Activation of CLEC-2 leads to proteolytic cleavage of GPVI and FcγRIIa. (A) 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. Platelet lysates were blotted and probed with an antibody against the cytoplasmic tail of GPVI, which detects both full-length and the cytoplasmic tail remnant of GPVI. The blot is representative for ≥3 independent experiments. (B) Flow cytometric analysis of the surface expression of GPVI. Washed platelets were preincubated with the calpain inhibitor E64d (100 µM), the metalloproteinase inhibitor GM6001 (100 µM), the Syk inhibitor PRT-060318 (PRT; 5 µM), or the Src family kinase inhibitor PP2 (20 µM) for 10 minutes at room temperature, followed by incubation with AYP1 Fab (2.5 µg/mL) cross-linked with 20 µg/mL α-mouse Fab-specific F(ab)2 fragments (xAYP1) for 15 minutes at 37°C. After treatment, platelets were incubated with Alexa Fluor 488-conjugated α-GPVI antibody 1G5 for 15 minutes at 37°C and immediately analyzed. (C) Washed platelets incubated under the conditions of B were blotted and probed with mouse α-human FcγRIIa-biotin. The blot is representative for ≥3 independent experiments. (D) Flow cytometric analysis of FcγRIIa surface expression on platelets after AYP1 Fab cross-linking. Data are presented as the ratio of MFI of treated over control platelets (n = 3).
