Berberine and Its Main Metabolite Berberrubine Inhibit Platelet Activation Through Suppressing the Class I PI3Kβ/Rasa3/Rap1 Pathway

Abstract

Background: Berberine (BBR), a natural product, was reported to inhibit platelet aggregation; however, the molecular mechanisms remain unclear. This study aims to investigate the effects and mechanisms of BBR in inhibiting platelet activation and thrombus formation.Methods: Flow cytometry, immunofluorescence, and Western blot were used to determine the inhibitory effects and mechanisms of BBR and its main metabolite berberrubine (M2) on platelet activation in vitro and ex vivo. Purified integrin αIIbβ3, class I PI3K kit, and molecular docking were used to identify the possible targets of BBR and M2. A carrageenan-induced mouse thrombosis model was used to evaluate the effects of BBR on thrombus formation in vivo.Results:In vitro, BBR and M2 significantly inhibited ADP-induced integrin αIIbβ3 activation, reduced the level of P-selectin on the platelet membrane, and suppressed the binding of fibrinogen to the platelets. In this process, BBR and M2 greatly suppressed the PI3K/Akt pathway and inhibited Rasa3 membrane translocation and Rap1 activation. Furthermore, BBR and M2 selectively inhibited class I PI3Kβ, perhaps through binding to its active site. The activities of BBR were stronger than those of M2. After oral administration, BBR significantly inhibited the PI3K/Akt pathway and Rap1 activation and suppressed ADP-induced platelet activation and carrageenan-induced thrombosis in mice without prolonging bleeding time.Conclusions: We reveal for the first time the possible targets and mechanisms of BBR and M2 in inhibiting platelet activation. Our research may support the future clinical application of BBR as an antiplatelet drug in the prevention or treatment of thrombotic diseases.

Palmatine Derivatives as Potential Antiplatelet Aggregation Agents via Protein Kinase G/Vasodilator-Stimulated Phosphoprotein and Phosphatidylinositol 3-Kinase/Akt Phosphorylation

“…Isolated PRP was treated with prostaglandin E1 (PGE1, 1 μM) and centrifuged at 1200 g for 8 min to obtain the precipitation of washed platelets (WPs). The WPs were resuspended in N -(2-Hydroxyethyl) piperazine- N ′-(2-ethanesulfonic acid) (HEPES)-modified Tyrode buffer as described previously, and the platelet concentrations were determined using a hemocytometer and adjusted appropriately. , …”

“…1% paraformaldehyde was used to fix the WPs for 15 min. After washing, platelets were incubated with the PE-labeled integrin αIIbβ3 (JON/A) antibody, FITC-labeled P-selectin antibody, or isotype IgGs for 15 min, respectively . The reactions were terminated with phosphate-buffered saline (PBS), and platelets were analyzed on an Agilent NovoCyte flow cytometer (Agilent Technologies Inc., San Diego, USA) immediately.…”

“…Target proteins were probed with specific primary antibodies and an appropriate secondary antibody. After signal development, the bands were scanned and quantified as described previously …”

“…Berberine (BBR, Figure ), an antidiarrheal drug in China for half a century, − was reported to inhibit platelet aggregation, suppress thrombus formation, and ameliorate related cardiovascular or cerebrovascular diseases in clinical studies with a good safety profile. − Our recent study proves that BBR suppresses platelet activation through blocking the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which plays a crucial role in platelet activation and aggregation and is considered as a useful target to treat thrombosis. , …”

“…Berberine (BBR, Figure 1), an antidiarrheal drug in China for half a century, 7−9 was reported to inhibit platelet aggregation, suppress thrombus formation, and ameliorate related cardiovascular or cerebrovascular diseases in clinical studies with a good safety profile. 10−14 Our recent study proves that BBR suppresses platelet activation through blocking the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, 15 which plays a crucial role in platelet activation and aggregation and is considered as a useful target to treat thrombosis. 16,17 Another natural medicine palmatine (PMT, Figure 1), an analogue of BBR, also exhibited various biological activities, such as antimicrobial activity, anti-inflammatory effects, and anticancer activities, with the advantage of a good safety profile.…”