Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway

Summary

Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A₂ mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y₁₂ receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

Keywords

Platelet aggregation - sulforaphane - phosphatidylinositol 3-kinase - ubiquitination

• References

• 1 Verkerk R, Schreiner M, Krumbein A. et al. Glucosinolates in Brassica vegetables: the influence of the food supply chain on intake, bioavailability and human health. Mol Nutr Food Res 2009; 53: S219.
• 2 Cheung KL, Kong AN. Molecular targets of dietary phenethyl isothiocyanate and sulforaphane for cancer chaemoprevention. AAPS J 2010; 12: 87-97.
• 3 Thimmulappa RK, Mai KH, Srisuma S. et al. Identification of Nrf2-regulated genes induced by the chaemopreventive agent sulforaphane by oligonucleotide microarray. Cancer Res 2002; 62: 5196-5203.
• 4 Clarke JD, Dashwood RH, Ho E. Multi-targeted prevention of cancer by sulforaphane. Cancer Lett 2008; 269: 291-304.
• 5 Keum YS. Regulation of the Keap1/Nrf2 system by chaemopreventive sulforaphane: implications of posttranslational modifications. Ann NY Acad Sci 2011; 1229: 184-189.
• 6 Lin HJ, Probst-Hensch NM, Louie AD. et al. Glutathione transferase null genotype, broccoli, and lower prevalence of colorectal adenomas. Cancer Epidemiol Biomarkers Prev 1998; 07: 647-652.
• 7 Cotton SC, Sharp L, Little J. et al. Glutathione S-transferase polymorphisms and colorectal cancer: a HuGE review. Am J Epidemiol 2000; 151: 7-32.
• 8 Zhang Y. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. Mutat Res 2004; 555: 173-190.
• 9 Dinkova-Kostova AT, Kostov RV. Glucosinolates and isothiocyanates in health and disease. Trends Mol Med 2012; 18: 337-347.
• 10 Wu L, Noyan Ashraf MH, Facci M. et al. Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system. Proc Natl Acad Sci USA 2004; 101: 7094-7099.
• 11 Piao CS, Gao S, Lee GH. et al. Sulforaphane protects ischaemic injury of hearts through antioxidant pathway and mitochondrial K(ATP) channels. Pharmacol Res 2010; 61: 342-348.
• 12 Zhao J, Kobori N, Aronowski J. et al. Sulforaphane reduces infarct volume following focal cerebral ischaemia in rodents. Neurosci Lett 2006; 393: 108-112.
• 13 Zakkar M, Van der Heiden K, Luong le A. et al. Activation of Nrf2 in endothelial cells protects arteries from exhibiting a proinflammatory state. Arterioscler Thromb Vasc Biol 2009; 29: 1851-1857.
• 14 Michelson AD. Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discov 2010; 09: 154-169.
• 15 Lievens D, von Hundelshausen P. Platelets in atherosclerosis. Thromb Haemost 2011; 106: 827-838.
• 16 Wei AH, Schoenwaelder SM, Andrews RK. et al. New insights into the haemostatic function of platelets. Br J Haematol 2009; 147: 415-430.
• 17 Li Z, Delaney MK, O’Brien KA. et al. Signalling during platelet adhesion and activation. Arterioscler Thromb Vasc Biol 2010; 30: 2341-2349.
• 18 Gratacap MP, Guillermet-Guibert J, Martin V. et al. Regulation and roles of PI3Kβ, a major actor in platelet signalling and functions. Adv Enzyme Regul 2011; 51: 106-116.
• 19 Jackson SF, Schoenwaelder SM. Type I phosphoinositide 3-kinases: potential antithrombotic targets?. Cell Mol Life Sci 2006; 63: 1085-1090.
• 20 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.
• 21 Séverin S, Gratacap MP, Lenain N. et al. Deficiency of Src homology 2 domaincontaining inositol 5-phosphatase 1 affects platelet responses and thrombus growth. J Clin Invest 2007; 117: 944-952.
• 22 Meyer dos Santos S, Klinkhardt U, Schneppenheim R. et al. Using ImageJ for the quantitative analysis of flow-based adhesion assays in real-time under physiologic flow conditions. Platelets 2010; 21: 60-66.
• 23 Wu CC, Wu CI, Wang WY. et al. Low concentrations of resveratrol potentiate the antiplatelet effect of prostaglandins. Planta Med 2007; 73: 439-443.
• 24 Pollock WK, Rink TJ, Irvine RF. Liberation of [³H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen. Biochem J 1986; 235: 869-877.
• 25 Mancini F, Rigacci S, Berti A. et al. The low-molecular-weight phosphotyrosine phosphatase is a negative regulator of FcgammaRIIA-mediated cell activation. Blood 2007; 110: 1871-1878.
• 26 Wang WY, Hsieh PW, Wu YC. et al. Synthesis and pharmacological evaluation of novel beta-nitrostyrene derivatives as tyrosine kinase inhibitors with potent antiplatelet activity. Biochem Pharmacol 2007; 74: 601-611.
• 27 Gresele P, Momi S, Falcinelli E. Anti-platelet therapy: phosphodiesterase inhibitors. Br J Clin Pharmacol 2011; 72: 634-646.
• 28 Trumel C, Payrastre B, Plantavid M. et al. A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase. Blood 1999; 94: 4156-4165.
• 29 Kim S, Jin J, Kunapuli SP. Relative contribution of G-protein-coupled pathways to proteaseactivated receptor-mediated Akt phosphorylation in platelets. Blood 2006; 107: 947-954.
• 30 Elzagallaai A, Rosé SD, Trifaró JM. Platelet secretion induced by phorbol esters stimulation is mediated through phosphorylation of MARCKS: a MARCKS-derived peptide blocks MARCKS phosphorylation and serotonin release without affecting pleckstrin phosphorylation. Blood 2000; 95: 894-902.
• 31 Fang D, Liu YC. Proteolysis-independent regulation of PI3K by Cbl-b-mediated ubiquitination in T cells. Nat Immunol 2001; 02: 870-875.
• 32 Fang D, Wang HY, Fang N. et al. Cbl-b, a RING-type E3 ubiquitin ligase, targets phosphatidylinositol 3-kinase for ubiquitination in T cells. J Biol Chem 2001; 276: 4872-4878.
• 33 Balasubramanian S, Chew YC, Eckert RL. Sulforaphane suppresses polycomb group protein level via a proteasome-dependent mechanism in skin cancer cells. Mol Pharmacol 2011; 80: 870-878.
• 34 Busso CS, Iwakuma T, Izumi T. Ubiquitination of mammalian AP endonuclease (APE1) regulated by the p53-MDM2 signalling pathway. Oncogene 2009; 28: 1616-1625.
• 35 Dangelmaier CA, Quinter PG, Jin J. et al. Rapid ubiquitination of Syk following GPVI activation in platelets. Blood 2005; 105: 3918-3924.
• 36 Hu R, Hebbar V, Kim BR. et al. In vivo pharmacokinetics and regulation of gene expression profiles by isothiocyanate sulforaphane in the rat. J Pharmacol Exp Ther 2004; 310: 263-271.
• 37 Jackson SP, Schoenwaelder SM, Goncalves I. et al. PI 3-kinase p110beta: a new target for antithrombotic therapy. Nat Med 2005; 11: 507-514.
• 38 Martin V, Guillermet-Guibert J, Chicanne G. et al. Deletion of the p110beta isoform of phosphoinositide 3-kinase in platelets reveals its central role in Akt activation and thrombus formation in vitro and in vivo. Blood 2010; 115: 2008-2013.
• 39 Garcia A, Kim S, Bhavaraju K. et al. Role of phosphoinositide 3-kinase beta in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways. Biochem J 2010; 429: 369-377.
• 40 Kovacsovics TJ, Bachelot C, Toker A. et al. Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. J Biol Chem 1995; 270: 11358-11366.
• 41 Schoenwaelder SM, Ono A, Nesbitt WS. et al. Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces. J Biol Chem 2010; 285: 2886-2896.
• 42 Pasquet JM, Bobe R, Gross B. et al. A collagen-related peptide regulates phospholipase Cγ2 via phosphatidylinositol 3-kinase in human platelets. Biochem J 1999; 342: 171-177.
• 43 Welchman RL, Gordon C, Mayer RJ. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat Rev Mol Cell Biol 2005; 06: 599-609.
• 44 Brophy TM, Raab M, Daxecker H. et al. RN181, a novel ubiquitin E3 ligase that interacts with the KVGFFKR motif of platelet integrin alpha(IIb)beta3. Biochem Biophys Res Commun 2008; 369: 1088-1093.
• 45 Lin AE, Mak TW. The role of E3 ligases in autoimmunity and the regulation of autoreactive T cells. Curr Opin Immunol 2007; 19: 665-673.
• 46 Schwartz AL, Ciechanover A. Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol 2009; 49: 73-96.
• 47 Komander D, Clague MJ, Urbé S. Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 2009; 10: 550-563.
• 48 Mi L, Hood BL, Stewart NA. et al. Identification of potential protein targets of isothiocyanates by proteomics. Chem Res Toxicol 2011; 24: 1735-1743.
• 49 Lee BH, Lee MJ, Park S. et al. Enhancement of proteasome activity by a smallmolecule inhibitor of USP14. Nature 2010; 467: 179-184.
• 50 Hussain S, Foreman O, Perkins SL. et al. The de-ubiquitinase UCH-L1 is an oncogene that drives the development of lymphoma in vivo by deregulating PHLPP1 and Akt signalling. Leukemia 2010; 24: 1641-1655.