Disulfide bond disruption by a β3-Cys549Arg mutation in six Jordanian families with Glanzmann thrombasthenia causes diminished production of constitutively active αllbβ3

alpha llb beta 3 integrin mediates platelet aggregation following its activation. Its absence or dysfunction causes Glanzmann thrombasthenia (GT), an inherited bleeding disorder that is rare worldwide but relatively frequent in several populations with high rates of consanguinity, including Arabs in Israel and Jordan. Cysteine residues in the beta 3 epidermal growth factor (EGF) domains are involved in alpha llb beta 3 formation and activation. In this study we present a novel Cys549Arg mutation in beta 3 identified in six Jordanian families, which in the homozygous state is manifested by severe GT The mutation is located in EGF-3 of beta 3 predicting disruption of a conserved disulfide bond between Cys549 and Cys558. Haplotype analysis disclosed a common founder whose age estimate was 120-150 years. Flow cytometry revealed 1-14% of normal alpha llb beta 3 expression at the patients' platelet surface. The Cys549Arg or artificial Cys549Ser mutations were introduced into a beta 3 expression vector. Co-transfection of baby hamster kidney cells with normal or mutant beta 3 along with normal alpha llb demonstrated reduced surface expression of alpha llb beta 3 by both mutants. The mutants were constitutively active as demonstrated by 20-fold increased binding of the ligand-mimetic antibody PAC-1. Immunoblotting and immunoprecipitation experiments showed reduced beta 3 and alpha llb beta 3 expression and a higher than normal ratio of pro-alpha llb to mature alpha llb. Immunofluorescence experiments showed that beta 3 and alpha llb beta 3 were mostly retained in the endoplasmic reticulum. In conclusion, the novel ancestral mutation found in a cluster of Jordanian GT patients disrupts a conserved Cys549-Cys558 bond which results in reduced production of constitutively active alpha llb beta 3.