Agonist-induced phosphorylation of Akt is reduced in Cdc42^−/− platelets.

<p>Washed platelets from Cdc42^+/+ and Cdc42^−/− mice were incubated with CRP (0.2 µg/ml) or thrombin (0.1 U/ml) for 5 minutes at 37°C with constant stirring in a Chrono-Log aggregometer. The reactions were terminated by adding 5× sample buffer, processed for Western blotting and probed for Akt, p-Akt and β-actin as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022117#s2" target="_blank">methods</a>.</p

Genetic targeting of Cdc42 inhibited release of p-selectin from α-granules and secretion of ATP from dense granules.

<p>A, Thrombin (0.2 U/ml) induced expression of P-selectin and B, CRP (0.2 µg/ml) induced secretion of ATP is inhibited in platelets from Cdc42^−/− mice compared with the platelets from Cdc42^+/+ mice. Bar graphs are the means ± SD (n = 3).</p

Deficiency of Cdc42 prolonged tail bleeding time.

<p>The tail bleeding times were assessed in Cdc42^+/+ (n = 8) and Cdc42^−/− (n = 8) mice as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022117#s2" target="_blank">methods</a>. Bar graphs are the means ± SEM of the bleeding times. Cdc42-deficient, as compared to Cdc42^+/+, mice exhibited significantly prolonged (<i>p</i><0.05) bleeding times.</p

Deficiency of Cdc42 inhibited platelet aggregation induced by collagen, CRP or thrombin.

<p>Washed platelets from Cdc42^+/+ and Cdc42^−/− were stimulated by addition of CRP (A), collagen (B) or thrombin (C) and aggregation was recorded using a Lumi-Aggregometer at 37°C and a stirring speed of 900 rpm. Platelets from Cdc42^−/− mice compared with the platelets from the Cdc42^+/+ mice exhibited diminished aggregation induced by all three agonists. The aggregation tracings are representative of three experiments.</p

Inducible genetic targeting of Cdc42 inhibited platelet spreading on immobilized CRP and fibrinogen.

<p>Spreading of washed platelets from Cdc42^+/+ and Cdc42^−/− mice on CRP or fibrinogen was quantified using Image J software (<a href="http://rsbweb.nih.gov/ij" target="_blank">http://rsbweb.nih.gov/ij</a>). Each bar represents mean surface are ± SEM of 100 platelets. The spreading of Cdc42^−/− platelets on immobilized CRP or fibrinogen was significantly (p<0.01) diminished as compared to the Cdc42^+/+ platelets.</p

Gene targeting of Cdc42 induced thrombocytopenia and blocked signaling downstream of Cdc42.

<p>A, Expression of Cdc42, Rac1 and RhoA in platelet lysates from the wild type and genetically targeted mice was probed by Western blotting of Cdc42, Rac1 and RhoA. Platelets from Cdc42 gene targeted mice as compared to the poly (I∶C) treated matching wild type mice showed a complete lack of Cdc42 GTPase. The expression of Rac1 and RhoA was not altered in Cdc42^−/− platelets. β-actin expression was used as a loading control. B, Platelet counts (Mean ± SEM) in the Cdc42^−/− mice (n = 11) were significantly lower (<i>p</i><0.05) than the platelet counts in the Cdc42^+/+ mice (n = 12). C, CRP (0.2 µg/ml) or thrombin (0.1 U/ml) induced phosphorylation of PAK1/2 is inhibited in the Cdc42^−/− mice platelets as compared to the platelets from Cdc42^+/+ mice. Phosphorylation of PAK1/2 was analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022117#s2" target="_blank">methods</a> section.</p

Inducible genetic targeting of Cdc42 inhibited filopodia formation on immobilized fibrinogen.

<p>Washed platelets from Cdc42^+/+ (A) and Cdc42^−/− (B) mice were layered over cover slips coated with fibrinogen (3.0 µg/ml) for 20 minutes. The DIC (top panel) and immuno-fluorescence (bottom panel) images show that platelets from Cdc42^−/− mice failed to form filopodia on immobilized fibrinogen.</p

α-PGG inhibited collagen-induced phosphorylation of Akt.

<p>Washed human platelets were stimulated with collagen (1.0 µg mL⁻¹) in the presence or absence of α-PGG (10 µM). Lysis buffer was added to samples at 6 min to terminate reactions. Total Akt and p-Akt were visualized after PAGE and Western blotting as described in the Experimental Procedures. The β-actin was used as a loading control.</p

α-PGG inhibited thrombin induced rise in cytosolic calcium.

<p>Changes in cytosolic calcium were quantified in Fura2/AM loaded platelets. Platelets were incubated with α-PGG (3 or 10 µM) prior to stimulation with thrombin (0.1 U mL⁻¹) and changes in calcium levels were recorded by fluorescence spectrometry as described in Experimental Procedures. The results are reported as means ± SE (n = 4).</p

Administration of α-PGG inhibited <i>ex vivo</i> platelet aggregation induced by ADP or collagen.

<p>A, ADP or B, collagen was added to platelet-rich plasma, prepared from murine blood drawn at 30 min after oral administration of α-PGG (20 mg kg⁻¹) or vehicle, to induce aggregation. The aggregation tracings are representative of three experiments.</p