Autocrine amplification of integrin αIIbβ3 activation and platelet adhesive responses by deoxyribose-1-phosphate

Summary

Using direct injection mass spectrometry (DIMS) we discovered that deoxyribose-1-phosphate (dRP) is released by platelets upon activation. Interestingly, the addition of exogenous dRP to human platelets significantly increased platelet aggregation and integrin αIIbβ3 activation in response to thrombin. In parallel, genetically modified platelets with double genetic deletion of thymidine phosphorylase and uridine phosphorylase were characterised by reduced release of dRP, impaired aggregation and decreased integrin αIIbβ3 activation in response to thrombin. In vitro platelet adhesion onto fibrinogen and collagen under physiological flow conditions was potentiated by treatment of human platelets with exogenous dRP and impaired in transgenic platelets with reduced dRP release. Human and mouse platelets responded to dRP treatment with a sizeable increase in reactive oxygen species (ROS) generation and the pre-treament with the antioxidant apocynin abolished the effect of dRP on aggregation and integrin activation. Experiments directly assessing the activation of the small G protein Rap1b and protein kinase C suggested that dRP increases the basal levels of activity of these two pivotal platelet-activating pathways in a redox-dependent manner. Taken together, we present evidence that dRP is a novel autocrine amplifier of platelet activity, which acts on platelet redox levels and modulates integrin αIIbβ3.

Keywords

Platelets - reactive oxygen species - deoxyribose-1-phosphate - integrin - redox signalling

• References

• 1 Nurden AT. Qualitative disorders of platelets and megakaryocytes. J Thromb Haemost 2005; 03: 1773-1782.
• 2 Linden MD, Furman MI, Frelinger AL. et al. Indices of platelet activation and the stability of coronary artery disease. J Thromb Haemost 2007; 05: 761-765.
• 3 Jennings LK. Role of platelets in atherothrombosis. Am J Cardiol 2009; 103 (Suppl. 03) 4A-10A.
• 4 Egbrink MG, Van Gestel MA, Broeders MA. et al. Regulation of microvascular thromboembolism in vivo. Microcirculation 2005; 12: 287-300.
• 5 von Hundelshausen P, Weber C. Platelets as immune cells: bridging inflammation and cardiovascular disease. Circ Res 2007; 100: 27-40.
• 6 Nurden AT. Platelets, inflammation and tissue regeneration. Thromb Haemost 2011; 105 (Suppl. 01) S13-S33.
• 7 Langlois S, Gingras D, Beliveau R. Membrane type 1-matrix metalloproteinase (MT1-MMP) cooperates with sphingosine 1-phosphate to induce endothelial cell migration and morphogenic differentiation. Blood 2004; 103: 3020-3028.
• 8 Panetti TS, Hannah DF, Avraamides C. et al. Extracellular matrix molecules regulate endothelial cell migration stimulated by lysophosphatidic acid. J Thromb Haemost 2004; 02: 1645-1656.
• 9 Kulkarni S, Woollard KJ, Thomas S. et al. Conversion of platelets from a proaggregatory to a proinflammatory adhesive phenotype: role of PAF in spatially regulating neutrophil adhesion and spreading. Blood 2007; 110: 1879-1886.
• 10 Pula G, Garonna E, Dunn WB. et al. Paracrine stimulation of endothelial cell motility and angiogenesis by platelet-derived deoxyribose-1-phosphate. Arte-rioscler Thromb Vasc Biol 2010; 30: 2631-2638.
• 11 Pula G, Mayr U, Evans C. et al. Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures. Circ Res 2009; 104: 32-40.
• 12 Brown NS, Jones A, Fujiyama C. et al. Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors. Cancer Res 2000; 60: 6298-6302.
• 13 Koh G, Lee DH, Woo JT. 2-Deoxy-D-ribose induces cellular damage by increasing oxidative stress and protein glycation in a pancreatic beta-cell line. Metabolism 2010; 59: 325-332.
• 14 Begonja AJ, Gambaryan S, Geiger J. et al. Platelet NAD(P)H-oxidase-generated ROS production regulates alphaIIbbeta3-integrin activation independent of the NO/cGMP pathway. Blood 2005; 106: 2757-2760.
• 15 Irani K, Pham Y, Coleman LD. et al. Priming of platelet alphaIIbbeta3 by ox-idants is associated with tyrosine phosphorylation of beta3. Arterioscler Thromb Vasc Biol 1998; 18: 1698-1706.
• 16 Gregg D, de Carvalho DD, Kovacic H. Integrins and coagulation: a role for ROS/redox signaling?. Antioxid Redox Signal 2004; 06: 757-764.
• 17 Lopez LC, Akman HO, Garcia-Cazorla A. et al. Unbalanced deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphorylase-defi-cient mice. Hum Mol Genet 2009; 18: 714-722.
• 18 Pula G, Schuh K, Nakayama K. et al. PKCdelta regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation. Blood 2006; 108: 4035-4044.
• 19 Pope GA, MacKenzie DA, Defernez M. et al. Metabolic footprinting as a tool for discriminating between brewing yeasts. Yeast 2007; 24: 667-679.
• 20 Campanella M, Seraphim A, Abeti R. et al. IF1, the endogenous regulator of the F(1)F(o)-ATPsynthase, defines mitochondrial volume fraction in HeLa cells by regulating autophagy. Biochim Biophys Acta 2009; 1787: 393-401.
• 21 Pula G, Crosby D, Baker J. et al. Functional interaction of protein kinase Calpha with the tyrosine kinases Syk and Src in human platelets. J Biol Chem 2005; 280: 7194-7205.
• 22 Hotchkiss KA, Ashton AW, Schwartz EL. Thymidine phosphorylase and 2-deo-xyribose stimulate human endothelial cell migration by specific activation of the integrins alpha 5 beta 1 and alpha V beta 3. J Biol Chem 2003; 278: 19272-1929.
• 23 Bijnsdorp IV, de Bruin M, Laan AC. et al. The role of platelet-derived endothe-lial cell growth factor/thymidine phosphorylase in tumor behavior. Nucleosides Nucleotides Nucleic Acids 2008; 27: 681-691.
• 24 Vara D, Campanella M, Pula G. The novel NOX inhibitor 2-acetylphenothiazine impairs collagen-dependent thrombus formation in a GPVI-dependent manner. Br J Pharmacol. 2012 Epub ahead of print
• 25 Dharmarajah J, Arthur JF, Sobey CG, Drummond GR. The anti-platelet effects of apocynin in mice are not mediated by inhibition of NADPH oxidase activity. Naunyn Schmiedebergs Arch Pharmacol 2010; 382: 377-384.
• 26 Gianni D, Nicolas N, Zhang H. et al. H A novel and specific NADPH oxidase-1 (Nox1) small-molecule inhibitor blocks the formation of functional invadopodia in human colon cancer cells. ACS Chem Biol 2010; 05: 981-993.
• 27 Seeliger H, Guba M, Koehl GE. et al. Blockage of 2-deoxy-D-ribose-induced angiogenesis with rapamycin counteracts a thymidine phosphorylase-based escape mechanism available for colon cancer under 5-fluorouracil therapy. Clin Cancer Res 2004; 105: 1843-1852.
• 28 Miyadera K, Sumizawa T, Haraguchi M. et al. Role of thymidine phosphorylase activity in the angiogenic effect of platelet derived endothelial cell growth fac-tor/thymidine phosphorylase. Cancer Res 1995; 55: 1687-1690.
• 29 Kahner BN, Shankar H, Murugappan S. et al. Nucleotide receptor signaling in platelets. J Thromb Haemost 2006; 04: 2317-2326.
• 30 Packham MA, Kinlough-Rathbone RL, Mustard JF. Thromboxane A2 causes feedback amplification involving extensive thromboxane A2 formation on close contact of human platelets in media with a low concentration of ionized calcium. Blood 1987; 70: 647-651.
• 31 Beigi R, Kobatake E, Aizawa M. et al. Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase. Am J Physiol 1999; 276: C267-C278.
• 32 Boyle JJ, Wilson B, Bicknell R. et al. Expression of angiogenic factor thymidine phosphorylase and angiogenesis in human atherosclerosis. J Pathol 2000; 192: 234-242.
• 33 Toi M, Atiqur Rahman M, Bando H. et al. Thymidine phosphorylase (platelet-derived endothelial-cell growth factor) in cancer biology and treatment. Lancet Oncol 2005; 06: 158-166.
• 34 Bronckaers A, Gago F, Balzarini J. et al. The dual role of thymidine phosphorylase in cancer development and chemotherapy. Med Res Rev 2009; 29: 903-953.
• 35 Ueda T, Tamaki M, Ogawa O. et al. Over expression of platelet-derived endothe-lial cell growth factor/thymidine phosphorylase in patients with interstitial cystitis and bladder carcinoma. J Urol 2002; 167: 347-351.
• 36 Savage B, Cattaneo M, Ruggeri ZM. Mechanisms of platelet aggregation. Curr Opin Hematol 2001; 08: 270-276.
• 37 Novak L, Deckmyn H, Damjanovich S. et al. Shear-dependent morphology of von Willebrand factor bound to immobilized collagen. Blood 2002; 99: 2070-2076.
• 38 Gawaz MP, Loftus JC, Bajt ML. et al. Ligand bridging mediates integrin alpha IIb beta 3 (platelet GPIIB-IIIA) dependent homotypic and heterotypic cell-cell interactions. J Clin Invest 1991; 88: 1128-1134.
• 39 Zaidi TN, McIntire LV, Farrell DH. et al. Adhesion of platelets to surface-bound fibrinogen under flow. Blood 1996; 88: 2967-2972.
• 40 Litvinov RI, Barsegov V, Schissler AJ. et al. Dissociation of bimolecular alphaIIb-beta3-fibrinogen complex under a constant tensile force. Biophys J 2011; 100: 165-173.
• 41 Siljander PR, Munnix IC, Smethurst PA. et al. Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood. Blood 2004; 103: 1333-1341.
• 42 Lepantalo A, Beer JH, Siljander P. et al. Variability in platelet responses to col-lagen--comparison between whole blood perfusions, traditional platelet function tests and PFA-100. Thromb Res 2001; 103: 123-133.
• 43 Ardestani A, Yazdanparast R, Nejad AS. 2-Deoxy-D-ribose-induced oxidative stress causes apoptosis in human monocytic cells: prevention by pyridoxal-5’-phosphate. Toxicol In Vitro 2008; 22: 968-979.
• 44 Krotz F, Sohn HY, Gloe T. et al. NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment. Blood 2002; 100: 917-924.
• 45 Salvemini D, de Nucci G, Sneddon JM. et al. Superoxide anions enhance platelet adhesion and aggregation. Br J Pharmacol 1989; 97: 1145-1150.
• 46 Essex DW, Li M. Redox control of platelet aggregation. Biochemistry 2003; 42: 129-136.
• 47 Chrzanowska-Wodnicka M, Smyth SS, Schoenwaelder SM. et al. Rap1b is required for normal platelet function and hemostasis in mice. J Clin Invest 2005; 115: 680-687.
• 48 Han J, Lim CJ, Watanabe N. et al. Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3. Curr Biol 2006; 16: 1796-1806.
• 49 Harper MT, Poole AW. Diverse functions of protein kinase C isoforms in platelet activation and thrombus formation. J Thromb Haemost 2010; 08: 454-462.
• 50 Cifuni SM, Wagner DD, Bergmeier W. CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin alphaIIbbeta3 in platelets. Blood 2008; 112: 1696-1703.