Fibrin induction of tissue plasminogen activator expression in corneal endothelial cells in vitro.
OBJECTIVE
Fibrin is deposited in the anterior chamber of the eye in response to injury and can damage corneal endothelial cells (CEC). Fibrin degradation is plasmin dependent and is regulated by the balance between plasminogen activators (PA), tissue-PA (t-PA), urokinase-PA (u-PA), and their inhibitors (PAI). Although several factors can modulate PA/PAI expression in cells, the effect of fibrin is inconclusive. We hypothesized that fibrin can regulate fibrinolysis in the anterior segment by modulating PA/PAI expression in CEC.


METHODS
Bovine CEC (BCEC) were treated for 3 to 72 hours with in situ polymerized fibrin (2 mg/ml) +/- 35S-methionine, cycloheximide, or actinomycin D. Polymerization was thrombin catalyzed, and control BCEC were incubated with or without thrombin or polymerization by-products. PA and PAI in conditioned medium, fibrin matrix, and cell fractions were analyzed by PA-specific zymographic and enzymatic assays.


RESULTS
Fibrin treatment induced a dramatic (> 20-fold) accumulation of extracellular, fibrin-bound PA. This activity was identified as t-PA by its Mw (70 kD) affinity for fibrin and sensitivity to inhibition by Erythrina. Induction of t-PA was not observed in control BCEC under any condition. Fibrin induction of t-PA was selective because the levels of u-PA (45 kD), PAI-1 (50 kD), or protein synthesis in general were unaffected. Fibrin induction of t-PA was not accompanied by changes in cellular t-PA levels and was dependent on both RNA and protein synthesis.


CONCLUSIONS
Fibrin selectively induces t-PA expression in CEC. Induced t-PA is released extracellularly and binds exclusively to the fibrin matrix. These findings suggest a role for fibrin and CEC in the regulation of fibrinolysis in the anterior segment of the eye.
Ramsby
M L
ML
Department of Pathology, University of Connecticut Health Center, Farmington 06030.
Kreutzer
D L
DL
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000187:drug effects*; D005136:Eye Proteins / Q000096:biosynthesis; D005337:Fibrin / Q000302:isolation & purification / Q000494:pharmacology*; D005342:Fibrinolysis / Q000187:drug effects; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D015849:Plasminogen Inactivators / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis / Q000187:drug effects; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D003470:Culture Media; D005136:Eye Proteins; D017395:Plasminogen Activator Inhibitor 1; D015849:Plasminogen Inactivators; D012313:RNA; D005337:Fibrin; D003513:Cycloheximide; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator