Betulinic Acid Inhibits Collagen-Induced Platelet Adhesion and Activation Through Inhibition of Spleen Tyrosine Kinase (SYK) Phosphorylation

Betulinic acid is a pentacyclic triterpenoid and is found in various plant species. Betulinic acid has been shown to exhibit several biological activities, such as antiviral, anticancer, immunomodulatory, anti-inflammatory, antimicrobial, anti-diabetic, anti-parasitic, and anti-HIV properties. Additionally, betulinic acid has shown antiplatelet effects against thrombin receptor activator peptide (TRAP), adenosine diphosphate (ADP), and arachidonic acid (AA)-induced platelet activation and aggregation. However, its effect on collagen-induced platelet activation and signaling pathways is not yet known. Therefore, we investigated the effect of betulinic acid on collagen-induced platelet adhesion and activation. Platelets were isolated from mice and labeled with CD41 PE-Cy7 at 37 °C for one hour and induced with various agonists to determine the platelet activation profile against different platelet agonists. Further, the concentration-dependent effect of betulinic acid (100 µM, 300 µM, 500 µM, or 1 mM) against collagen-induced platelet activation was assessed by flow cytometry. Moreover, platelets were labeled with CD41 PE-Cy7 and phospho-spleen tyrosine kinase (SYK) PE at 37 °C for one hour and incubated with betulinic acid (500 µM) or cytochalasin D (1 µg/mL) for 20 minutes, followed by the addition of collagen (25 µg/mL). Thereafter, samples were analyzed by flow cytometer (BD FACSCanto^TM II) to determine their platelet activation and SYK phosphorylation. Further, washed platelets labeled with CD41 PE-Cy7 were incubated with phalloidin-fluorescein isothiocyanate (FITC) and pre-treated with either betulinic acid (500 µM) or cytochalasin D (1 µg/mL), followed by 30 minutes of incubation on a collagen-coated cover slip for platelet adhesion studies by confocal microscopy. Analysis of data was performed by one-way ANOVA, followed by post-hoc Dunnett's test concerning the control group using GraphPad Prism statistical software (GraphPad Software, San Diego, CA). All the experiments were performed in duplicates of ≥3 independent experiments. All the data were reported as mean ± SEM in all the groups. P < 0.5 was considered to be statistically significant. Betulinic acid inhibited the collagen-induced platelet activation in a concentration-dependent manner at 500 µM and 1 mM (6091 ± 901 vs 3569 + 291, 6091 ± 901<i> </i>vs<i> </i>3305 + 623)and decreased the SYK phosphorylation by inhibiting protein tyrosine kinase SYK in flow cytometry studies. Further, it inhibited the platelet adhesion on the collagen-coated surface in imaging studies. Betulinic acid inhibited the collagen-induced platelet activation and reduced SYK phosphorylation from 82.1% to 49.8%. It has shown better inhibition than cytochalasin D (60.2%). Moreover, betulinic acid exhibited a platelet inhibitory effect on platelet adhesion on collagen-coated surfaces. These studies show that betulinic acid inhibits collagen-induced platelet activation through a reduction in SYK phosphorylation, corroborating the antiplatelet activities of betulinic acid.