Genetic variation at the human tissue-type plasminogen activator locus

Abstract

Tissue-type plasminogen activator (tPA) is the key initiator of intravascular fibrinolysis. Family studies suggest that tPA release is regulated by hereditary factors. The aim of the present thesis was therefore to test the hypothesis that a neutral polymorphism at the tPA locus is associated with vascular release of tPA, and if so search the tPA locus for functional polymorphisms that could explain this finding. An identified genetic marker of vascular tPA release was then investigated in an association study of myocardial infarction.Vascular tPA release rates were assessed in 51 healthy males by the perfused forearm model. By this invasive procedure blood samples were simultaneously drawn from the brachial artery and vein. Net release of tPA was defined as the venoarterial plasma concentration difference times local plasma flow. In an initial study, subjects were typed for an Alu insertion polymorphism in intron eight of the tPA gene. A significant association with both basal and stimulated tPA release rates was observed. As the Alu polymorphism is neutral, this finding prompted us to search the tPA locus for putative functional variations, that could explain our observation. Regulatory and coding regions of the tPA gene were re-sequenced in 30 chromosomes and eight single nucleotide polymorphisms (SNPs) were discovered. All SNPs were typed in the 51 subjects who had taken part in invasive experi-ments as well as in 240 subjects in whom plasma levels of tPA had been determined. The results showed a high level of linkage disequilibrium among the majority of SNPs and thus, only a few common haplotypes were observed. Three of the SNPs showed a significant association with vascular release rates of tPA, but were not significantly associated with plasma levels of tPA. The closest association was observed for tPA -7,351C>T, which is located in an enhancer region upstream of the gene. Initial in vitro studies suggested a preferential binding of the transcription factor Sp1 to the C allele, which is the allele that was associated with a high tPA release rate. Since it was recently shown that Sp1 mediates induction of tPA gene transcription through the enhancer, this finding indicates that tPA -7,351C>T is functional. This SNP was therefore tested in a prospective nested case-control study on myocardial infarction in northern Sweden, which included 61 cases with a first myocardial infarction and 120 controls matched for age, sex, and geographical region. The results showed that both tPA -7,351C>T and plasma tPA antigen were associated with myocardial infarction and this association was independent of traditional risk factors. Carriers of the T allele had an increased risk of myocardial infarction.In conclusion, eight novel SNPs were identified at the tPA locus. The enhancer -7,351C>T variant, which was associated both with vascular tPA release rates and myocardial infraction, is of putative functional importance. Thus, the genetic variation at this locus appears important for physiological regulation of tPA.