There are three objectives for this project. First, regulation of proteolysis by human trabecular cells (HTC) will be characterized. Using an immunoassay for tissue plasminogen activator (TPA), as well as nucleotide genetic probes, regulation of synthesis of TPA will be studied. Based on our previous observation that HTC incubated with fibrinogen or fibrin have increased TPA activity, the effects of exposing HTC to the components of the fibrinolytic system (fibrinogen and fibrin, plasminogen and plasmin) on synthesis and release of TPA will be determined. If we demonstrate that fibrinogen or fibrin induces synthesis of TPA, we will determine whether the intact molecule or fragments are necessary. Fibrinogen will be digested with plasmin, and degradation products purified by HPLC. HTC will be incubated with fragments X, Y, D and E, then TPA measured. It will also be determined whether fragments of intact fibrinogen must be internalized in order to stimulate synthesis of TPA. Metabolic inhibitors will be used as well as exposure of HTC to agonists at 4 degrees C to inhibit internalization. The second objective will be to determine the effects of specifically inhibiting TPA on growth of HTC. Based on our inability to demonstrate the inhibitor of TPA (TPA-I) in HTC, TPA-I will be purified and its effect on HTC growth rate determined. Also, the effect of TPA-I on cell-surface proteins will be determined in that it is possible that unregulated TPA results either directly or indirectly in proteolysis of cell surface proteins. Cell surface proteins will be radiolabelled either with (125I) using lactoperoxidase or with endogenous (3H)-amino acids. Autoradiograms of 2-dimensional SDS-polylacrylamide gels will be analyzed following growth of cells in the presence or absence of TPA. If TPA hydrolyses a cell surface protein, appearance of a new protein would be expected in cells incubated with TPA-I. A third series of experiments relating to urokinase (UK) metabolism in bovine corneal endothelia (BCE) cells and human keratocytes will be initiated. Based on preliminary experiments, we will determine whether there is a glycoaminoglycan (GAG) component to the receptor for UK. Cells will be treated with heparinase and heparitinase, then binding of (125I)-UK measured. If there is a GAG component to the receptor, binding should be diminished. Ultimately, the receptor will be purified by affinity chromatography using the UK-binding protein secreted by BCE cells and HPLC.
| Shuman, M A; Polansky, J R; Merkel, C et al. (1988) Tissue plasminogen activator in cultured human trabecular meshwork cells. Predominance of enzyme over plasminogen activator inhibitor. Invest Ophthalmol Vis Sci 29:401-5 |
| Shuman, M A; Merkel 3rd, C H (1985) Urokinase binding to bovine corneal endothelial cells. Exp Eye Res 41:371-82 |
