Aggregation and ATP secretion of WT and PDI-null platelets and the rescue effect of recombinant PDI. Platelets isolated from WT (black line) and PDI CKO (gray line) mice were stimulated with 0.05 U/mL thrombin (A), 0.5 μg/mL collagen (B), and 2.5 μM ADP (C). (i) Platelet aggregation and quantitative graphs. (ii) ATP secretion. In some experiments, PDI-null platelets were pretreated with 50 μg/mL wtPDI (black dotted line) or dmPDI (gray dotted line) and activated with an agonist. Quantitative results of aggregation and ATP secretion are presented as mean ± SD (n = 4). **P < .01 vs WT platelets after ANOVA and Dunnett test; ##P < .01 vs PDI-null platelets treated with wtPDI after Student t test. (D,E) Mouse platelets were pretreated with His-tagged wtPDI or dmPDI and activated with or without thrombin in the presence of 1 mM EGTA under a stirring condition. Platelets were washed with HEPES-Tyrode buffer (D) or carbonate buffer (0.1 M Na₂CO₃, pH 9.0) (E). Binding of recombinant PDI was analyzed by flow cytometry using a Dylight 488–conjugated anti-polyHis antibody. PDI binding is shown as a fold increase by the ratio of the geometric mean intensity value of the anti-His antibody on PDI-treated vs untreated platelets (mean ± SD, n = 3). *P < .05; **P < .01 vs resting platelets; and #P < .05 vs activated WT platelets treated with wtPDI or dmPDI after Student t test.