Mediators of Induction of Augmented Expression of Plasminogen Activator Inhibitor Type-1 in Hep G2 Cells by Platelets

Summary

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. We have shown previously that PAI-1 biosynthesis in cultured cells depends on several factors in serum. Because platelets are richly endowed with specific growth factors and because the release reaction is an integral part of thrombosis, the present study was performed to determine whether platelets augment PAI-1 production and if so, to define mediators responsible. Hep G2 cells were used to determine whether platelet lysates increased PAI-1 synthesis in a dose and time-dependent manner. In cells labeled metabolically with ³⁵S-methionine for 6 h, an increase in labeled PAI-1 was elicited indicative of de novo synthesis as well as increased secretion of PAI-1 mediated by platelet lysates. Steady state levels of both the 3.2 and 2.2 kb forms of PAI-1 mRNA increased after 2 h and peaked in 3-5 h in a dose-dependent fashion as well. Incubation of Hep G2 cells with collagen activated platelets resulted in a similar induction of PAI-1 mRNA. The increase in PAI-1 mRNA occurred with exposure of the cells to platelet lysates for intervals as brief as 15 min and was not inhibited by cycloheximide indicating its independence of new protein synthesis. In order to identify the factors in platelets responsible for the induction of PAI-1 synthesis in the Hep G2 cell model system, neutralizing antibodies were used to inhibit specific platelet associated growth factors. Antibodies to transforming growth factor-β (TGF-β) and to the epidermal growth factor (EGF)/transforming growth factor alpha (TGF-α) receptor inhibited the platelet lysate-mediated increase in PAI-1 protein by 77%. The results indicate that physiologic concentrations of platelet derived TGF-β and EGF/TGF-α increase PAI-1 expression in Hep G2 cells and suggest that the augmentation of PAI-1 synthesis induced by platelets is mediated by specific platelet associated growth factors that may contribute to observed increases in circulating PAI-1 levels in patients with disorders manifested by thrombosis.

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