Vasodilator-stimulated phosphoprotein-phosphorylation by ginsenoside Ro inhibits fibrinogen binding to αIIb/β3 in thrombin-induced human platelets

Abstract

Background: Glycoprotein IIb/IIIa (αIIb/β₃) is involved in platelet adhesion, and triggers a series of intracellular signaling cascades, leading to platelet shape change, granule secretion, and clot retraction. In this study, we evaluated the effect of ginsenoside Ro (G-Ro) on the binding of fibrinogen to αIIb/β₃.

Methods: We investigated the effect of G-Ro on regulation of signaling molecules affecting the binding of fibrinogen to αIIb/β₃, and its final reaction, clot retraction.

Results: We found that G-Ro dose-dependently inhibited thrombin-induced platelet aggregation and attenuated the binding of fibrinogen to αIIb/β₃ by phosphorylating cyclic adenosine monophosphate (cAMP)-dependently vasodilator-stimulated phosphoprotein (VASP; Ser¹⁵⁷). In addition, G-Ro strongly abrogated the clot retraction reflecting the intensification of thrombus.

Conclusion: We demonstrate that G-Ro is a beneficial novel compound inhibiting αIIb/β₃-mediated fibrinogen binding, and may prevent platelet aggregation-mediated thrombotic disease.

Keywords: VASP (Ser157); cAMP; clot retraction; fibrinogen binding; ginsenoside Ro.

Fig. 1

Chemical structure of ginsenoside Ro. Ginsenoside Ro (G-Ro), an oleanane-type saponin, is contained in Panax ginseng Meyer , , and is composed of oleanolic acid as aglycone, and two glucose and one glucuronic acid as sugar component .

Fig. 2

Effects of ginsenoside Ro (G-Ro) on thrombin-induced human platelet aggregation. Measurement of platelet aggregation was carried out as described in the “Materials and methods” section. Data are presented as mean ± SD (n = 4). * p < 0.05 versus the thrombin-stimulated human platelets.

Fig. 3

Effects of ginsenoside Ro (G-Ro) on vasodilator-stimulated phosphoprotein (VASP) phosphorylation. (A) Effects of G-Ro on VASP phosphorylation. Lane 1, intact platelets (base); Lane 2, thrombin (0.05 U/mL); Lane 3, thrombin (0.05 U/mL) + G-Ro (100μM); Lane 4, thrombin (0.05 U/mL) + G-Ro (200μM); and Lane 5, thrombin (0.05 U/mL) + G-Ro (300μM). (B) The ratio of phosphorylation of VASP (Ser¹⁵⁷)-50 kDa to β-actin by G-Ro. Its ratio is from Fig. 3A. (C) Effects of G-Ro on VASP (Ser²³⁹)-50 kDa phosphorylation. Lane 1, intact platelets (base); Lane 2, thrombin (0.05 U/mL); Lane 3, thrombin (0.05 U/mL) + G-Ro (100 μM); Lane 4, thrombin (0.05 U/mL) + G-Ro (200μM); Lane 5, thrombin (0.05 U/mL) + G-Ro (300 μM); and Lane 6, thrombin (0.05 U/mL) + 8-Br-cGMP (1mM). (D) Effects of G-Ro on VASP (Ser¹⁵⁷)-50 kDa phosphorylation in the presence of an A-kinase inhibitor (Rp-8-Br-cAMPS). Lane 1, intact platelets (base); Lane 2, thrombin (0.05 U/mL); Lane 3, thrombin (0.05 U/mL) + G-Ro (300μM); Lane 4, thrombin (0.05 U/mL) + G-Ro (300μM) + Rp-8-Br-cAMPS (250 μM); and Lane 5, thrombin (0.05 U/mL) + pCPT-cAMP (1mM). Western blotting was performed as described in the “Materials and methods” section. Data are presented as mean ± SD (n = 4). * p < 0.05 versus the thrombin-stimulated human platelets. ** p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300μM).

Fig. 4

Effects of ginsenoside Ro (G-Ro) on thrombin-induced fibrinogen binding. (A) The flow cytometry histograms on fibrinogen binding. a, intact platelets (base); b, thrombin (0.05 U/mL); c, thrombin (0.05 U/mL) + G-Ro (50μM); d, thrombin (0.05 U/mL) + G-Ro (100 μM); e, thrombin (0.05 U/mL) + G-Ro (200μM); f, thrombin (0.05 U/mL) + G-Ro (300 μM); g, thrombin (0.05 U/mL) + eptifibatide (50μM); and h, thrombin (0.05 U/mL) + GR 144053 (50μM). (B) Effects of G-Ro on thrombin-induced fibrinogen binding. Its binding degree (%) is from Fig. 4A. Determination of fibrinogen binding to glycoprotein IIb/IIIa (αIIb/β₃) was carried out as described in the “Materials and methods” section. Data are presented as mean ± standard deviation (n = 4). * p < 0.05 versus the thrombin-stimulated human platelets. FL1-H, fluorescent light-1 height.

Fig. 5

Effects of ginsenoside Ro (G-Ro) on thrombin-induced fibrinogen binding in the presence of an A-kinase inhibitor (Rp-8-Br-cAMPS). (A) The flow cytometry histograms on fibrinogen binding. Thrombin (0.05 U/mL) + G-Ro (300μM) + Rp-8-Br-cAMP (250μM). (B) Effects of G-Ro on thrombin-induced fibrinogen binding in the presence of the A-kinase inhibitor (Rp-8-Br-cAMPS). Its binding degree (%) is from Fig. 4, Fig. 5A. Determination of fibrinogen binding to αIIb/β₃ was carried out as described in the “Materials and methods” section. Data are presented as mean ± SD (n = 4). * p < 0.05 versus the thrombin-stimulated human platelets. ** p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300μM). FL1-H, fluorescent light-1 height.

Fig. 6

Effects of ginsenoside Ro (G-Ro) on fibrin clot retraction. (A) Photographs of fibrin clot. (B) Effects of G-Ro on thrombin-retracted fibrin clot. Quantification of fibrin clot retraction was performed as described in the “Materials and methods” section. ¹⁾ (control − thrombin)/control × 100. ²⁾ [control − (thrombin + G-Ro)]/control × 100. ³⁾ [thrombin − (thrombin + G-Ro)]/thrombin × 100. Data are presented as mean ± standard deviation (n = 4). * p < 0.05 versus the thrombin-stimulated human platelets.