Tetherin/BST-2 negatively regulates P2Y₁₂R internalization in both cell lines and mouse platelets. (A) Representative live TIRF microscopy images of HEK293 cells coexpressing mCherry-HA-P2Y₁₂R (red) and either GFP control (green; top) or tetherin/BST-2-GFP before (green; middle) and after agonist stimulation (ADP; 20 μM; 60 minutes; green; bottom). (B) Fluorescence intensity profiles from lines shown in images from panel A assessing either mCherry (receptor) or GFP (either control or tetherin/BST-2) surface expression. (C-D) A time course series (0-60 minutes) of the intensity profiles was assessed (n = 6 cells over the course of 6 independent experiments), and the ADP-stimulated change in surface mCherry-HA-P2Y₁₂R (C) or tetherin/BST-2-GFP (D) was quantified (percentage change after agonist stimulation; mean ± standard error of the mean [SEM]). (E) Assessment of FLAG-P2Y₁₂R recycling by flow cytometry. Receptor-expressing cells prelabeled with anti-FLAG(M1)-FITC were stimulated with ADP (20 μM; 60 minutes). Surface antibody and ADP were removed by washing with a Ca²⁺-free buffer containing apyrase (0.2 U/mL). P2Y₁₂R recycling was then monitored by flow cytometry. Data represent percentage of recycled P2Y₁₂R (*P < .05; 1-way analysis of variance [ANOVA]). (F-G) P2Y₁₂, but not P2Y₁R internalization, was negatively regulated by tetherin/BST-2 in mouse platelets. BST-2^−/− and BST-2^+/+ mouse platelets were stimulated with ADP (10 μM). P2Y₁ and P2Y₁₂ surface receptor levels were measured in fixed, washed platelets, with[³H]-2MeSADP (100 nM) in the presence of either the P2Y₁R antagonist A3P5P (1 mM) or the P2Y₁₂R antagonist AR-C69931MX (1 μM). Data are expressed as a percentage of surface receptor and represent means ± SEM of 3 independent experiments. P2Y₁₂ but not P2Y₁R surface loss was potentiated in BST-2^−/− vs BST-2^+/+ mouse platelets (P < .05; 2-way ANOVA).