Qiliqiangxin capsule attenuates platelet activation and thrombosis by suppressing Ca2+ influx and PKC signaling

Fig. 1. QLQX treatment inhibits platelet activation in patients with CHF. (A) 4-week QLQX administration inhibited platelet aggregation in response to ADP (2 µM) and collagen (0.8 µg/mL) in PRP from patients with CHF. (B-C) 4-week QLQX administration suppressed platelet spreading on immobilized fibrinogen (100 µg/mL) (B) and clot retraction induced by thrombin (1.0 U/mL) (C) in patients with CHF. Scale bar = 10 μm. (D) Whole blood from patients with CHF exhibited reduced platelet adhesion and ex vivo thrombus formation on immobilized collagen (100 µg/mL) at a shear rate of 1000 s− 1 after 4-week QLQX administration. Scale bar = 200 μm. Statistical analyses were performed using a paired two-tailed Student’s t-test. n = 10. ***P < 0.001

Fig. 2. QLQX shows no significant pharmacological toxicity. (A) Administration of QLQX to mice at a dose of 0.468 g/kg/day (o.g.) for 7 days did not induce obvious liver or kidney damage. This was determined by analyzing serum biomarkers: alanine aminotransferase (ALT) and aspartate transaminase (AST) for hepatic injury, and uric acid (UA) and blood urea nitrogen (BUN) for renal injury. (B) Administering QLQX to mice at a dose of 0.468 g/kg/day for seven days did not affect blood cell count and platelet parameters. RBC, red blood cells; WBC, white blood cells; PLT, platelets; MPV, mean platelet volume. Statistical analyses were performed using an unpaired two-tailed Student’s t test in (A) and Mann-Whitney U test in (B). n = 10. ns, not significant

Fig. 3. QLQX inhibits platelet activation in mice. (A-B) QLQX treatment did not affect platelet ultrastructure in mice. Representative transmission electron microscopy (TEM) images of platelets (A, left; Scale bar = 5 μm–1 μm) and quantitative analysis of platelet area (A, right) and platelet α-granules and dense granules (B). Wild-type mice were treated with QLQX (0.468 g/kg/day, o.g.) or vehicle control for 7 days before analysis. n = 10. (C-D) QLQX administration (0.468 g/kg/day, 7 days, o.g.) in mice did not affect expression levels of platelet surface receptors, including αIIb (CD41), β3 (CD61), GPVI, and GPIbα (CD42b) as assessed by flow cytometry. Representative (C) and summary data (D) are shown, with values expressed as MFI. n = 5. (E) QLQX administration (0.468 g/kg/day, 7 days, o.g.) in mice inhibited platelet integrin αIIbβ3 activation (JON/A) and P-selectin exposure (CD62P) as detected by flow cytometry. n = 5. (F) Oral administration of QLQX to mice at doses of 0.234, 0.468, and 0.936 g/kg/day for 7 days concentration-dependently inhibited platelet aggregation induced by ADP (10, 20 µM), thrombin (0.03, 0.05 U/mL), and collagen (0.5, 1.0 µg/mL). n = 5. Statistical analyses…

Fig. 4. QLQX suppresses mouse platelet spreading, clot retraction, and ex vivo thrombus formation. (A) QLQX administration inhibited mouse platelet adherence and spreading on immobilized fibrinogen (100 µg/mL). Scale bar = 10 μm. (B) QLQX administration inhibited clot retraction induced by thrombin (1.0 U/mL) in mouse platelets. (C) QLQX administration inhibited ex vivo thrombus formation over an immobilized collagen surface at a shear rate of 1000 s− 1 in whole blood. Scale bar = 200 μm. Wild-type mice were randomly assigned to receive either QLQX (0.468 g/kg/day) or vehicle by oral gavage for 7 days. Statistical analyses were performed using two-way ANOVA followed by Sidak’s multiple comparisons test. n = 5. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5. QLQX inhibits in vivo thrombus formation and microvascular obstruction in myocardial ischemia-reperfusion (I/R) injury. (A) QLQX administration inhibited FeCl3-induced thrombus formation in mice. Scale bar = 200 μm. (B-C) QLQX administration inhibited collagen and epinephrine-induced pulmonary embolism formation. H&E staining of lung sections, the arrows indicate emboli (B), and immunohistochemistry staining of platelets using CD42b antibody in lung sections (C). Scale bar = 200 μm. (D-E) QLQX administration decreased the infarct area/AAR ratio (D) and myocardial I/R injury-induced microthrombi (CD42b+) in the cardiac tissue (E) from mice after myocardial I/R injury at 48 h. Scale bar = 100 μm. Wild-type mice were randomly assigned to receive either QLQX (0.468 g/kg/day) or vehicle by oral gavage for 7 days. Data were analysed by unpaired two-tailed Student’s t test in (A, C-E) and Mann-Whitney U test in (B). n = 10. ns, not significant, *P < 0.05, ***P < 0.001

Fig. 6. QLQX shows no significant effect on bleeding and coagulation parameters. (A) QLQX administration did not significantly alter bleeding time or blood loss. (B) QLQX administration did not significantly affect plasma prothrombin time (PT) or activated partial thromboplastin time (APTT). Wild-type mice were randomly assigned to receive either QLQX (0.468 g/kg/day) or the vehicle control by oral gavage for seven days. Data were analysed by an unpaired two-tailed Student’s t-test. n = 10. ns, not significant

Fig. 7. QLQX suppresses platelet Ca2+ influx and PKC phosphorylation (A-B) Platelet RNA sequencing (RNA-seq) was performed on platelets of QLQX- or vehicle-treated mice. n = 3. KEGG enrichment pathway annotation results are shown, with the six first-level KEGG pathways distinguished in different colors (A). Bubble plot of KEGG enrichment analysis shows the top 15 KEGG pathway enrichment results for differentially expressed genes (B). (C) QLQX administration inhibited Ca2+ influx in washed platelets stimulated with 10 μM ADP, 0.05 U/mL thrombin, or 0.5 μg/mL collagen-related peptide (CRP). n = 5. (D) Western blot analysis shows that QLQX inhibits PKC substrates phosphorylation in washed platelets stimulated with 10 μM ADP, 0.05 U/mL thrombin, or 0.5 μg/mL collagen. n = 5. Wild-type mice were randomly assigned to receive either QLQX (0.468 g/kg/day) or the vehicle control by oral gavage for seven days. Statistical analyses were performed using two-way ANOVA followed by Sidak’s multiple comparisons test in (C-D). **P < 0.01, ***P < 0.001