Figure 3.
ERp46 is critical for aggregation of mouse platelets and interacts with αIIbβ3 (A) ERp46-deficient platelets have defective thrombin (0.02 U/mL-induced activation of αIIbβ3 detected by the JON/A activation-dependent antibody). (B) P-selectin expression is decreased in thrombin-stimulated ERp46-null platelets. (A-B) left panels, representative histogram; right panels, combined results; mean ± SEM, n = 5 for each group, ***P < .001, Student t test. (C-E) Representative aggregation and ATP release tracings (left panels) and combined results (right) showing the defects in ERp46-deficient platelets using (C) thrombin, (D) CRP, or aggregation for (E) ADP; mean ± SEM, n = 4 (thrombin), n = 6 (CRP), n = 5 (ADP), **P < .01, ***P < .001, ****P < .0001, Student t test. Aggregation and ATP secretion were monitored in the lumi-aggregometer. (F) Anti-ERp46 inhibits CHAMP-induced aggregation of human platelets; mean ± SEM, n = 4, ****P < .0001, ANOVA. Normal rabbit IgG (90 μg/mL) was used as a control. (G) CHAMP-induced aggregation of ERp46-null mouse platelets is decreased; mean ± SEM, n = 5, ****P < .0001, Student t test. CHAMP was added to final concentration of 3 μM and aggregation was performed in the presence of indomethacin (100 µM) and Apyrase (4 U/mL). (H) ERp46 interaction with αIIbβ3 by surface plasmon resonance. Recombinant full-length human αIIbβ3 (25 μg/mL) was immobilized on the surface of a CM5 chip. Different concentrations of WT ERp46 without or with MnCl2 (1 mM) were infused over the chip in the running buffer. The equilibrium dissociation constant was calculated based on the Kon and Koff values with Biacore T200 evaluation software. (I) ERp46 interacts with β3 integrins on mouse platelets. Binding of Alexa Fluor 488-conjugated ERp46 to Mn2+-treated WT and β3-null mouse platelets. Representative histogram (left panels); cumulative data (right panels); mean ± SEM, n = 5 for each group, ***P < .001, ****P < .0001, ANOVA. Washed mouse platelets (3 ×108/mL) were preincubated with Alexa Fluor 488 ERp46 (30 μg/mL) for 10 minutes at room temperature and then treated with Mn2+ (12 mM) for 5 minutes at room temperature. Surface binding of Alexa Fluor 488 ERp46 was detected by flow cytometry. (J) Stimulation-dependent association of ERp46 with integrin β3. Platelets (1 × 109/mL) were stimulated in the presence of EGTA, apyrase, and indomethacin at varying concentrations of convulxin (30, 60, or 120 ng/mL) for 90 seconds. Following sample lysis, proteins were precipitated with mouse anti-β3 antibody SZ21 and protein G–resin. Immunoblotting with goat anti-ERp46 antibody and polyclonal rabbit anti-β3 antibody (Abcam) showed interacting proteins.

ERp46 is critical for aggregation of mouse platelets and interacts with αIIbβ3 (A) ERp46-deficient platelets have defective thrombin (0.02 U/mL-induced activation of αIIbβ3 detected by the JON/A activation-dependent antibody). (B) P-selectin expression is decreased in thrombin-stimulated ERp46-null platelets. (A-B) left panels, representative histogram; right panels, combined results; mean ± SEM, n = 5 for each group, ***P < .001, Student t test. (C-E) Representative aggregation and ATP release tracings (left panels) and combined results (right) showing the defects in ERp46-deficient platelets using (C) thrombin, (D) CRP, or aggregation for (E) ADP; mean ± SEM, n = 4 (thrombin), n = 6 (CRP), n = 5 (ADP), **P < .01, ***P < .001, ****P < .0001, Student t test. Aggregation and ATP secretion were monitored in the lumi-aggregometer. (F) Anti-ERp46 inhibits CHAMP-induced aggregation of human platelets; mean ± SEM, n = 4, ****P < .0001, ANOVA. Normal rabbit IgG (90 μg/mL) was used as a control. (G) CHAMP-induced aggregation of ERp46-null mouse platelets is decreased; mean ± SEM, n = 5, ****P < .0001, Student t test. CHAMP was added to final concentration of 3 μM and aggregation was performed in the presence of indomethacin (100 µM) and Apyrase (4 U/mL). (H) ERp46 interaction with αIIbβ3 by surface plasmon resonance. Recombinant full-length human αIIbβ3 (25 μg/mL) was immobilized on the surface of a CM5 chip. Different concentrations of WT ERp46 without or with MnCl2 (1 mM) were infused over the chip in the running buffer. The equilibrium dissociation constant was calculated based on the Kon and Koff values with Biacore T200 evaluation software. (I) ERp46 interacts with β3 integrins on mouse platelets. Binding of Alexa Fluor 488-conjugated ERp46 to Mn2+-treated WT and β3-null mouse platelets. Representative histogram (left panels); cumulative data (right panels); mean ± SEM, n = 5 for each group, ***P < .001, ****P < .0001, ANOVA. Washed mouse platelets (3 ×108/mL) were preincubated with Alexa Fluor 488 ERp46 (30 μg/mL) for 10 minutes at room temperature and then treated with Mn2+ (12 mM) for 5 minutes at room temperature. Surface binding of Alexa Fluor 488 ERp46 was detected by flow cytometry. (J) Stimulation-dependent association of ERp46 with integrin β3. Platelets (1 × 109/mL) were stimulated in the presence of EGTA, apyrase, and indomethacin at varying concentrations of convulxin (30, 60, or 120 ng/mL) for 90 seconds. Following sample lysis, proteins were precipitated with mouse anti-β3 antibody SZ21 and protein G–resin. Immunoblotting with goat anti-ERp46 antibody and polyclonal rabbit anti-β3 antibody (Abcam) showed interacting proteins.

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