Polymorphism of human platelet membrane glycoprotein IIb associated with the Baka/Bakb alloantigen system.
The human Baka/Bakb alloantigen system has been implicated in the pathogenesis of post-transfusion purpura and neonatal alloimmune thrombocytopenic purpura. Human alloantisera specific for either the Baka or Bakb allele have been shown to react exclusively with the heavy chain of membrane glycoprotein (GP) IIb. To investigate the structure of the Bak epitopes, we used the polymerase chain reaction (PCR) to amplify GPIIb cDNA synthesized from platelet RNA samples prepared from individuals of known serologic phenotype. Subsequent DNA sequence analysis of amplified GPIIb cDNAs derived from one Baka homozygous individual and one Bakb homozygous individual revealed a single nucleotide base difference near the 3' end of the mRNA encoding the GPIIb heavy chain. Short 13 base allele-specific oligonucleotides (ASO) containing the putative phenotype-specific base in the middle were then synthesized, end-labeled with digoxigenin-11-dUTP using terminal transferase, and used as probes in subsequent dot-blot hybridization experiments. Platelet RNA was prepared from a panel made up of four Baka/a, three Bakb/b, and two Baka/b individuals, and the mRNA encoding GPIIb was amplified using PCR and spotted onto nylon membranes. ASO hybridization showed that the nucleotide base difference identified above segregated with Bak phenotype in all nine individuals examined (P = .002). The base pair substitution results in an amino acid polymorphism at residue 843 of the mature heavy chain. The Baka form of GPIIb encodes an isoleucine at this position, whereas the Bakb allele contains a serine. Identification of the polymorphism associated with this clinically important alloantigen system should permit new therapeutic and diagnostic approaches for treating and managing patients with alloimmune thrombocytopenic disorders.
Lyman
S
S
Blood Center of Southeastern Wisconsin, Milwaukee 53233.
Aster
R H
RH
Visentin
G P
GP
Newman
P J
PJ
D000595:Amino Acid Sequence; D016824:Antigens, Human Platelet; D001483:Base Sequence; D001792:Blood Platelets / Q000276:immunology* / Q000502:physiology; D006801:Humans; D007519:Isoantigens / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010980:Platelet Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000235:genetics
C044561:3a alloantigen, human; D016824:Antigens, Human Platelet; D007519:Isoantigens; D015345:Oligonucleotide Probes; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger