Figure 3
Figure 3. Increased GPVI/ITAM signaling in Slap−/−/Slap2−/− platelets occurs independently of elevated GPVI expression levels. (A) Analysis of GPVI expression levels in WT, Slap−/−/Slap2−/−/Gp6+/−, and Slap−/−/Slap2−/−/Gp6+/+ platelets. Diluted whole blood was stained with fluorescein isothiocyanate-labeled JAQ1 antibody and platelets were analyzed by flow cytometry (n = 4 mice per group). (B) Detection of integrin αIIbβ3 activation (binding of JON/A-PE) (top) and α-granule release (bottom) in response to the indicated agonists in WT, Slap−/−/Slap2−/−/Gp6+/−, and Slap−/−/Slap2−/−/Gp6+/+ platelets. Results are expressed as mean fluorescence intensity ± SD (n = 4 mice per group). (C) Washed platelets were activated with CRP and light transmission was monitored on a Fibrintimer 4-channel aggregometer. (D) WT, Slap−/−/Slap2−/−/Gp6+/− (HET), and Slap−/−/Slap2−/−/Gp6+/+ (HOM) platelets were stimulated with 0.5 μg/mL convulxin for the indicated time points. Whole-cell lysates were western-blotted and probed with the anti-phosphotyrosine antibody 4G10 or with phospho-specific antibodies. Staining of the respective nonphosphorylated proteins and actin served as loading control. (E) WT and Slap−/−/Slap2−/− platelets were stimulated with 1 μg/mL rhodocytin and analysis of tyrosine phosphorylation patterns was performed as described for (D). (F) SLAP/SLAP2 attenuate Lyn binding to activated GPVI. Washed WT and Slap−/−/Slap2−/− (DKO) platelets were left unstimulated or stimulated with 0.5 μg/mL convulxin for 20 seconds, lysed, and proteins immunoprecipitated (IP) with Lyn were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and immunoblotted (WB) for GPVI or Lyn. An immunoblot of whole-cell lysate (WCL) loaded at 0.4% of Lyn immunoprecipitation input is also shown. Note that GPVI is detected as an ∼65 kDa monomer and an ∼120 kDa dimer, respectively. Results in all panels are representative of 3 independent experiments. *P < .05; **P < .01; ***P < .001. A/U, ADP + U-46619; CVX, convulxin; CRP, collagen-related peptide; DKO, double knockout; FITC, fluorescein isothiocyanate; HOM, Slap−/−Slap2−/−Gp6+/+ platelets; PE, phycoerythrin; RC, rhodocytin; thr., thrombin; WB, western blot; WCL, whole-cell lysate.

Increased GPVI/ITAM signaling in Slap−/−/Slap2−/− platelets occurs independently of elevated GPVI expression levels. (A) Analysis of GPVI expression levels in WT, Slap−/−/Slap2−/−/Gp6+/−, and Slap−/−/Slap2−/−/Gp6+/+ platelets. Diluted whole blood was stained with fluorescein isothiocyanate-labeled JAQ1 antibody and platelets were analyzed by flow cytometry (n = 4 mice per group). (B) Detection of integrin αIIbβ3 activation (binding of JON/A-PE) (top) and α-granule release (bottom) in response to the indicated agonists in WT, Slap−/−/Slap2−/−/Gp6+/−, and Slap−/−/Slap2−/−/Gp6+/+ platelets. Results are expressed as mean fluorescence intensity ± SD (n = 4 mice per group). (C) Washed platelets were activated with CRP and light transmission was monitored on a Fibrintimer 4-channel aggregometer. (D) WT, Slap−/−/Slap2−/−/Gp6+/− (HET), and Slap−/−/Slap2−/−/Gp6+/+ (HOM) platelets were stimulated with 0.5 μg/mL convulxin for the indicated time points. Whole-cell lysates were western-blotted and probed with the anti-phosphotyrosine antibody 4G10 or with phospho-specific antibodies. Staining of the respective nonphosphorylated proteins and actin served as loading control. (E) WT and Slap−/−/Slap2−/− platelets were stimulated with 1 μg/mL rhodocytin and analysis of tyrosine phosphorylation patterns was performed as described for (D). (F) SLAP/SLAP2 attenuate Lyn binding to activated GPVI. Washed WT and Slap−/−/Slap2−/− (DKO) platelets were left unstimulated or stimulated with 0.5 μg/mL convulxin for 20 seconds, lysed, and proteins immunoprecipitated (IP) with Lyn were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and immunoblotted (WB) for GPVI or Lyn. An immunoblot of whole-cell lysate (WCL) loaded at 0.4% of Lyn immunoprecipitation input is also shown. Note that GPVI is detected as an ∼65 kDa monomer and an ∼120 kDa dimer, respectively. Results in all panels are representative of 3 independent experiments. *P < .05; **P < .01; ***P < .001. A/U, ADP + U-46619; CVX, convulxin; CRP, collagen-related peptide; DKO, double knockout; FITC, fluorescein isothiocyanate; HOM, Slap−/−Slap2−/−Gp6+/+ platelets; PE, phycoerythrin; RC, rhodocytin; thr., thrombin; WB, western blot; WCL, whole-cell lysate.

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