The Regulation of Human PAI-1 Levels in Blood

Plasminogen activator inhibitor type 1 (PAI-1) is the major inhibitor of fibrinolysis. Plasma PAI-1 levels show clear circadian oscillation, peaking in the morning. Expression of the PAI-1 gene may be directly influenced by the –675 4G/5G polymorphism in the PAI-1 promoter. The aim of the present study was to investigate the regulation of PAI-1, using monocytes and platelets as cell models, under conditions that would simulate events occurring within a forming thrombus. Blood from donors genotyped for the 4G/5G polymorphism was used. Direct stimulation of monocyte with lipopolysaccharide (LPS), induced a small increase in Pai-1 expression (710-fold) above baseline, however, monocyte Pai-1 expression was markedly increased (4700-fold) by interaction with platelets activated by cross-linked collagen related peptide (XL-CRP). Furthermore, the induction of monocyte PAI-1 expression by platelets was not dependent upon cell-cell contact, but appeared to be mediated by both exogenous and endogenous release of TGF-β1. The induction of monocyte Pai-1 expression by either LPS or CRP-XL was not associated with the 4G/5G polymorphism, but the amount of PAI-1 released into plasma following stimulation was influenced by the polymorphism. Five peripheral clock genes examined were expressed in monocytes under resting conditions. LPS induced the expression of Dec1 and Bmal2 but rapidly switched off the expression of mPer2 and clock in monocytes, whereas the stimulation of monocytes by CRP-XL did not affect the expression of clock genes, apart from the down-regulation of Clock and up-regulation of mPer2. Therefore, the acute induction of PAI-1 in blood cells does not appear to be dependent on the circadian clock. Platelet count significantly increased during the afternoon compared to the morning. But platelet Pai-1 mRNA abundance did not exhibit significant diurnal variation nor was it influenced by the 4G/5G polymorphism. Although anucleate, platelets appear to retain the ability to synthesise PAI-1, which could be partially inhibited by cycloheximide, both the synthesis of PAI-1, and release from α-granules of activated platelets, was related to the 4G/5G polymorphism, with the highest level of PAI-1 synthesised and released by platelets from 4G homozygous subjects. In conclusion, PAI-1 in circulatory cells is regulated by circadian factors under resting conditions, but circadian influences may be masked following stimulation. The 4G/5G polymorphism regulates the amount of PAI-1 released by both stimulated monocytes and platelets, and may therefore play a role in the formation or resolution of a thrombus.