Alboaggregin-A binds and signals through GPVI.
(A) Human washed platelets (5 × 108/mL) in the presence of EGTA (1 mM) and indomethacin (10 μM) were stimulated by convulxin (Cvx; 1 μg/mL); alboaggregin-A (Alb-A; 1 μg/mL); or vWF ristocetin (vWF; 10 μg/mL; rist; 1mg/mL). (A) Reactions were stopped by the addition of equal volumes of Laemmli sample buffer. (B) Experiments were stopped by addition of an equal volume of cold lysis buffer, and the FcR γ-chain was precipitated from lysates by means of the tandem SH2 domains of Syk expressed as a glutathione-S-transferase fusion protein. Proteins were separated by 8% to 18% gradient SDS-PAGE and Western blotted for phosphotyrosine with mAb 4G10. (C) K562 cells (2 × 106/mL) transfected with either GPVI/FcR γ-chain (FID10/γ) or FcR γ-chain alone (PRC/γ) were stimulated with alboaggregin-A (Alb-A; 1 μg/mL) or convulxin (Cvx; 1 μg/mL). Following separation by SDS-PAGE, blots were probed for phosphotyrosine with mAb, 4G10.
