A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth

Abstract

To uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34^lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.

Graphical abstract

Figure 1.

Defective platelet production and granule distribution in Vps34-deficient platelets. (A) Whole blood platelet count was measured by using a HORIBA ABX Micros 60 analyzer (mean ± SEM; n = 38 mice for WT and 49 for Pf4-Cre-Pik3c3^lox/lox mice [Vps34]); ***P < .001 vs WT according to 2-tailed Student t test) (left). Quantification of the percentage of mice with a mean platelet volume ranging from 4 to 7 µm³ and from 7 to 10 µm³ (mean ± SEM; n = 38 mice for WT and 49 for Pf4-Cre-Pik3c3^lox/lox mice [Vps34]) (middle). TEM of resting platelets (right). Images are representative of 5 mice of each genotype. Scale bar represents 2 µm. (B) Mice were intravenously injected with a dylight⁴⁸⁸–anti-GPIbβ immunoglobulin derivative antibody. The percentage of labeled platelets in blood samples was measured at various time points after injection. (C) Thrombocytopenia in mice was induced by intraperitoneal injection of anti-GPIbα antibody (left). The platelet count was measured in blood samples collected 6 hours after injection (time = 0) and at various time points. The mouse serum TPO level was quantified by immunoassay (middle). Platelets were incubated with 50 ng/mL of TPO at the indicated times and after fixation with a rat antibody against the extracellular domain of Mpl and an anti-rat Alexa Fluor⁴⁸⁸ antibody (right). The graph is expressed as the percentage of the mean fluorescence intensity (MFI) resting (0) values after flow cytometry analysis (mean ± SEM; n = 4-6 mice of each genotype, *P < .05 vs WT according to 2-way ANOVA). (D) TEM of resting platelets. Images are representative of 5 mice of each genotype. Scale bar represents 1.5 µm. Arrows indicate α-granules. Platelet α- and dense (δ)-granule numbers and mean areas were measured on TEM images by using ImageJ software (mean ± SEM; n = 5 mice of each genotype; *P < .05; ***P < .001 vs WT according to 2-tailed Student t test).

Figure 2.

Vps34 is critical for MK migration and granule biogenesis. (A) TEM of MKs from native bone marrow sections. Images are representative of 5 mice of each genotype. Scale bar represents 5 µm (upper images) and 1 µm (lower images). The α-granule number and mean area were quantified on 70-µm² field of TEM images by using ImageJ software (mean ± SEM; n = 5 mice of each genotype; **P < .01; ***P < .001 vs WT according to 2-tailed Student t test). (B) WT or Vps34-deficient (Vps34) MKs were exposed to a SDF1α gradient within the Dunn chamber. Migration paths over 6 hours of 5 representative MKs from 8 independent experiments in each graph were traced. The intersection of the x-axis and y-axis was taken to be the starting point of each cell path, whereas the source of the SDF1α was at the top. The accumulated distance and directionality from WT MKs in the presence of dimethyl sulfoxide (WT) or 1 μM VPS34-IN1 or from Vps34-deficient (Vps34) MKs were analyzed by using the ImageJ software manual tracking plug-in (mean ± SEM; n = 30-50 MKs from 8 independent experiments; **P < .01 vs WT according to 1-way ANOVA). (C) Representative confocal images of immunostained native bone marrow. MKs and platelets are labeled by vWF staining (green). FABP4/A-FABP staining (red) labels sinusoid vessels. Nucleus staining was done by 4′,6-diamidino-2-phenylindole (blue). Scale bars represent 50 μm. The graph represents the percentage of platelets inside and outside the sinusoids (mean ± SEM; n = 20 images from 4 mice of each genotype, ***P < .001 vs WT according to 2-tailed Student t test).

Figure 3.

Defective intracellular trafficking and PI3P production in Vps34-deficient MKs. (A-C) MK uptake of transferrin-Alexa Fluor⁵⁴⁶ (A) or fibrinogen-Alexa Fluor⁴⁸⁸ (B-C) was observed by confocal (A) or superresolution structured illumination microscopy (B-C) at different incubation time points. (A-B) Graphs represent fluorescence intensity quantified on a MK z-stack by ImageJ or Imaris software. (C) Representative 3-dimensional surface rendering of z-stacks acquired after 960 minutes of fibrinogen uptake are shown. Scale bar represents 5 µm. Graphs represent fibrinogen-positive structure number and volume quantified over time on 3-dimensional images using Imaris software (mean ± SEM; n = 10-60 MKs from 3 mice of each genotype; *P < .05; ***P < .001 vs WT according to 2-way ANOVA). (D-E) Fixed MKs stained with anti-clathrin, anti-EEA1, anti-Rab11, or anti-LAMP1 antibodies followed by corresponding secondary Alexa Fluor⁴⁸⁸ antibodies were observed by confocal microscopy. Live MKs were stained with LysoTracker Deep Red, fixed, and observed by confocal microscopy. Representative images of a z-stack are shown. Scale bar represents 5 µm. Graphs represent the structure number and area analyzed on a z-stack with ImageJ software (mean ± SEM; n = 30-50 MKs from 3 mice of each genotype; *P < .05; ***P < .001 vs WT according to 2-tailed Student t test). (F) PI3P mass assay performed on MKs as described in “Methods” (mean ± SEM; n = 5; **P < .01 vs WT according to 1-sample Student t test). (G) MKs stained with anti-PI3P and secondary Alexa Fluor⁴⁸⁸ antibodies were observed by confocal microscopy, and fluorescence intensity was quantified by using ImageJ software (mean ± SEM; n = 40 MKs from 3 mice of each genotype; ***P < .001 vs WT according to 2-tailed Student t test).

Figure 4.

Vps34 regulates stimulation-dependent PI3P production in platelets. (A) PI3P content was analyzed by mass assay in washed resting WT platelets treated for 1 hour with dimethyl sulfoxide (WT) or 1 μM VPS34-IN1 and Vps34-deficient platelets (Vps34) (mean ± SEM; n = 3-5; **P < .01 vs WT according to 1-sample Student t test). (B) PI3P content from WT platelets treated for 1 hour with dimethyl sulfoxide (WT) or 1 μM VPS34-IN1 and from Vps34-deficient platelets (Vps34) in resting or stimulated (CRP, 10 µg/mL; thrombin, 0.5 UI/mL) conditions was assessed by mass assay (mean ± SEM; n = 5; *P < .05; **P < .01 vs WT according to 2-way ANOVA). (C) HPLC analysis of the PI3P level of resting and stimulated (CRP, 10 µg/mL; thrombin, 0.5 IU/mL) Pi-labeled platelets (mean ± SD; n = 2). (D) Vps34 was immunoprecipitated from resting or activated (CRP, 10 µg/mL; thrombin, 0.5 IU/mL) washed platelets and assayed for lipid kinase activity in vitro. Graph represents Vps34 activity (fold increase) normalized to the levels of immunoprecipitated kinase in each condition as assessed by immunoblot and densitometry analysis (mean ± SEM; n = 5; ***P < .001 vs resting according to 1-sample Student t test).

Figure 5.

Vps34 plays an important role in thrombosis via its kinase activity. (A) Tail bleeding time (n = 30 mice of each genotype) was measured as described in “Methods.” (B) The thrombotic response of mice to carotid injury after exposure to 7.5% ferric chloride for 3 minutes was assessed by a flow probe. Graph represents the percentage of mice without occlusion 30 minutes after injury (n = 15 mice of each genotype; P = .0002 according to 1-sample Student t test). (C-D) DiOC₆-labeled platelets in mouse whole blood were perfused through collagen-coated microcapillaries at a physiological arterial shear rate of 500 s⁻¹ (C) or at an arteriolar shear rate of 1500 s⁻¹ (D). Scale bar represents 20 µm. The surface covered by fluorescent platelets and thrombus volume were analyzed using ImageJ software (mean ± SEM; n = 8 mice of each genotype; *P < .05; **P < .01 vs WT according to 2-tailed Student t test and 1-sample Student t test). (E) Healthy human donor whole blood was treated with 1 µM VPS34-IN1, 1 µM SAR405, or vehicle (dimethyl sulfoxide) for 1 hour. Then, DiOC₆-labeled whole blood was perfused through collagen-coated microcapillaries at a physiological shear rate of 1500 s⁻¹. Scale bar represents 20 µm. The surface covered by fluorescent platelets and thrombus volume were analyzed using ImageJ software (mean ± SEM; n = 3-5 healthy donors depending on the inhibitor; *P < .05; **P < .01 vs vehicle according to 2-way ANOVA and 1-sample Student t test).

Figure 6.

Vps34 kinase activity regulates platelet secretion. (A) Kinetics of ATP secretion of washed platelets under resting or stimulated conditions (CRP, 1 µg/mL; thrombin, 0.1 IU/mL) were recorded by measuring the luminescence from the firefly luciferin-luciferase reaction by lumi-aggregometry using the Chrono-log aggregometer. Graphs represent the percentage of WT maximal secretion at 80 seconds (mean ± SEM; n = 6-15 mice of each genotype depending on the agonist; *P < .05; **P < .01; ***P < .001 vs WT according to 2-way ANOVA). (B) Kinetics of vWF secretion of washed platelets under resting or CRP (1 µg/mL) or thrombin (0.1 IU/mL) stimulated conditions was analyzed by enzyme-linked immunosorbent assay . The results are expressed as the fold increase compared with resting WT platelets (mean ± SEM; n = 6 mice of each genotype; *P < .05 vs resting WT according to 2-way ANOVA). The kinetics of ATP secretion (C) and vWF secretion (D) of washed platelets treated 1 hour with vehicle (dimethyl sulfoxide) or 1 μM VPS34-IN1 and stimulated with CRP (1 µg/mL) or thrombin (0.1 IU/mL) were analyzed as described above (mean ± SEM; n = 6; *P < .05 vs resting WT according to 2-way ANOVA). (E) Washed platelets were spread on a fibrinogen-coated surface for 20 minutes in the presence or absence of apyrase (2 IU/mL), and the platelet surface was measured using ImageJ software (mean ± SEM; n = 3 mice per genotype; **P < .01 vs WT according to 2-way ANOVA). (F) Whole blood from WT mice treated for 1 hour with dimethyl sulfoxide (WT) or 1 μM VPS34-IN1 or from Pf4-Cre-Pik3c3^lox/lox mice was perfused through a collagen-coated microcapillary in the presence of fluoxetine (25 µM) at a physiological shear rate of 1500 s⁻¹. The serotonin content was quantified in plasma by immunoassay (mean ± SEM normalized by thrombus volume; n = 3-5 per condition; ***P < .001 vs WT according to 1-way ANOVA). (G) Unlabeled whole blood from WT (WT > WT) or Pf4-Cre-Pik3c3^lox/lox (WT > Vps34) mice were perfused through collagen-coated microcapillaries at 1500 s⁻¹ for 1 minute, and were then replaced by DiOC₆-labeled WT whole blood perfused at the same shear rate. The surface covered by fluorescent platelets was analyzed by using ImageJ software (mean ± SEM; n = 5 mice of each genotype; ***P < .001 vs WT > WT according to 2-way ANOVA test).