Tetherin/BST-2 negatively regulates P2Y₁₂R activities in both platelets and cell lines. (A) PCR and RT-PCR were used to genotype mice. RT-PCR was performed on total RNA isolated from BST^+/+ and tetherin/BST-2^−/− mouse platelets, with primers specific for glyceraldehyde-3-phosphate dehydrogenase or tetherin/BST-2. (B) Washed platelets from humans or BST-2^+/+ and BST-2^−/− mice were solubilized and immunoblotted with tetherin/BST-2 or tubulin-specific antibodies. Data are representative of at least 4 independent experiments. (C-D) ADP-stimulated platelet aggregation was significantly enhanced in BST-2^−/− vs BST-2^+/+ mice in either platelet-rich plasma (PRP) (C) or washed platelets (D). Representative traces of platelet aggregation in PRP or washed platelets are shown and are representative of 5 independent experiments. Data from washed platelets were quantified and expressed as maximum platelet aggregation (right). Data represent mean ± standard error of the mean (SEM) of 5 independent experiments. Maximum platelet aggregation was significantly enhanced in BST-2^−/− vs BST-2^+/+ mice. *P < .05; 2-tailed Student t test. (E) P2Y₁₂R activity in BST-2^−/− vs BST-2^+/+ mouse platelets, as assessed by inhibition of forskolin (1 μM; 10 minutes)-induced cAMP production by ADP (0.1 nM-100 μM; 10 minutes). Data are means ± SEM of 4 independent experiments with ADP-stimulated inhibition of adenylyl cyclase activity significantly enhanced in BST-2^−/− vs BST-2^+/+ mouse platelets (2-way analysis of variance [ANOVA]; P < .01). (F) ADP (0.1-100 μM)-stimulated calcium responses were unchanged in BST-2^+/+ vs BST-2^−/− mouse platelets. Data are expressed as peak calcium responses (ratio of 340 nm/380 nm readings with Fura-2) and represent the mean ± SEM of 3 independent experiments. (G-I) ADP-induced inhibition of forskolin-stimulated cAMP production and ERK1/2 activation were assessed in cells transiently cotransfected with mCherry-HA–tagged P2Y₁₂R and either tetherin/BST-2-GFP or GFP control vectors. (G) In P2Y₁₂R-expressing cells, ADP-induced (0.1 nM-100 μM; 10 minutes) inhibition of forskolin-stimulated (1 μM; 10 minutes) cAMP production was significantly attenuated by tetherin/BST-2-GFP overexpression. P < .01; 2-way ANOVA. Data are the mean ± SEM of 4 independent experiments. (H-I) Activity of ADP (20 μM)-stimulated ERK1/2 activation was also assessed by immunoblot analysis with a p-ERK specific antibody. Total ERK and α-tubulin served as protein-loading controls. (H) Representative blots of at least 4 independent experiments. (I) Data expressed as p-ERK levels normalized to total ERK. Mean ± SEM; n = 4. P2Y₁₂R-stimulated ERK activity was significantly attenuated by tetherin/BST-2-GFP overexpression (*P < .05; 2-way ANOVA). (J) Fluorescence intensity profile of mCherry-HA-P2Y₁₂R surface expression taken from TIRF microscopy images of HEK293 cells coexpressing tetherin/BST-2-GFP or GFP control (n = 6 cells over the course of 6 independent experiments).