Mutating MIDAS residue D119A eliminates binding of normal αIIbβ3-HEK and αIlb(FF)β3-HEK cells to immobilized fibrinogen and binding of αIlb(FF)β3-HEK cells to ‘D98,’ but does affect adhesion of normal αIIbβ3-HEK cells to ‘D98’ in the presence of EDTA. HEK293 cells (2 × 103/µL; 50 µL) expressing normal αIIbβ3, the αIlbβ3 constitutively active mutant αIlb(FF)β3, the αIlbβ3(D119A) MIDAS-disrupting mutant, or the combined αIlb(FF)β3(D119A) mutant were labeled with calcein (10 µM) and then added to microtiter wells precoated with fibrinogen or ‘D98’ (each at 10 µg/mL coating concentration) for 1 hour at 22°C in the absence and presence of EDTA (10 mM). The fluorescent signal of adherent cells was measured after washing away the nonadherent cells. (A) The normal αIlbβ3-HEK and αIlb(FF)β3-HEK cells adhered to fibrinogen, whereas the αIlbβ3(D119A)-HEK MIDAS mutant and the combined αIlb(FF)β3(D119A)-HEK mutant did not adhere to fibrinogen (n = 3; P ≤ .01). (B) αIlb(FF)β3-HEK cells adhered to ‘D98,’ whereas the same cells with the MIDAS-disrupting mutation β3(D119A), did not adhere (n = 3; P = .004). (C) EDTA treatment (10 mM) of αIlbβ3(D119A)-HEK cells increased their adhesion to ‘D98’ to the level of normal αIlbβ3-HEK cells (n = 4; P > .05). Data reported as mean ± SD. Expression levels for each cell line were measured based on mAb 7E3 binding and are expressed as GMFI: normal αIIbβ3 53 ± 26 AFU; αIIbβ3(D119A) 36 ± 11 AFU; αIIb(FF)β3 25 ± 5 AFU; αIIb(FF)β3(D119A) 27 ± 2 AFU.