Characterization of Integrin αIIbβ3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets

Figures

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  Figure 1. The effect of integrin αIIbβ3 on PKCδ phosphorylation in platelets. (A) Washed murine platelets were stimulated at 37 °C with 200 µM AYPGKF and 100 nM 2-MeSADP in the presence and absence of 1 µM GR144053 and probed with antibodies indicated. The blots are representative of three independent experiments and are presented as mean ± standard error (SE). ** p < 0.01 and *** p < 0.005. (B) Platelets were plated on either bovine serum albumin (BSA) (5 mg/mL) or fibrinogen (100 μg/mL) for 45 min, and lysates were probed with anti-phospho-Pyk2 (Tyr402), anti-phospho-PKCδ (Tyr311), anti-phospho-Akt (Ser473) or anti-β-actin (lane loading control) antibodies by western blotting. The blots shown are representative of three independent experiments and are presented as mean ± SE. * p < 0.05 and ** p < 0.01.

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  Figure 2. The effect of Src and Pyk2 inhibition on αIIbβ3-mediated PKCδ phosphorylation. (A) Fibrinogen-adherent platelets in the presence of 10 µM PP2 or 10 µM PP3 were probed with anti-phospho-PKCδ (Tyr311) or anti-β-actin antibodies. The blots shown are representative of three independent experiments and are presented as mean ± SE. * p < 0.05. (B) Fibrinogen-adherent platelets in the presence of Pyk2 inhibitor TAT-Pyk2-CT or TAT-GFP (control) were probed with anti-phospho-PKCδ (Tyr311) or anti-β-actin antibodies by Western blotting. The blots shown are representative of three independent experiments and are presented as mean ± SE. * p < 0.05.

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  Figure 3. The effect of PKCδ on Pyk2 and Akt phosphorylation by αIIbβ3-mediated outside-in signaling. Washed platelets from PKCδ -/- mice and PKCδ +/+ littermates were plated on either BSA (5 mg/mL) or fibrinogen (100 μg/mL) for 45 min and probed with anti-phospho-Pyk2 (Tyr402), anti-phospho-PKCδ (Tyr311), anti-phospho-Akt (Ser473) or anti-β-actin antibodies by western blotting. The blots are representative of three experiments and are mean ± SE. * p < 0.05.

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  Figure 4. Role of PKCδ in platelet spreading on fibrinogen and clot retraction. (A) Washed platelets from PKCδ -/- mice and PKCδ +/+ littermates were plated on either BSA (5 mg/mL) or fibrinogen (100 μg/mL) for 45 min, stained with rhodamine phalloidin, and analyzed by fluorescence microscopy. Scale bar, 5 µm. (B) Platelets from PKCδ -/- mice and PKCδ +/+ littermates were mixed with CaCl2 (1 mM) and fibrinogen (2 mg/mL). Clot retraction was initiated by adding 0.1 U/mL of thrombin and allowed to proceed at 37 °C. Photographs of the clots were taken by a digital camera at 20, 30, and 40 min. The data shown are representative of three independent experiments.