The Novel S527F Mutation in the Integrin β3 Chain Induces a High Affinity αIIbβ3 Receptor by Hindering Adoption of the Bent Conformation

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor alpha IIb beta 3 in a patient with an ill defined platelet disorder: one in the beta 3 gene (S527F) and two in the alpha IIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant alpha IIb beta 3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the beta 3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated alpha IIb beta 3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of alpha IIb beta 3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the alpha IIb beta 3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe(527) in beta 3 and the calf-1 domain in alpha IIb and by decreased flexibility between I-EGF2 and I-EGF3.