Inhibitory effects of total saponin from Korean red ginseng via vasodilator-stimulated phosphoprotein-Ser157 phosphorylation on thrombin-induced platelet aggregation원문보기
In this study, we have investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-induced platelet aggregation. TSKRG dose-dependently inhibited thrombin-induced platelet aggregation with $IC_{50}$ value of about 81.1 ${\mu}g/mL$. In addition, TSKRG d...
In this study, we have investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-induced platelet aggregation. TSKRG dose-dependently inhibited thrombin-induced platelet aggregation with $IC_{50}$ value of about 81.1 ${\mu}g/mL$. In addition, TSKRG dose-dependently decreased thrombin-elevated the level of cytosolic-free $Ca^{2+}$ ($[Ca^{2+}]_i$), one of aggregation-inducing molecules. Of two $Ca^{2+}$-antagonistic cyclic nucleotides as aggregation-inhibiting molecules, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), TSKRG significantly dose-dependently elevated intracellular level of cAMP, but not cGMP. In addition, TSKRG dose-dependently inhibited thrombin-elevated adenosine triphosphate (ATP) release from platelets. These results suggest that the suppression of $[Ca^{2+}]_i$ elevation, and of ATP release by TSKRG are associated with upregulation of cAMP. TSKRG elevated the phosphorylation of vasodilator-stimulated phosphoprotein (VASP)-$Ser^{157}$, a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-$Ser^{239}$, a cGMP-dependent protein kinase substrate, in thrombin-activated platelets. We demonstrate that TSKRG involves in increase of cAMP level and subsequent elevation of VASP-$Ser^{157}$ phosphorylation through A-kinase activation to inhibit $[Ca^{2+}]_i$ mobilization and ATP release in thrombin-induced platelet aggregation. These results strongly indicate that TSKRG is a beneficial herbal substance elevating cAMP level in thrombin-platelet interaction, which may result in preventing of platelet aggregation-mediated thrombotic diseases.
In this study, we have investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-induced platelet aggregation. TSKRG dose-dependently inhibited thrombin-induced platelet aggregation with $IC_{50}$ value of about 81.1 ${\mu}g/mL$. In addition, TSKRG dose-dependently decreased thrombin-elevated the level of cytosolic-free $Ca^{2+}$ ($[Ca^{2+}]_i$), one of aggregation-inducing molecules. Of two $Ca^{2+}$-antagonistic cyclic nucleotides as aggregation-inhibiting molecules, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), TSKRG significantly dose-dependently elevated intracellular level of cAMP, but not cGMP. In addition, TSKRG dose-dependently inhibited thrombin-elevated adenosine triphosphate (ATP) release from platelets. These results suggest that the suppression of $[Ca^{2+}]_i$ elevation, and of ATP release by TSKRG are associated with upregulation of cAMP. TSKRG elevated the phosphorylation of vasodilator-stimulated phosphoprotein (VASP)-$Ser^{157}$, a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-$Ser^{239}$, a cGMP-dependent protein kinase substrate, in thrombin-activated platelets. We demonstrate that TSKRG involves in increase of cAMP level and subsequent elevation of VASP-$Ser^{157}$ phosphorylation through A-kinase activation to inhibit $[Ca^{2+}]_i$ mobilization and ATP release in thrombin-induced platelet aggregation. These results strongly indicate that TSKRG is a beneficial herbal substance elevating cAMP level in thrombin-platelet interaction, which may result in preventing of platelet aggregation-mediated thrombotic diseases.
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제안 방법
As described above, since TXA2 is produced when [Ca2+]i level is increased by agonists, TSKRG that decreases TXA2 production must decline [Ca2+]i level to have antiplatelet effect. In this study, we investigated whether TSKRG reduces thrombin-elevated [Ca2+]i level, increases Ca2+- antagonistic cAMP and cGMP level, stimulates the pVASP, and inhibits the release of ATP, a cAMP precursor, to evaluate antiplatelet effect of TSKRG.
대상 데이터
TSKRG was dissolved with 100% methanol and then analyzed by HPLC [22,23]. An Dionex-Ultimate 3000 series HPLC apparatus (Dionex, Sunnyvale, CA, USA), equipped with vacuum degaser, quaternary gradient pump, and autosampler was used. A Zorbax ODS C18 column (250 mm×4.
ATP assay kit is purchased from Biomedical Research Service Center (Buffalo, NY, USA). Anti-VASP, anti-phosphor-VASP (Ser157), anti-phosphor-VASP (Ser239), and anti-rabbit IgG-horseradish peroxidase conjugate (HRP) or anti-goat IgG-HRP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyvinylidene difluoride (PVDF) membrane was from GE Healthcare (Piseataway, NJ, USA).
데이터처리
The experimental results are expressed as means±SEM accompanied by the number of observations. Data were assessed by ANOVA. If this analysis indicated significant differences among the group means, then each group was compared by the Newman-Keuls method.
이론/모형
Inhibition rate by TSKRG was recorded as percentage of the thrombin-induced aggregation rate. IC50 value of TSKRG was calculated by 4-parameter log fit method [25]. Data are expressed as means±SEM (n=4).
5 s from 340 to 380 nm; the emission wavelength was set at 510 nm. The [Ca2+]i values were calculated using the method of Schaeffer[21].
성능/효과
In conclusion, the most important result of this study is that TSKRG significantly decreases the level of [Ca2+]i , platelet-aggregating molecule, and inhibition of [Ca2+]i by TSKRG depends on up-regulation of Ca2+-antagonistic intracellular cAMP level. Furthermore, our findings showed that TSKRG-elevated cAMP stimulates the phosphorylation of an A-kinase substrate VASP (Ser157)-50 kDa, which may contribute to attenuating [Ca2+]i level to inhibit thrombin-induced platelet aggregation.
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