All C57BL/6N mice (males, 6–7 weeks old, 18–20 g) and BALB/c mice (males, 6–7 weeks old, 18–20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were kept in a room at a temperature of 22–24°C and humidity of 45% with a 12-h day and night cycle (lighting time 8:00–20:00). All animal experiments were reviewed and approved by the Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC) (No. 00005787, No. 00005788, No. 00005789). After the experiments, all mice were anesthetized with 2% isoflurane inhalation and sacrificed with cervical dislocation to perform animal euthanasia. To protect animals used for scientific purposes, all animal procedures which were performed conformed to the guidelines from EU Directive 2010/63/EU for animal experiments.

Sodium citrate tribasic dihydrate, prostaglandin E1 (PGE1), N-[2-Hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid (HEPES), berberine chloride, ADP, 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate (#T4444), aspirin (Asp), and a stop reagent for the TMB substrate (#S5814) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Berberrubine chloride (M2) was obtained from Chengdu Herbpurify CO., LTD. (Chengdu, Sichuan, China). PE-labeled rat anti-mouse JON/A monoclonal antibody (#M023-2), PE-labeled rat IgG polyclonal antibody (#P190-2), FITC-labeled rat anti-mouse p-selectin (CD62P) monoclonal antibody (#M130-1), and FITC-labeled rat IgG polyclonal antibody (#P190-1) were purchased from Emfret Analytics (Würzburg, Germany). Eight-well chambered glass coverslips (#155411PK), FluoroNunc 96-well plates, Calcein-AM (#C3099), TRITC–phalloidin (#R415), the M-PER™ Mammalian Protein Extraction Reagent (#78501), the Mem-PER™ Plus Membrane Protein Extraction Kit (#89842), and the active Rap1 pull-down and detection kit (#16120) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, United States). Mouse fibrinogen (#CT15) was obtained from Oxford Biomedical Research (Oxford, United Kingdom). Human GPIIbIIIa (#GP2b3a) was obtained from Enzyme Research Laboratories (South Bend, IN, United States). Fibrinogen plasminogen-depleted (#341578) and rabbit anti-FcRγ polyclonal antibodies (#06–727) were purchased from Millipore (Billerica, MA, United States). Fibrinogen antibody (HRP) (#60R-1012) was purchased from Fitzgerald (Acton, MA, United States). Tirofiban hydrochloride monohydrate (#HY-17369) and clopidogrel (Clop) hydrogen sulfate were obtained from MedChemExpress (Shanghai, China). Rabbit anti-p-Akt (Ser⁴⁷³) (#4058), rabbit anti-Akt (#9272), rabbit anti-ERK (#9102) antibodies, and wortmannin (Wtm) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, United States). Mouse anti-Rasa3 monoclonal antibody (#SC-398283) and integrin β3 antibody (B-7) (#SC-46655) were sourced from Santa Cruz Biotechnology, Inc. (Dallas, TX, United States). ADP-Glo lipid kinase systems (#V1691) were purchased from Promega Corporation (Madison, WI, United States). TGX221 was purchased from Cayman Chemical Company (Ann Arbor, MI, United States).

Mouse washed platelets (WPs) were prepared as described previously (Blue et al., 2008). Briefly, mice were anaesthetized with 2% isoflurane inhalation, and retro-orbital blood samples were collected into the sodium citrate solution (blood volume: sodium citrate solution, 9:1). The anticoagulated whole blood sample was centrifuged at 22°C at 650 g for 4 min to obtain platelet-rich plasma (PRP). Then, PGE1 (final concentration 1 μM) was added to PRP and mixed well, and the PRP was centrifuged at 22°C at 1,200 g for 8 min to obtain the precipitation of WPs. Then, the WPs were resuspended in HEPES-modified Tyrode buffer (HBMT; 10 mM HEPES, 138 mM NaCl, 12 mM NaHCO₃, 2.7 mM KCl, 0.4 mM NaH₂PO₄, 0.1% glucose, 0.35% BSA, 2 mM CaCl₂, and 1 mM MgCl₂, pH = 7.4). The platelet concentration was adjusted to 3 × 10⁸/ml by counting with a hemocytometer.

The methods of flow cytometry were performed as described previously (Deng et al., 2016). Briefly, the suspension of the WPs was divided into several groups and treated with the vehicle control (0.1% DMSO) or different compounds at 37°C for 10 min and then stimulated with 10 μM of ADP for 10 min. After treatment, the WPs were fixed with 1% paraformaldehyde at room temperature for 15 min. The platelet suspension was centrifuged at 22°C at 1,200 g for 8 min, and then the precipitation was resuspended in HBMT. Each group was divided into four parts, and they were incubated with the PE-conjugated JON/A antibody (selectively binding to the high-affinity conformation of mouse integrin αIIbβ3), FITC-labeled rat anti-mouse P-selectin monoclonal antibody, or the negative control IgGs for 15 min. After terminating the reaction with 400 μl PBS, the platelets were analyzed using a BD FACSCalibur flow cytometer (BD-Biosciences, Heidelberg, Germany) or BD Accuri C6 flow cytometer (BD-Biosciences, Heidelberg, Germany). All data were analyzed using FlowJo software (BD-Biosciences, Heidelberg, Germany).

Immunofluorescence was performed as described before with slight modifications (Blue et al., 2008). Briefly, eight-chambered glass coverslips (Nunc) were coated with 200 μl fibrinogen (50 μg/ml). After incubating at 4°C overnight, each chamber was washed three times with Tris/saline (100 mM sodium chloride, 50 mM Tris/HCl, pH = 7.4). And then, each chamber was blocked with HBMT at room temperature for 1 h and washed two times with Tris/saline. After treatment, WPs were added to the eight-well chambered glass coverslips coated with fibrinogen and incubated at room temperature for 1 h, then each chamber was washed with HBMT (containing 2 mM CaCl₂ and 1 mM MgCl₂) four times. The adherent platelets were fixed with 1% paraformaldehyde at room temperature for 15 min and washed with PBS three times. Then, the platelets were treated with 0.1% Triton X-100 for 15 min and washed with PBS for another three times. After being blocked with 1% BSA/PBS at room temperature for 1 h, the adherent platelets were incubated with the TRITC–phalloidin working solution at room temperature for 1 h and washed with PBS again three times. Finally, immunofluorescence images were taken using an Olympus IX71 fluorescent inverted microscope with a 40× objective and 10× eyepiece.

Platelet adhesion to fibrinogen was performed as described before with slight modifications (Blue et al., 2008; Su et al., 2016). Briefly, human fibrinogen was diluted and dissolved into the fibrinogen solution with a final concentration of 50 g/ml. 200 μl of the fibrinogen solution (50 μg/ml) was added to FluoroNunc 96-well plates. After incubating at 4°C overnight, the plates were washed with 200 μl of Tris/saline three times. Then, the wells were blocked with 200 μl of HBMT containing 2% BSA for 1 h at room temperature and washed with Tris/saline two times. During the period, WPs were treated with different concentrations of BBR or M2 or the vehicle control (0.1% DMSO) at 37°C for 10 min and then stimulated with 10 μM ADP for 10 min. After treatment, WPs were labeled with calcein-AM at a final concentration of 16 μM at room temperature for 30 min in the absence of light. And then, 100 μl labeled platelets were added into coated FluoroNunc 96-well plates and incubated at room temperature for 1 h. After washing four times with 200 μl HBMT containing 2 mM CaCl₂ and 1 mM MgCl₂, 100 μl HBMT containing 2 mM CaCl₂ and 1 mM MgCl₂ was added. The fluorescence intensity was measured using the EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, United States) to represent the relative number of adherent platelets (excitation wavelengths/emission wavelengths: 490 nm/515 nm).

After treatment, the WPs were centrifuged at 1,200 g at 22°C for 8 min to obtain the precipitation. The M-PER™ Mammalian Protein Extraction Reagent was added to obtain whole cell lysis, and the Mem-PER™ Plus Membrane Protein Extraction Kit was used to obtain membrane proteins and cytosolic proteins following the kit's instructions. Western blot was performed as described before (Wang et al., 2016). Briefly, 20 μl of the cell lysate was used for 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins were transferred from the gels onto the PVDF membranes. After blocking, the blots of target proteins were detected with specific primary antibodies and appropriate secondary antibodies. The signals were developed using the ECL kit (EMD Millipore Corporation). After scanning and quantification, the levels of p-AKT (Ser⁴⁷³) were normalized to those of AKT and plotted as indicated. The ratios of the Rasa3 protein in the membrane to FcRγ and those in the cytoplasm to ERK were calculated. The original scans of Western blot are shown in Supplementary Figures S1–4.

The Rap1 activation assay was performed following the kit's instructions; GTPγS (positive control) and GDP (negative control) were used to ensure that the pull-down procedures worked properly. Briefly, after treatment, the WPs precipitation was prepared and split with 300 μl lysis/binding/wash buffer on ice for 5 min. The supernatant was collected by centrifuging the lysate at 4°C for 15 min and added to the spin cup containing the glutathione resin and GST-RalGDS-RBD. The reaction mixture was vortexed and incubated at 4°C for 1 h with gentle rocking. The spin cups were centrifuged with collection tubes at 6,000 g for 1 min, and the resin was washed three times. After adding 50 μl 2× reducing sample buffer (1 part β-mercaptoethanol to 20 parts 2× SDS sample buffer), the samples were vortexed and incubated at room temperature for 2 min and then centrifuged at 6,000 g for 2 min. The spin cup containing the resin was removed and discarded. The eluted samples were heated for 5 min at 95–100°C. Western blot was used to detect the pull-down of GTP-Rap1 (Rap1 active form). The original scans of Western blot are shown in Supplementary Figures S1, 3.

The purified integrin αIIbβ3 binding assay was performed as described previously (Blue et al., 2008; Su et al., 2016). Briefly, 96-well plates were coated with the integrin β3 antibody (B-7) (10 μg/ml) at 4°C overnight and, then, were blocked with 3.5% BSA at room temperature for 1 h. Purified integrin αIIbβ3 was diluted in buffer A (50 mM Tris/HCl, 100 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1% BSA, and 0.0035% Triton X-100) to 10 μg/ml, added to the wells, and captured by the integrin β3 antibody (B-7) for 2 h at 37°C. The wells were then washed three times with buffer A. Fibrinogen, prepared in buffer A (20 μg/ml), was incubated in the well plates for 2 h at 37°C with or without the studied compounds or control solutions. The wells were washed three times with buffer A and then incubated with a horseradish peroxidase (HRP)–conjugated polyclonal anti-fibrinogen antibody (1:1,000 in buffer A) for 1 h at room temperature. For color development, the wells were washed three times. 100 μl of a peroxidase substrate (TMB) was added, and the reaction was terminated after 30 min by adding 100 μl of the stop reagent for the TMB substrate. Finally, the absorbance value was determined by spectrophotometry at 450 nm.

The effects of BBR and M2 on different isoforms of class I PI3K were determined following the kit's instructions. Briefly, different isoforms of recombinant class I PI3K enzymes were mixed with the lipid kinase substrate which contained 1 mg of phosphoinositol-4, 5-bisphosphate (PIP2) and 3 mg of phosphatidylserine. Together with the studied compounds, the reactions were incubated at room temperature for 20 min. After incubation, the ATP solution (25 μM) was added to each well. The assay plate was covered and incubated at room temperature for 1 h. The ADP-Glo reagent containing 10 mM of MgCl₂ was used to stop the reaction and deplete the unconsumed ATP. Then, the kinase detection reagent was added to each well and incubated at room temperature for 40 min to convert ADP to ATP. The amount of newly synthesized ATP was detected with a coupled luciferin/luciferase reaction and used to represent class I PI3K activity.

For the ATP competition experiments, different isoforms of class I PI3K were incubated with the substrate and 1 μM of BBR or M2 for 20 min, and lipid kinase reactions were performed in the presence of different concentrations of ATP (6.25–400 μM).

The 2D structures of BBR and M2 were drawn using ChemBioDraw 2014 and converted to 3D structures in MOE through energy minimization. The 3D structure of the mouse protein PI3K p110β was downloaded from the RCSB Protein Data Bank (PDB ID was 2Y3A). Prior to docking, the force field of AMBER10:EHT and the implicit solvation model of the reaction field (R-field) were selected. MOE-Dock was used for molecular docking simulations of molecules with proteins. The docking workflow followed the “induced fit” protocol, in which the side chains of the receptor pocket were allowed to move according to ligand conformations, with a constraint on their positions. The weight used for tethering side chain atoms to their original positions was 10. For each ligand, all docked poses were ranked by London dG scoring first, then a force field refinement was carried out on the top 20 poses followed by the rescoring of GBVI/WSA dG. The conformations with the lowest free energies of binding were selected as the best (probable) binding modes. Molecular graphics were generated by PyMOL.

C57BL/6N mice were randomly divided into five groups (n = 5 each), and each group received the following treatment for 14 days: vehicle group—intragastric (i.g.) administration of normal saline (NS, 0.1 ml/10 g), and the WPs were incubated with the vehicle control (0.1% DMSO); vehicle + ADP group—i.g., administration of NS, and the WPs were stimulated with ADP (10 μM); BBR100 + ADP group—i.g., administration of BBR (100 mg/kg), and the WPs were stimulated with ADP; BBR200 + ADP group—i.g., administration of BBR (200 mg/kg), and the WPs were stimulated with ADP; Clop10 + ADP group—i.g., administration of Clop (10 mg/kg) and the WPs were stimulated with ADP.

After treatment, the PE-conjugated JON/A antibody and FITC-labeled P-selectin antibody binding of the platelets were determined by flow cytometry. The proteins were extracted, and p-Akt and GTP-Rap1 levels were determined by the pull-down assay and Western blot as described above.

The mice tail bleeding time was determined according to a method described previously with modifications (Song et al., 2019). Briefly, 30 C57BL/6N mice were randomly divided into six groups (n = 5 each). Before drug administration, the mice were anaesthetized with 2% isoflurane inhalation, and then the tail bleeding time was determined. A 5-mm tail tip of each mouse was cut off using a surgical blade, and the transected tail tip was transferred onto a clean filter paper. The tail bleeding time was recorded every 30 s s until the bleeding completely stopped.

Twenty-five hours before the second measurement, the mice were orally administrated with BBR (200 mg/kg), Clop (10 mg/kg), Asp (100 mg/kg), BBR (200 mg/kg) + Clop (10 mg/kg), BBR (200 mg/kg) + Asp (100 mg/kg), or vehicle (1% carboxymethyl cellulose sodium). All mice were given the same volume at 0.1 ml/10 g. One hour before the second measurement, all groups' mice were given the studied compounds or the vehicle control once again in the same way as described above. One hour after the second dose administration, the mice were anaesthetized with 2% isoflurane inhalation, and then the tail bleeding time was measured again.

The methods of carrageenan-induced thrombus formation in mice were performed as described previously with modifications (Li et al., 2019). Forty BALB/c mice were randomly divided into the following six groups: vehicle group (n = 5), which was i.g. administered with NS (0.1ml/10 g), vehicle + carrageenan group (n = 7), BBR 50 mg/kg + carrageenan group (n = 7), BBR 100 mg/kg + carrageenan group (n = 7), BBR 200 mg/kg + carrageenan group (n = 7), and Clop 10 mg/kg + carrageenan group (n = 7).

The treatment lasted for 12 days. On day 12, the mice in the vehicle control group were intraperitoneally (i.p.) injected with NS (50μl/10 g), while the mice in the other groups were i.p. injected with 0.5% carrageenan solution (50 μl/10 g, final dose 50 mg/kg). Two days after carrageenan injection, the mice were anaesthetized with 2% isoflurane inhalation. The tails of mice were photographed quickly; the original images of mice tail thrombosis are shown in Supplementary Figure S4, and both the length of thrombus in and the full length of the tails of the mice were measured using a steel ruler. The thrombosis rate of the tails of the mice was calculated by the following formula: thrombus length/whole tail length × 100.

After photography and measurement, the blood samples were collected through retro-orbital puncture, and the tails of the mice were harvested and fixed in 4% paraformaldehyde. For the tails of the mice, paraffin sections were prepared at 2, 4, and 6 cm away from the tail tips for hematoxylin and eosin (H&E) staining. The thrombus areas were calculated using Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, United States) and presented as percentages of the whole mice tail vessels. The WPs were prepared, and the proteins were extracted for Western blot to detect the p-AKT/AKT level. The original scans of Western blot are shown in Supplementary Figure S4.

For in vitro experiments, the values are expressed as mean ± standard deviation (SD) of three to five repeated experiments. For ex vivo and in vivo experiments, the values are expressed as mean ± SD of five or seven mice in each group. After validation of the test for homogeneity of variance, one-way ANOVA followed by the Newman–Keuls test for multiple comparisons was used to analyze significant differences among multiple studying groups. In all experiments, p < 0.05 was considered to be statistically significant.

As shown in Figure 2, compared with the vehicle control group (p < 0.05, p < 0.01, or p < 0.001), the percentages of positive platelets with PE-conjugated JON/A antibody binding or FITC-conjugated P-selectin antibody binding on the surface increased significantly when platelets were stimulated by ADP. Compared with the ADP-treated platelets (p < 0.05 or p < 0.01), pretreatment with different concentrations of BBR (0.5–5.0 μM) (Figures 2A–D) or M2 (0.5–5.0 μM) (Figures 2E–H) significantly decreased the percentages of positive platelets.

The inhibitory effects of BBR and M2 on platelet activation at the same concentration were compared. Compared with the ADP group, 0.5 μM of BBR or M2 reduced the proportions of positive platelets binding to the PE-conjugated JON/A antibody by about 43.7% (p < 0.001) and 32.1% (p < 0.01), respectively (Figures 2I,J) and reduced the proportions of positive platelets binding to the FITC-conjugated P-selectin antibody by about 36.5% (p < 0.001) and 26.6% (p < 0.01), respectively (Figures 2K,L). These results indicated that both BBR and M2 could significantly inhibit platelet activation induced by ADP, and BBR has a stronger inhibitory effect on platelet activation than M2 at the same concentration (p < 0.001 or p < 0.01). As a positive control, TGX221 potently suppressed platelet activation induced by ADP, and the proportions of positive platelets almost returned to normal levels after TGX221 treatment (Figures 2I–L).

As shown in Figure 3A, TRITC–phalloidin staining indicated that as compared to the vehicle control group, a large number of the WPs bound to coated fibrinogen after stimulation with ADP, as indicated by a significant increase of the red-stained area. BBR (0.5–5.0 μM) or M2 (0.5–5.0 μM) inhibited the binding of the platelets to fibrinogen, as indicated by an obvious reduction of the red-stained area after pretreatment. To quantitatively analyze the inhibitory effects of BBR and M2, the platelets were stained with calcein-AM, incubated with coated fibrinogen, and fluorescence intensities were determined. As shown in Figure 3B, as low as 0.5 μM of BBR or M2 was able to cause a significant reduction of the binding of platelets to fibrinogen (p < 0.01 vs ADP-induced platelets), and when the concentration of BBR or M2 reached 5.0 μM, a greater extent of binding inhibition was observed (p < 0.01 or p < 0.001 vs ADP-induced platelets). Similar to the inhibitory effects on platelet activation, BBR exhibited a more potent effect to inhibit the binding of fibrinogen to the platelets than did M2 at the same concentration (p < 0.05) (Figure 3B).

To explore whether or not BBR and M2 could directly inhibit the binding of integrin αIIbβ3 to fibrinogen, purified αIIbβ3 was used in our experiments. As shown in Figure 3C, neither BBR nor M2 had any influence on the binding of fibrinogen to purified integrin αIIbβ3. For comparison, tirofiban, a positive control used in the experiments, suppressed the binding of fibrinogen to purified integrin αIIbβ3 significantly (p < 0.001 vs vehicle control) (Figure 3C).

As the activation of PI3K/Akt and Rap1 is crucial for ADP-induced platelet activation (Zhu et al., 2017; Stefanini and Bergmeier, 2019), the effects of BBR and M2 on these molecules were determined. As shown in Figure 4, compared with the vehicle control group (p < 0.05, p < 0.01, or p < 0.001), the phosphorylation of Akt, which reflected the activation of PI3K, and the level of GTP-Rap1 (Rap1 active form) in the platelets simultaneously increased after induction with 10 μM of ADP. BBR and M2 had no effect on the levels of total Akt and Rap1. However, compared with the ADP treatment group, pretreatment with BBR (0.5–5.0 μM) (p < 0.01 or p < 0.001) (Figures 4A–C) or M2 (0.5–5.0 μM) (p < 0.05, p < 0.01, or p < 0.001) (Figures 4D–F) significantly inhibited Akt phosphorylation and reduced the GTP-Rap1 level upon ADP stimulation. Moreover, the effects of BBR were stronger than those of M2 (p < 0.05 or p < 0.01) when administered at the same concentration (Figures 4G–I). As a positive control, TGX221 had potent inhibitory effects on Akt phosphorylation and Rap1 activation stimulated by ADP (p < 0.001 vs ADP treatment group) (Figures 4G–I).

As the Rap1 GTPase-activating protein (GAP) Rasa3 was involved in the modulation of Rap1 activation in the platelets (Battram et al., 2017), the effects of BBR and M2 on Rasa3 were investigated in our experiments. As shown in Figure 5, compared with the vehicle control (p < 0.05 or p < 0.01), the membrane content of Rasa3 increased significantly, while that of cytosol decreased significantly after ADP stimulation, which indicated a membrane translocation of Rasa3. Different concentrations of BBR (Figures 5A,B) or M2 (Figures 5C,D) significantly inhibited the translocation of Rasa3 from the cytoplasm to membrane in the platelets upon ADP stimulation (p < 0.05 or p < 0.01 vs ADP treatment group). The inhibitory effect of BBR on Rasa3 membrane translocation was stronger than that of M2 when administered at the same concentration (p < 0.05) (Figures 5E,F). Actually, 0.5 μM of BBR showed an inhibitory effect similar to that of 100 nM of Wtm, which was used as a positive control in this experiment (Figures 5E,F).

The ADP-Glo lipid kinase systems were used to detect the influences of BBR and M2 on the activities of different class I PI3K isoforms. As shown in Figure 6A, both BBR and M2 significantly inhibited class I PI3Kβ activity (p < 0.001 vs vehicle control), and BBR had a stronger inhibitory effect than M2 when administered at the same concentration (p < 0.05 or p < 0.01). As a positive control, Wtm also potently inhibited class I PI3Kβ activity (p < 0.001 vs vehicle control) (Figure 6A).

Interestingly, neither BBR nor M2 inhibited the activities of class I PI3Kα, PI3Kγ, or PI3Kδ (Figure 6B), which were greatly inhibited by Wtm (p < 0.01 or p < 0.001 vs vehicle control). These results indicate that BBR and M2 are isoform-selective class I PI3Kβ inhibitors.

The class I PI3Kβ protein contains an active site and an ATP catalytic site (Zhang et al., 2011). To explore the mechanisms of BBR and M2 to inhibit class I PI3Kβ, we observed whether or not their inhibitory effects were competitively inhibited by increasing ATP concentrations. As shown in Figures 6C,D, the inhibitory effects of BBR and M2 on class I PI3Kβ activity were not influenced when ATP concentration increased from 6.25 to 400 μM (p < 0.05 or p < 0.01 vs vehicle control).

Then, docking simulation studies were carried out to investigate the binding modes of BBR and M2 with PI3K p110β. As known inhibitors of class I PI3Kβ, TGX211 and Wtm were used as positive controls for the active site (Marshall et al., 2015) and catalytic site (Yano et al., 1993) binding, respectively. The docking scores of BBR, M2, and TGX221 for the active site of PI3K p110β are shown in Table 1, which are −7.24, −6.93, and −7.88 kcal/mol, respectively. The binding mode of BBR with the active site of PI3K p110β is illustrated in Figure 7A. The oxygen atom of the methoxy radical of BBR, regarded as a hydrogen bond acceptor, forms a hydrogen bond with the nitrogen atom of the amino group of Lys799 in the active site of PI3K p110β. The carbon atom of the dioxolane ring of BBR, regarded as a hydrogen-bond donor, forms a hydrogen bond with the sulfur atom of the thioether group of Met920 in the active site of PI3K p110β. The carbon atom of isoquinoline of BBR forms H-π conjugate with the benzene ring of Tyr833 in the active site of PI3K p110β. The binding mode of M2 with the active site of PI3K p110β is illustrated in Figure 7B. The carbon atom of the dioxolane ring of M2, regarded as a hydrogen-bond donor, forms a hydrogen bond with the sulfur atom of the thioether group of Met920 in the active site of PI3K p110β. The carbon atom of isoquinoline of M2 forms H-π conjugate with the benzene ring of Tyr833 in the active site of PI3K p110β. As a positive control, the binding mode of TGX211 with the active site of PI3K p110β is illustrated in Figure 7C. The oxygen atom of the six-membered heterocycles of TGX211, regarded as a hydrogen-bond acceptor, forms a hydrogen bond with the nitrogen atom of the amino group of Lys799 in the active site of PI3K p110β. The oxygen atom of carbonyl of TGX211, regarded as a hydrogen-bond acceptor, forms a hydrogen bond with the oxygen atom of the phenolic hydroxyl group of Tyr833 in the active site of PI3K p110β. The nitrogen atom of TGX211, regarded as a hydrogen-bond donor, forms a hydrogen bond with the sulfur atom of the thioether group of Met920 in the active site of PI3K p110β. The carbon atom TGX211 forms a H-π conjugate with the benzene ring of Tyr833 in the active site of PI3K p110β.

The docking scores of BBR, M2, and Wtm for the catalytic site of PI3K p110β are shown in Table 1, which are −5.42, −5.14, and −6.12 kcal/mol, respectively. The binding mode of BBR with the catalytic site of PI3K p110β is illustrated in Figure 7D. The carbon atom of the dioxolane ring of BBR, regarded as a hydrogen-bond donor, forms a hydrogen bond with the oxygen atom of the carboxyl group of Glu948 in the catalytic site of PI3K p110β. The binding mode of M2 with the catalytic site of PI3K p110β is illustrated in Figure 7E. Similar to BBR, the carbon atom of the dioxolane ring of M2, regarded as a hydrogen-bond donor, forms a hydrogen bond with the oxygen atom of the carboxyl group of Glu948 in the catalytic site of PI3K p110β. The binding mode of Wtm with the catalytic site of PI3K p110β is illustrated in Figure 7F. The oxygen atom of carbonyl of Wtm, regarded as a hydrogen-bond acceptor, forms hydrogen bonds with the nitrogen atoms of the backbone of Asp913 and Phe939 in the catalytic site of PI3K p110β. The carbon atom of Wtm, regarded as a hydrogen-bond donor, forms a hydrogen bond with the oxygen atom of the carboxyl group of Glu948 in the catalytic site of PI3K p110β. Taken together, the computational results indicate that BBR and M2 are more likely to bind to the PI3K p110β active site rather than the catalytic site, which is in agreement with the results of the ATP competition experiment (Figures 6C,D).

To explore the suppressive effects of BBR on platelet activation ex vivo, the mice were orally administrated with 100 mg/kg or 200 mg/kg BBR for 14 days. After treatment, WPs were prepared and stimulated with 10 μM of ADP. Compared with the ADP-stimulated group (p < 0.05 or p < 0.001), 100 mg/kg and 200 mg/kg of BBR significantly reduced the process of positive platelets with PE-conjugated JON/A antibody binding (Figures 8A,B) or FITC-conjugated P-selectin antibody binding (Figures 8C,D) in a dose-dependent manner.

Compared with the ADP-stimulated group (p < 0.01 or p < 0.001), the oral treatment of BBR also inhibited the phosphorylation of Akt and suppressed Rap1 activation (Figures 8E–G) dose-dependently. The ex vivo inhibitory effects of BBR at 200 mg/kg on platelet activation (Figures 8A–D), Akt phosphorylation (Figures 8E,F), and Rap1 activation (Figures 8E,G) were similar to those of Clop at 10 mg/kg (Clop10), which was used as a positive control in this animal experiment.

As shown in Figure 8I, there was no statistically significant difference observed among the studied groups before treatment. After oral administration with 100 mg/kg of Asp or 10 mg/kg of Clop, the tail bleeding time in the mice was prolonged significantly as compared to the vehicle control group (p < 0.01). For comparison purposes, 200 mg/kg of BBR had no influence on the tail bleeding time in the mice. Moreover, when BBR was used in combination with Asp or Clop, it did not further prolong the tail bleeding time in the mice (Figure 8I).

A carrageenan-induced thrombosis model was used to investigate the inhibitory effects of BBR on thrombus formation. The animal experimental scheme is shown in Figure 9A. After i.p. injection, carrageenan induced obvious thrombus formation in the tails of the mice (Figure 9B) with an average thrombosis rate of more than 80% (p < 0.001 vs vehicle control) (Figure 9C). The oral administration of BBR at 50, 100, or 200 mg/kg effectively inhibited carrageenan-induced thrombosis (Figure 9B) and reduced the thrombosis rate in the tails of mice dose-dependently (p < 0.001 vs vehicle + carrageenan group) (Figure 9C). Clop at 10 mg/kg inhibited thrombus formation and reduced the thrombosis rate potently (p < 0.001 vs vehicle + carrageenan group), and the effects were similar to those of BBR at 200 mg/kg (Figures 9B,C).

The tails of the mice were subjected to pathological examination, and the results showed that in the vehicle + carrageenan group, the tail vessel lumen was almost completely occupied by thrombus (Figure 9D), and the percentages of the thrombus area exceeded 60% at 2, 4, and 6 cm from the tail tips (p < 0.001 vs vehicle control) (Figures 9E–G). BBR at different doses greatly reduced the thrombus area at different locations in the tails of the mice (Figures 9D–G). For instance, at 4-cm position from the tail tips, the thrombus area was reduced by about 29.6, 55.9, and 75.5% after receiving a low, middle, or high dose of BBR treatment, respectively (p < 0.05 or p < 0.001 vs vehicle + carrageenan group) (Figure 9F). Furthermore, there was no thrombus observed at the 6-cm position after different doses of BBR treatment (p < 0.001 vs vehicle + carrageenan group) (Figures 9D,G). As a positive control, Clop reduced the thrombus area to an extent similar to that of BBR at 200 mg/kg (Figures 9D–G).

Accompanied by thrombus formation, carrageenan significantly activated the PI3K/Akt signaling pathway in the platelets, as indicated by a significant increase in the p-Akt level after injection (p < 0.001 vs vehicle control) (Figure 9H). The oral administration of BBR (50, 100, and 200 mg/kg) or Clop significantly inhibited the phosphorylation of Akt in platelets (p < 0.05, p < 0.01, or p < 0.001 vs vehicle + carrageenan group) (Figure 9H).