Species-Specific Differences in Human and Mouse Integrin Alpha-2 Expression.

Variation in the expression of integrin α 2 β 1 leads to differences in collagen-induced platelet responses in vitro , reflecting the contribution of this receptor to hemostasis in vivo . In mice, there are two ITGA2 haplotypes, distinguished by several nucleotide substitutions in the 5′-regulatory/promoter region in linkage disequilibrium (LD) with a variable number of CA repeats in intron 1. Haplotype 1, present in seven strains (C3H/HeJ, C57L/J, DBA/2J, BALB/cJ, C57BL/6J, 129X1/SvJ, SM/J) contains 21 CA repeats at one site in intron 1. Haplotype 2, represented in four strains (SJL/J, SWR/J, A/J and FVB/NJ), contains only 7 CA repeats in that position. The strain AKR/J carries a unique haplotype, arising from a single crossover between the 5′-regulatory region of haplotype 1 and intron 1 of haplotype 2 downstream from the CA repeat site. No difference was observed in transcription rate initiated by the two 5′-regulatory/promoter haplotypes. Nonetheless, in every strain expressing haplotype 2 (7 CA repeats), a two-fold decrease in platelet surface α 2 β 1 is found. The difference in CA repeat length affects the binding of heterogeneous nuclear ribonucleotide binding protein L (hnRNP L), which influences the rate and fidelity of mRNA splicing. This is the sole determinant of the two-fold difference in platelet α 2 β 1 level between mouse strains. In humans, control of ITGA2 transcription and α 2 expression is more complex. The two oldest human haplotypes, as determine by HapMap analyses (Di Paola J et al., J. Thromb. Haemost., 2005; 3:1511–1521), are distinguished by the single nucleotide polymorphism (SNP) C-52T, and we have reported that -52T is associated with at least a five-fold decrease in rate of ITGA2 transcription in vitro . In this study, we have identified a difference in CA repeat length at two sites in human ITGA2 . One site is located in the 5′ regulatory region (−512 to −492) upstream from C-52T, where haplotype −52C bears 12 CA repeats, while haplotype −52T bears 10 or 11 CA repeats. A comparable CA repeat site is not present in the murine ITGA2 5′-regulatory region. The presence of 12 repeats increases transcription rate in vitro by two- to three-fold independently of the nucleotide substitution at −52. An additional difference in CA repeat length is seen at the second site, located downstream and within the 5′ region of intron 1. However, unlike the findings in mice, the intron 1 CA repeat length in human ITGA2 does not significantly alter the rate or fidelity of mRNA splicing. Increased CA repeat length was shown to enhance transcription of the human matrix metalloproteinase 9 gene ( MMP9 ), and nuclear CA repeat-binding proteins were detected, but none has since been identified (Shimajiri S. et al., 1999; 455:70–74). Such proteins are likely involved in the enhancement of ITGA2 transcription observed in our study and are distinct from hnRNP L or other mRNA binding proteins that regulate mRNA splicing. Thus, CA repeat length polymorphisms play a role in post-transcriptional regulation of mRNA splicing in mice and transcriptional regulation in humans. The human ITGA2 promoter SNP C-52T and the polymorphic CA repeat site at −512 to −492 define the two oldest human haplotypes and synergistically modulate the expression of human integrin α 2 β 1 .