Pharmacological actions of miltirone in the modulation of platelet function

Abstract

Salvia miltiorrhiza Bunge contains various active constituents, some of which have been developed as commercially available medicine. Moreover, some other ingredients in Salvia miltiorrhiza play roles in anti-platelet activity. The aim of the present study was to investigate the effects and the underlying mechanism of miltirone, a lipophilic compound of Salvia miltiorrhiza Bunge. The ability of miltirone to modulate platelet function was investigated by a variety of in vitro and in vivo experiments. Platelet aggregation and dense granule secretion induced by various agonists were measured with platelet aggregometer. Clot retraction and spreading were imaged by digital camera and fluorescence microscope. Ferric chloride-induced carotid injury model and pulmonary thromboembolism model were used to check miltirone antithrombotic effect in vivo. To elucidate the mechanisms of anti-platelet activity of miltirone, flow cytometry and western blotting were performed. Miltirone (2, 4, 8 µM) was shown to suppress platelet aggregation, dense granule, and α granule secretion in a dose-dependent manner. Meanwhile, miltirone inhibited the clot retraction and spreading of washed platelets. It reduced the phosphorylation of PLCγ2, PKC, Akt, GSK3β and ERK1/2 in the downstream signal pathway of collagen receptor. It also reduced the phosphorylation of Src and FAK in the integrin αIIbβ3-mediated “outside-in” signaling, while it did not suppress the phosphorylation of β3. In addition, miltirone prolonged the occlusion time and reduced collagen/epinephrine-induced pulmonary thrombi. Miltirone suppresses platelet “inside-out” and “outside-in” signaling by affecting PLCγ2/PKC/ERK1/2, PI3K/Akt, and Src/FAK signaling. Therefore, miltirone might represent a potential anti-platelet candidate for the prevention of thrombotic disorders.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. 1.

    Holinstat M. Normal platelet function. Cancer Metastasis Rev. 2017;36:195–8.

  2. 2.

    Joshi S, Whiteheart SW. The nuts and bolts of the platelet release reaction. Platelets. 2017;28:129–37.

  3. 3.

    Yeung J, Holinstat M. Newer agents in antiplatelet therapy: a review. J Blood Med. 2012;3:33–42.

  4. 4.

    Bryniarski L, Pelc-Nowicka A, Zabojszcz M, Mirek-Bryniarska E. Dual anti-platelet therapy and antithrombotic treatment: recommendations and controversies. Cardiol J. 2009;16:179–89.

  5. 5.

    Guthrie R. Review and management of side effects associated with antiplatelet therapy for prevention of recurrent cerebrovascular events. Adv Ther. 2011;28:473–82.

  6. 6.

    Alexopoulos D, Bhatt DL, Hamm CW, Steg PG, Stone GW. Early P2Y12 inhibition in ST-segment elevation myocardial infarction: Bridging the gap. Am Heart J. 2015;170:3–12.

  7. 7.

    Hankey GJ, Eikelboom JW. Aspirin resistance. Lancet. 2006;367:606–17.

  8. 8.

    Siddique A, Butt M, Shantsila E, Lip GYH. New antiplatelet drugs: beyond aspirin and clopidogrel. Int J Clin Pract. 2009;63:776–89.

  9. 9.

    Coccheri S. Antiplatelet drugs-do we need new options? With a reappraisal direct thromboxane inhibitors. Drugs. 2010;70:887–908.

  10. 10.

    Betz JM, Brown PN, Roman MC. Accuracy, precision, and reliability of chemical measurements in natural products research. Fitoterapia. 2011;82:44–52.

  11. 11.

    Valli G, Giardina EGV. Benefits, adverse effects and drug interactions of herbal therapies with cardiovascular effects. J Am Coll Cardiol. 2002;39:1083–95.

  12. 12.

    Maione F, De Feo V, Caiazzo E, De Martino L, Cicala C, Mascolo N. Tanshinone IIA, a major component of Salvia milthorriza Bunge, inhibits platelet activation via Erk-2 signaling pathway. J Ethnopharmacol. 2014;155:1236–42.

  13. 13.

    Liu J-Q, Lee T-F, Miedzyblocki M, Chan GCF, Bigam DL, Cheung P-Y. Effects oftanshinone IIA, a major component ofSalvia miltiorrhiza, on platelet aggregation in healthy newborn piglets. J Ethnopharmacol. 2011;137:44–9.

  14. 14.

    Wang H, Gu J, Hou X, Chen J, Yang N, Liu Y, et al. Anti-inflammatory effect of miltirone on inflammatory bowel disease via TLR4/NF-kappaB/I0GAP2 signaling pathway. Biomed Pharm. 2017;85:531–40.

  15. 15.

    Zhou L, Jiang L, Xu M, Liu Q, Gao N, Li P, et al. Miltirone exhibits antileukemic activity by ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction pathways. Sci Rep. 2016;6:20585.

  16. 16.

    Wang L, Hu T, Shen J, Zhang L, Li LF, Chan RL, et al. Miltirone induced mitochondrial dysfunction and ROS-dependent apoptosis in colon cancer cells. Life Sci. 2016;151:224–34.

  17. 17.

    Colombo G, Serra S, Vacca G, Orru A, Maccioni P, Morazzoni P, et al. Identification of miltirone as active ingredient of Salvia mil-tiorrhiza responsible for the reducing effect of root extracts on alcohol intake in rats. Alcohol Clin Exp Res. 2006;30:754–62.

  18. 18.

    Liu G, Xie W, He AD, Da XW, Liang ML, Yao GQ, et al. Antiplatelet activity of chrysin via inhibiting platelet alphaIIbbeta3-mediated signaling pathway. Mol. Nutr. Food Res. 2016;60:1984–93.

  19. 19.

    Liang ML, Da XW, He AD, Yao GQ, Xie W, Liu G, et al. Pentamethylquercetin (PM0) reduces thrombus formation by inhibiting platelet function. Sci Rep. 2015;5:11142.

  20. 20.

    Vaiyapuri S, Roweth H, Ali MS, Unsworth AJ, Stainer AR, Flora GD, et al. Pharmacological actions of nobiletin in the modulation of platelet function. BJP. 2015;172:4133–45.

  21. 21.

    Li W, Tang X, Yi W, Li Q, Ren L, Liu X, et al. Glaucocalyxin A inhibits platelet activation and thrombus formation preferentially via GPVI signaling pathway. PLoS One. 2013;8:e85120.

  22. 22.

    Sonkar VK, Kulkarni PP, Dash D. Amyloid beta peptide stimulates platelet activation through RhoA-dependent modulation of actomyosin organization. FASEB J. 2014;28:1819–29.

  23. 23.

    Tyagi T, Ahmad S, Gupta N, Sahu A, Ahmad Y, Nair V, et al. Altered expression ofplatelet proteins and calpain activitymediate hypoxia-induced prothrombotic phenotype. Blood. 2014;123:1250–60.

  24. 24.

    Watson SP, Auger JM, McCarty OJ, Pearce AC. GPVI and integrin alphaIIb beta3 signaling in platelets. J Thromb Haemost. 2005;3:1752–62.

  25. 25.

    Su XL, Su W, Wang Y, Wang YH, Ming X, Kong Y. The pyrrolidinoindoline alkaloid Psm2 inhibits platelet aggregation and thrombus formation by affecting PI3K/Akt signaling. Acta Pharmacol Sin. 2016;37:1208–17.

  26. 26.

    Watson SP, Herbert JM, Pollitt AY. GPVI and CLEC-2 in hemostasis and vascular integrity. J Thromb Haemost. 2010;8:1456–67.

  27. 27.

    Eckly A, Rinckel JY, Proamer F, Ulas N, Joshi S, Whiteheart SW, et al. Respective contributions of single and compound granule fusion to secretion byactivated platelets. Blood. 2016;128:2538–49.

  28. 28.

    Heijnen H, van der Sluijs P. Platelet secretory behaviour: as diverse as the granules.. or not? J Thromb Haemost. 2015;13:2141–51.

  29. 29.

    Calderwood DA. Integrin activation. J Cell Sci. 2004;117:657–66.

  30. 30.

    Shen B, Delaney MK, Du X. Inside-out, outside-in, and inside-outside-in: G protein signaling in integrin-mediated cell adhesion, spreading, and retraction. Curr Opin Cell Biol. 2012;24:600–6.

  31. 31.

    Phillips DR, Nannizzi-Alamio L, Prasad KSS. Beta 3 tyrosine phosphoryla-tion in alpha IIb beta 3 (platelet membrane GP IIb-IIIa) outside-in integrin signaling. Thromb Haemost. 2001;86:246–58.

  32. 32.

    Qi H, Huang Y, Yang Y, Dou G, Wan F, Zhang W, et al. Anti-platelet activity of panaxatriol saponins is mediated by suppression of intracellular calcium mobilization and ERK2/p38 activation. BMC Complement Altern Med. 2016;16:174.

  33. 33.

    Borst O, Walker B, Munzer P, Russo A, Schmid E, Faggio C, et al. Skepinone-L, a novel potent and highly selective inhibitor of p38 MAP kinase, effectively impairs platelet activation and thrombus formation. Cell Physiol Biochem. 2013;31:914–24.

  34. 34.

    Adam F, Kauskot A, Rosa JP, Bryckaert M. Mitogen-activated protein kinases in hemostasis and thrombosis. J Thromb Haemost. 2008;6:2007–16.

  35. 35.

    Kuliopulos A, Mohanlal R, Covic L. Effect of selective inhibition of the p38 MAP kinase pathway on platelet aggregation. Thromb Haemost. 2004;92:1387–93.

  36. 36.

    Saklatvala J, Rawlinson L, Waller RJ, Sarsfield S, Lee JC, Morton LF, et al. Role for p38 mitogen-activated protein kinase in platelet aggregation caused by collagen or a thromboxane analogue. J Biol Chem. 1996;271:6586–9.

Download references

Acknowledgements

This study was supported by grants from The National Natural Science Fundation of China (No. 81273574 to Z.-Y.M.) and Chinese herb key project by Health and Family Planning Commission of Hubei Province (to Z.-Y.M.)

Author information

Z.-Y.M. and W.S. designed the study and wrote the manuscript. Y.-y.M. and S.M. prepared mouse platelets. R.-p.Y. and Y.Z. performed the evaluation of platelet function and analyzed data. All authors reviewed the manuscript. D.S., M.L., and R.M.A. approved the final version of the manuscript.

Correspondence to Zhang-yin Ming.

Ethics declarations

Competing interests

The authors declare no competing interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Keywords

  • miltirone
  • anti-platelet
  • glycoprotein VI pathway
  • integrin αIIbβ3