Figure 2
Figure 2. Increased integrin activation, α− and dense-granule release, and aggregation in Slap−/−/Slap2−/− platelets upon GPVI or CLEC-2 stimulation. (A) Analysis of SLAP and SLAP2 expression in WT, Slap−/−, and Slap2−/− platelets by western blot. Actin expression was used as loading control and for quantification. Arrowheads indicate both SLAP2 isoforms (25 kDa and 28 kDa) in WT platelets. (B) Western blot analysis of GPVI and CLEC-2 expression in Slap−/−/Slap2−/− platelets. GPIIIa expression was used as loading control and for quantification. (A-B) Equal protein amounts were loaded and expression levels were quantified by densitometry with ImageJ software (National Institutes of Health) on 10 (A) or 12 (B) samples per genotype. Results represent mean ± SD. Expression levels were adjusted such that SLAP-to-actin, GPVI-to-GPIIIa, or CLEC-2-to-GPIIIa ratio in WT platelets was set to 1. (C) Flow cytometric analysis of activated integrin αIIbβ3 (binding of JON/A-PE) (left) and degranulation-dependent P-selectin exposure (right) upon stimulation with the indicated agonists in WT and Slap−/−/Slap2−/− platelets. Results are expressed as mean fluorescence intensity ± SD (n = 4 mice per group) and are representative of 6 independent experiments. Dividing lines indicate separate measurements. (D) Washed platelets were activated with the indicated agonists and light transmission was recorded on a Fibrintimer 4-channel aggregometer. ADP measurements were performed in platelet-rich plasma (PRP). Aggregation traces representative of 3 independent experiments with n = 4 are depicted. Arrows indicate addition of the respective agonist. (E) Washed platelets were incubated with Luciferase-Luciferin reagent and ATP release was measured in a Lumi-aggregometer upon stimulation with thrombin, CRP, or rhodocytin. Results are representative of 2 individual experiments with n = 4. Results represent mean ± SD. *P < .05; **P < .01; ***P < .001. A/U, ADP + U-46619; CRP, collagen-related peptide, CVX, convulxin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PRP, platelet-rich plasma; RC, rhodocytin; thr., thrombin; U46, U-46619.

Increased integrin activation, α− and dense-granule release, and aggregation in Slap−/−/Slap2−/− platelets upon GPVI or CLEC-2 stimulation. (A) Analysis of SLAP and SLAP2 expression in WT, Slap−/−, and Slap2−/− platelets by western blot. Actin expression was used as loading control and for quantification. Arrowheads indicate both SLAP2 isoforms (25 kDa and 28 kDa) in WT platelets. (B) Western blot analysis of GPVI and CLEC-2 expression in Slap−/−/Slap2−/− platelets. GPIIIa expression was used as loading control and for quantification. (A-B) Equal protein amounts were loaded and expression levels were quantified by densitometry with ImageJ software (National Institutes of Health) on 10 (A) or 12 (B) samples per genotype. Results represent mean ± SD. Expression levels were adjusted such that SLAP-to-actin, GPVI-to-GPIIIa, or CLEC-2-to-GPIIIa ratio in WT platelets was set to 1. (C) Flow cytometric analysis of activated integrin αIIbβ3 (binding of JON/A-PE) (left) and degranulation-dependent P-selectin exposure (right) upon stimulation with the indicated agonists in WT and Slap−/−/Slap2−/− platelets. Results are expressed as mean fluorescence intensity ± SD (n = 4 mice per group) and are representative of 6 independent experiments. Dividing lines indicate separate measurements. (D) Washed platelets were activated with the indicated agonists and light transmission was recorded on a Fibrintimer 4-channel aggregometer. ADP measurements were performed in platelet-rich plasma (PRP). Aggregation traces representative of 3 independent experiments with n = 4 are depicted. Arrows indicate addition of the respective agonist. (E) Washed platelets were incubated with Luciferase-Luciferin reagent and ATP release was measured in a Lumi-aggregometer upon stimulation with thrombin, CRP, or rhodocytin. Results are representative of 2 individual experiments with n = 4. Results represent mean ± SD. *P < .05; **P < .01; ***P < .001. A/U, ADP + U-46619; CRP, collagen-related peptide, CVX, convulxin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PRP, platelet-rich plasma; RC, rhodocytin; thr., thrombin; U46, U-46619.

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