Collagen Receptors/
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, The Integrin Subunit a2 GeneNucleotide polymorphisms in the a2 gene define multiple alleles that are associated with differences in platelet a2b1 density.
Kritzik M, Savage B, Nugent DJ, Santoso S, Ruggeri ZM, Kunicki TJ
Blood 1998 Oct 1;92(7):2382-8
Three allelic differences in the a2 gene are associated with expression levels of the a2b1 integrin on the platelet surface. We have previously defined two linked silent polymorphisms in the a2 gene coding region at nucleotides 807 (C or T) and 873 (G or A). We have now identified one rarer nucleotide polymorphism in the coding region at nucleotide 837 (T or C) and four additional linked polymorphisms within the introns that flank these coding sequences. Moreover, we have determined that the alloantigenic Br polymorphism, which resides in a distal coding region at nucleotide 1648, is also linked to the 837 polymorphism. Thus, three a2 gene alleles, defined by eight nucleotide polymorphisms, have now been discovered. Allele 1 (807T/837T/873A/Brb) is associated with increased levels of a2b1; allele 2 (807C/837T/873G/Brb) and allele 3 (807C/837C/873G/Bra) are each associated with lower levels of a2b1. Finally, we also show here that the rate of platelet attachment to type I collagen in whole blood under conditions of high shear rate (1,500/s) is proportional to the density of a2b1 on the platelet surface. Thus, the density of platelet a2b1 could have an important impact on platelet adhesion to collagen in whole blood and therefore on platelet function in vivo, contributing to an increased risk of thrombosis or to bleeding in relevant disease states.
Characterization of Inherited Differences in Transcription of the Human Integrin alpha 2 Gene.
Jacquelin B, Rozenshteyn D, Kanaji S, Koziol JA, Nurden AT, Kunicki TJ.
J Biol Chem. 2001 Jun 29;276(26):23518-24. Epub 2001 Apr 19.
Inherited, single-base substitutions are found at only two positions, C(-)52T and C(-)92G, within the proximal 5'-regulatory region (within -1096 to +48) of the human integrin alpha(2) gene. We recently reported that the T(-)52 substitution results in decreased binding of transcription factor Sp1 to adjacent binding sites, decreased transcription of the alpha(2) gene, and reduced densities of platelet alpha(2)beta(1). In this study, we identify an additional Sp1-binding site at position -107 to -99 and show that the adjacent dimorphic sequence C(-)92G also influences the rate of gene transcription. In the erythroleukemia cell line Dami, transfected promoter-luciferase constructs bearing the G(-)92 sequence exhibit roughly a 3-fold decrease in activity relative to the C(-)92 constructs. In transfected CHRF-288-11 megakaryocytic cells, the corresponding activity decreases by 5-fold. DNase I footprinting of the promoter region with Dami nuclear extracts showed a protected segment at -107 to -99 that can be deprotected by coincubation with molar excess of a consensus Sp1 oligonucleotide. Gel mobility shift assays and supershift assays with specific antibodies indicate that Sp1 binds to this region of the alpha(2) gene promoter. Mutation of the Sp1 binding element within -107 to -99 in constructs containing either C(-)92 or G(-)92 abolishes basal promoter activity and eliminates the binding of Sp1. The G(-)92 sequence has a gene frequency of 0.15 in a typical Caucasian population, and the presence of this allele correlates with reduced densities of platelet alpha(2)beta(1). The combined substitution G(-)92/T(-)52 has an additive influence on gene transcription, resulting in an 8-fold decrease in transfected Dami cells or a 20-fold decrease in transfected CHRF-288-11 cells. In summary, the natural dimorphism C(-)92G within the proximal 5'-regulatory region of the human integrin alpha(2) gene contributes to the regulation of integrin alpha(2)beta(1) expression on megakaryocytes and blood platelets and must thereby modulate collagen-related platelet function in vivo.
Allele-dependent transcriptional regulation of the human integrin alpha2 gene.
Jacquelin B, Tarantino MD, Kritzik M, Rozenshteyn D, Koziol JA, Nurden AT, Kunicki TJ.
Blood. 2001 Mar 15;97(6):1721-6.
Genetically controlled variation in alpha2beta1 expression by human blood platelets was previously described. Sixty-two haplotype sequences corresponding to the proximal 5' regulatory region (-1096 to +1) of the alpha2 gene were compared, and a dimorphic sequence -52C>T was identified that is located precisely between 2 tandem Sp1/Sp3 binding elements previously shown to be absolutely required for transcriptional activity of this gene in epithelial cell lines and the erythroleukemic cell line K562. The gene frequency of -52T in a random Caucasian population is approximately 0.35, and the expression of -52T correlates directly with reduced densities of platelet alpha2beta1. In mobility shift analyses, the -52T substitution attenuates complex formation with both Sp1 and Sp3. When transfected into the erythroleukemia cell line Dami, promoter-luciferase constructs bearing the -52T sequence exhibit a 5-fold decrease in activity relative to the -52C construct. In transfected CHRF-288-11 megakaryocytic cells, the corresponding activity decreases by 10-fold. The -52T sequence appears to be in linkage disequilibrium with the previously defined allele A3 (807C; HPA-5b), known to be associated with diminished expression of platelet alpha2beta1. In summary, a natural dimorphism has been identified within the proximal 5' regulatory region of the human integrin alpha2 gene that is responsible for decreased expression levels of the integrin alpha2beta1 on blood platelets through a mechanism that is probably mediated by the nuclear regulatory proteins Sp1 and Sp3.
Collagen Receptors : ITGA2 created by Thomas J. Kunicki tomk@scripps.edu
Scripps Research Institute