Establishment of the stable cell line expressing human platelet GPIb/IX receptor and its utilization for platelet function

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The platelet GPIb/IX complex, a surface receptor for von Willebrand factor (vWF), is composed of three-independent gene products of GPIb-alpha, -beta and IX. Those components linked together and form a functional receptor for vWF. So far, genetic analyses of the patients with Bernard-Soulier syndrome have shown that the leucine rich repeat (LRR) in the GPIb-alpha is essential for an efficient surface expression of the complex. LRR is a short 24 amino acid stretch containing well preserved leucine residues, and each GP possesses at least one of this consensus motif. Little is known, however, about the importance of the LRR within GPIb-beta because of the absence of the clinical case. To assess the importance of the GPIb-beta LRR for the expression of GPIb/IX complex, four amino acid residues within the LRR of the GPIb-beta have been chosen and site-directed mutagenesis was performed to obtain series of mutants; Leu35, Leu45 and Leu50 → Ala, Val or Phe, Asn40 → Ala, Thr or Cys. The obtai … More ned plasmids were co-transfected with GPIb-alpha and GPIX, and the expression of GPIb/IX complex was analyzed by FACS and Northern blotting.I. FACS analyses of the GPIb/IX receptor transfected with the mutant plasmids: All Asn40-mutants drastically decreased the surface expression of the complex. Similar results were obtained by using Leu → Ala or Phe mutants, however, Leu → Val mutants always showed 40-80% surface expression of the complex when analyzed by FACS.II. Northern blot analyses of the mutants: Northern blotting analyses showed that GPIb-beta mRNA level of the mutants were almost the same as control, indicating post-transcriptional events were causative for the decreased receptor expression. These data have suggested that Leu residues and Asn40 within the LRR of the GPIb-beta are important for the efficient expression of the GPIb/IX complex, although Leu → Val mutants showed moderate complex expression probably due to the structural similarity of both amino acids.III. Establishment of stable transformant expressing human platelet GPIb/IX receptor: We have utilized the above described plasmids to establish the stable transformants. The transformants possess the platelet-like aggregatory potential by ristocetin, mimic the human platelet GPIb/IX complex. However, they gradually lost the activity by long-term cultivation. Some modifications are necessary to maintain the aggregatory potential of the transformants. Less