Ayurvedic preparations of Raudra Rasa inhibit agonist-mediated platelet activation and restrict thrombogenicity without affecting cell viability

Abstract

Ayurveda is considered to be one of the most ancient forms of medicine still practiced. The Ayurvedic preparation Raudra Rasa and its derivatives have been widely employed against cancer since the 12th century, but the effect of these traditional formulations on platelet function and signaling has not previously been examined. Here we demonstrate that Raudra Rasa and its derivatives significantly reduce thrombin-induced integrin activation and granule secretion in platelets, as observed by reduced PAC-1 binding and P-selectin externalization, respectively. These formulations also inhibited thrombin-stimulated phosphatidylserine exposure, mitochondrial reactive oxygen species generation, and mitochondrial transmembrane potential in platelets. Consistent with the above, Raudra Rasa significantly reduced thrombin-induced tyrosine phosphorylation of the platelet proteins, as well as phosphorylation of the enzymes AKT and GSK-3β. In summary, Raudra Rasa inhibits agonist-mediated platelet activation without affecting cell viability, suggesting it may have therapeutic potential as an anti-platelet/anti-thrombotic agent.

Keywords: AKT; CRR; MRR; RRE; mROS; platelets.

Fig. 1

CRR, MRR, and RRE attenuate integrin activation in human platelets in a dose‐dependent manner. A,C,E: histograms showing binding of PAC‐1 to platelets pretreated with either CRR (100–200 μg·mL⁻¹) or MRR (50–200 μg·mL⁻¹) or RRE (100 μg·mL⁻¹ to 2.5 mg·mL⁻¹) or vehicle for 30 min at RT followed by stimulation with thrombin (Thr, 0.25 U·mL⁻¹) for 5 min at 37 °C, as indicated. B (n = 6), D (n = 6), and F (n = 6), the corresponding bar charts showing mean fluorescence intensity (MFI) of PAC‐1 binding to platelets. Data are presented as mean ± SEM and analyzed by RM one‐way ANOVA with Dunnett's multiple comparisons test.

Fig. 2

CRR, MRR, and RRE attenuate P‐selectin externalization in human platelets in a dose‐dependent manner. A,C,E: histograms showing binding of P‐selectin antibody (anti‐CD62P) to platelets pretreated with either CRR (100–200 μg·mL⁻¹) or MRR (50–200 μg·mL⁻¹) or RRE (100 μg·mL⁻¹ to 2.5 mg·mL⁻¹) or vehicle for 30 min at RT followed by stimulation with thrombin (0.25 U·mL⁻¹) for 5 min at 37 °C, as indicated. B (n = 5), D (n = 5), and F (n = 4), the corresponding bar charts showing mean fluorescence intensity (MFI) of P‐selectin antibody binding to platelets. Data are presented as mean ± SEM and analyzed by RM one‐way ANOVA with Dunnett's multiple comparisons test.

Fig. 3

CRR, MRR, and RRE attenuate mitochondrial transmembrane potential in human platelets in a dose‐dependent manner. (A,C,E) histograms showing binding of MitoTracker Red dye to platelets pretreated with either CRR (100–200 μg·mL⁻¹) or MRR (50–200 μg·mL⁻¹) or RRE (100 μg·mL⁻¹ to 2.5 mg·mL⁻¹) or vehicle for 30 min at RT followed by stimulation with thrombin (0.25 U·mL⁻¹) for 5 min at 37 °C, as indicated. B (n = 5), D (n = 6), and F (n = 5), the corresponding bar charts showing mean fluorescence intensity (MFI) of MitoTracker Red dye binding to platelets. Data are presented as mean ± SEM and analyzed by RM one‐way ANOVA with Dunnett's multiple comparisons test.

Fig. 4

Effect of CRR, MRR, and RRE on thrombin‐induced AKT and GSK‐3β phosphorylation in human platelets. Platelets were pretreated with either CRR (200 μg·mL⁻¹) or MRR (100 and 200 μg·mL⁻¹) or RRE (2.5 mg·mL⁻¹) or vehicle for 30 min at RT followed by stimulation with thrombin (0.25 U·mL⁻¹) for 5 min at 37 °C, as indicated. (A,E,I) Western analysis showing expression of p‐AKT. (C,G,K) Western analysis showing expression of p‐GSK‐3β. B (n = 4), F (n = 4), and J (n = 4), the corresponding densitometric analyses of p‐AKT normalized with AKT. D (n = 4), H (n = 4), and L (n = 7), the corresponding densitometric analyses of p‐GSK‐3β normalized with GSK‐3β. Data are presented as mean ± SEM and analyzed by RM one‐way ANOVA with Dunnett's multiple comparisons test.

Fig. 5

Effect of CRR, MRR, and RRE on thrombin‐induced tyrosine phosphoproteome in human platelets. (A–C) Western analysis of platelets preincubated with either CRR (200 μg·mL⁻¹) or MRR (100 or 200 μg·mL⁻¹) or RRE (2.5 mg·mL⁻¹) or vehicle for 30 min at RT, followed by stimulation with thrombin (0.25 U·mL⁻¹) for 5 min at 37 °C, as indicated. D (n = 4), E (n = 4), and F (n = 4), the corresponding densitometric analyses of phosphotyrosine normalized with β‐Actin. Data are presented as mean ± SEM and analyzed by RM one‐way ANOVA with Dunnett's multiple comparisons test.

Fig. 6

CRR, MRR, and RRE attenuate thrombin‐induced phosphatidylserine exposure in human platelets. A,C,E: dotplot showing binding of annexin V to platelets pretreated with either CRR (100 or 200 μg·mL⁻¹) or MRR (100 and 200 μg·mL⁻¹) or RRE (1.25 or 2.5 mg·mL⁻¹) or vehicle for 30 min at RT, followed by stimulation with thrombin (0.25 U·mL⁻¹) for 5 min at 37 °C, as indicated. B (n = 7), D (n = 6), and F (n = 6), the corresponding bar charts showing mean per cent of annexin V binding to platelets. Data are presented as mean ± SEM and analyzed by RM one‐way ANOVA with Dunnett's multiple comparisons test.

Fig. 7

Effect of pretreatment with CRR, MRR, and RRE on thrombin‐induced mitochondrial ROS generation in human platelets. A,C,E: histograms of MitoSOX Red dye binding to platelets pretreated with either CRR (100 or 200 μg·mL⁻¹) or MRR (100 μg·mL⁻¹) or RRE (100 μg·mL⁻¹) or vehicle for 30 min at RT, followed by stimulation with thrombin (0.25 U·mL⁻¹) for 5 min at 37 °C, as indicated. B (n = 7), D (n = 8), and F (n = 4), the corresponding bar charts showing mean fluorescence intensity (MFI) of MitoSOX Red dye binding to platelets. Data are presented as mean ± SEM and analyzed by RM one‐way ANOVA with Dunnett's multiple comparisons test.

Fig. 8

Effect of CRR, MRR, and RRE on human platelets viability. Bar diagram representing cell viability of platelets preincubated with either vehicle or CRR (100–200 μg·mL⁻¹) or MRR (50–200 μg·mL⁻¹) or RRE (100 μg·mL⁻¹ to 2.5 mg·mL⁻¹) for 30 min at RT, followed by stimulation with thrombin (0.25 U·mL⁻¹) for 5 min at 37 °C, as indicated (n = 4). Data are presented as mean ± SEM. ns (nonsignificant) compared to either resting or thrombin‐treated platelets and analyzed by RM one‐way ANOVA with Dunnett's multiple comparisons test.