Washed human platelets were prepared from a healthy donor. One ml of platelet suspension (363,000 per µL) was untreated or activated with 20 µM ADP for 2 min. Platelet releasate was collected by centrifugation for 15 min at 1000 g. The platelet pellet was lysed with 50 µL 2% NP40, 30 mM Hepes, 150 mM NaCl, 2 mM EDTA, pH 7.4 buffer containing proteinase inhibitor cocktail and clarified by centrifugation at 10,000 g for 20 min at 4°C. One µL of platelet lysate and 10 µL of releasate were resolved on reducing SDS-PAGE and the ERp5 immunoblotted with 1 µg/mL rabbit anti-ERp5 polyclonal antibody. Platelet ERp5 levels were calculated by reference to a standard curve of recombinant ERp5 concentrations. Densitometry of the chemiluminescent bands was performed by ImageQuant TL software (Biorad). Molecular size markers are shown at left.