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

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

<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 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

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

Unaltered thrombotic and hemostatic function in Cd84-deficient mice.

<p>(A) The abdominal aorta was injured by firm compression with a forceps and blood flow was monitored for 30 min. Each symbol represents one animal. (B) Small mesenteric arterioles were injured by topical application of FeCl₃ and occlusive thrombus formation was monitored using intravital microscopy. Each symbol represents one mesenteric arteriole. The horizontal dotted line indicates the mean time to vessel occlusion. (C) Representative images of the FeCl₃-induced injury model of mesenteric arterioles in <i>Cd84^+/+</i> and <i>Cd84⁻</i>^/<i>−</i> mice, asterisk indicates stable occlusion of the vessel. (D) 1 mm tail tip was amputated and tail bleeding times of <i>Cd84^+/+</i> and <i>Cd84^−/−</i> mice were monitored. Each symbol represents one animal. The horizontal dotted line indicates the mean time to vessel occlusion.</p

CD84-deficient mice display normal platelet count and size.

<p>(A) CD84 targeting strategy: This scheme illustrates the detection of wild-type and <i>Cd84^−/−</i> (targeted) alleles. Upon homologous recombination, the pWH9 cassette containing a neomycin resistance gene disrupts the <i>Cd84</i> gene. An external probe (EP) recognizes a sequence downstream of 3′ arm in intron 2. With the pWH9 cassette, a new <i>Bam</i>HI restriction site is introduced, enabling the determination of a wild-type and <i>Cd84^−/−</i> band by Southern blot analysis. (B) Analysis of CD84 expression in wild-type (<i>Cd84^+/+</i>) and <i>Cd84^−/−</i> platelets by Western blot. Expression of GPIIIa was used as loading control. (C) Peripheral platelet counts and (D) platelet volume of wild-type and <i>Cd84^−/−</i> mice measured with a blood cell counter. (E) Determination of the platelet life span in wild-type and <i>Cd84^−/−</i> mice. Mice were injected with a DyLight 488-conjugated anti-GPIX Ig derivate 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.</p