Identification of CalDAG-GEFI as an intracellular target for the vicinal dithiol binding agent phenylarsine oxide in human platelets

Summary

CalDAG-GEFI, a guanine nucleotide exchange factor activating Rap1, is known to play a key role in Ca²⁺-dependent glycoprotein (GP)IIb/IIIa activation and platelet aggregation. Although inhibition of CalDAG-GEFI could be a potential strategy for antiplatelet therapy, no inhibitor of this protein has been identified. In the present study, phenylarsine oxide (PAO), a vicinal dithiol blocker, potently prevented Rap1 activation in thrombin-stimulated human platelets without significantly inhibiting intracellular Ca²⁺ mobilisation and protein kinase C activation. PAO also prevented the Ca²⁺ ionophore-induced Rap1 activation and platelet aggregation, which are dependent on CalDAG-GEFI. In the biotin-streptavidin pull-down assay, CalDAG-GEFI was efficiently pull-downed by streptavidin beads from the lysates of biotin-conjugated PAO-treated platelets, suggesting that PAO binds to intracellular CalDAG-GEFI with high affinity. The above effects of PAO were reversed by a vicinal dithiol compound 2,3-dimercaptopropanol. In addition, CalDAG-GEFI formed disulfide-linked oligomers in platelets treated with the thiol-oxidant diamide, indicating that CalDAG-GEFI contains redox-sensitive thiols. In a purified recombinant protein system, PAO directly inhibited CalDAG-GEFI-stimulated GTP binding to Rap1. Using CalDAG-GEFI and Rap1-overexpressed human embryonic kidney 293T cells, we further confirmed that PAO abolished Ca²⁺-mediated Rap1 activation. Taken together, these results have demonstrated that CalDAG-GEFI is one of the targets of action of PAO, and propose an important role of vicinal cysteines for the functions of CalDAG-GEFI.

Keywords

CalDAG-GEFI - Rap1 - platelet aggregation - phenylarsine oxide - vicinal dithiol

• References

• 1 Jackson SP. Arterial thrombosis- insidious, unpredictable and deadly. Nat Med 2011; 17: 1423-1436.
• 2 Lievens D, von Hundelshausen P. Platelets in atherosclerosis. Thromb Haemost 2011; 106: 827-838.
• 3 Michelson AD. Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discov 2010; 09: 154-169.
• 4 Eikelboom JW, Hirsh J, Spencer FA. et al. Antiplatelet drugs: antithrombotic therapy and prevention of thrombosis, 9th ed: American college of chest physicians evidence-based clinical practice guidelines. Chest 2012; 141: e89S-119S.
• 5 Li Z, Delaney MK, O’Brien KA. et al. Signalling during platelet adhesion and activation. Arterioscler Thromb Vasc Biol 2010; 30: 2341-2349.
• 6 Broos K, De Meyer SF, Feys HB. et al. Blood platelet biochemistry. Thromb Res 2012; 129: 245-249.
• 7 Wei AH, Schoenwaelder SM, Andrews RK. et al. New insights into the haemostatic function of platelets. Br J Haematol 2009; 147: 415-430.
• 8 Tao L, Zhang Y, Xi X. et al. Recent advances in the understanding of the molecular mechanisms regulating platelet integrin αIIbβ3 activation. Protein Cell 2010; 01: 627-637.
• 9 Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end game. Nat Rev Mol Cell Biol 2010; 11: 288-300.
• 10 Stefanini L, Bergmeier W. CalDAG-GEFI and platelet activation. Platelets 2010; 21: 239-243.
• 11 Cifuni SM, Wagner DD, Bergmeier W. CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin αIIbβ3 in platelets. Blood 2008; 112: 1696-1703.
• 12 Watanabe N, Bodin L, Pandey M. et al. Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin αIIbβ3. J Cell Biol 2008; 181: 1211-1222.
• 13 Crittenden JR, Bergmeier W, Zhang Y. et al. CalDAG-GEFI integrates signalling for platelet aggregation and thrombus formation. Nat Med 2004; 10: 982-986.
• 14 Stolla M, Stefanini L, Roden RC. et al. The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy. Blood 2011; 117: 1005-1013.
• 15 Essex DW. Redox control of platelet function. Antioxid Redox Signal 2009; 11: 1191-1225.
• 16 Jordan PA, Gibbins JM. Extracellular disulfide exchange and the regulation of cellular function. Antioxid Redox Signal 2006; 08: 312-324.
• 17 Essex DW, Li M. Redox control of platelet aggregation. Biochemistry 2003; 42: 129-136.
• 18 Manickam N, Ahmad SS, Essex DW. Vicinal thiols are required for activation of the αIIbβ3 platelet integrin. J Thromb Haemost 2011; 09: 1207-1215.
• 19 Bennett TA, Edwards BS, Sklar LA. et al. Sulfhydryl regulation of L-selectin shedding: phenylarsine oxide promotes activation-independent L-selectin shedding from leukocytes. J Immunol 2000; 164: 4120-4129.
• 20 Root P, Sliskovic I, Mutus B. Platelet cell-surface protein disulphide-isomerase mediated S-nitrosoglutathione consumption. Biochem J 2004; 382: 575-580.
• 21 Wu CC, Wang WY, Wei CK. et al. Combined blockade of thrombin anion binding exosite-1 and PAR4 produces synergistic antiplatelet effect in human platelets. Thromb Haemost 2011; 105: 88-95.
• 22 Wang HC, Lee AY, Chou WC. et al. Inhibition of ATR-dependent signalling by protoapigenone and its derivative sensitizes cancer cells to interstrand crosslink-generating agents in vitro and in vivo. Mol Cancer Ther 2012; 11: 1443-1453.
• 23 Wu CC, Wu SY, Liao CY. et al. The roles and mechanisms of PAR4 and P2Y12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation. Br J Pharmacol 2010; 161: 643-658.
• 24 Glaven JA, Whitehead IP, Nomanbhoy T. et al. Lfc and Lsc oncoproteins represent two new guanine nucleotide exchange factors for the Rho GTP-binding protein. J Biol Chem 1996; 271: 27374-27381.
• 25 Bouquier N, Fromont S, Zeeh JC. et al. Aptamer-derived peptides as potent inhibitors of the oncogenic RhoGEF Tgat. Chem Biol 2009; 16: 391-400.
• 26 Mao JH, Sun XY, Liu JX. et al. As4S4 targets RING-type E3 ligase c-CBL to induce degradation of BCR-ABL in chronic myelogenous leukemia. Proc Natl Acad Sci USA 2010; 107: 21683-21688.
• 27 Zhang XW, Yan XJ, Zhou ZR. et al. Arsenic trioxide controls the fate of the PML-RARα oncoprotein by directly binding PML. Science 2010; 328: 240-243.
• 28 Phelps DC, Hatefi Y. Inhibition of D-(-)-beta-hydroxybutyrate dehydrogenase by modifiers of disulfides, thiols, and vicinal dithiols. Biochemistry 1981; 20: 453-458.
• 29 Bogumil R, Ullrich V. Phenylarsine oxide affinity chromatography to identify proteins involved in redox regulation: dithiol-disulfide equilibrium in serine/ threonine phosphatase calcineurin. Methods Enzymol 2002; 348: 271-280.
• 30 Kosower NS, Kosower EM. Diamide: an oxidant probe for thiols. Methods Enzymol 1995; 251: 123-133.
• 31 Bokoch GM, Quilliam LA. Guanine nucleotide binding properties of rap1 purified from human neutrophils. Biochem J 1990; 267: 407-411.
• 32 Sugatani J, Steinhelper ME, Saito K. et al. Potential involvement of vicinal sulf-hydryls in stimulus-induced rabbit platelet activation. J Biol Chem 1987; 262: 16995-17001.
• 33 Greenwalt DE, Tandon NN. Platelet shape change and Ca2+ mobilization induced by collagen, but not thrombin or ADP, are inhibited by phenylarsine oxide. Br J Haematol 1994; 88: 830-838.
• 34 Guidetti GF, Manganaro D, Consonni A. et al. Phosphorylation of the guanine-nucleotide-exchange factor CalDAG-GEFI by protein kinase A regulates Ca2+-dependent activation of platelet Rap1b GTPase. Biochem J 2013; 453: 115-123.
• 35 Subramanian H, Zahedi RP, Sickmann A. et al. Phosphorylation of CalDAG-GEFI by protein kinase A prevents Rap1b activation. J Thromb Haemost 2013; 11: 1574-1582.
• 36 Donoghue N, Yam PT, Jiang XM. et al. Presence of closely spaced protein thiols on the surface of mammalian cells. Protein Sci 2000; 09: 2436-2445.
• 37 Kazanietz MG. Novel “nonkinase” phorbol ester receptors: the C1 domain connection. Mol Pharmacol 2002; 61: 759-767.
• 38 Johnson JE, Goulding RE, Ding Z. et al. Differential membrane binding and dia-cylglycerol recognition by C1 domains of RasGRPs. Biochem J 2007; 406: 223-236.