Normal integrin outside-in signaling in <i>Efhd2^-/-</i> platelets.

<p>(A-C) Washed platelets of <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> mice were allowed to spread on fibrinogen (100 µg/mL) for 30 min after stimulation with 0.01 U/mL thrombin. (A) Statistical evaluation of the percentage of spread platelets at different spreading stages and (B) representative differential interference contrast (DIC) images of 2 individual experiments. 1: roundish, 2: only filopodia, 3: filopodia and lamellipodia, 4: fully spread. (C) Visualization of filamentous actin (red) in spread (30 min) <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets by cofocal microscopy. (D) Clot retraction of prp upon activation with 4 U/mL thrombin in the presence of 20 mmol/L CaCl₂ at the indicated time points (n = 6).</p

Normal αIIbβ3 activation and α-granule release in <i>Efhd2^-/-</i> platelets.

<p>Flow cytometric analysis of integrin αIIbβ3 activation (A) and degranulation-dependent P-selectin exposure (B) in response to the indicated agonists in <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets. Results are mean fluorescence intensities (MFI) ± SD of 4 mice per group and are representative of 4 individual experiments. CRP: collagen-related peptide, CVX: convulxin, and RC: rhodocytin.</p

<i>Efhd2</i>^-/- mice display normal hematologic parameters.

<p>White blood cell count (WBC), red blood cells (RBC), hemoglobin (HGB) and hematocrit (HCT) were determined with a hematologic analyzer (Sysmex) (n = 5 vs. 5, two independent experiments, n.s. = not significant).</p><p><i>Efhd2</i>^-/- mice display normal hematologic parameters.</p

Normal adhesion and aggregate formation of <i>Efhd2^-/-</i> platelets on collagen under flow.

<p>Whole blood from <i>Efhd2^+/+</i> or <i>Efhd2^-/-</i> mice was perfused over a collagen-coated surface (0.2 mg/mL) at a shear rate of 1000 s⁻¹ (A) or 1700 s⁻¹ (B). Representative images of aggregate formation on collagen after 4 minutes of perfusion time. Mean surface coverage (left) and relative thrombus volume expressed as integrated fluorescence intensity (IFI) (right) ± SD of 5 <i>Efhd2^+/+</i> and 5 <i>Efhd2^-/-</i> mice.</p

Unaltered aggregation response of <i>Efhd2^-/-</i> platelets.

<p>Washed platelets from <i>Efhd2^+/+</i> (black line) and <i>Efhd2^-/-</i> (gray line) mice were activated with the indicated agonist concentrations and light transmission was recorded on a Fibrintimer 4-channel aggregometer. ADP measurements were performed in prp. Representative aggregation traces of at least 3 individual experiments are depicted.</p

Normal Ca²⁺-mobilization but slightly increased procoagulant activity in EFhd2-deficient platelets upon stimulation.

<p>(A) Maximal increase of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) of <i>Efhd2^+/+</i> (black bars) and <i>Efhd2^-/-</i> platelets (gray bars) after activation with the indicated agonists (thrombin 0.1 U/ml, CRP 4 µg/ml). (B) Flow cytometric analysis of phosphatidylserine (PS) exposure in response to the indicated agonists in <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets. Washed platelets were stained with Annexin-V-DyLight-488 in the presence of Tyrodes-HEPES buffer containing 3 mmol/L Ca²⁺. The values represent the mean fluorescence intensity (MFI) ± SD for 5 mice per group in 3 independent experiments. Thr: thrombin, CRP: collagen related peptide, CVX: convulxin, RC: rhodocytin, n.s.: not significant, *<i>p</i><0.05.</p

EFhd2 is dispensable for platelet production.

<p>(A) Analysis of EFhd2 expression in <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets by Western blot. Expression of GPIIIa was used as loading control. (B) Peripheral platelet counts and (C) platelet volume of <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> mice measured with a blood cell counter are depicted. Results are mean ± SD of 7 mice per group. (D) Determination of the platelet life span in wild-type and <i>Efhd2^-/-</i> mice. Mice were injected with a DyLight 488-conjugated anti-GPIX derivate (0.5 µg/g body weight) to label platelets <i>in vivo</i>. Results are% of fluorescently labeled platelets at the indicated days after injection as determined by flow cytometry. Values are mean ± SD of 5 mice per group. (E) Determination of MK numbers per visual field (294×221 µm) in hematoxylin and eosin stained BM sections. Values are mean ± SD (n = 5).</p

Normal expression levels of specific proteins in EFhd2-deficient platelets.

<p>(A) Analysis of EFhd1, Rac1, mDia1, actin and tubulin expression in <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets by Western blot. (B) Analysis of phospho-cofilin, phospho-Syk, and phospho-PAK1/2 expression in <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets by Western blot. Staining of the respective non-phosphorylated proteins served as loading controls.</p

Deletion of p38α ameliorates tubular but not glomerular damage during anti-GBM nephritis.

<p>(A) Semi-quantitative scoring of tubular damage. p38α deletion significantly diminishes tubular damage (mean score 0.6±0.1, p<0.05 vs. control; n = 7) as compared to wild type mice (mean score 0.9±0.01; n = 7). Tubules of wild type mice (B) are dilated, whereas tubules of <i>MxCre-p38α^Δ/Δ</i> (C) mice are still tightly packed. (D) KIM-1 mRNA level is dramatically increased in wild type mice (wild type 84.23±32.57; n = 4 vs. <i>p38α^Δ/Δ</i> 2.675±1.248; n = 4) indicating high tubular damage. (E) Vimentin shows clear upregulation of its mRNA in wild type mice (2.900±0.1732; n = 4) whereas it is reduced nearly to the baseline level in <i>MxCre-p38α^Δ/Δ</i> mice (1.375±0.3497; n = 4). Dashed line indicates RNA base level of control mice. (F–H) Analysis of crescent formation during anti GBM nephritis. Similar amounts of glomeruli revealed crescent formation in wild type (n = 6) and <i>MxCre-p38α^Δ/Δ</i> mice (n = 6) (wild type: mean 3.3±1.2% vs. <i>MxCre-p38α^Δ/Δ</i>: 4.2±1.5, p = ns). (I–K) Fibrotic tissue remodelling occurred in both wild type (n = 7) and <i>MxCre-p38α^Δ/Δ</i> mice (n = 7) (wild-type: mean 1.409±0.1132 vs <i>MxCre-p38α^Δ/Δ</i>: 1.615±0.1617, p = ns). Sirius red staining was performed 14 days after induction of anti-GBM nephritis. (L–M) p38α deletion affects the immune response to sheep IgG by decreased murine IgG depositions in the glomeruli.</p

Inflammatory gene expression during anti-GBM nephritis is partially p38α dependent.

<p>qPCRs from renal tissue of wild-type and <i>MxCre-p38α^Δ/Δ</i> mice 14 days after injection with anti-GBM antibodies. There is no difference in the mRNA expression of pro-inflammatory cytokines like TNF, IL-1β and IL-6. Furthermore there is a significant regulation of IL-8, IL-12 and IL-18. There are no significant changes in anti-inflammatory cytokines like IL-10, IL-13 and TGFβ1. MCP-1 is down-regulated in <i>MxCre-p38α^Δ/Δ</i> mice. n = 4/group measured in duplicates (A). <b>Leukocyte infiltration is regulated by corresponding chemokines:</b> Chemokines participating in (B) macrophage and (C) neutrophil recruitment are remarkably downregulated in <i>MxCre-p38α^Δ/Δ</i> mice. (D) Chemotactic regulation of T cells is unaffected in <i>MxCre-p38α^Δ/Δ</i> mice. In each group the RNA of five mice was pooled and then analyzed by RT-PCR with a RT² Profiler™ PCR Array kit. mRNA expression was normalized to beta-actin of wildtype mice.</p