Figure 4.
Platelet-specific PKM2-deficient mice were less susceptible to experimental arterial thrombosis. (A) The left panel shows representative microphotographs of carotid artery thrombus (5% FeCl3 injury) as visualized by upright intravital microscopy in male mice. Platelets were labeled ex vivo with calcein green; white lines delineate the arteries. The middle panel shows time to occlusion (n = 8 mice per group). The right panel shows the rate of thrombus growth (n = 8 mice per group). The rate of thrombus growth over a period of 8 minutes was calculated by dividing the area of the thrombus at the time (n) by the area of the same thrombus at the time (0) (defined as the time point at which the thrombus diameter first reached 30 μm). Slopes over time showed that the rate of thrombus growth in the PKM2fl/flPF4Cre+ mice (slope, 3.488) decreased compared with PKM2fl/fl mice (slope, 4.692). *P < .05. Two-way ANOVA with Tukey’s multiple comparisons test. (B) The left panel shows representative microphotographs of mesenteric artery thrombus (laser injury model) as visualized by upright intravital microscopy in male mice. The right panel shows mean fluorescence intensity over time (n = 10-12 vessels from 4 mice per genotype). *P < .05 compared with vehicle control. Mann-Whitney U test. (C) The left panel shows representative microphotographs of carotid artery thrombus (5% FeCl3 injury) as visualized by upright intravital microscopy in female mice. The middle panel shows time to occlusion (n = 10-11 mice per group). The right panel shows the rate of thrombus growth (n = 8 mice per group). Slopes over time showed that the rate of thrombus growth in the PKM2fl/flPF4Cre+ mice (slope, 2.723) decreased compared with PKM2fl/fl mice (slope, 5.175). *P < .05. Two-way ANOVA with Tukey’s multiple comparisons test. (D) Tail bleeding assay in male and female mice. The tail-transection bleeding time was determined as the time taken for the initial cessation of bleeding after transection. Each symbol represents a single mouse; n = 10 to 16 mice per group. Mann-Whitney U test. (E) The left panel shows representative microphotographs of carotid artery thrombus (5% FeCl3 injury) as visualized by upright intravital microscopy in male mice infused with vehicle or ML265. The middle panel shows time to occlusion, and the right panel shows the rate of thrombus growth. Slopes over time showed that the rate of thrombus growth in the ML265-pretreated mice (slope, 1.531) decreased compared with vehicle control (slope, 4.862). Values are expressed as ± SEM. n = 8 to 11 mice per group. *P < .05. Two-way ANOVA with Tukey’s multiple comparisons test. (F) Tail bleeding assay in male mice pretreated with vehicle or ML265. The horizontal bar shows the mean of each group ± SEM, n = 10 to 11 per group. Mann-Whitney U test. NS, not significant.

Platelet-specific PKM2-deficient mice were less susceptible to experimental arterial thrombosis. (A) The left panel shows representative microphotographs of carotid artery thrombus (5% FeCl3 injury) as visualized by upright intravital microscopy in male mice. Platelets were labeled ex vivo with calcein green; white lines delineate the arteries. The middle panel shows time to occlusion (n = 8 mice per group). The right panel shows the rate of thrombus growth (n = 8 mice per group). The rate of thrombus growth over a period of 8 minutes was calculated by dividing the area of the thrombus at the time (n) by the area of the same thrombus at the time (0) (defined as the time point at which the thrombus diameter first reached 30 μm). Slopes over time showed that the rate of thrombus growth in the PKM2fl/flPF4Cre+ mice (slope, 3.488) decreased compared with PKM2fl/fl mice (slope, 4.692). *P < .05. Two-way ANOVA with Tukey’s multiple comparisons test. (B) The left panel shows representative microphotographs of mesenteric artery thrombus (laser injury model) as visualized by upright intravital microscopy in male mice. The right panel shows mean fluorescence intensity over time (n = 10-12 vessels from 4 mice per genotype). *P < .05 compared with vehicle control. Mann-Whitney U test. (C) The left panel shows representative microphotographs of carotid artery thrombus (5% FeCl3 injury) as visualized by upright intravital microscopy in female mice. The middle panel shows time to occlusion (n = 10-11 mice per group). The right panel shows the rate of thrombus growth (n = 8 mice per group). Slopes over time showed that the rate of thrombus growth in the PKM2fl/flPF4Cre+ mice (slope, 2.723) decreased compared with PKM2fl/fl mice (slope, 5.175). *P < .05. Two-way ANOVA with Tukey’s multiple comparisons test. (D) Tail bleeding assay in male and female mice. The tail-transection bleeding time was determined as the time taken for the initial cessation of bleeding after transection. Each symbol represents a single mouse; n = 10 to 16 mice per group. Mann-Whitney U test. (E) The left panel shows representative microphotographs of carotid artery thrombus (5% FeCl3 injury) as visualized by upright intravital microscopy in male mice infused with vehicle or ML265. The middle panel shows time to occlusion, and the right panel shows the rate of thrombus growth. Slopes over time showed that the rate of thrombus growth in the ML265-pretreated mice (slope, 1.531) decreased compared with vehicle control (slope, 4.862). Values are expressed as ± SEM. n = 8 to 11 mice per group. *P < .05. Two-way ANOVA with Tukey’s multiple comparisons test. (F) Tail bleeding assay in male mice pretreated with vehicle or ML265. The horizontal bar shows the mean of each group ± SEM, n = 10 to 11 per group. Mann-Whitney U test. NS, not significant.

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