A single F153S & beta;3 mutation causes constitutive integrin & alpha;IIb & beta;3 activation in a variant form of Glanzmann thrombasthenia

This report identifies a novel variant form of the inherited bleeding disorder Glanzmann thrombasthenia, exhibiting only mild bleeding in a physically active individual. The platelets cannot aggregate ex vivo with physiologic agonists of activation, although microfluidic analysis with whole blood displays moderate ex vivo platelet adhesion and aggregation consistent with mild bleeding. Immunocytometry shows reduced expression of & alpha;IIb & beta;3 on quiescent platelets that spontaneously bind/store fibrinogen, and activationdependent antibodies (ligand-induced binding site-319.4 and PAC-1) report & beta;3 extension suggesting an intrinsic activation phenotype. Genetic analysis reveals a single F153S & beta;3 substitution within the & beta;I-domain from a heterozygous T556C nucleotide substitution of ITGB3 exon 4 in conjunction with a previously reported IVS5(+1)G>A splice site mutation with undetectable platelet messenger RNA accounting for hemizygous expression of S153 & beta;3. F153 is completely conserved among & beta;3 of several species and all human & beta;-integrin subunits suggesting that it may play a vital role in integrin structure/function. Mutagenesis of & alpha;IIbF153S & beta;3 also displays reduced levels of a constitutively activated & alpha;IIb-S153 & beta;3 on HEK293T cells. The overall structural analysis suggests that a bulky aromatic, nonpolar amino acid (F,W)153 & beta;3 is critical for maintaining the resting conformation of & alpha;2- and & alpha;1-helices of the & beta;I-domain because small amino acid substitutions (S,A) facilitate an unhindered inward movement of the & alpha;2- and & alpha;1-helices of the & beta;I-domain toward the constitutively active & alpha;IIb & beta;3 conformation, while a bulky aromatic, polar amino acid (Y) hinders such movements and restrains & alpha;IIb & beta;3 activation. The data collectively demonstrate that disruption of F153 & beta;3 can significantly alter normal integrin/platelet function, although reduced expression of & alpha;IIbS153 & beta;3 may be compensated by a hyperactive conformation that promotes viable hemostasis.