Panax notoginseng saponins inhibit platelet activation and thrombosis while preserving hemostasis: involvement of platelet oxylipin metabolic reprogramming

Figures

• 

  Fig. 1. PNS inhibited GPαIIbβ3 activation, platelet α granule release and platelet aggregation ex vivo. A, B Expression of P-selectin in platelets at rest and stimulated with thrombin analyzed by flow cytometry. C, D GPIIb-IIIa activation in platelets at rest and stimulated with thrombin analyzed by flow cytometry. E, F Max platelet aggregation in response to AA and ADP measured by light transmission aggregometry. Data were mean ± SEM. Each dot represents one rat (all n=12). Statistical significance was determined using the Kruskal-Wallis H test followed by all pairwise multiple comparisons. Horizontal lines with asterisks (*) indicate statistical significance between the connected groups (*p < 0.05, **p < 0.01)

• 

  Fig. 2. PNS ameliorated in vivo thrombosis, infarct expansion and attenuated ischemic-induced myocardial injury and heart failure. A, B The time to first thrombus (thrombosis time) and time to vessel occlusion (occlusion time) in mice mesenteric arterioles. Each dot represents one mouse. C Representative images of TTC staining. D Infarct size. Each dot represents one rat. E Representative images of myocardial histopathology by H&E staining (× 100, Scale bar: 200 μm; × 200, Scale bar: 100 μm). F Myocardial histopathological score for quantitative representation of cardiac injury. Each dot represents one rat. G Representative images of Masson's trichrome staining. H Representative images of echocardiography. I, J LVEF and FS analyzed in the parasternal short-axis plane. Each dot represents one rat (all n=12). Data were median (interquartile range) in (A, B and F) were mean ± SEM in (D, I and J). Statistical significance was determined using the Kruskal-Wallis H test followed by all pairwise multiple comparisons. Horizontal lines with asterisks (*) indicate statistical significance between the connected groups (*p < 0.05, ** p < 0.01)

• 

  Fig. 3. PNS preserved hemostasis in tail bleeding mice. A-C TT, APTT and bleeding time. D-F red blood cell count, hemoglobin concentration and platelet count. G, H Serum level of ALT and AST. I, J Serum level of creatinine and blood urea nitrogen. P=NS, not statistically significant. Data were mean ± SEM in (A, B, D-J) and was median (interquartile range in (C). Each dot represents one mouse (all n=8). Statistical significance was determined using one-way ANOVA with LSD as post hoc correction or APTT, and the Kruskal-Wallis H test followed by all pairwise multiple comparisons for all other parameters. Horizontal lines with asterisks (*) indicate statistical significance between the connected groups (*p < 0.05, ** p < 0.01)

• 

  Fig. 4. Lipidomic analysis unraveled perturbation of oxylipin metabolism post AMI in rat platelets. A OPLS-DA score plot demonstrated distinct intraplatelet oxylipin profile in AMI rats compared with those of sham control. B Validation of OPLS-DA model by permutation testing. C VIP score plot. D Heatmap provided a metabolic snapshot of the oxylipin profile in the two groups and visualized the metabolic perturbation under AMI condition. n = 12 for each group

• 

  Fig. 5. Lipidomic analysis uncovered significantly altered oxylipins that were trigger by AMI. Intraplatelet concentration of (A) AA-COX-1 derived oxylipins, (B) AA-5-LOX derived oxylipins, (C) 12-HETE and (D) 14,15-diHETrE were shown in box plots. Each dot represents one rat (n = 12). Data were presented as median (interquartile range) in all panels. Exact p values are annotated above the horizontal lines connecting the two groups

• 

  Fig. 6. Platelet lipidomic analysis revealed remodeling of oxylipin metabolism in response to PNS intervention. A OPLS-DA score plot illustrated distinct intraplatelet oxylipin profile in rats treated with high dose of PNS compared with AMI control. B Validation of OPLS-DA model by permutation testing. C VIP score plot. D Heatmap visualized the metabolic reprograming after PNS treatment. n = 12 for each group

• 

  Fig. 7. Oxylipins that were markedly altered after PNS intervention suggested attenuation of thrombo-inflammation in AMI state. A AA-COX-1 derived oxylipins, (B) 12-HETE, (C) AA-5-LOX derived oxylipins and (D) EPA-COX-1 derived oxylipins were shown in box plots. Each dot represents one rat (n = 12). Data were presented as median (interquartile range) in all panels. Exact p values are annotated above the horizontal lines connecting the two groups

• 

  Fig. 8. Mechanistic model illustrated that PNS inhibited platelet activation and attenuated arterial thrombosis while preserving basal hemostasis. AMI-induced platelet lipidomic perturbations that fueled thrombo-inflammation were shifted toward an antithrombotic phenotype by PNS, accompanied by mitigation of inflammatory responses