Identification of genomic loci regulating platelet plasminogen activator inhibitor-1 in mice

Abstract

Background: Plasminogen activator inhibitor-1 (PAI-1, Serpine1) is an important circulating fibrinolysis inhibitor. PAI-1 exists in 2 pools, packaged within platelet α-granules and freely circulating in plasma. Elevated plasma PAI-1 levels are associated with cardiovascular disease. However, little is known about the regulation of platelet PAI-1 (pPAI-1).

Objectives: We investigated the genetic control of pPAI-1 levels in mice and humans.

Methods: We measured pPAI-1 antigen levels via enzyme-linked immunosorbent assay in platelets isolated from 10 inbred mouse strains, including LEWES/EiJ (LEWES) and C57BL/6J (B6). LEWES and B6 were crossed to produce the F1 generation, B6LEWESF1. B6LEWESF1 mice were intercrossed to produce B6LEWESF2 mice. These mice were subjected to genome-wide genetic marker genotyping followed by quantitative trait locus analysis to identify pPAI-1 regulatory loci.

Results: We identified differences in pPAI-1 between several laboratory strains, with LEWES having pPAI-1 levels more than 10-fold higher than those in B6. Quantitative trait locus analysis of B6LEWESF2 offspring identified a major pPAI-1 regulatory locus on chromosome 5 from 136.1 to 137.6 Mb (logarithm of the odds score, 16.2). Significant pPAI-1 modifier loci on chromosomes 6 and 13 were also identified.

Conclusion: Identification of pPAI-1 genomic regulatory elements provides insights into platelet/megakaryocyte-specific and cell type-specific gene expression. This information can be used to design more precise therapeutic targets for diseases where PAI-1 plays a role.

Keywords: blood platelets; fibrinolysis; gene expression regulation; megakaryocytes; quantitative trait loci; serpin E1.

Figure 1:. Mouse Strain-Specific pPAI-1 Expression.

(A) Quantification of total pPAI-1 derived from inbred laboratory and wild-derived mouse strains was performed by mouse PAI-1 total antigen ELISA assay. Prior to lysis, platelet samples were normalized to 400,000 platelets/μL. *The reduced pPAI-1 levels in two LEWES/EiJ mouse strain mice may reflect commercially obtained mice that were contaminated with WSB/EiJ strain mice. (B) Genome-wide summary neighbor joining phylogeny of 63 inbred strains depicting the relationships of the inbred mouse strains analyzed by PAI-1 ELISA. The tree was rooted at the most recent common ancestor of M. m. castaneus and M. m. musculus wild-derived strains. Mouse strains analyzed here are depicted in larger font. (C) Plasma PAI-1 antigen levels measured by ELISA. Grey dots and text represent inbred laboratory strains and red dots and text represent wild-derived strains.

Figure 2:. Semidominant Serpine1 Regulatory Locus.

(A) Total pPAI-1 from platelet lysates normalized for total protein (150 μg/mL). (B) LEWES pPAI-1 sex differences. (C) RT-PCR of platelet Serpine1 expression (NTC=no template control). Actb expression serves as control. (D) qPCR of platelet Serpine1 expression. *p<0.05, ****p<0.0001.

Figure 3:. RT-qPCR data of relative Serpine1 mRNA expression in B6 and LEWES whole liver and adipose tissue samples.

No significant differences were detected in Serpine1 expression (n=3 per group, p=NS)

Figure 4:. QTL Analysis of pPAI-1 Levels in B6LEWESF2 mice.

(A-D) LOD curves from the QTL analysis for pPAI-1 levels, with close-ups of the significant peaks on Chromosome 5 (B), 6 (C), and 13 (D). The horizontal solid and dashed lines represent the thresholds for significant QTL (LOD=4.01) and suggestive QTL (LOD=2.65), respectively. (E) Effect plot of the strain alleles at SNP marker UNC050425680 (136.1 Mb) on Chromosome 5 (B/B: 3.3 ± 0.6, B/L: 37.2 ± 2.3, L/L: 57.7 ± 6.7 fg/ug total protein, ****p<0.0001). (F) Effect plot of the allelic sex differences at SNP marker UNC050425680 on Chromosome 5 (B/B F: 3.0 ± 1.02, B/B M: 3.6 ± 1.9, B/L F: 39.3 ± 2.9, B/L M: 35.4 ± 3.5, L/L F: 62.0 ± 10.7, L/L M: 53.8 ± 8.2 fg/ug total protein). (G) LOD curve of Chromosome 8 from the QTL with sex as an interactive covariate. Black curve is the single QTL analysis and red curve is the sex as a covariate QTL. Dashed line represents the threshold for suggestive QTL (LOD=3.76). (H) Effect plot of the allelic sex differences at SNP marker UNC081450048 (126.9 Mb) on Chromosome 8 (B/B F: 22.2 ± 4.9, B/B M: 46.9 ± 9.6, B/L F: 31.3 ± 5.3, B/L M: 26.6 ± 5.8, L/L F: 59.3 ± 8.6, L/L M: 34.1 ± 4.3 fg/ug total protein, *p<0.05). B/B=homozygous B6, B/L=heterozygous, and L/L=homozygous LEWES.