Tyrosine phosphorylation of CLEC-2 and signaling molecules downstream GPVI upon rhodocytin stimulation. (A) Human washed platelets (2 × 108/mL) were stimulated by 300 nM rhodocytin and platelet aggregation was monitored using an aggregometer. (B-E) Washed human platelets (300 or 500 μL at 1 × 109/mL) were stimulated with (B) 300 nM rhodocytin, (C-E) 50 nM rhodocytin, 50 μ g/mL collagen, or 100 μM SFLLRN for the indicated times. In panel D, platelets were pretreated with 30 μM PP3 or PP2, prior to stimulation with rhodocytin. Reactions were terminated by addition of an equal volume of 2 × lysis buffer. Platelet lysates were precleared and detergent-insoluble debris was removed by centrifugation. Antibodies against CLEC-2 (B-D), PLCγ2, Syk, Btk, SLP-76, or LAT (E) were added to the resultant supernatant and incubated overnight with protein A or G Sepharose. Precipitated proteins were separated by SDS-PAGE and Western blotted with the indicated antibodies. The data are representative of 2 to 5 experiments.
