Thrombin Modulation of Plasminogen Activator Inhibitor-1 (PAI-1) in Cultured Human Vascular Smooth Muscle Cells

Abstract

The effects of highly purified human thrombin (αThr) on fibrinolytic potential of cultured human vascular smooth muscle cells (HSMC) from saphenous, mammary and umbilical vessels were studied. Human mammary artery and saphenous vein were obtained from patients undergoing coronary bypass surgery. HSMCs from mammary artery, saphenous and umbilical veins, were cultured and used between second and sixth passages. For experimental purpose cells were made quiescent (48 hr in MEM containing 0.4% foetal calf serum) and then incubated in serum free medium with or without different concentrations of αThr. PAI-1 antigen levels were measured in conditioned medium (CM) and in extracellular matrix by ELISA. Experiments performed incubating HSMCs with thrombin for 16 hr showed that thrombin treatment increased PAI-1 antigen levels in CM of smooth muscle cells derived from saphenous veins and mammary arteries but not from umbilical veins. In particular, treatment of HSMCs with αThr (1.25 IU/ml) increased PAI-1 antigen levels in CM of cells derived from saphenous veins (+125 ± 18 SE % over control, n=4) and mammary arteries (+82 ± 20 SE% over control, n=3). PAI-1 antigen levels in the extracellular matrix showed a similar pattern, with an increase in PAI-1 associated with matrix from HSMCs obtained from saphenous and mammary vessels and no change in the umbilical vein after overnight incubation. The effect of αThr on these cells was dose-dependent in the range of 0.0125–1.25 IU/ml. Time course experiments showed that thrombin affected, with a different behavior, PAI-1 antigen release in HSMCs of all three districts considered. The effect of thrombin on HSMCs obtained from umbilical vein was evident only after 30–60 minutes incubation. On the other hand in HSMCs obtained from saphenous vein PAI-1 increased from 8 reaching a plateau after 16 hr incubation. Preincubation of αThr with hirudin or PPACK prevented the stimulatory effect on PAI-1 production, indicating that intact active site was required for this effect. The increase in PAI-1 release was accompanied by concomitant changes in PAI-1 mRNA levels in HSMCs from saphenous vein and mammary artery as determined by Northern blot analysis. In conclusion, at sites of vascular injury or denudation, smooth muscle cells interaction with thrombin may result in an increase of PAI-1 production, predisposing the vessels to thrombus formation.