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DREAM plays an important role in platelet activation and thrombogenesis

Kyungho Kim, Alan Tseng, Andrew Barazia, Joseph E. Italiano and Jaehyung Cho

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    DREAM is required for platelet thrombus formation and hemostasis at the site of vascular injury in mice. Fluorescence intravital microscopy was performed as described in “Methods.” Following laser-induced injury of cremaster muscle arterioles, platelet accumulation was visualized by infusion of DyLight 649–labeled anti-mouse CD42c antibodies into WT or DREAM KO mice. (A) Representative images are shown over the course of 240 seconds after injury. Arrows show direction of blood flow. (B) The integrated median fluorescence intensities of anti-CD42c antibodies (F platelet) were obtained from 42 to 44 thrombi in 6 WT or 6 KO mice and are presented as a function of time. (C) Fluorescence intensities of anti-CD42c antibodies in WT and DREAM KO mice were compared at 30, 90, and 180 seconds after arteriolar injury. (D) Tail bleeding time was monitored by cutting 5 mm of the tail of WT (●) and DREAM KO (▪) mice (n = 13 mice). (E) Blood loss during the bleeding time assay was determined by measuring the absorbance at 575 nm of hemoglobin (Hb). **P < .01 and ***P < .001 vs WT mice after the Mann-Whitney U test. Horizontal bars represent the median value of the fluorescence intensities of anti-CD42c antibodies, bleeding times, or Hb content.

  • Figure 2.

    Both hematopoietic and nonhematopoietic cell DREAMs participate in thrombosis and hemostasis, and platelet DREAM is important for thrombogenesis. WT or DREAM KO transplant control mice and DREAM bone marrow chimeras (hematopoietic [HP] or non-HP DREAM KO) were used for intravital microscopy as described in Figure 1. (A) Representative images. Arrows show direction of blood flow. (B-C) F platelet and fluorescence intensities of anti-CD42c antibodies in 4 different groups are presented as described in Figure 1 (32-38 thrombi in 5 mice of each group). **P < .01 and ***P < .001 vs WT control mice and #P < .05, ##P < .01, and ###P < .001 between 2 groups after the Mann-Whitney U test. (D) Cremaster arteriolar thrombosis was induced by FeCl3 injury. Time to occlusion was plotted (n = 8-10). (E-F) Tail bleeding time and blood loss were measured as described in Figure 1D-E (n = 8). (G-I) Fluorescently labeled WT or DREAM KO platelets, 108 in 100 µL of saline, were infused into thrombocytopenic WT mice. Three thrombi were generated following laser injury. The same labeled platelets were reinfused into the mouse to generate 3 additional thrombi. Accumulation of infused platelets was monitored and analyzed as described above. Horizontal bars represent the median value of the fluorescence intensities of anti-CD42c antibodies (C,I), time to occlusion (D), bleeding times (E), or Hb content (F). *P < .05 and ***P < .001 vs WT platelets after the Mann-Whitney U test (n = 4-5 mice for each group) or after the Dunn test (D-F).

  • Figure 3.

    Platelet DREAM regulates aggregation and ATP secretion following stimulation with numerous agonists. (A) Lysates of gel-filtered mouse and human platelets (hPLT) were immunoblotted. Representative blots were obtained from 3 independent experiments. (B) WT and DREAM KO platelets were treated with or without 0.5 µg/mL CRP, followed by transmission electron micrographs. Original magnification was ×4800 and extension was ×13 000. The arrows show α-granules. Bar, 500 nm. (C-G) Platelet aggregation and ATP secretion were induced by 0.5 µg/mL CRP, 100 nM A23187, 10 µM ADP, 0.0125 U/mL thrombin, and 0.25 µM U46619. In the ADP-induced aggregation assay, 30 µg/mL human FG was added to the platelet suspension before ADP stimulation. (I) Platelet aggregation and quantitative graphs. (II) ATP secretion was monitored with platelet aggregation with a luciferin/luciferase reagent. Data represent the mean ± SD (n = 3). (H-I) WT and DREAM KO platelets were pretreated with (H) 1 U/mL apyrase or (I) 1 µM ADP, and aggregation was induced by CRP, respectively. (J) WT and DREAM KO platelets were pretreated with 1 mM aspirin, followed by stimulation with 0.0125 U/mL thrombin. Platelet aggregation and quantitative graphs are shown. Data represent the mean ± SD (n = 3). **P < .01 and ***P < .001 vs WT platelets after the Student t test.

  • Figure 4.

    Platelet DREAM plays a critical role in PI3K activation through GPVI-, integrin-, or A23187-mediated signaling. (A) WT and DREAM KO platelets were stimulated by 0.5 µg/mL CRP for 1, 2, and 5 minutes under stirring conditions (1000 rpm) in an aggregometer. An equal amount of cell lysate protein (50 µg) was immunoblotted, followed by densitometry (arbitrary unit [AU], n = 5). (B-D) WT and DREAM KO platelets were stimulated with 0.5 µg/mL CRP for 2 minutes under stirring conditions. The lysates were immunoprecipitated with control IgG or antibodies against (B) LAT, (C) DREAM, or (D) total PI3K p85 (t-PI3K p85) followed by immunoblotting and densitometry (n = 4). (E-F) Human platelets were stimulated with 0.5 µg/mL CRP for 2 minutes under stirring conditions, followed by coimmunoprecipitation as shown in panels C and D. (G-H) WT and DREAM KO platelets were stimulated by 10 µM ADP for 1, 2, or 5 minutes in the (G) absence or (H) presence of 30 µg/mL FG under stirring conditions (n = 4). (I-J) WT and DREAM KO platelets were pretreated (I) without or (J) with 20 µg/mL eptifibatide following stimulation with 100 nM A23187 for 1, 2, or 5 minutes with stirring. An equal amount of lysate protein was immunoblotted, followed by densitometry (n = 4). *P < .05 and **P < .01 vs WT platelets at each time point (or resting platelets for panels C-F) after the Student t test.

  • Figure 5.

    DREAM is important for platelet activation through PI3K-Iβ activity. (A-C) WT and DREAM KO platelets were pretreated (A-B) without or (C) with 0.05% dimethyl sulfoxide (DMSO) (vehicle), 50 µM LY294002 (LY), 0.1 µM wortmannin (Wort), or an isoform-specific inhibitor for PI3K-Iα (0.5 µM PIK75) or PI3K-Iβ (0.5 µM TGX221), followed by stimulation with 0.5 (for panel C), 1, or 2 µg/mL CRP. P-selectin exposure or αIIbβ3 integrin activation was analyzed by flow cytometry. The data are presented as the geometric mean fluorescence intensity (MFI) value (n = 3). (D-E) WT and DREAM KO platelets were incubated with a Ca2+ dye and treated with 0.5 µg/mL CRP or 100 nM A23187 in the absence of exogenous Ca2+ and the presence of 1 mM EGTA. Cytosolic Ca2+ levels were measured and quantified by area under the curve (AUC) (n = 3). (F-G) WT and DREAM KO platelets were incubated with vehicle or a PI3K inhibitor, followed by the Ca2+ assay as described above (n = 4). *P < .05, **P < .01, and ***P < .001 vs WT platelets after Student t test (for panels A-B and D-E) or vehicle-treated WT (white bars) or DREAM KO platelets (gray bars) after ANOVA and the Tukey test (for C and F-G). #P < .05 and ##P < .01 between 2 groups. (H-I) WT and DREAM KO platelets were pretreated with vehicle or a PI3K inhibitor as described above, followed by stimulation with 1 µg/mL CRP or 100 nM A23187. (J-K) WT and DREAM KO platelets were pretreated with vehicle or a PI3K inhibitor and then incubated (J) without or (K) with 1 mM aspirin, followed by stimulation with 0.0125 U/mL thrombin. Representative results of aggregation are presented (n = 3).

  • Figure 6.

    DREAM regulates Ca2+ mobilization in MEG-01 cells and its regulatory function requires Ca2+ binding and PI3K-Iβ activity. Control or DREAM siRNA (#3 or #4) was transfected into MEG-01 cells. (A-B) After 48 hours, cell lysates were immunoblotted, followed by densitometry (arbitrary unit [AU], n = 4). (C) After 48 hours, cells were incubated with a Ca2+ dye and treated with 100 nM A23187, followed by measurement of cytosolic Ca2+ levels (n = 4). *P < .05 vs control after the Student t test. (D) A schematic of DREAM siRNA-resistant wt and EF-hand mutant DREAM. (E) DREAM siRNA #4 was transfected into MEG-01 cells. After 4 hours, cells were transfected with a vector expressing siRNA #4–resistant wt or EF-hand mutant DREAM. After 48 hours, DREAM overexpression was verified by immunoblotting (n = 4). (F) The cells described in panel E were used for Ca2+ assays. **P < .01 and ***P < .001 vs control after the Student t test (for siRNA#4) or wt DREAM-overexpressing cells (for EF-hand mutant groups) after ANOVA and the Tukey test. ##P < .01 between 2 groups. (G) Control or DREAM siRNA #4 was transfected into MEG-01 cells. After 48 hours, cells were incubated with a Ca2+ dye and treated with 0.05% DMSO, 25 µM LY294002 (LY), 0.1 µM wortmannin (Wort), 0.5 µM PIK75, or 0.5 µM TGX221. Ca2+ release was measured following stimulation with 100 nM A23187 (n = 4). (H) Control or DREAM knockdown MEG-01 cells were pretreated with vehicle or a PI3K inhibitor and stimulated with A23187 for 5 minutes. Immunoblotting was performed as described in “Methods” (n = 3). *P < .05, **P < .01, and ***P < .001 vs control (white bars in panel G) or DREAM siRNA-treated (gray bars in panel G) cells (vehicle group), or control siRNA-treated, stimulated cells (for panel H) after ANOVA and the Tukey test. #P < .05 and ###P < .001 between 2 groups.

  • Figure 7.

    Model of a novel role for platelet DREAM. Megakaryocyte DREAM mRNA and protein are transferred to platelets during thrombopoiesis. In platelets, DREAM regulates platelet activation by modulating PI3K-Iβ activity (for example, in GPVI-mediated signaling). ADP signaling further activates αIIbβ3 integrin and amplifies integrin outside-in signaling which also activates PI3K-Iβ through DREAM.

Tables

  • Table 1.

    The number of circulating blood cells in WT and DREAM KO mice

    WBC, 109/LNE, 109/LLY, 109/LMO, 109/LRBC, 109/LPLT, 109/LMPV, fL
    WT5.0 ± 1.60.7 ± 0.23.3 ± 1.80.1 ± 0.09.0 ± 1.21138 ± 1677.3 ± 1.6
    DREAM KO3.7 ± 1.50.5 ± 0.21.7 ± 0.70.1 ± 0.09.2 ± 1.01154.3 ± 1598.3 ± 0.3
    • Blood cells from WT and DREAM KO mice were counted using an automated hematology analyzer (ADVIA 120; Siemens AG). Data represent the mean ± SD (n = 6 mice per group).

    • LY, lymphocyte; MO, monocyte; MPV, mean platelet volume; NE, neutrophil; PLT, platelet; RBC, red blood cell; WBC, white blood cell.

  • Table 2.

    The number of circulating blood cells in control and DREAM bone marrow chimeric mice

    WBC, 109/LNE, 109/LLY, 109/LMO, 109/LRBC, 109/LPLT, 109/LMPV, fL
    WT4.2 ± 1.30.7 ± 0.23.2 ± 1.50.1 ± 0.09.0 ± 1.31088 ± 1677.6 ± 1.6
    HP DREAM KO3.1 ± 0.70.7 ± 0.23.0 ± 0.50.1 ± 0.010.5 ± 1.41011 ± 1787.4 ± 1.8
    Non-HP DREAM KO3.3 ± 0.51.0 ± 0.33.8 ± 0.80.1 ± 0.09.9 ± 1.01054 ± 1598.3 ± 1.3
    DREAM KO3.2 ± 0.60.6 ± 0.22.7 ± 0.70.1 ± 0.09.5 ± 1.0889 ± 1107.3 ± 1.5
    • Blood cells from WT control, DREAM KO control, and their bone marrow chimeric mice (HP and Non-HP DREAM KO) were counted using an automated hematology analyzer (ADVIA 120; Siemens AG). Data represent the mean ± SD (n = 6 mice per group).

    • HP, hematopoietic; Non-HP, nonhematopoietic. Other abbreviations are explained in Table 1.