TMD-tail mini-integrins activate native integrin α_IIbβ₃. (A) Tac-α_IIb^TM or Tac-α_IIb was transfected into CHO cells expressing α_IIbβ₃ (A5 cells). Twenty-four hours after transfection, cells were stained with PAC1 (activation-specific anti-α_IIbβ₃ antibody) and 7G7B6 (anti-Tac antibody) in the presence or absence of a selective inhibitor of ligand binding to α_IIbβ₃, Ro-43-5054, and then analyzed by flow cytometry to generate the dot plots shown (left). Geometric means of PAC1 binding in cells expressing different quantities of Tac constructs were plotted as larger red dots. Specific PAC1 binding was calculated by subtracting the geometric means of PAC1 binding in the presence of Ro-43-5054 from that in the absence of Ro-43-5054 in each range, and plotted as a line graph (right). (B) The effects of Tac-α_IIb^TMΔGFFKR and Tac-α_IIb^TMLXXXL on α_IIbβ₃ activation were analyzed, as in panel A. (C) The effects of Tac-β₃^TM, Tac-β₃, and Tac-β₃^TM(Y747A) on α_IIbβ₃ activation were analyzed, as in panel A. The activating effects of each Tac constructs were tested at least 3 times, and representative data are shown.