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S. S. Twining, D. J. Warejcka; The Effect of Thrombin on the Plasminogen-Plasmin System in the Cornea. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2940.
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Thrombin is a protease that generates fibrin from fibrinogen and acts as a signaling molecule on cells via the protease activated receptors (PARs). In the cornea, fibrin is found very early at the site of injury, and the cells of the cornea synthesize and secrete all the components of the coagulation cascade necessary to generate thrombin. In addition, the cornea synthesizes plasminogen (PG) and the plasminogen activators (PAs), which eventually degrade the fibrin and are important in cell migration and ECM remodeling during wound healing. The purpose of this study is to determine whether thrombin affects the PG-PA system in the wound healing cells of the stroma
Primary human corneal stromal cells were cultured on collagen in serum-free media with supplements and were converted to fibroblasts with FGF-2 or myofibroblasts with TGF-β. Fibroblasts and myofibroblasts were treated with 0, 0.1, 1 or 10 units/ml human α-thrombin for 1, 4, 8 or 24 hours. Supernatant proteins were separated on 10% SDS-PAGE, transblotted to nitrocellulose membranes, then probed with monoclonal antibodies to uPA, tPA, and PAI-1. Zymograms were performed using SDS gels containing casein and plasminogen. Cells and supernates were incubated with plasminogen and FTC-casein for 24 hours, then the TCA-precipitable casein was removed and the digested FTC-casein was quantified using a fluorescent plate reader
PAI-1 levels in supernates from myofibroblasts increase with thrombin treatment in a concentration dependent manner. Fibroblasts make very little PAI-1, but thrombin does increase this amount in 24 hour supernates. Zymograms show that uPA in supernates from myofibroblasts is decreased 1, 4, and 8 hours after thrombin treatment. In fibroblasts, tPA is decreased in supernates after 1, 4 and 8 hours after thrombin treatment. After 24 hours, uPA in myofibroblasts and tPA in fibroblasts return to control levels. Thrombin treated fibroblasts, incubated with FTC-casein and fibrinogen, generate 38% (4 hours) to 81%(24 hours) of the plasmin generated by control fibroblasts. Myofibroblasts do not generate plasmin on the cell surface. Supernates from thrombin treated fibroblasts generate 28% of the plasmin of control supernates in the FTC-casein assay.
Thrombin inhibits the secretion or activity of plasminogen activators synthesized by stromal fibroblasts and myofibroblasts. Thrombin affects the amount of PAs both on the cell surface of fibroblasts and the amount in the supernates. This decrease in PAs may be due to the increase in PAI-1 that is secreted by cells treated with thrombin.
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