Figure 6.
ERp46 generates thiols in the β3 subunit of αIIbβ3. (A) Reductase activity of ERp46 compared with ERp57, PDI, ERp5, ERp72, and inactivated ERp46 [ERp46(aaaaaa)] (30 nM each) in the Di-E-GSSG assay. (B-C) ERp46 generates thiols in β3 more effectively than PDI, ERp57, ERp72, or ERp5. ERp46 or other PDIs (1 μM) were incubated with αIIbβ3 (0.5 μM) (Abcam) for 20 minutes at 37°C. IodoTMT (400 μM) was then added for 1 hour at 37°C and labeling of thiols performed in 1% SDS with 5 mM ethylenediaminetetraacetic acid . These studies were performed in the absence of added GSH. (D-E) The active site cysteines of ERp46 generate thiols in αIIbβ3. Shown is the labeling of β3 with WT ERp46 or inactivated ERp46 [ERp46(aaaaaa)]. ERp46 or ERp46(aaaaaa) (1 μM) was incubated with αIIbβ3 (0.5 μM) and GSH (100 μM), and labeling was performed with iodoTMT (500 μM). (F-G) Effect of fibrinogen (Fbgn) on thiol generation by ERp46 and PDI in αIIbβ3. ERp46 or PDI (1 μM) were incubated with αIIbβ3 (0.5 μM) with GSH (100 μM). In some samples, fibrinogen (0.5 μM) was added. Thiols were labeled with iodoTMT (500 μM). Blotting for iodoTMT was performed using the anti-TMT antibody. (H-I) Effect of fibrinogen on thiol generation by ERp46 and PDI in αIIbβ3 in platelets. ERp46 or PDI (1 μM) were incubated with human platelets (4 × 108 platelets/mL) without GSH. In some samples, fibrinogen (1 μM) was added. Thiols were labeled with MPB and blotting was performed as described in supplemental Methods. The intensity of each band was calculated using the Image J program, and the ratio of iodoTMT (TMT) or MPB to β3 protein was compared with the untreated sample. Shown are the blots for β3 (H-96 or B-7 for D), PDI and ERp46 (rabbit antibodies), and the β chain of fibrinogen; mean ± SEM, n = 4 (B-C), n = 3 (D-E), n = 3 (F-G), n = 7 (H-I); *P < .05, **P < .01, ***P < .001, ****P < .0001, ANOVA. (J-K) Addition of ERp46 followed by PDI (ERp46/PDI) maximally generates thiols in β3. αIIbβ3 was treated with 1 μM PDI or ERp46 for 40 minutes, except when added sequentially they were added for 20 minutes each. Addition of PDI followed by ERp46 (PDI/ERp46) gave similar results to ERp46 alone or PDI plus ERp46 added simultaneously. Labeling was with iodoTMT (400 μM) in the absence of GSH; mean ± SEM, n = 4; ****P < .0001, ANOVA. (L-M) Addition of ERp46 followed by PDI (ERp46/PDI) provides maximal enhancement of platelet aggregation. 1.5 μM PDI or ERp46 were added for 10 minutes to platelets, except when added sequentially they were added for 5 minutes each. Aggregation was induced with thrombin (0.012 U/mL). The aggregation tracings for ERp46 alone, PDI plus ERp46 added simultaneously, or PDI followed by ERp46 (PDI/ERp46) are overlapping; mean ± SEM, n = 5; *P < .05, ****P < .0001, ANOVA. ns, not significant.

ERp46 generates thiols in the β3 subunit of αIIbβ3. (A) Reductase activity of ERp46 compared with ERp57, PDI, ERp5, ERp72, and inactivated ERp46 [ERp46(aaaaaa)] (30 nM each) in the Di-E-GSSG assay. (B-C) ERp46 generates thiols in β3 more effectively than PDI, ERp57, ERp72, or ERp5. ERp46 or other PDIs (1 μM) were incubated with αIIbβ3 (0.5 μM) (Abcam) for 20 minutes at 37°C. IodoTMT (400 μM) was then added for 1 hour at 37°C and labeling of thiols performed in 1% SDS with 5 mM ethylenediaminetetraacetic acid . These studies were performed in the absence of added GSH. (D-E) The active site cysteines of ERp46 generate thiols in αIIbβ3. Shown is the labeling of β3 with WT ERp46 or inactivated ERp46 [ERp46(aaaaaa)]. ERp46 or ERp46(aaaaaa) (1 μM) was incubated with αIIbβ3 (0.5 μM) and GSH (100 μM), and labeling was performed with iodoTMT (500 μM). (F-G) Effect of fibrinogen (Fbgn) on thiol generation by ERp46 and PDI in αIIbβ3. ERp46 or PDI (1 μM) were incubated with αIIbβ3 (0.5 μM) with GSH (100 μM). In some samples, fibrinogen (0.5 μM) was added. Thiols were labeled with iodoTMT (500 μM). Blotting for iodoTMT was performed using the anti-TMT antibody. (H-I) Effect of fibrinogen on thiol generation by ERp46 and PDI in αIIbβ3 in platelets. ERp46 or PDI (1 μM) were incubated with human platelets (4 × 108 platelets/mL) without GSH. In some samples, fibrinogen (1 μM) was added. Thiols were labeled with MPB and blotting was performed as described in supplemental Methods. The intensity of each band was calculated using the Image J program, and the ratio of iodoTMT (TMT) or MPB to β3 protein was compared with the untreated sample. Shown are the blots for β3 (H-96 or B-7 for D), PDI and ERp46 (rabbit antibodies), and the β chain of fibrinogen; mean ± SEM, n = 4 (B-C), n = 3 (D-E), n = 3 (F-G), n = 7 (H-I); *P < .05, **P < .01, ***P < .001, ****P < .0001, ANOVA. (J-K) Addition of ERp46 followed by PDI (ERp46/PDI) maximally generates thiols in β3. αIIbβ3 was treated with 1 μM PDI or ERp46 for 40 minutes, except when added sequentially they were added for 20 minutes each. Addition of PDI followed by ERp46 (PDI/ERp46) gave similar results to ERp46 alone or PDI plus ERp46 added simultaneously. Labeling was with iodoTMT (400 μM) in the absence of GSH; mean ± SEM, n = 4; ****P < .0001, ANOVA. (L-M) Addition of ERp46 followed by PDI (ERp46/PDI) provides maximal enhancement of platelet aggregation. 1.5 μM PDI or ERp46 were added for 10 minutes to platelets, except when added sequentially they were added for 5 minutes each. Aggregation was induced with thrombin (0.012 U/mL). The aggregation tracings for ERp46 alone, PDI plus ERp46 added simultaneously, or PDI followed by ERp46 (PDI/ERp46) are overlapping; mean ± SEM, n = 5; *P < .05, ****P < .0001, ANOVA. ns, not significant.

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