Disulfide bond disruption by a beta 3-Cys549Arg mutation in six Jordanian families with Glanzmann thrombasthenia causes diminished production of constitutively active alpha IIb beta 3.
Alpha IIb 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 IIb 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 IIb 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 IIb demonstrated reduced surface expression of alpha IIb 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 IIb beta 3 expression and a higher than normal ratio of pro-alpha IIb to mature alpha IIb. Immunofluorescence experiments showed that beta 3 and alpha IIb 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 IIb beta 3.
Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer, Israel 52621.
Ronit
R
Mor-Cohen
Nurit
N
Rosenberg
Hava
H
Peretz
Meytal
M
Landau
Barry S
BS
Coller
Abdalla
A
Awidi
Uri
U
Seligsohn
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry; D005190:Family; D005260:Female; D018703:Founder Effect; D005838:Genotype; D006239:Haplotypes; D006720:Homozygote; D006801:Humans; D039421:Integrin alpha2 / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D007597:Jordan; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D010641:Phenotype; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012720:Severity of Illness Index; D013915:Thrombasthenia / Q000097:blood / Q000235:genetics*; D014162:Transfection
D004220:Disulfides; C056564:ITGA2B protein, human; D039421:Integrin alpha2; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D003545:Cysteine