Effect of R21D10 on PDI, fibrinogen, and PAC-1 binding, P-selectin expression, and platelet aggregation. Washed platelets were incubated with 40 μg/mL R21D10 for 15 minutes at room temperature and then activated with T6 for 10 minutes. Alexa Flour 488–labeled PDI (A) or Alexa Flour 488–labeled fibrinogen (Fg) (B) was added to the activated platelets, and binding of PDI and Fg was detected by flow cytometry. To assess nonspecific PDI binding, labeled PDI was tested in the presence of 10-fold excess unlabeled PDI (ulPDI). Data are presented as the mean ± standard deviation. Data are from 6 separate experiments with blood from 6 donors. (C-D) The effect of R21D10 on PAC-1 binding and P-selectin expression was detected as above. Data are from 3 separate experiments with blood from 3 donors. (E) Washed platelets were incubated with different concentrations of R21D10 for 15 minutes and aggregation was induced by adding T6. Data reported are from 4 separate experiments with blood from 4 donors. (F) PRP was incubated with different concentrations of R21D10 and aggregation was induced by ADP (5 μM). The data reported are from 4 separate experiments with blood from 4 donors. ns, not significant; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Cntl, control.