Abstract
Aim:
Psm2, one of the pyrrolidinoindoline alkaloids isolated from whole Selaginella moellendorffii plants, has shown a potent antiplatelet activity. In this study, we further evaluated the antiplatelet effects of Psm2, and elucidated the underlying mechanisms.
Methods:
Human platelet aggregation in vitro and rat platelet aggregation ex vivo were investigated. Agonist-induced platelet aggregation was measured using a light transmission aggregometer. The antithrombotic effects of Psm2 were evaluated in arteriovenous shunt thrombosis model in rats. To elucidate the mechanisms underlying the antiplatelet activity of Psm2, ELISAs, Western blotting and molecular docking were performed. The bleeding risk of Psm2 administration was assessed in a mouse tail cutting model, and the cytotoxicity of Psm2 was measured with MTT assay in EA.hy926 cells.
Results:
Psm2 dose-dependently inhibited human platelet aggregation induced by ADP, U4619, thrombin and collagen with IC50 values of 0.64, 0.37, 0.35 and 0.87 mg/mL, respectively. Psm2 (1, 3, 10 mg/kg) administered to rats significantly inhibited platelet aggregation ex vivo induced by ADP. Psm2 (1, 3, 10 mg/mL, iv) administered to rats with the A–V shunt dose-dependently decreased the thrombus formation. Psm2 inhibited platelet adhesion to fibrinogen and collagen with IC50 values of 84.5 and 96.5 mg/mL, respectively, but did not affect the binding of fibrinogen to GPIIb/IIIa. Furthermore, Psm2 inhibited AktSer473 phosphorylation, but did not affect MAPK signaling and Src kinase activation. Molecular docking showed that Psm2 bound to phosphatidylinositol 3-kinase β (PI3Kβ) with a binding free energy of −13.265 kcal/mol. In addition, Psm2 did not cause toxicity in EA.hy926 cells and produced only slight bleeding in a mouse tail cutting model.
Conclusion:
Psm2 inhibits platelet aggregation and thrombus formation by affecting PI3K/Akt signaling. Psm2 may be a lead compound or drug candidate that could be developed for the prevention or treatment of thrombotic diseases.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Chen M, Ye X, Ming X, Chen Y, Wang Y, Su X, et al. A novel direct factor Xa inhibitory peptide with anti-platelet aggregation activity from Agkistrodon acutus venom hydrolysates. Sci Rep 2015; 5: 10846.
Ye X, Chen M, Chen Y, Su X, Wang Y, Su W, et al. Isolation and characterization of a novel antithrombotic peptide from enzymatic hydrolysate of Agkistrodon acutus venom. Int J Pert Res Ther 2015; 21: 343–51.
Peng L, Xu X, Shen D, Zhang Y, Song J, Yan X, et al. Purification and partial characterization of a novel phosphodiesterase from the venom of Trimeresurus stejnegeri: inhibition of platelet aggregation. Biochimie 2011; 93: 1601–9.
Lu WQ, Qiu Y, Li TJ, Tao X, Sun LN, Chen WS . Antiplatelet and antithrombotic activities of timosaponin B-II, an extract of Anemarrhena asphodeloides. Clin Exp Pharmacol P 2011; 38: 430–4.
Michelson AD . Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discov 2010; 9: 154–69.
Pan C, Wei X, Ye J, Liu G, Zhang S, Zhang Y, et al. BF066, a novel dual target antiplatelet agent without significant bleeding. PLoS One 2012; 7: e40451.
Johnson S . Known knowns and known unknowns: risks associated with combination antithrombotic therapy. Thromb Res 2008; 123: S7–11.
Jackson S, Yap C, Anderson K . Phosphoinositide 3-kinases and the regulation of platelet function. Biochem Soc T 2004; 32: 387–92.
Chen J, De S, Damron DS, Chen WS, Hay N, Byzova TV . Impaired platelet responses to thrombin and collagen in AKT-1–deficient mice. Blood 2004; 104: 1703–10.
Huang Z, Zeng C, Zhu L, Jiang L, Li N, Hu H . Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3-kinase. J Thromb Haemost 2010; 8: 1383–93.
Sheu J, Hung W, Wu C, Lee Y, Yen M . Antithrombotic effect of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, on platelet plug formation in in vivo experiments. Br J Haematol 2000; 110: 110–5.
Wu ZY, Zhou TY, Xiao PG . Xinghua Bencao Gangyao. v 3. Shanghai: Shanghai Scientific and Technological Press; 1990. p 626–31.
Zhang XC, Nooteboom HP, Kato M . Selaginellaceae. In: Wu ZY, Raven PH, Hong DY, editors. Flora of China. v 2–3. Pteridophytes. Beijing: Beijing and Missouri Botanical Garden Press; 2013. p 37–46.
Wang YH, Long CL, Yang FM, Wang X, Sun QY, Wang HS, et al. Pyrrolidinoindoline alkaloids from Selaginella moellendorfii. J Nat Prod 2009; 72: 1151–4.
Klafke JZ, da Silva MA, Rossato MF, Trevisan G, Walker CIB, Leal CAM, et al. Antiplatelet, antithrombotic, and fibrinolytic activities of Campomanesia xanthocarpa. Evid-Based Compl Alt 2012; 2012: 1–8.
Shi S, Zhou H, Zhang Y, Huang K . Hyphenated HSCCC–DPPH for rapid preparative isolation and screening of antioxidants from Selaginella moellendorffii. Chromatographia 2008; 68: 173–8.
Fan HY, Fu FH, Yang MY, Xu H, Zhang AH, Liu K . Antiplatelet and antithrombotic activities of salvianolic acid A. Thromb Res 2010; 126: e17–22.
Huang J, Wang S, Luo X, Xie Y, Shi X . Cinnamaldehyde reduction of platelet aggregation and thrombosis in rodents. Thromb Res 2007; 119: 337–42.
Gadi D, Bnouham M, Aziz M, Ziyyat A, Legssyer A, Legrand C, et al. Parsley extract inhibits in vitro and ex vivo platelet aggregation and prolongs bleeding time in rats. J Ethnopharmacol 2009; 125: 170–4.
Umar A, Guerin V, Renard M, Boisseau M, Garreau C, Begaud B, et al. Effects of armagnac extracts on human platelet function in vitro and on rat arteriovenous shunt thrombosis in vivo. Thromb Res 2003; 110: 135–40.
Umetsu T, Sanai K . Effect of 1-methyl-2-mercapto-5-(3-pyridyl)-imidazole (KC-6141), an anti-aggregating compound, on experimental thrombosis in rats. Thromb Haemost 1978; 39: 74–83.
Zhu L, Zhao L, Wang H, Wang Y, Pan D, Yao J, et al. Oroxylin A reverses P-glycoprotein-mediated multidrug resistance of MCF7/ADR cells by G2/M arrest. Toxicol Lett 2013; 219: 107–15.
Zhu B, Zhao L, Zhu L, Wang H, Sha Y, Yao J, et al. Oroxylin a reverses CAM-DR of HEPG2 cells by suppressing integrin β1 and its related pathway. Toxicol Appl Pharm 2012; 259: 387–94.
Liu Y, Jennings NL, Dart AM, Du XJ . Standardizing a simpler, more sensitive and accurate tail bleeding assay in mice. World J Exp Med 2012; 2: 30–6.
Yang T, Jia M, Mei Q, Shang P . Effects of Angelica polysaccharide on blood coagulation and platelet aggregation. Zhong Yao Cai 2002; 25: 344–5.
Blue R, Murcia M, Karan C, Jiroušková M, Coller BS . Application of high-throughput screening to identify a novel αIIb-specific small-molecule inhibitor of αIIbβ3-mediated platelet interaction with fibrinogen. Blood 2008; 111: 1248–56.
Chadderdon RC, Cappello M . The hookworm platelet inhibitor: functional blockade of integrins GPIIb/IIIa (αIIbβ3) and GPIa/IIa (α2β1) inhibits platelet aggregation and adhesion in vitro. J Infect Dis 1999; 179: 1235–41.
Da Silva M, Lucena S, Aguilar I, Rodríguez-Acosta A, Salazar AM, Sánchez EE, et al. Anti-platelet effect of cumanastatin 1, a disintegrin isolated from venom of South American Crotalus rattlesnake. Thromb Res 2009; 123: 731–9.
Kong Y, Wang Y, Yang W, Xie Z, Li Z . LX0702, a novel snake venom peptide derivative, inhibits thrombus formation via affecting the binding of fibrinogen with GPIIb/IIIa. J Pharmacol Sci 2015; 127: 462–6.
Ma D, Xu X, An S, Liu H, Yang X, Andersen JF, et al. A novel family of RGD-containing disintegrin (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets integrins αIIbβ3 and αVβ3 and inhibits platelet aggregation and angiogenesis. Thromb Haemost 2011; 105: 1032–45.
Prevost N, Woulfe D, Tanaka T, Brass LF . Interactions between Eph kinases and ephrins provide a mechanism to support platelet aggregation once cell-to-cell contact has occurred. Proc Natl Acad Sci U S A 2002; 99: 9219–24.
Yi W, Li Q, Shen J, Ren L, Liu X, Wang Q, et al. Modulation of platelet activation and thrombus formation using a Pan-PI3K inhibitor S14161. PLoS One 2014; 9: e102394.
Pinson JA, Zheng Z, Miller MS, Chalmers DK, Jennings IG, Thompson PE . l-Aminoacyl-triazine derivatives are isoform-selective PI3Kβ inhibitors that target nonconserved Asp862 of PI3Kβ. ACS Med Chem Lett 2012; 4: 206–10.
Li Z, Delaney MK, O'Brien KA, Du X . Signaling during platelet adhesion and activation. Arterioscl Throm Vas 2010; 30: 2341–9.
Gao C, Boylan B, Bougie D, Gill JC, Birenbaum J, Newman DK, et al. Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcγRIIa and the integrin β3 cytoplasmic domain. J Clin Invest 2009; 119: 504–11.
Savage B, Saldívar E, Ruggeri ZM . Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996; 84: 289–97.
Jackson SP, Schoenwaelder SM, Goncalves I, Nesbitt WS, Yap CL, Wright CE, et al. PI 3-kinase p110β: a new target for antithrombotic therapy. Nat Med 2005; 11: 507–14.
Francischetti IM . Platelet aggregation inhibitors from hematophagous animals. Toxicon 2010; 56: 1130–44.
Hyun KW, Jeong SC, Lee DH, Park JS, Lee JS . Isolation and characterization of a novel platelet aggregation inhibitory peptide from the medicinal mushroom, Inonotus obliquus. Peptides 2006; 27: 1173–8.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (No 81273375), the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Jiangsu Provincial Qing Lan Project.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Su, Xl., Su, W., Wang, Y. et al. The pyrrolidinoindoline alkaloid Psm2 inhibits platelet aggregation and thrombus formation by affecting PI3K/Akt signaling. Acta Pharmacol Sin 37, 1208–1217 (2016). https://doi.org/10.1038/aps.2016.52
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/aps.2016.52
Keywords
This article is cited by
-
Fibroblast growth factor-21 as a novel metabolic factor for regulating thrombotic homeostasis
Scientific Reports (2022)
-
Pharmacological actions of miltirone in the modulation of platelet function
Acta Pharmacologica Sinica (2019)
-
Amides, Isoquinoline Alkaloids and Dipeptides from the Aerial Parts of Piper mullesua
Natural Products and Bioprospecting (2018)
