The a and a′ active sites of ERp72 are critical for platelet aggregation and ATP release. (A-C) Effect of preincubating human platelets (2 × 10⁸ platelets/mL) with single active site mutants of ERp72, ERp72(oo-ss-ss), ERp72(ss-oo-ss), and ERp72(ss-ss-oo). Submaximal aggregation (baseline) was stimulated with collagen (1 µg/mL). The concentrations for each type of protein are indicated. Representative aggregation tracings with ATP release for the 3 single active site mutants (A) and corresponding cumulative data for aggregation (B) and secretion (C) with wild-type ERp72(ss-ss-ss) and the double active site mutant ERp72(ss-oo-oo) results added to the cumulative data; mean ± SEM, n = 3, *P < .05, analysis of variance. (D-F) Correction of the aggregation and secretion defects of ERp72-null platelets (2 × 10⁸ platelets/mL) from Pf4-Cre/ERp72^fl/fl mice compared with wild type (ERp72^fl/fl) littermate mice by ERp72 mutants. The mutant proteins were added 5 minutes prior to the addition of thrombin (0.015 U/mL). Representative tracings (D) and cumulative data for aggregation (E) and secretion (F); mean ± SEM, n = 3, *P < .05, **P < .01, ***P < .001, analysis of variance. ERp72^fl/fl, PDI^fl/fl, and ERp57^fl/fl represent the Cre-negative littermate controls. NS, not significant.