Secretion of ERp5 from platelets and endothelial cells. (A) Detection of ERp5 in lysate and supernatant of human and mouse (C57BL/6) platelets. Equal number of platelets were stimulated with thrombin (+IIa) at a dose of 0.5 U/mL or maintained in the resting state (−IIa). The supernatant (containing the platelet releasate) was separated from the platelets by centrifugation, and the platelets were lysed. Platelet lysates were probed for ERp5 and GAPDH. Platelet supernatants were probed for ERp5. (B) Expression of ERp5 on the surface of mouse platelets. Washed platelets were prepared from C57BL/6 mouse blood and activated with mouse thrombin (0.5 U/mL). Resting (−IIa) and activated (+IIa) platelets were incubated with monoclonal anti-human ERp5 antibody or isotype control (IgG) antibody, both labeled with Alexa Fluor 647. (C) Detection of ERp5 in the supernatant and lysate of cultured HUVECs before (−IIa) and after (+IIa) stimulation with 0.5 U/mL thrombin. HUVEC lysate was probed for ERp5 and GAPDH. HUVEC supernatant was probed for ERp5. (D) Release of ERp5 from thrombin-stimulated HUVECs over time as determined by densitometry compared to ERp5 control. N = 3; error bars represent standard deviation; **P < .005. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pg, picograms.