Fig. 5. TC21 activation by GPVI receptor agonists downstream of Src kinases.

Washed platelets were stimulated with 2.5 μg/ml CRP or 75 ng/ml CVX (A), or 500 μM AYPGKF or 100 μg/ml fucoidan (B) as shown, lysed, and GTP-bound TC21 (upper panels) was extracted from platelet lysates (lower panels, total TC21) using GST-RAF-1-RBD coupled to Gluthatione-sepharose beads. (C) TC21 activation induced by 5 μg/ml CRP for 60 sec was evaluated in the presence of 10 μM PP2 or PP3 as indicated. (D) Washed platelets were incubated with 1 μm OXSI-2 or DMSO vehicle for 5 min, then stimulated with CRP as shown and evaluated for total and GTP-bound TC21 as in (A). (E) TC21 was immunoprecipitated from IgG-sepharose pre-cleared lysates of GPVI-stimulated platelets, with 2 μg of α-TC21 antibodies. Washed immunoprecipitate fractions were subject to western blotting with α-TC21 and α-GPVI antibodies as indicated. (F) FcγRIIa was cross-linked with 2.5 μg/ml IV.3 antibodies followed by 50 μg/ml anti-mouse IgG(F_(ab)) for the indicated times. GTP-bound, activated TC21 was precipitated from platelet lysates and detected by western blotting as above. All data representative of three independent experiments each.