ABSTRACT. Platelet glycoprotein IIb-IIIa (GPIIb-IIIa, alphaIIbbeta3) is expressed on the cell surface of the human erythroleukemia (HEL) cell line. Previous studies have demonstrated differences in GPIIb-IIIa ligand binding properties of HEL cells when compared to platelets. Although the mRNA sequences for GPIIb and GPIIIa are identical in platelets and HEL cells, cell specific differences in the conformation states of the GPIIb-IIIa complex may exist and may explain in part the contrasting functional properties. Two monoclonal antibodies (mAbs), an anti-GPIIb mAb C3 and an anti-GPIIIa mAb D3, were used to determine whether differences in GPIIb-IIIa conformational states could be measured. Initial studies in a purified system showed that the mAbs' binding to isolated GPIIb-IIIa conformers was increased to the active GPIIb-IIIa and to dissociated receptor subunits when compared to the inactive form. Furthermore, soluble active GPIIb-IIIa was a much better inhibitor of D3 binding to the immobilized receptor compared to soluble inactive GPIIb-IIIa. Extending these studies with intact cells, we detected at least two classes of binding sites for each mAb on each cell type. Differences in Bmax and in the relative affinities of the mAbs were identified and may represent subpopulations of GPIIb-IIIa conformations. Total HEL cell and platelet GPIIb-IIIa was determined in our binding assays using a radiolabeled GPIIb-IIIa complex specific mAb, 10E5. HEL cells express approximately five times more GPIIb-IIIa on a per cell basis. The percent of total GPIIb-IIIa that represented each class of mAb binding sites was determined. In summary, the relative differences in GPIIb-IIIa conformation found on platelets and HEL cells may be related to cell-specific ligand binding properties and activation states of the receptor.

Keywords: platelets, integrins, HEL cells, GPIIb-IIIa.

Reprint requests to: Lisa K. Jennings, Ph.D., Department of Medicine, Room A303 Coleman Bldg., 956 Court Avenue, Memphis, Tennessee 38163, USA, phone: (901) 448-5067, fax: (901) 448-5854, e-mail: ljennings@utmem1.utmem.edu.

ABSTRACT. The genetic basis of idiopathic hemochromatosis, a common disorder of iron metabolism, has remained an enigma for over two decades. In an attempt to refine the chromosomal localization of this gene, we have conducted a linkage disequilibrium mapping study utilizing a large group of unrelated American patients. The 12 microsatellites used as genetic markers in this analysis include a series of recently described polymorphic dinucleotide (D6S1558, D6S1545 and D6S1554) and tetranucleotide (D6S1016 and D6S1281) repeats which map between D6S105 and D6S299. Haplotype reconstructions indicate that a core genotype, composed of D6S464 allele 3/D6S1260 allele 4/D6S1558 allele 5, exists on a majority of disease chromosomes. Stringent statistical measures of marker-disease disequilibrium suggest that only associations with D6S1260 are significant and furthermore, aid in the assignment of refined centromeric and telomeric limits for the likely location of the hemochromatosis gene. In summary, the genetic data presented in this report predict that the hemochromatosis locus resides between D6S464 and D6S1558, most likely very close to marker D6S1260. Because a single yeast artificial chromosome clone contains all three of the above loci, a thorough search for coding sequences in this region is likely to identify the gene mutated in this common disorder.

Keywords: hemochromatosis, linkage disequilibrium mapping, chromosome 6, MHC class I region.

Reprint requests to: Michael J. Chorney, Ph.D., Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA, phone: (717) 531-4604, fax: (717) 531-3562, e-mail: mjc18@psu.edu.

Communicated on February 16, 1996 by Ernest Beutler, M.D. and Carol West, Department of Molecular and Experimental Medicine, Division of Biochemistry, The Scripps Research Institute, 10666 N. Torrey Pines Rd., SBR3, La Jolla, California USA 92037.