High Dose of Metformin Decreases Susceptibility to Occlusive Arterial Thrombosis in Diabetic Mice

Figures

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  Figure 1. Diabetic mouse model development and oral Metformin studies methodologic scheme. Created with BioRender.

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  Figure 2. DM mice FeCl3 arterial thrombosis and flow cytometry analysis on 7-day oral Metformin (200mg/kg) treated mice. A) Effect of Metformin in vivo, decreased hyperglycemia in DM mice after 7 days with no effect in healthy mice(these mice did not exhibit hyperglycemia); student T-test B) Metformin shortens time to thrombus formation in normal mice while there is an apparent protective effect in DM mice; One way ANOVA: p 0.0258 with multiple comparisons. C) Representative image of FeCl3 surgical procedure with black arrow pointing to area of thrombus formation (white). D-E) Metformin increased platelet activation via P-selectin expression mostly with Collagen stimulation in DM mice after Metformin treatment; One-way ANOVA: p <0.0001 with multiple comparisons. F) In healthy mice Metformin increases platelet adhesion (GpIIb/IIIa expression) under Collagen stimulation with a minimal effect under ADP simulation; One-way ANOVA: p 0.0005 with multiple comparisons. G) In DM mice, Metformin decreased GpIIb/IIIa expression under Collagen stimulation with an inverse effect with ADP showing increase in GpIIb/IIIa expression; One-way ANOVA: p 0.0003 (G), p <0.0001 with multiple comparisons (H).

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  Figure 3. Whole blood 7-day oral Metformin (200mg/kg) aggregometry and flow cytometry with ADP/Ca+ and Collagen/Ca+ in healthy mice. A-C) Whole blood aggregometry with Collagen (1ug/mL). Data showed increased aggregation by AUC with Metformin treatment orally 200mg/kg twice daily compared to mice given vehicle for 7 days twice a day. Lag time is increased in Metformin treated mice; One-way ANOVA: p <0.0001 (A), p 0.0057 (B) with multiple comparisons. D-F) Whole blood aggregometry with ADP (20uM) showed increased aggregation by AUC in mice given Metformin orally 200mg/kg twice daily compared to mice given vehicle for 7 days twice a day. Paradoxically to our earlier findings in increased lag time in Metformin treated mice stimulated with Collagen, when we used ADP as an agonist for aggregometry lag time increase effect was lost; One-way ANOVA: p <0.0001 (D), p 0.1754 with multiple comparisons (E).

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  Figure 4. FeCl3 arterial thrombosis, bleeding time on 7-day Metformin treated mice. A) Metformin treatment results in faster time of occlusive thrombosis compared to vehicle treated mice*. B-C) Representative images of the readout of the time of occlusive thrombosis in the FeCl3 model in 7-day vehicle and Metformin treated mice. D) Oral Metformin treatment results in shorter tail bleeding time compared to vehicle treated mice. E-F) Representative images show amount of blood collected during tail bleeding assays and sediment of cells after centrifugation. G L-M) Platelet activation marker analysis with urinary Thromboxane and correlation with plasma Cystatin C levels for acute kidney injury development analysis showed no differences between vehicle and Metformin treated mice for 7 days. *Data was represented again in figure 2 to compare DM mice to healthy mice and is shown in this panel to show the effect of Metformin in normal mice.

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  Figure 5. Platelet mitochondrial respiration after 7-Day Metformin Treatment. Due to chronic disease state due to diabetes, DM mice have higher threshold of platelet mitochondrial bioenergetics compared to healthy mice. Results show that DM mice do not evidence any differences in platelet bioenergetic parameters. Contrarily, healthy mice treated with Metformin show increase in maximal respiration and spare respiratory capacity. There also appeared to be an increase in basal respiration, proton leak and ATP production in Metformin treated healthy mice but there were no significant statistical differences found. Two-side t-test was performed: p<0.01 (C-D).