein kinases C but not Raf-1 or B-Raf. FEBS Lett 2002; 531: 475-482.
• 17 Begonja AJ, Geiger J, Rukoyatkina N. et al. Thrombin stimulation of p38 MAP kinase in human platelets is mediated by ADP and thromboxane A2 and inhibited by cGMP/cGMP-dependent protein kinase. Blood 2007; 109: 616-618.
• 18 Adany R, Bardos H. Factor XIII subunit A as an intracellular transglutaminase. Cell Mol Life Sci 2003; 60: 1049-1060.
• 19 Watson SP, Bahou WF, Fitzgerald D. et al. Mapping the platelet proteome: a report of the ISTH Platelet Physiology Subcommittee. J Thromb Haemost 2005; 03: 2098-2101.
• 20 Muszbek L, Yee VC, Hevessy Z. Blood coagulation factor XIII: structure and function. Thromb Res 1999; 94: 271-305.
• 21 Zhu Y, Tassi L, Lane W. et al. Specific binding of the transglutaminase, platelet factor XIII, to HSP27. J Biol Chem 1994; 269: 22379-22384.
• 22 Siebenlist KR, Meh DA, Mosesson MW. Protransglutaminase (factor XIII) mediated crosslinking of fibrinogen and fibrin. Thromb Haemost 2001; 86: 1221-1228.
• 23 Shen L, Lorand L. Contribution of fibrin stabilization to clot strength. Supplementation of factor XIIIdeficient plasma with the purified zymogen. J Clin Invest 1983; 71: 1336-1341.
• 24 Jackson SP, Yap CL, Anderson KE. Phosphoinositide 3-kinases and the regulation of platelet function. Biochem Soc Trans 2004; 32: 387-392.
• 25 Aliprantis AO, Yang RB, Mark MR. et al. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 1999; 285: 736-739.
• 26 Hazeki K, Nigorikawa K, Hazeki O. Role of phosphoinositide 3-kinase in innate immunity. Biol Pharm Bull 2007; 30: 1617-1623.
• 27 Liu F, Morris S, Epps J. et al. Demonstration of an activation regulated NF-kappaB/I-kappaBalpha complex in human platelets. Thromb Res 2002; 106: 199-203.
• 28 Zhang J, Banfic H, Straforini F. et al. A type II phosphoinositide 3-kinase is stimulated via activated integrin in platelets. A source of phosphatidylinositol 3-phosphate. J Biol Chem 1998; 273: 14081-14084.
• 29 Flevaris P, Li Z, Zhang G. et al. Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signalling pathway. Blood 2009; 113: 893-901.
• 30 Maguire PB, Wynne KJ, Harney DF. et al. Identification of the phosphotyrosine proteome from thrombin activated platelets. Proteomics 2002; 02: 642-648.
• 31 Garcia A, Prabhakar S, Brock CJ. et al. Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry. Proteomics 2004; 04: 656-668.
• 32 Garcia A, Prabhakar S, Hughan S. et al. Differential proteome analysis of TRAP-activated platelets: involvement of DOK-2 and phosphorylation of RGS proteins. Blood 2004; 103: 2088-2095.
• 33 Zahedi RP, Begonja AJ, Gambaryan S. et al. Phosphoproteomics of human platelets: A quest for novel activation pathways. Biochim Biophys Acta 2006; 1764: 1963-1976.
• 34 Garcia A. Proteome analysis of signalling cascades in human platelets. Blood Cells Mol Dis 2006; 36: 152-156.
• 35 Maynard DM, Heijnen HF, Horne MK. et al. Proteomic analysis of platelet alpha-granules using mass spectrometry. J Thromb Haemost 2007; 05: 1945-1955.
• 36 Clark EA, Shattil SJ, Brugge JS. Regulation of protein tyrosine kinases in platelets. Trends Biochem Sci 1994; 19: 464-469.
• 37 Shattil SJ, Haimovich B, Cunningham M. et al. Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signalling through integrin and agonist receptors. J Biol Chem 1994; 269: 14738-14745.
• 38 Ohmori T, Yatomi Y, Asazuma N. et al. Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on alphaIIbbeta3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2. Biochem J 2000; 347: 561-569.
• 39 Fox JE. Cytoskeletal proteins and platelet signalling. Thromb Haemost 2001; 86: 198-213.
• 40 Zhu Y, O’Neill S, Saklatvala J. et al. Phosphorylated HSP27 associates with the activation-dependent cytoskeleton in human platelets. Blood 1994; 84: 3715-3723.
• 41 Reed GL. Platelet secretion. In: Platelets. 2nd ed. New York: Elsevier Science; 2007: 309-318.
• 42 Flaumenhaft R. Molecular basis of platelet granule secretion. Arterioscler Thromb Vasc Biol 2003; 23: 1152-1160.
• 43 Sehgal S, Storrie B. Evidence that differential packaging of the major platelet granule proteins von Willebrand factor and fibrinogen can support their differential release. J Thromb Haemost 2007; 05: 2009-2016.
• 44 von Hundelshausen P, Petersen F, Brandt E. Platelet-derived chemokines in vascular biology. Thromb Haemost 2007; 97: 704-713.
• 45 Marx G, Korner G, Mou X, Gorodetsky R. Packaging zinc, fibrinogen, and factor XIII in platelet alphagranules. J Cell Physiol 1993; 156: 437-442.
• 46 Lopaciuk S, Lovette KM, McDonagh J, McDonagh Chuang HY. Subcellular distribution of fibrinogen and factor XIII in human blood platelets. Thromb Res 1976; 08: 453-465.
• 47 Yin HL, Kwiatkowski DJ, Mole JE. et al. Structure and biosynthesis of cytoplasmic and secreted variants of gelsolin. J Biol Chem 1984; 259: 5271-5276.
• 48 Hartwig JH. The platelet cytoskeleton. In: Platelets. 2nd ed. New York: Elsevier Science; 2007: 75-97.
• 49 Sixma JJ, van den Berg A, Jockusch BM. et al. Immunoelectron microscopic localization of actin, alphaactinin, actin-binding protein and myosin in resting and activated human blood platelets. Eur J Cell Biol 1989; 48: 271-281.
• 50 Lawler J, Derick LH, Connolly JE. et al. The structure of human platelet thrombospondin. J Biol Chem 1985; 260: 3762-3772.