Figure 2.
ML265 treatment regulates dimeric PKM2 formation to inhibit multiple aspects of human platelet functions. (A) Human platelet-rich plasma pretreated with vehicle or ML265 and stimulated with convulxin (5 ng/mL), collagen (2.5 μg), TRAP (10 μM), and ADP (5 μM). 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, vehicle-control; black, 50 μM ML265; red, 100 μM ML265). Values are mean ± SEM, n = 5 individual donors per group. One-way ANOVA followed by Tukey’s multiple comparisons test. Effect of dimeric PKM2 inhibition on integrin αIIbβ3 activation (B), P-selectin exposure (C), and ATP secretion (D) from dense granules in stimulated-platelets with convulxin (5 ng/mL), TRAP (50 μM), and thrombin (0.1 U/mL). Values are mean ± SEM, n = 4 to 5 individual donors per group. *P < .05 vs resting platelets, †P < .05 vs vehicle. Two-way ANOVA (B and C) and one-way ANOVA (D) with Tukey’s multiple comparisons test. (E) Clot retraction was measured for 1 hour in platelet-rich plasma, supplemented with red blood cells, after adding 0.25 U/mL of thrombin in the presence of a vehicle or ML265 (50 and 100 μM). The left panels show representative images at different time points; the right panel shows the quantification of clot size with time. Values are mean ± SEM, n = 4 individual donors per group. Two-way ANOVA with Tukey’s multiple comparisons test. (F) Human whole blood pretreated with vehicle or ML265 (150 μM) 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. The middle panel shows the thrombus growth on the collagen matrix over time. Slopes over time showed that the rate of thrombus growth in ML265-treated whole blood (slope, 37.92) was decreased compared with vehicle control (slope, 237.3). Values are mean ± SEM, n = 3 individual donors 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. Values are mean ± SEM, n = 11 areas per group; Mann-Whitney U test. MFI, mean fluorescence intensity; PE, phycoerythrin.

ML265 treatment regulates dimeric PKM2 formation to inhibit multiple aspects of human platelet functions. (A) Human platelet-rich plasma pretreated with vehicle or ML265 and stimulated with convulxin (5 ng/mL), collagen (2.5 μg), TRAP (10 μM), and ADP (5 μM). 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, vehicle-control; black, 50 μM ML265; red, 100 μM ML265). Values are mean ± SEM, n = 5 individual donors per group. One-way ANOVA followed by Tukey’s multiple comparisons test. Effect of dimeric PKM2 inhibition on integrin αIIbβ3 activation (B), P-selectin exposure (C), and ATP secretion (D) from dense granules in stimulated-platelets with convulxin (5 ng/mL), TRAP (50 μM), and thrombin (0.1 U/mL). Values are mean ± SEM, n = 4 to 5 individual donors per group. *P < .05 vs resting platelets, †P < .05 vs vehicle. Two-way ANOVA (B and C) and one-way ANOVA (D) with Tukey’s multiple comparisons test. (E) Clot retraction was measured for 1 hour in platelet-rich plasma, supplemented with red blood cells, after adding 0.25 U/mL of thrombin in the presence of a vehicle or ML265 (50 and 100 μM). The left panels show representative images at different time points; the right panel shows the quantification of clot size with time. Values are mean ± SEM, n = 4 individual donors per group. Two-way ANOVA with Tukey’s multiple comparisons test. (F) Human whole blood pretreated with vehicle or ML265 (150 μM) 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. The middle panel shows the thrombus growth on the collagen matrix over time. Slopes over time showed that the rate of thrombus growth in ML265-treated whole blood (slope, 37.92) was decreased compared with vehicle control (slope, 237.3). Values are mean ± SEM, n = 3 individual donors 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. Values are mean ± SEM, n = 11 areas per group; Mann-Whitney U test. MFI, mean fluorescence intensity; PE, phycoerythrin.

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