| Summary: | 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. ...
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