ERp72 is critical for platelet aggregation and interacts with β3 integrins. (A) Western blots of platelet lysates using a polyclonal rabbit anti–ERp72 antibody and antibodies against ERp57 and PDI. Shown are the PLCγ2 loading controls for ERp72. Blots are representative of 3 separate experiments, with separate loading controls run for ERp57 and PDI (not depicted). (B) ERp72-deficient platelets have defective thrombin (0.025 U/mL)–induced activation of αIIbβ3 (detected by the JON/A activation-dependent antibody). (C) P-selectin expression is decreased in thrombin-stimulated ERp72-null platelets. (B and C) Representative histogram (left) and combined results (right); mean ± SEM, n = 6 for each group, ***P < .001, Student t test. (D and E) Representative aggregation and ATP release tracings (left) and combined results (right) showing the defect in ERp72-deficient platelets using convulxin (15 ng/mL) or thrombin (0.025 U/mL); mean ± SEM, n = 3, *P < .05, **P < .01, Student t test. Aggregation and ATP secretion were monitored in the lumiaggregometer. (F and G) ERp72 interacts with β3 integrins on mouse platelets. Binding of Alexa Fluor 488–conjugated ERp72 to nonactivated (NA) and thrombin-activated (F) or Mn²⁺-treated (G) wild-type and β3-null mouse platelets. Cumulative data for ERp72 binding to thrombin-activated platelets (right panel of F) and to Mn²⁺-treated platelets (right panel of G); mean ± SEM, n = 3 for each group, ***P < .001, analysis of variance. Washed mouse platelets (3 × 10⁸/mL) were preincubated with Alexa Fluor 488 ERp72 (30 μg/mL) for 10 minutes at room temperature and then activated by thrombin (0.1 U/mL) (F) or treated with Mn²⁺ (12 mM) (G) for 5 minutes at room temperature. Surface binding of Alexa Fluor 488 ERp72 was detected by flow cytometry. ATP, adenosine triphosphate.