Abstract
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.
Highlights
This work characterizes a mutant integrin a1&3 (glycoprotein (GP)IIb-IIIa)froma thrombasthenicpatient, ET, whose platelets fail to aggregate in response to stimuli
Analysis of platelet cDNA synthesis andpolymerase chain reaction (PCR) Products-5 pl of each PCR sample or control of thrombasthenia contain normal levels of a& but fail to sample was analyzed on a 1.2% agarose gel to verify the presence of aggregate followingplatelet activation(13)
Pools of 50 individual positive characterize the defect of (YIlbP3 on ET platelets, the binding ofmAb PACl was assessed by flow cytometry (Fig. 1).monoclonal antibodies (mAb) PACl binds preferentially to activated platelets and is directed against a fibrinogen binding site (19, 32, 33)
Summary
Analysis of PCR Products-5 pl of each PCR sample or control of thrombasthenia contain normal levels of a& but fail to sample was analyzed on a 1.2% agarose gel to verify the presence of aggregate followingplatelet activation(13). Verify the absence of any other substitutions To distinguish between these possibilities, the ability of the Stable Cell Lines-A stably transfected CHO cell line expressing platelets to bind an activation-independent RGD peptide the mutant receptor was established by electroporationusingthe expression constructs CD3(R214 + Q ) and CD2bNeo,a CDM8 construct encoding the full-length allband the neomycin resistance genes. Control platelets exhibited a saturable increase in antibody binding such that half-maximal expression of the LIBSl epitope was observed at 20 pM GRGDSP and 120 pM L10. At 200 p~ GRGDSP, anti-LIBS1 binding to ET platelets was less than that seen with control platelets in the presence of 4 pM peptide, suggesting that the affinity of E T Detergent extracts of control and ET platelets were chromatographed iomnmobilized KYGRGDS.
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