A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site.
This work characterizes a mutant integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen gamma chain (gamma 402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant alpha IIb beta 3 and the reduced binding of mutant alpha IIb beta 3 to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-alpha IIb beta 3, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G----A base change which encoded substitution of R214 by Q in mature beta 3. Introduction of this point mutation into recombinant wild type alpha IIb beta 3 expressed in Chinese hamster ovary cells reproduced the ET platelet alpha IIb beta 3 deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of beta 3(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified alpha IIb beta 3. These findings suggest that substitution of beta 3 R214 by Q is responsible for the functional defect in alpha IIb beta 3 and that R214 is proximal to or part of a ligand binding domain in alpha IIb beta 3.
Bajt
M L
ML
Committee on Vascular Biology, Scripps Research Institute, La Jolla, California 92037.
Ginsberg
M H
MH
Frelinger
A L
AL
Berndt
M C
MC
Loftus
J C
JC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013915:Thrombasthenia / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D012313:RNA; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; D004247:DNA