CLEC-2 is not shed or internalized following activation. (A-B) Washed platelets were incubated with vehicle control (veh; dimethylsulfoxide), NEM (2 mM), W7 (150 µM), CRP (10 µg/mL), IV.3 Fab (5 µg/mL) cross-linked with 20 µg/mL α-mouse Fab-specific F(ab)2 fragments (xIV.3), rhodocytin (100 nM), or AYP1 Fab (2.5 µg/mL) cross-linked with 20 µg/mL α-mouse Fab-specific F(ab)2 fragments (xAYP1) for 1 hour at 37°C. (A) Platelet lysates were blotted and probed with AYP2. Membranes were subsequently stripped and reprobed with an antibody against α-tubulin. Quantification is presented as the mean ratio of treated over control platelets (n = 3). (B) Flow cytometric analysis of the surface expression of CLEC-2. After treatment, platelets were incubated with Alexa Fluor 488-conjugated AYP1 for 15 minutes at 37°C and immediately analyzed. Alexa Fluor 488-conjugated AYP1 Fab was used to assess CLEC-2 expression after AYP1 Fab cross-linking. (C) Analysis of surface-bound rhodocytin and cross-linked AYP1 Fab. Washed platelets were incubated with rhodocytin or xAYP1 for indicated times at 37°C. Surface-bound rhodocytin was determined by incubation with rabbit α-rhodocytin antibody, followed by incubation with F(ab)2 fragments of FITC-conjugated swine α-rabbit IgG. Surface-bound xAYP1 was assessed by incubation with F(ab)2 fragments of polyclonal FITC-conjugated sheep α-mouse IgG. Data are presented as the ratio of MFI of treated over control platelets (n = 5).
