PURPOSE: To investigate the effect of thrombin on the urokinase plasminogen activator receptor (u-PAR) in retinal pigment epithelial (RPE) cells. METHODS: The authors analyzed u-PAR mRNA by Northern blot hybridization. Retinal pigment epithelial cell surface u-PAR was assayed by measuring the amount of functional urokinase plasminogen activator (u-PA) bound to cells at saturation. Retinal pigment epithelial cells were derived from fetal retinal tissue and established in primary cell culture. RESULTS: Thrombin increased u-PAR mRNA 4-fold in RPE cells examined by Northern blot hybridization, whereas the amount of thrombin receptor mRNA was unchanged. Thrombin stimulated u-PA binding to RPE cells 2.5- to 5-fold in a time- and dose-dependent manner. Hirudin, a thrombin antagonist, completely blocked the effects of thrombin on u-PAR expression in RPE cells. Phosphatidylinositol phospholipase C treatment of RPE cells resulted in the abolition of thrombin-induced u-PA binding. Recombinant soluble u-PAR competitively inhibited two-chain u-PA binding to the surface of thrombin-treated RPE cells. A thrombin receptor agonist peptide (SFLLRNPNDKYEPF) also induced a 2.5-fold increase in binding of u-PA to the surface of RPE cells. CONCLUSION: Thrombin increases u-PAR expression by RPE cells by a mechanism involving activation of the seven transmembrane thrombin receptor.
PURPOSE: To investigate the effect of thrombin on the urokinase plasminogen activator receptor (u-PAR) in retinal pigment epithelial (RPE) cells. METHODS: The authors analyzed u-PAR mRNA by Northern blot hybridization. Retinal pigment epithelial cell surface u-PAR was assayed by measuring the amount of functional urokinase plasminogen activator (u-PA) bound to cells at saturation. Retinal pigment epithelial cells were derived from fetal retinal tissue and established in primary cell culture. RESULTS:Thrombin increased u-PAR mRNA 4-fold in RPE cells examined by Northern blot hybridization, whereas the amount of thrombin receptor mRNA was unchanged. Thrombin stimulated u-PA binding to RPE cells 2.5- to 5-fold in a time- and dose-dependent manner. Hirudin, a thrombin antagonist, completely blocked the effects of thrombin on u-PAR expression in RPE cells. Phosphatidylinositol phospholipase C treatment of RPE cells resulted in the abolition of thrombin-induced u-PA binding. Recombinant soluble u-PAR competitively inhibited two-chain u-PA binding to the surface of thrombin-treated RPE cells. A thrombin receptor agonist peptide (SFLLRNPNDKYEPF) also induced a 2.5-fold increase in binding of u-PA to the surface of RPE cells. CONCLUSION:Thrombin increases u-PAR expression by RPE cells by a mechanism involving activation of the seven transmembrane thrombin receptor.
Authors: Martin Scholz; Jens-Uwe Vogel; Gerold Höver; Susanna Prösch; Ruslan Kotchetkov; Jaroslav Cinatl; Frank Koch; Hans Wilhelm Doerr; Jindrich Cinatl Journal: Med Microbiol Immunol Date: 2003-09-12 Impact factor: 3.402