Effect of AYP1 on CLEC-2 signaling. (A-B) Washed platelets were preincubated with 2.5 µg/mL AYP1 Fab fragments for 15 minutes at room temperature and stimulated with (A) 100 nM rhodocytin or (B) 10 µg/mL hPDPN-Fc for 15 minutes at 37°C. (A) Platelet activation by rhodocytin was determined by flow cytometric analysis of P-selectin expression and fibrinogen binding and (B) podoplanin binding using a phycoerythrin-conjugated α-human podoplanin antibody. (C) Aggregation of washed platelets induced by 100 nM rhodocytin or AYP1 Fab (2.5 µg/mL) cross-linked with 20 µg/mL α-mouse Fab-specific F(ab)2 fragments. Single incubation with either AYP1 Fab or α-mouse F(ab)2 fragments did not trigger aggregation. The traces are representative of 3 independent experiments. (D) Flow cytometric analysis of P-selectin expression and fibrinogen binding induced by cross-linking AYP1 Fab. Washed platelets were incubated with AYP1 Fab, α-mouse F(ab)2, or both for 15 minutes at 37°C and immediately analyzed. Preincubation with either the Syk inhibitor PRT-060318 (PRT; 5 µM) or the Src family kinase inhibitor PP2 (20 µM) for 10 minutes at room temperature prevented platelet activation induced by cross-linking AYP1 Fab. Data are presented as the ratio of MFI of treated over control platelets (n = 4).
