The a°, a, and a′ active sites of ERp46 each contribute to aggregation and ATP release of mouse and human platelets. (A) Schematic diagram of the ERp46 variants. (B) Characterization of variant ERp46 proteins in the Di-E-GSSG assay. ERp46(cc-cc-cc), WT ERp46; ERp46(cc-cc-aa) had the a′ CGHC motif inactivated; ERp46(cc-aa aa) had the a and a′ active sites inactivated; ERp46(aa-aa-aa) had all 3 sites inactivated. (C-E) Correction of the aggregation and secretion defects of ERp46-null platelets (2 × 10⁸ platelets/mL) by ERp46 variants (100 nM). (F) Effect of preincubating human platelets (2 × 10⁸ platelets/mL) with ERp46 variants (1 μM). Submaximal aggregation (baseline) was stimulated with thrombin (0.015 U/mL). The ERp46 variants were added 5 minutes prior to the addition of thrombin. Representative aggregation and ATP release tracings (left panels) and combined results (right); mean ± SEM, n = 3, mouse platelets; n = 5, human platelets, *P < .05, **P < .01, ***P < .001, ****P < .0001, ANOVA. ns, not significant.