Platelet mass is enhanced in PKCε deficient mice.

<p>A) Western blot analysis of PKCε, PKCα, and PKCδ in PKCε^+/+ and PKCε^-/- mouse megakaryocytes (n = 3). Actin was used to assess loading. B) Platelet counts from PKCε^-/- and WT littermate control (PKCε^+/+) whole blood (n = 9). C) Reticulated “new” platelets expressed as a percent of total blood cells in PKCε^+/+ and PKCε^-/- mice (n = 6). D) Platelet clearance in PKCε^+/+ and PKCε^-/- mice (n = 3). E) Representative images of femur sections stained with H&E. White arrows denote megakaryocytes. Images were captures used a Nikon E1000 microscope at 200X magnification. F) Quantitation of megakaryocytes per field of view (FOV). * p < 0.05, n = 11.</p

Blood cell counts in PKCε^+/+ and PKCε^-/- mice.

<p>Blood cell counts in PKCε^+/+ and PKCε^-/- mice.</p

PKC-epsilon deficiency alters progenitor cell populations in favor of megakaryopoiesis

<div><p>Background</p><p>It has long been postulated that Protein Kinase C (PKC) is an important regulator of megakaryopoiesis. Recent contributions to the literature have outlined the functions of several individual PKC isoforms with regard to megakaryocyte differentiation and platelet production. However, the exact role of PKCε remains elusive.</p><p>Objective</p><p>To delineate the role of PKCε in megakaryopoiesis.</p><p>Approach and results</p><p>We used a PKCε knockout mouse model to examine the effect of PKCε deficiency on platelet mass, megakaryocyte mass, and bone marrow progenitor cell distribution. We also investigated platelet recovery in PKCε null mice and TPO-mediated signaling in PKCε null megakaryocytes. PKCε null mice have higher platelet counts due to increased platelet production compared to WT littermate controls (p<0.05, n = 8). Furthermore, PKCε null mice have more bone marrow megakaryocyte progenitor cells than WT littermate control mice. Additionally, thrombopoietin-mediated signaling is perturbed in PKCε null mice as Akt and ERK1/2 phosphorylation are enhanced in PKCε null megakaryocytes stimulated with thrombopoietin. Finally, in response to immune-induced thrombocytopenia, PKCε null mice recovered faster and had higher rebound thrombocytosis than WT littermate control mice.</p><p>Conclusions</p><p>Enhanced platelet recovery could be due to an increase in megakaryocyte progenitor cells found in PKCε null mice as well as enhanced thrombopoietin-mediated signaling observed in PKCε deficient megakaryocytes. These data suggest that PKCε is a negative regulator of megakaryopoiesis.</p></div

Platelet recovery and rebound thrombocytosis is enhanced in PKCε^-/- mice following immune-induced thrombocytopenia.

<p>A) PKCε^+/+ and PKCε^-/- Mice were injected (I.P.) with 50 μg/kg anti-mouse CD41 antibody at day 0. Blood was collected daily via submandibular puncture for 5 days and again on day 7 and platelets were enumerated. * p < 0.05 compared to corresponding PKCε^+/+ time point, n = 7. B) Representative images of proplatelet producing megakayrocytes. C) Quantitation of proplatelet production taken from several fields per experiment, expressed as a percentage of total megakaryocytes. *p < 0.05, n = 11.</p

PKCε^-/- mice have a reduced LSK population, but a heightened megakaryocyte progenitor cell population.

<p>A) Schematic showing gates used to define each progenitor cell population. LK cells are defined as Lineage (Lin) negative cells that stain C-Kit+, Sca-1-. LSK cells stain Lin-, Sca-1+, C-Kit+. Megakaryocyte progenitors (MkP) are from the LK population and stain CD41+, CD150+. The LSK population is used to define multipotent progenitor cells (MPP), which stain CD105- and CD150-, as well as hematopoietic stem cells (HSC), which stain CD105+ and CD150+. B-F) Quantification of each progenitor cell population as defined in A in PKCε^+/+ and PKCε^-/- mouse bone marrow expressed as a percentage of total bone marrow cells. * p < 0.05 compared to PKCε^+/+, n = 4.</p

Megakaryocyte number is enhanced in PKCε^-/- bone marrow cultured with exogenous TPO.

<p>A) Megakaryocyte DNA content in PKCε^-/- and PKCε^+/+ bone marrow cultured in the presence of 50 ng/mL TPO. B) Megakaryocyte number is cultured bone marrow from PKCε^-/- and PKCε^+/+ mice supplemented with 50 ng/mL TPO. * p < 0.05 compared to PKCε^+/+, n = 7.</p

Treatment with the PKCδ inhibitor decreased sepsis-induced platelet-leukocyte aggregate formation.

<p>Blood samples were incubated with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression and cell shape and data were analyzed as a percentage of aggregates expressing both CD61 and CD11b (<b>A</b>) Representative dot plot images of percentage of neutrophil-platelet aggregates in rat blood samples of Sham, CLP plus vehicle and CLP plus PKCδ inhibitor. (<b>B</b>) Graph showing the percentage of aggregate formation of the data represented in (A) (*p <0.05 sham versus CLP, **<i>p</i> < 0.01; CLP plus vehicle versus CLP plus PKCδ inhibitor, n = 4).</p

PKCδ inhibitor decreases sepsis-mediated activation of platelet PKCδ.

<p>Rat platelets were isolated from blood samples of the following groups: Sham, vehicle-treated CLP and CLP treated with with PKCδ inhibitor. PKD2 phosphorylation was measured by Western Blotting analysis using phospho-specific antibody against PKD2. A representative image (top) and the ratio between phospho-PKD2 and loading control band (bottom) are shown. (*<i>p</i> < 0.05; Sham versus CLP plus vehicle; CLP plus vehicle versus CLP plus PKCδ inhibitor, n = 5).</p

The effect of PKCδ inhibition on sepsis-induced lung injury, neutrophil sequestration and platelet influx in the lung and BALF.

<p>(<b>A</b>) Photomicrographs of hematoxylin- and eosin-stained tissue sections were obtained 24 hours after Sham or CLP surgery. Representative images of lung tissue specimens are shown for sham, CLP and CLP plus PKCδ inhibitor (Magnification 20; <i>n</i> = 5). (<b>B</b>) MPO analysis was performed in lung samples of sham and CLP rats. Values are expressed as units per gram of tissue (*<i>p</i> < 0.05; sham versus CLP plus vehicle and CLP plus vehicle versus CLP plus PKCδ inhibitor, n = 6). (<b>C</b>) Platelet accumulation in the lung was studied thought fluorescence microscopy (blue) DAPI; Green CD61, platelets, n = 4). (<b>D</b>) Fluorescence mean of CD61 staining values (**<i>p</i><0.01 Sham versus CLP plus vehicle and CLP plus vehicle versus CLP plus PKCδ inhibitor, n = 4). (<b>E</b>) Platelet counts were analyzed in the BAL fluid of Sham and CLP animals. Cells were counted using a Hemavet^® Multispecies Hematology System (*<i>p</i> < 0.05; Sham versus CLP plus vehicle and CLP plus vehicle versus CLP plus PKCδ inhibitor, n = 6).</p

Treatment with the PKCδ inhibitor alters platelet secretion into the lungs of septic rats.

<p>PF4 concentration in the BALF was analyzed by ELISA. Data are expressed as International Units (IU)/ml (*<i>p</i> < 0.05; Sham versus CLP plus vehicle and CLP plus vehicle versus CLP plus PKCδ inhibitor, n = 6).</p