Platelet surface ERp57 protein increases with platelet activation. Surface expression was detected with the polyclonal anti-ERp57 antibody (Ab1) in response to convulxin (A) and SFLLRN (B). Surface expression was also detected by the monoclonal anti-ERp57 antibodies Mab1 (C) or Mab2 (D) using convulxin. Left panels show a representative result with non-activated (NA) and activated (Act) platelets; right panels show the MFI from several experiments ± SE (A, n = 8; B, n = 4; C, n = 5; D, n = 5). The platelets were incubated with convulxin or SFLLRN for 5 minutes before the analysis. When primary monoclonal antibodies were used, secondary PE-labeled antimouse antibodies were used. (E) Time course of surface expression of ERp57 in platelets using thrombin (3 U/mL) ± SE (n = 3). Left panels, representative flow histograms; right panel MFI ± SE (n = 3). (F) Mab1 binds to ERp57^fl/fl platelets but not to ERp57-deficient platelets. Shown is binding of Mab1 to nonactivated platelets and activated platelets that express (ERp57^fl/fl) or lack ERp57 (Pf4-Cre/ERp57^fl/fl). The platelets were activated with convulxin (600 ng/mL). The curves with nonactivated and activated ERp57-deficient platelets are superimposable. (G) Cumulative data showing binding of Mab1 to mouse platelets activated with convulxin (1 µg/mL) or thrombin (3 U/mL) ± SE (n = 3).