Disulfide bond disruption by a β3-Cys549Arg mutation in six Jordanian families with Glanzmann thrombasthenia causes diminished production of constitutively active αIIbβ3

Abstract

SummaryαIIbβ3 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 β3 epidermal growth factor (EGF) domains are involved in αIIbβ3 formation and activation. In this study we present a novel Cys549Arg mutation in β3 identified in six Jordanian families, which in the homozygous state is manifested by severe GT. The mutation is located in EGF-3 of β3 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 αIIbβ3 expression at the patients' platelet surface. The Cys549Arg or artificial Cys549Ser mutations were introduced into a β3 expression vector. Co-transfection of baby hamster kidney cells with normal or mutant β3 along with normal αIIb demonstrated reduced surface expression of αIIbβ3 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 β3 and αIIbβ3 expression and a higher than normal ratio of pro-αIIb to mature αIIb. Immunofluorescence experiments showed that β3 and αIIbβ3 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 αIIbβ3.

Atypical structure and function of integrin αVβ8

“…Cysteine residues in the extracellular domain are correlated with the integrin structure. Mutations in the conserved cysteines in the EGF domains of the β 3 lower leg have been found to cause extension (Kamata et al, 2004; Mor‐Cohen et al, 2007). Indeed, sequence alignment has shown that, compared with other β integrins, β 8 lacks two pairs of conserved cysteines that form disulfide bonds within the EGF3 and β‐tail domains, rendering β 8 unable to stabilize its bent conformation (Cormier et al, 2018).…”