A naturally occurring mutation near the amino terminus of alpha IIb defines a new region involved in ligand binding to alpha IIb beta 3

Decreased expression of functional alpha IIb beta 3 complexes on the platel et surface produces Glanzmann thrombasthenia, We have identified mutations of alpha IIb(P145) in 3 ethnically distinct families affected by Glanzmann thrombasthenia, Affected Mennonite and Dutch patients were homozygous and d oubly heterozygous, respectively, for a P(145)A substitution, whereas a Chi nese patient was doubly heterozygous for a (PL)-L-145 substitution. The mut ations affect expression levels of surface alpha IIb beta 3 receptors on th eir platelets, which was confirmed by co-transfection of alpha IIb(P145A) a nd beta 3 cDNA constructs in COS-1 cells. Each mutation also impaired the a bility of alpha IIb beta 3 on affected platelets to interact with ligands. Moreover, when alpha IIb(P145A) and beta 3 were stably coexpressed in Chine se hamster ovary cells, alpha IIb beta 3 was readily detected on the cell s urface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate-fibrinogen after alpha IIb beta 3 activation by the activating monoclonal antibody PT25-2, Nonetheless, incub ating affected platelets with the peptide LSARLAF, which binds to alpha IIb , induced PF4 secretion, indicating that the mutant alpha IIb beta 3 retain ed the ability to mediate outside-in signaling. These studies indicate that mutations involving alpha IIb(P145) impair surface expression of alpha IIb beta 3 and that the alpha IIb(P145A) mutation abrogates ligand binding to the activated integrin, A comparative analysis of other alpha IIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the alpha IIb and may define a domain influ encing ligand binding. (C) 2000 by The American Society of Hematology.