Involvement of the cysteine-rich domain of glycoprotein IIIa in the expression of the human platelet alloantigen, PlA1: evidence for heterogeneity in the humoral response.
To assess the potential contribution of the cysteine-rich domain of human platelet glycoprotein (GP) IIIa to the structure of the PlA1 epitope, we used site-directed mutagenesis to substitute alanines for cysteines at key positions potentially involved in PlA1 alloantigen formation. One of these GPIIIa isoforms in which alanine replaced Cys435 reacted normally with a series of well-characterized murine MoAbs directed against the 250 amino-terminal residues of GPIIIa, whereas binding of MoAbs specific for residues 348-692 was diminished or lost. Interestingly, of eight PlA1-specific antibodies tested, two recognized Ala435GPIIIa, two did not, and four bound to it less well than to wild-type (WT) GPIIIa. However, disruption of disulfide bonds located at or near the N-terminus of GPIIIa abolished the binding of all the anti-PlA1 alloantibodies tested. These findings provide evidence that the humoral response to the PlA1 antigen is (1) heterogenous, ie, the binding site on GPIIIa for human anti-PlA1 antibodies differs from one individual to another and (2) complex--although some anti-PlA1 alloantibodies bind to an epitope comprised solely of the amino-terminus of GPIIIa, others combine most efficiently with a more complex determinant that involves the cysteine-rich domain of GPIIIa. These findings may have implications for diagnostic and therapeutic use of synthetic or recombinant PlA1 mimetics.
Valentin
N
N
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee 53201, USA.
Visentin
G P
GP
Newman
P J
PJ
D000483:Alleles; D000911:Antibodies, Monoclonal / Q000276:immunology; D016824:Antigens, Human Platelet / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003545:Cysteine; D006801:Humans; D039661:Integrin beta3; D007518:Isoantibodies / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D016824:Antigens, Human Platelet; C061248:ITGB3 protein, human; D039661:Integrin beta3; D007518:Isoantibodies; D010980:Platelet Membrane Glycoproteins; D003545:Cysteine