Figure 3.
Platelet-specific deletion of PKM2 downregulates a range of platelet functions. (A) The upper panel shows the western blot of PKM2 in platelets from PKM2fl/fl and PKM2fl/flPF4Cre+ mice. The individual lanes are from 3 different mice per group. The lower panel shows the transmission emission microscopy of platelets. The inset in the boxed region is magnified and shown in a microphotograph. Scale bar, 0.5 μm. (B) Platelet-rich plasma or washed platelets from PKM2fl/fl and PKM2fl/flPF4Cre+ were stimulated with different agonists, including convulxin, collagen, PAR4, thrombin, and ADP. Results are expressed as the percent change in light transmission with respect to the blank (platelet-poor plasma/buffer without platelets), set at 100%. The upper panel in each bar graph denotes the representative aggregation curves (blue and red, PKM2fl/fl; black and green, PKM2fl/flPF4Cre+. Values are mean ± SEM, n = 3 to 6 mice per group. *P < .05 vs control. One-way ANOVA followed by Tukey’s multiple comparisons test. Effect of lack of PKM2 on integrin αIIbβ3 activation (C), P-selectin exposure (D), and ATP secretion (E) from dense granules in stimulated platelets with agonists, including convulxin, PAR4, and thrombin. Values are mean ± SEM, n = 3 to 7 mice per group. *P < .05 vs resting platelets, †P < .05 vs PKM2fl/fl. Two-way ANOVA (C and D) and one-way ANOVA (E) followed by Tukey’s multiple comparisons test. (F) Clot retraction was measured for 1 hour in platelet-rich plasma, supplemented with red blood cells, after adding 0.25 U/mL of thrombin. The left panels show the representative images at different time points, and the right panel shows the quantification of the clot size. Platelet-rich plasma was pooled from 5 mice in each group. Values are mean ± SEM, with n = 3 experiments per group. *P < .05 vs PKM2fl/fl, two-way ANOVA with Holm-Šídák’s multiple comparisons test. (G) Mouse whole blood from PKM2fl/fl or PKM2fl/flPF4Cre+ was perfused over a collagen-coated (100 μg/mL) surface for 5 minutes at a shear rate of 1500 s−1 in a BioFlux Microfluidic flow chamber system from Fluxion Biosciences. The left panel shows the representative image at the end of the assay, and the middle panel shows the thrombus growth on the collagen matrix over time. Slopes over time showed that the rate of thrombus growth in the PKM2fl/flPF4Cre+ mice (slope, 58.40) was decreased compared with PKM2fl/fl mice (slope, 108.7). Values are mean ± SEM, n = 3 mice per group. *P < .05. Two-way ANOVA with Tukey’s multiple comparisons test. The right panel shows the surface area covered by fluorescent platelets after 5 minutes of perfusion. Three to 4 areas from different areas of the flow chamber were analyzed from each blood sample; n = 11 areas per group. Mann-Whitney U test. AU, arbitrary unit; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; NS, not significant; PE, phycoerythrin.

Platelet-specific deletion of PKM2 downregulates a range of platelet functions. (A) The upper panel shows the western blot of PKM2 in platelets from PKM2fl/fl and PKM2fl/flPF4Cre+ mice. The individual lanes are from 3 different mice per group. The lower panel shows the transmission emission microscopy of platelets. The inset in the boxed region is magnified and shown in a microphotograph. Scale bar, 0.5 μm. (B) Platelet-rich plasma or washed platelets from PKM2fl/fl and PKM2fl/flPF4Cre+ were stimulated with different agonists, including convulxin, collagen, PAR4, thrombin, and ADP. Results are expressed as the percent change in light transmission with respect to the blank (platelet-poor plasma/buffer without platelets), set at 100%. The upper panel in each bar graph denotes the representative aggregation curves (blue and red, PKM2fl/fl; black and green, PKM2fl/flPF4Cre+. Values are mean ± SEM, n = 3 to 6 mice per group. *P < .05 vs control. One-way ANOVA followed by Tukey’s multiple comparisons test. Effect of lack of PKM2 on integrin αIIbβ3 activation (C), P-selectin exposure (D), and ATP secretion (E) from dense granules in stimulated platelets with agonists, including convulxin, PAR4, and thrombin. Values are mean ± SEM, n = 3 to 7 mice per group. *P < .05 vs resting platelets, †P < .05 vs PKM2fl/fl. Two-way ANOVA (C and D) and one-way ANOVA (E) followed by Tukey’s multiple comparisons test. (F) Clot retraction was measured for 1 hour in platelet-rich plasma, supplemented with red blood cells, after adding 0.25 U/mL of thrombin. The left panels show the representative images at different time points, and the right panel shows the quantification of the clot size. Platelet-rich plasma was pooled from 5 mice in each group. Values are mean ± SEM, with n = 3 experiments per group. *P < .05 vs PKM2fl/fl, two-way ANOVA with Holm-Šídák’s multiple comparisons test. (G) Mouse whole blood from PKM2fl/fl or PKM2fl/flPF4Cre+ was perfused over a collagen-coated (100 μg/mL) surface for 5 minutes at a shear rate of 1500 s−1 in a BioFlux Microfluidic flow chamber system from Fluxion Biosciences. The left panel shows the representative image at the end of the assay, and the middle panel shows the thrombus growth on the collagen matrix over time. Slopes over time showed that the rate of thrombus growth in the PKM2fl/flPF4Cre+ mice (slope, 58.40) was decreased compared with PKM2fl/fl mice (slope, 108.7). Values are mean ± SEM, n = 3 mice per group. *P < .05. Two-way ANOVA with Tukey’s multiple comparisons test. The right panel shows the surface area covered by fluorescent platelets after 5 minutes of perfusion. Three to 4 areas from different areas of the flow chamber were analyzed from each blood sample; n = 11 areas per group. Mann-Whitney U test. AU, arbitrary unit; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; NS, not significant; PE, phycoerythrin.

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