Inhibition of platelet aggregation by an inhibitory antibody for ERP5. Platelets (4 × 10⁸/mL) were incubated with anti-ERP5 IgG or control IgG at the given concentrations for 2.5 minutes prior to addition of agonist. Prior to the addition of antibodies, platelets were preincubated with a saturating concentration of an F(ab′) fragment of the IV.3 protein to prevent signaling through the Fc_γRIIa receptor. Control IgG was purified from the preimmune serum of the animal in which antibodies were raised. (A) Concentration effect of inhibitory antibodies. (i) Concentration effect of 2.5 μg/mL collagen incubated with preimmune IgG (36 μg/mL); anti-ERP5 antibody (6 μg/mL); or anti-ERP5 antibody (36 μg/mL). (ii) Concentration effect of 30 ng/mL convulxin incubated with preimmune IgG (24 μg/mL); anti-ERP5 antibody (12 μg/mL); or anti-ERP5 antibody (24 μg/mL). (B) Additive effect of inhibitory antibodies for ERP5 and PDI. (i) Additive effect of 4.0 μg/mL collagen incubated with preimmune IgG (36 μg/mL); anti-ERP5 (12 μg/mL) plus preimmune IgG (19 μg/mL); anti-PDI (6 μg/mL) plus preimmune IgG (19 μg/mL); or anti-ERP5 (12 μg/mL) plus anti-PDI (6 μg/mL). (ii) Additive effect of 150 ng/mL convulxin incubated with preimmune IgG (36 μg/mL); anti-ERP5 (12 μg/mL) plus preimmune IgG (19 μg/mL); anti-PDI (6 μg/mL) plus preimmune IgG (19 μg/mL); or anti-ERP5 (12 μg/mL) plus anti-PDI (6 μg/mL). Traces shown are representative of those observed for at least 3 different donors. (C) Mobilization of calcium from intracellular stores. Ca²⁺ release was measured by Fura-2 fluorescence in platelets stimulated with convulxin (300 ng/mL, ▪) or collagen (4 μg/mL, ▦). Prior to stimulation, platelets were incubated with anti-ERP5 antibodies or control antibodies (24 μg/mL) from preimmune sera. Data are presented as mean ± SE for 3 separate experiments.