The metabolic enzyme pyruvate kinase M2 regulates platelet function and arterial thrombosis

Abstract

Very little is known about the role of metabolic regulatory mechanisms in platelet activation and thrombosis. Dimeric pyruvate kinase M2 (PKM2) is a crucial regulator of aerobic glycolysis that facilitates the production of lactate and metabolic reprogramming. Herein, we report that limiting PKM2 dimer formation, using the small molecule inhibitor ML265, negatively regulates lactate production and glucose uptake in human and murine stimulated platelets. Furthermore, limiting PKM2 dimer formation reduced agonist-induced platelet activation, aggregation, clot retraction, and thrombus formation under arterial shear stress in vitro in both human and murine platelets. Mechanistically, limiting PKM2 dimerization downregulated phosphatidylinositol 3-kinase (PI3K)-mediated protein kinase B or serine/threonine-specific protein kinase (Akt)/glycogen synthase kinase 3 (GSK3) signaling in human and murine platelets. To provide further evidence for the role of PKM2 in platelet function, we generated a megakaryocyte or platelet-specific PKM2-/- mutant strain (PKM2fl/flPF4Cre+). Platelet-specific PKM2-deficient mice exhibited impaired agonist-induced platelet activation, aggregation, clot retraction, and PI3K-mediated Akt/GSK3 signaling and were less susceptible to arterial thrombosis in FeCl3 injury-induced carotid- and laser injury-induced mesenteric artery thrombosis models, without altering hemostasis. Wild-type mice treated with ML265 were less susceptible to arterial thrombosis with unaltered tail bleeding times. These findings reveal a major role for PKM2 in coordinating multiple aspects of platelet function, from metabolism to cellular signaling to thrombosis, and implicate PKM2 as a potential target for antithrombotic therapeutic intervention.

Graphical abstract

Figure 1.

Human platelet activation is associated with an increase in dimeric PKM2 formation. (A) Western blot of PKM2 in human and murine platelets. (B) The left panel shows a representative image of the nonreducing (native) western blot of PKM2 dimer and tetramer formation in human platelets pretreated with vehicle or ML265 and stimulated with convulxin (100 ng/mL) or thrombin (0.1 U/mL). The red box represents PKM2 dimers. The right panel presents the dimer/tetramer ratio densitometry analysis. Values are mean ± SEM, n = 4 individual donors per group. Two-way ANOVA followed by Tukey’s multiple comparisons test. (C) Lactate production in human platelets with convulxin (100 ng/mL). Values are mean ± SEM, n = 4 individual donors per group. One-way ANOVA followed by Tukey’s multiple comparisons test. (D) Effect of PKM2 inhibition on glucose uptake. Values are mean ± SEM, n = 3 individual donors per group. *P < .05 vs resting platelets, ^†P < .05 vs activated platelets (vehicle). Two-way ANOVA followed by Tukey’s multiple comparisons test. RP, resting platelets.

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.

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 PKM2^fl/fl and PKM2^fl/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 PKM2^fl/fl and PKM2^fl/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, PKM2^fl/fl; black and green, PKM2^fl/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 PKM2^fl/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 PKM2^fl/fl, two-way ANOVA with Holm-Šídák’s multiple comparisons test. (G) Mouse whole blood from PKM2^fl/fl or PKM2^fl/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 PKM2^fl/flPF4Cre⁺ mice (slope, 58.40) was decreased compared with PKM2^fl/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.

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 PKM2^fl/flPF4Cre⁺ mice (slope, 3.488) decreased compared with PKM2^fl/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 PKM2^fl/flPF4Cre⁺ mice (slope, 2.723) decreased compared with PKM2^fl/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.

Figure 5.

PKM2 regulates PI3K-mediated Akt/GSK3β signaling in platelets. (A) Human and mouse platelets were pretreated with vehicle or ML265 (50 and 100 μM) for 10 minutes at room temperature before stimulation with convulxin (100 ng/mL) or thrombin (0.1 U/ml) for 10 minutes. The left panels show representative western blots for phospho-Akt (Ser 473), total Akt, phospho-GSK3β, and total GSK3β. The middle and right panels show densitometry analysis of immunoblots. Values are mean ± SEM, n = 4 per group. *P < .05 vs resting platelets, ^†P < .05 vs activated platelets (vehicle). Two-way ANOVA with Tukey’s multiple comparisons test. (B) Platelets from PKM2^fl/fl or PKM2^fl/flPF4Cre⁺ were stimulated with convulxin (100 ng/mL) or thrombin (0.1 U/mL) for 10 minutes. The left panels show representative western blots for phospho-Akt (Ser 473), total Akt, phospho-GSK3β, and total GSK3β; the middle and right panels show densitometry analysis of immunoblots. Values are mean ± SEM, n = 3 to 4 per group. *P < .05 vs resting platelets, ^†P < .05 vs PKM2^fl/fl. Two-way ANOVA with Tukey’s multiple comparisons test. RP, resting platelets.

Figure 6.

Summary scheme graphic showing a link between PKM2-mediated cellular signaling, platelet function, and thrombosis. In the presence of excess glucose, dimeric PKM2 helps in glucose uptake, and lactate production enhances platelet activation and aggregation via PI3K-mediated Akt/GSK3β signaling to submaximal stimulus-response to GPVI agonists (collagen and convulxin) and GPCR agonists (ADP and thrombin). Glut1, glucose transporter 1; Glut3, glucose transporter 3; PEP, phosphoenolpyruvate; TCA, tricarboxylic acid.