Little is known about the role of metabolic regulatory mechanisms in the pathobiology of deep vein thrombosis (DVT). Recent studies have demonstrated the involvement of the metabolic enzyme pyruvate kinase M2 (PKM2) in platelet function; however, whether platelet PKM2 contributes to DVT has not been investigated yet. Using platelet-specific PKM2^−/− (PKM2^Plt-KO) or wild-type (WT) mice orally administered ML265 (a small molecule that limits PKM2 dimers by stabilizing PKM2 tetramers), we found reduced thrombus burden at 48 h post-surgery in the inferior vena cava (IVC) stenosis model compared with littermate controls. This reduction was associated with lower levels of CitH3, a marker of neutrophil extracellular traps (NETs), in the harvested thrombi and improved IVC wall contraction and relaxation responses (assessed by myography). Mechanistically, thrombin-stimulated platelets from PKM2^Plt-KO mice or ML265-pretreated platelets from WT mice showed reduced SNAP23 phosphorylation and diminished PF4 release (a marker of α-granule exocytosis). The releasate collected from thrombin-stimulated platelets was less effective at inducing NETosis, compared to respective controls. Utilizing ML265-pretreated human whole blood perfused over a tissue factor-coated surface at a venous shear rate, we found that the area covered by platelet-leukocyte aggregates was profoundly reduced compared to vehicle control. Consistent with murine data, human platelets pretreated with ML265 and stimulated with thrombin exhibited decreased PF4 release and generated releasates that were less potent in inducing NETosis. These findings reveal for the first time that targeting PKM2 genetically or pharmacologically reduces SNAP23-mediated α-granule exocytosis in platelets, platelet releasate-induced NETosis, and susceptibility to DVT.

Materials

ML265 was purchased from Cayman (CAS# 1221186-53-3). Apyrase, EGTA (ethylene glycol tetraacetic acid), prostaglandin I₂ (PGI₂), thrombin, and protease inhibitors were purchased from Sigma (St. Louis, MO, USA). Convulxin (CAS # 37206-04-5) was purchased from SantaCruz Biotech. Primary anti-PF4 antibodies (rabbit anti-mouse #ab182988, 1:1000; mouse anti-human #ab49735, 1:1000), anti-CitH3 (#ab5103, 1:1000) from Abcam and goat anti-rabbit IgG, HRP-linked secondary antibody (#7074, 1:2500) were from Cell Signaling Technologies; goat anti-mouse IgG, HRP-linked secondary antibody (#P0447, 1:2500) from Dako. SNAP23 and phospho-SNAP23 antibodies were gifted by Makoto Itakura, Department of Biochemistry, Kitasato University School of Medicine, Japan. Super Signal West Pico chemiluminescent substrate (#34580), Femto Maximum Sensitivity Substrate (#34096), and PVDF membrane (#IPVH00010) were the products from Thermo Scientific and Millipore, respectively. Hoechst dye (#33342) was purchased from ThermoFisher Scientific. Sytox™ green (#S7020) and prolong gold antifade mountant (#P36934) were purchased from Invitrogen. Zombie Violet Fixable Viability Kit (#423114), BV605-conjugated anti-mouse CD41 (#133921), and Alexa Fluor 647–conjugated Annexin V (#640943) were purchased from Biolegend. FITC-conjugated CD62P (#553744) was purchased from BD Pharmingen. All other reagents were of analytical grade.

Methods

Human subjects

Human platelets were obtained from healthy volunteers who provided informed consent and had not taken any antiplatelet medication within the preceding two weeks. Blood collection was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board at the University of Iowa.

Mice

Male mice, aged 12–13 weeks, on the C57BL/6J background were used throughout the study. Wild-type mice were obtained from the Jackson Laboratory. Platelet-specific PKM2-deficient mice (PKM2^fl/flPF4Cre⁺) were generated by crossing PKM2^fl/fl with PF4Cre⁺ mice at the University of Iowa animal facility; littermate PKM2^fl/flPF4Cre^– mice were used as control. For simplicity, from now on PKM2^fl/flPF4Cre⁺ mice will be referred to as PKM2^Plt-KO mice and PKM2^fl/fl controls as PKM2^WT. Mice were genotyped by polymerase chain reaction . Mice were kept in standard animal housing conditions with controlled temperature and humidity, and they had ad libitum access to a standard chow diet and water. The University of Iowa Animal Care and Use Committee approved all experiments.

Human and mouse platelet isolation

Briefly, human venous blood was collected into 10 mL tubes containing acid citric dextrose solution A (ACD-A). Mouse blood from anaesthetized mice was drawn from the retro-orbital plexus and collected into 1.5 mL polypropylene tubes containing enoxaparin (0.3 mg/mL; Sanofi-Aventis, US LLC).

ML265 preparation and administration for in vivo experiments

For in vitro and ex vivo assays, ML265 was dissolved in DMSO and used at final concentrations of 50 or 100 μM, with the final concentration of DMSO maintained at 0.1 % in all samples. For in vivo studies, ML265 was suspended in 0.1 % v/v Tween-80 and 0.5 % w/v carboxymethyl cellulose (CMC). The mice received ML265 at a dose of 50 mg/kg body weight via oral gavage 30 min before inducing DVT and then every 12 h until the animals were sacrificed. Control mice received the same volume of the vehicle (0.1 % v/v Tween-80 and 0.5 % w/v CMC) at the same time points. The appropriate concentrations of ML265 were determined based on our previous study.

Myography

Myography was performed using Myograph System 610M (Danish Myo technology) was used to determine the contraction and relaxation in veins. Briefly, IVC was excised, sufficiently cleaned, and placed into a dish filled with physiologic saline solution (PSS; prepared by dissolving 4.37 g NaCl, 4.47 g KCl, 0.16 g KH₂PO₄, 0.29 g MgSO₄·7H₂O, 1.25 g NaHCO₃, 1.00 g dextrose, 0.0097 g EDTA, and 0.235 g CaCl₂ in 1 L of water) to ensure vessel reactivity. Vessels were then cleaned and mounted on the wire myograph with the chambers filled with PSS and bubbled with a gas mixture of O₂ and CO₂. Myograph channels were zeroed for force readings to establish a baseline, and a wake-up protocol was utilized to ensure viability. The vessels were then subjected to 2-mN tension and allowed to equilibrate for 30 min. Vessels were then exposed to high-potassium (96 mM) saline solution (KPSS; prepared by dissolving 2.26 g NaCl, 7.16 g KCl, 0.16 g KH₂PO₄, 0.29 g MgSO₄·7H₂O, 1.25 g NaHCO₃, 1.00 g dextrose, 0.0097 g EDTA, and 0.235 g CaCl₂ in 1 L of water) to elicit contraction responses. This response was carried out for 15 min. The vessels were then washed 3 times for 10 min each with PSS. The wake-up protocol was performed twice. Next, vessels were contracted utilizing phenylephrine (0.5 mM) and then subjected to acetylcholine (1.62 mM) to relax for 10 minutes. The maximum relaxation point was considered when the curve plateau was maintained for 2 minutes.

NETosis assay

Freshly isolated neutrophils from the mouse bone marrow or human whole blood were seeded onto poly-L-lysine-coated coverslips (1 × 10⁴ cells/coverslip). The cells were incubated at 37 °C for 1 h in a CO₂ incubator and then co-incubated with thrombin-stimulated platelets (4 × 10⁸ cells/mL) or their releasates for an additional 4 h in the same conditions. In this assay, platelets were treated with 0.1 U/mL thrombin for 10 min, centrifuged at 5000g for 5 mins at room temperature, and the resulting supernatant (releasate) was collected for further experiments. Ice-cold PBS was added to stop the reaction, and the coverslips were placed on ice for 10 min. The cells were fixed for 15 min in ice-cold PBS containing 2 % paraformaldehyde at room temperature. The fixed cells were then washed with ice-cold PBS. For specific staining of extracellular nuclear structures and nuclei, cells were incubated at room temperature with Sytox Green (Sigma) dye and Hoechst (Thermo Scientific), respectively, for 30 min at room temperature. Coverslips were washed with PBS and mounted onto glass slides using a drop of the mounting medium (ProLong™ Gold Antifade Mountant, ThermoFisher) before fluorescence microscopy analysis.

NETs quantification: NETs were imaged using fluorescence microscopy and analyzed with ImageJ (NIH, Bethesda, MD, USA). Images were converted to 8-bit grayscale, and a global threshold was applied. The total number of neutrophils based on size (~20 μm in diameter) was quantified from Hoechst staining using the Analyze Particles tool with a size range of 20–2000 μm². NET-positive cells (forming extracellular traps; web-like structure) were identified as irregular structures with a size range of ~50–2000 μm². For quantification, two 20x magnification fields were analyzed (coverslip edges were avoided). The percentage of NET-positive neutrophils was calculated by dividing the number of NET-positive cells by the total neutrophil count and multiplying by 100.

Flow cytometry

Mouse whole blood samples were collected through the retro-orbital plexus into ACD-A 48 h post-DVT surgery. Samples were diluted 1:20 in the Tyrode buffer (20 mM HEPES, 138 mM NaCl, 2.9 mM KCl, 1 mM MgCl₂, 0.36 mM NaH₂PO₄, supplemented with 5 mM glucose and 0.2 % bovine serum albumin, pH 7.4), incubated with 2.1 mM CaCl₂ for 10 min, stained with the antibody cocktail (Zombie Violet, CD41-BV605, CD62P-FITC, and Annexin V-Alexa Fluor 647) for 20 min in the dark, fixed in 0.2 % paraformaldehyde for 10 min in the dark, and analyzed using Cytek Aurora spectral flow cytometer (Cytek Biosciences Inc., Fremont, CA, USA). Platelet population of interest was characterized as Live^{+CD41+CD62P+AnnexinV+} cells.⁶ Antibodies were used at the final concentrations recommended by the manufacturers. All steps were performed at room temperature.

Bioflux flow chamber assay

Thrombosis assays with human whole blood were performed using BioFluxTM 200 (Fluxion Biosciences, USA) microfluidics flow chamber. The channels were coated with tissue factor (TF) (Innovin stock diluted 1:9 in HBSS) for 1 h at room temperature and then blocked with 1 % Bovine serum albumin (BSA) for 20 min. The whole blood was incubated with DiOC6 (5 μM) for 10 min. The whole blood was then pretreated with ML265 or vehicle (DMSO) for 10 min. Next, whole blood was treated with 10 mM of CaCl₂ and immediately perfused over the TF-coated plate at a venous shear stress of 500 s⁻¹ for 8 min. The fluorescently labeled platelets/thrombi on the TF-coated surface were analyzed with ImageJ (NIH, Bethesda, MD, USA).

Western blot analysis

Briefly, immediately after harvesting, a DVT thrombus was placed in a Petri dish containing PBS on ice to prevent protein degradation, transferred to a pre-chilled microcentrifuge tube containing an ice-cold RIPA lysis buffer, and homogenized by using micro-tube homogenizer system. The homogenate was centrifuged at 6000 × g for 10 min at 4 °C to remove cellular debris, and the supernatant was collected for further analysis. Thrombus lysate and platelet lysate proteins were separated on precast 4–20 % SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gradient gels and electrophoretically transferred to PVDF (polyvinylidene fluoride) membrane using Bio-Rad western blotting system. For PKM2 dimer and tetramer study, handcast 6 % native gels (without SDS) are used. PVDF membranes were blocked with 5 % BSA or skimmed milk in 10 mM Tris-HCl, 150 mM NaCl, pH 8.0 (TBS) containing 0.05 % Tween-20 for one hour at room temperature. Blots were incubated with a primary antibody overnight at 4 °C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were developed using enhanced chemiluminescence and quantified using Image J software from NIH (Bethesda, MD, USA).

Ponceau Staining: PVDF membranes were rinsed with distilled water, then incubated in the Ponceau S solution for 30 min at room temperature on a shaker to stain the protein bands. After staining, membranes were washed in distilled water for 2–3 min until the red protein bands were clearly visible against the clear background.

Plasma recalcification assay

Briefly, the blood was collected from mice in 3.8% trisodium citrate. The platelet-poor plasma (PPP) was obtained by centrifuging the blood at 2650 rpm for 5 min and 30 s. The recalcification of plasma was initiated by adding 25 mM CaCl₂. The optical density of plasma at 405 nm was observed using a spectrophotometer (Spectramax ID3, Molecular Devices) every minute up to 10 min to estimate fibrin formation in PPP.

Deep venous thrombosis (DVT) model

Briefly, mice were anesthetized and underwent a midline laparotomy. A spacer (30-gauge, 3-mm-long needle) was positioned on the exposed IVC, and a permanent narrowing ligature (with 5-0 nonabsorbable silk sutures) was tied around the IVC and spacer immediately caudal to the junction of the left renal vein. Next, the spacer was removed to restrict the IVC blood flow by 80-90 %. All visible side branches were ligated. Mice were then allowed to recover and were sacrificed 48 h post-surgery (the time required for complete thrombus formation in this model). Control and experimental mice were operated on in batches on the same day to minimize day-to-day variation. Thrombi were isolated from the vessels, and their length and weight were measured. In some mice, thrombi were further processed for western blotting.

Microfluidic studies using custom-made DVT device

DVT was simulated ex vivo using a custom-fabricated microfluidic device consisting of a 150-µm wide channel expanding into a 300 µm-wide channel at an angle of 150° to define two model valve pockets. TF (undiluted) adsorbed into the wide channel and pockets by careful backfilling via pipette aspiration and allowed to incubate for 1 h at room temperature. The device was flushed with 2 % BSA and then blocked with 2 % BSA for 1 h. Citrated whole blood was pre-treated with ML265 (150 μM) or vehicle and incubated with DiOC6 (1 µM) to visualize platelets and leukocytes for 15 min at 37 °C. Whole blood and recalcification buffer (75 mM CaCl₂ and 37.5 mM MgCl₂ in HEPES-buffered saline, pH 7.4) were mixed upstream of the inlet of the device in T-junction at a volumetric ratio of 9:1 (blood : buffer) and perfused through the device at a flow rate of 372 µL/min for 30 min using two syringe pumps (Harvard Apparatus PhD 2000 and New Era NE300), corresponding to a Reynolds number (the ratio of inertial forces/viscous forces) of 10, enabling secondary flows into the valve pockets. Platelet and leukocyte accumulation was captured by confocal microscopy (Olympus IX83 with Yokogawa CSU-W1 Confocal Scanner Unit, 20x) every 10 s. Kinetic data all utilized the same combined region of interest (ROI) based on the dimensions of the valve pockets in the device. Fluorescent images were thresholded using the ‘Max Entropy’ algorithm in ImageJ and processed into binary masks. The area covered over time within each valve pocket ROI was measured using ImageJ and normalized to the total area of each valve pocket ROI. Final images (20x) at the end of the experiment (25 min) were used for endpoint thrombus size measurements, and fraction of total valve pocket area was reported.

Statistical analysis

Continuous variables were presented as mean ± standard error (SEM) or as median with interquartile range and range, as specified. Categorical variables were presented as counts, proportions, and/or percentages. For continuous variables, differences between two groups were evaluated using the Mann–Whitney U test or Student’s t-test, and differences between three or more groups using one- or two-way ANOVA followed by a post hoc multiple comparisons test. For categorical variables, differences between two groups were evaluated using the Fisher’s exact test. Two-tailed P values less than 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism software, version 10.6.0.