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Diacylglycerol kinase ζ is a negative regulator of GPVI-mediated platelet activation

Alyssa J. Moroi, Nicole M. Zwifelhofer, Matthew J. Riese, Debra K. Newman and Peter J. Newman

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    DGKζ negatively regulates GPVI-mediated platelet activation but enhances thrombin (Thr)-mediated platelet activation. (A) Whole lysates of washed platelets and megakaryocytes (MKs) from WT and DGKζ-KO mice were analyzed by western blotting for DGKζ and actin. The blot shown on the left is representative of 3 to 4 independent experiments. Quantification of band densities (right) is reported as the mean ± SEM (n = 3-4). (B) Platelet counts (left) and mean platelet volumes (MPVs; right) observed in WT and DGKζ-KO mice. Values represent means ± SEM (n = 20). (Ci) Aggregation of washed platelets from WT and DGKζ-KO mice was measured by lumiaggregometry in response to high (top) and low (bottom) doses of CRP, collagen (Col), Thr, adenosine 5′-diphosphate (ADP), and U46619. Each aggregation tracing is representative of 3 to 5 independent experiments. (ii) Dose-response curve of maximal platelet aggregation after CRP, Col, and Thr stimulation. Values represent the mean ± SEM of maximal platelet aggregation observed in 3 to 4 independent experiments. (D) P-selectin exposure (left) and Jon/A binding as a reporter of activation of integrin αIIbβ3 (right) were measured by flow cytometry in washed platelets after 20 minutes of stimulation with CRP, Thr, ADP, and U46619 (U46) at the indicated concentrations. Values represent the mean ± SEM of mean fluorescence intensity (MFI) observed in 5 independent experiments. (E) Quantification of peak adenosine triphosphate (ATP) released from platelets stimulated with CRP (2.5 μg/mL), Col (5 μg/mL), and Thr (0.5 U/mL) relative to WT. ATP release was measured by lumiaggregometry. Values represent the mean ± SEM observed in 3 to 5 independent experiments. Statistical analysis was performed by the unpaired Student t test. *P < .05, **P <.01, ***P < .001, ****P < .0001 of DGKζ-KO as compared with WT. n.s., not significant.

  • Figure 2.

    DGKζ-KO platelets exhibit enhanced platelet spreading on substrates for both GPVI and integrin αIIbβ3. Washed platelets from WT and DGKζ-KO mice were allowed to adhere on immobilized CRP (50 μg/mL), collagen (50 μg/mL), or fibrinogen (30 μg/mL) for the indicated times at 37°C. After washing with phosphate-buffered saline to remove unbound platelets, adherent platelets were fixed and stained with phalloidin-TRITC. (A) Images of spread platelets representative of 3 independent experiments are shown. Bars represent 10 μm. (B) The average area covered by individual platelets was quantified from at least 4 images per substrate and 250 to 500 platelets per time point. Spreading area is reported as the mean ± SEM. Statistical analysis was performed using the unpaired Student t test. ****P < .0001 of DGKζ-KO as compared with WT.

  • Figure 3.

    DGKζ-KO mice exhibit normal hemostasis but faster time to platelet plug formation after arterial injury. (A) Bleeding assay was performed by tail tip amputation followed by immersion in phosphate-buffered saline at 37°C. Symbols represent times to cessation of bleeding for individual animals, and error bars represent the mean ± SEM (n = 10-12). (B) ROTEM was performed using whole blood collected from WT and DGKζ-KO mice and adjusted to the indicated platelet count. Results are shown as maximal clot firmness (MCF; n = 5; left) and clot formation time (CFT; n = 5; right). Symbols represent MCF and CFT for individual animals, and bars represent the mean ± SEM. (C-D) The right carotid artery was injured by applying 10% FeCl3 for 3 minutes. Blood flow was monitored with a Doppler flow probe until 3 minutes after full occlusion. (C) Representative flow traces for WT and DGKζ-KO mice. (D) Symbols represent times to occlusion in individual animals, and bars represent the mean ± SEM (n = 10). Statistical analysis was performed by the unpaired Student t test. *P < .05 of DGKζ-KO as compared with WT.

  • Figure 4.

    DGKζ deficiency improves platelet adhesion to collagen under conditions of flow. Whole blood collected from WT and DGKζ-KO mice was labeled with mepacrine and flowed over collagen-coated microchannels. (A) Representative images of platelet coverage under conditions of arterial shear (1500 s−1). Images were taken under ×20 objective. (B) Surface area coverage at the indicated time points was quantified and reported as the mean ± SEM (n = 6). (C) Microchannels were washed after whole blood perfusion for 300 seconds and fixed, permeabilized, and stained with phalloidin-TRITC; Z stack images were then taken. (i) Representative Z stack images. Bars represent 100 μm. The volume (ii) and height (iii) of each platelet thrombus at 300 seconds were quantified and reported as the mean ± SEM (n = 5; 76-116 thrombi per sample). Statistical analysis was performed by the unpaired Student t test. *P < .05, **P < .01, ***P < .001 of DGKζ-KO as compared with WT.

  • Figure 5.

    Increased expression of GPVI in DGKζ-KO platelets. (A) Platelet surface receptor expression of GPVI (i-ii), integrin α2 (iii), integrin αIIb (iv), PECAM-1 (v), GPIbα (vi), and CLEC-2 (vii) was measured in diluted whole blood samples from WT and DGKζ-KO mice by flow cytometry. Mean fluorescence intensity (MFI) is reported as the mean ± SEM (n = 10). (B) Whole lysates of washed platelets from WT and DGKζ-KO mice were analyzed by western blotting for GPVI, GPIIIa, FcRγ chain, and actin. The blots are representative of 4 independent experiments. Quantification of band densities (right) is reported as the mean ± SEM (n = 4). Statistical analysis was performed using the unpaired Student t test. *P < .05, ****P < .0001 of DGKζ-KO as compared with WT platelets. FITC, fluorescein isothiocyanate; IgG, immunoglobulin G.

  • Figure 6.

    DGKζ deficiency enhances surface expression of GPVI without increased messenger RNA (mRNA) in murine megakaryocytes. (A) Bone marrow–derived hematopoietic stem cells from WT and DGKζ-KO mice were cultured in TPO plus SCF for 0, 1, 5, or 8 days to obtain megakaryocytes, and surface expression of GPVI, integrin α2, integrin αIIb, and GPIbα was measured by flow cytometry. Mean fluorescence intensity (MFI) is reported as the mean ± SEM (n = 6). (B) Mature megakaryocytes were enriched by using a BSA density gradient, and RNA was extracted. Quantitative RT-PCR was performed, and relative expression levels of GPVI, FcRγ chain, integrin α2, integrin αIIb, GPIbα, and DGKζ in DGKζ-KO megakaryocytes are shown relative to WT expression levels (n = 5). Hypoxanthine-guanine phosphoribosyltransferase was used for normalization. *P < .05, **P < .01 of DGKζ-KO as compared with WT megakaryocytes. N.D., not detectable.

  • Figure 7.

    DGKζ deficiency enhances signaling in response to GPVI activation. WT and DGKζ-KO washed platelets were stimulated with 1 μg/mL of CRP for 90 and 300 seconds under stirring conditions in an aggregometer. Lysates were analyzed for DGKζ, phosphorylated ERK (P-ERK; Thr202/Tyr204), ERK2, P-Lyn (Tyr 507), Lyn, P-PLCγ2 (Tyr 1217), PLCγ2, P-Akt (Thr 308), Akt, and actin. (A) Blot shown is representative of 3 independent experiments. (B) Quantification of band densities normalized to actin over all experiments is reported as the mean ± SEM (n = 3). Statistical analysis was performed using the unpaired Student t test. *P < .05, **P < .01, ****P < .0001 of DGKζ-KO as compared with WT platelets.

Tables

  • Table 1.

    Blood parameters of WT and DGKζ-KO mice (n = 20)

    Blood parameterMean ± SEM
    Platelet count, 106/mLPlatelet volume, μm3WBC count, 106/mLRBC count, 109/mLHematocrit, %
    WT469.7 ± 16.25.5 ± 0.12.9 ± 0.37.0 ± 0.233.4 ± 0.8
    DGKζ-KO466.8 ± 24.25.7 ± 0.12.7 ± 0.26.9 ± 0.233.9 ± 0.9
    • RBC, red blood cell; WBC, white blood cell.