Analysis of GPIb and GPIX in HEK 293T cells transfected with GPIb, GPIbβ, and GPIX.

HEK 293T cells were transiently transfected with wild-type GPIbα, GPIbβ, and GPIX or with wild-type GPIbα and GPIX and mutant W21stop-GPIbβ. The cells were analyzed with an anti-GPIbα polyclonal antibody, the anti-GPIX antibody FMC25, or the complex-specific antibody AK1 (each graph is representative of 4 different experiments). (A) In cells transfected with wild-type GPIbα, GPIbβ, and GPIX, there was a significant increase in fluorescence in cells in reaction to the anti-GPIbα polyclonal antibody (boldface lines) compared with mock-transfected cells (shaded area). In cells transfected with W21stop-GPIbβ and wild-type GPIbα and GPIX, GPIbα was detectable on the cell surface (thin line) but in significantly smaller amounts than in triple wild-type transfections. (B) In cells transfected with wild-type GPIbα, GPIbβ, and GPIX, GPIX was readily detectable on the cell surface (boldface line). However, GPIX was not detectable when W21stop-GPIbβ was transfected with wild-type GPIbα and GPIX (thin line). Mock-transfected cells are shown for comparison (shaded area). (C) There was a marked increase in surface fluorescence in cells transfected with wild-type GPIbα, GPIbβ, and GPIX in reaction to the complex-specific antibody, AK1 (boldface lines), compared with mock-transfected cells (shaded area) and cells transfected with wild-type GPIbα, GPIX, and mutant GPIbβ (thin line). Thus, GPIbα that was expressed on the cell surface (A) in the transfections involving the mutant GPIbβ was not recognized by AK1, thereby confirming the lack of surface expression of GPIX or complex formation.