Effects of fibrin on the secretion of plasminogen activator inhibitor-1 from endothelial cells and on protein kinase C.

We previously demonstrated that cultured human umbilical vein endothelial cells (HUVECs) overlaid with a fibrin clot induced a slight increase in tissue-type plasminogen activator (t-PA) secretion and marked reduction in plasminogen activator inhibitor-1 (PAI-1) secretion. In this study, the intracellular signal transduction after fibrin stimulation was further investigated by analyzing cyclic AMP (cAMP) and protein kinase C (PK-C). When HUVECs were stimulated by fibrin clots, t-PA mRNA increased to 130% but PAI-1 mRNA decreased to 42%. These changes concurred with the data on the protein levels of t-PA and PAI-1 as previously reported. The effect of fibrin on t-PA production in HUVECs was not significantly altered after the elevation of cAMP by either forskolin or dibutyryl cAMP. Furthermore, an effect of fibrin on t-PA production did not appear when the cells were treated by phorbol 12-myristate 13-acetate (PMA) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). The suppressive effect of fibrin on PAI-1 secretion from HUVECs was not altered by elevation of cAMP. Regarding the activation of PK-C by PMA, PAI-1 secretion was enhanced, but was suppressed by fibrin stimulation. H-7 suppressed PAI-1 secretion and further stimulation by fibrin almost completely abolished PAI-1 secretion. These changes were well associated with mRNA levels of t-PA and PAI-1. These results suggested that fibrin on HUVECs preferably down-regulates PK-C resulting in a decrease of PAI-1 in both the protein and mRNA levels and that effect of fibrin on t-PA secretion is neither involved in PK-C nor cAMP pathway.