THE CYTOPLASMIC DOMAIN OF GLYCOPROTEIN (GP) IB-ALPHA CONSTRAINS THE LATERAL DIFFUSION OF THE GP IB-IX COMPLEX AND MODULATES VON-WILLEBRAND-FACTOR BINDING

To study the role of the glycoprotein (GP) Ib alpha cytoplasmic domain in the mobility of the GP Ib-IX complex within the plasma membrane an d in its ability to bind vWf, we established eight cell lines expressi ng GP Ib-IX complexes (these complexes lack CP V but function normally as receptors for vWf) that contain either wild-type GP Ib alpha or on e of a series of GP Ib alpha truncation mutants missing different leng ths of the cytoplasmic domain. To test the mobility of these complexes within the plasma membrane, we used the technique of fluorescence rec overy after photobleaching after labeling them with a fluorescein-conj ugated anti-GP Ib alpha monoclonal antibody. Fluorescence recovery wit hin a bleached area on the cell surface was evaluated by scanning the cell surface with a low-intensity laser for 3 min after bleaching and then extrapolating the recovery values to infinite time, Fluorescence recovery in cells expressing wild-type GP Ib alpha was negligible, How ever, when only six amino acids were removed from the GP Ib alpha carb oxyl terminus (t604 mutant, polypeptide length of 604 vs 610 residues for wild-type GP Ib alpha), complex mobility increased greatly, as jud ged by a more rapid recovery of fluorescence in the bleached area (48% recovery), The mobility increased further in the t594 mutant and rema ined approximately the same through the t534 mutant (55-67% recovery), A further increase in mobility was observed with the t518 mutant (> 8 0% recovery), which lacks almost all of the GP Ib alpha cytoplasmic do main. The ristocetin-dependent binding of the mutant cell lines was al so evaluated, Binding of vWf to cells expressing any of the mutant com plexes was markedly lower than that to cells expressing the wild-type complex. These studies demonstrate that the cytoplasmic domain of GP r od fixes the position of the GP Ib-IX complex on the platelet surface and that this orientation is an important determinant of the complex's ability to bind vWf.