On the Tissue-type Plasminogen Activator (t-PA) -7,351C>T Enhancer Polymorphism. Importance for endothelial t-PA gene expression and arterial thrombotic disease
Local endothelial release of tissue-type plasminogen activator (t-PA) is an important thromboprotective mechanism. Earlier work by our group has identified a common single nucleotide polymorphism (SNP) at the t-PA locus (-7,351C>T), located within a GC-box in the retinoic acid (RA) and steroid hormone responsive t-PA enhancer. This SNP was associated with local t-PA release in vivo, as subjects homozygous for the wild-type -7,351C allele had twice the t-PA release rates compared to carriers of the mutant T allele. The aim of the present thesis was to elucidate the physiological and pathophysiological relevance of this t-PA variant. TaqMan genotyping assays were designed for a set of SNPs in hemostatic genes to facilitate association studies on these SNPs and thrombotic disease. Specificity and reproducibility was confirmed by DNA sequencing and repeated genotyping. The pathophysiologial relevance of the t-PA -7,351C>T SNP was initially addressed in a prospective study on myocardial infarction (MI) in northern Sweden. An independent association for the t-PA -7,351C>T SNP was found, with a greater risk of MI in T allele carriers. In a large case-control study on ischemic stroke from western Sweden we did, however, not detect a similar association. This study also included a genetic variant of the main inhibitor of t-PA, the plasminogen activator inhibitor type-1 (PAI-1) -675 4G>5G SNP. A reduced risk of ischemic stroke was observed for the combined t-PA CC and PAI-1 4G4G genotype.In vitro studies were performed to functionally characterize the t-PA -7,351C>T SNP. Gel shift analysis using nuclear extracts derived form various cell types, including endothelial cells (ECs) and neuronal-like cells, revealed a strongly reduced binding affinity of transcription factors Sp1 and Sp3 to the T allele, which is interesting in view of the role for Sp1 in gene regulation and enhancer action. Transient transfections demonstrated a lower transcriptional activity in the T enhancer variant after stimulation with RA. An interaction between Sp1 and the RA receptor was also observed. ECs carrying the T allele showed a reduced t-PA induction, both at the mRNA and protein level, in response to RA and protein kinase C (PKC) activation. The combination of RA and PKC activation produced a synergistic t-PA response, resulting in a 2-fold difference in t-PA expression between genotypes.In conclusion, the t-PA -7,351C>T SNP affects endothelial t-PA gene expression at the level of transcription. The reduced expression seen with the mutant T allele may explain our finding of an increased risk for MI in individuals carrying this allele. The t-PA -7,351C>T SNP did not show a significant association to ischemic stroke, but a reduced risk was observed in subjects with the combined t-PA CC and PAI-1 4G4G genotype, supporting a differentiated and more complex role for t-PA and PAI-1 in the brain as compared to the heart
