Inhibition of fibrinogen binding in platelets following blocking of cell-surface ERP5. Binding of FITC-labeled fibrinogen was measured with the use of flow cytometry on platelets stimulated with the agonists convulxin or collagen. Prior to stimulation, platelets were incubated with anti-ERP5 antibodies, anti-PDI antibodies, or control antibodies from preimmune sera. (A) Histogram for fluorescence of FITC-fibrinogen labeled platelets in response to the agonist convulxin (100 ng/mL). Control IgG (12 μg/mL) is indicated by a filled histogram; anti-ERP5 antibody (12 μg/mL), by an empty histogram; and anti-PDI antibody (33 μg/mL), by a dotted-line empty histogram. (Bi) Residual binding of FITC-fibrinogen following incubation of platelets with preimmune IgG (12 μg/mL), anti-ERP5 antibody (12 μg/mL), or anti-PDI antibody (33 μg/mL). Agonists were 100 ng/mL convulxin. (Bii) Additive effect of inhibitory antibodies on residual binding of FITC-fibrinogen following incubation of platelets 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). Agonists were 300 ng/mL convulxin. (Ci) Residual binding of FITC-fibrinogen following incubation of platelets with antibodies as described for panel Bi. Agonists were 10 μg/mL collagen. (Cii) Additive effect of inhibitory antibodies on residual binding of FITC-fibrinogen following incubation of platelets with antibodies as described for panel Bii. Agonists were 4 μg/mL collagen. Data are presented as mean ± SE for 4 separate experiments (*P < .05; **P < .005).