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P. T. Logan, D. Abourbih, C. Martins, D. Faingold, S. Bakalian, M. N. Burnier, Jr.; In vitro and in vivo Classification of the Protease Activated Receptor Family and the Consequences of Thrombin Induced Receptor Activation in Uveal Melanoma. Invest. Ophthalmol. Vis. Sci. 2008;49(13):74.
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Thrombin is a serine protease that cleaves Protease Activated Receptor 1 (PAR-1), PAR-3, and PAR-4, allowing for the tethered end to self-activate the G-protein coupled receptor. While the role of thrombin and PARs in blood clotting and other physiological processes are well described, the function of the protease-activated receptors in the oncogenesis and pathogenesis of malignancies, particularly uveal melanoma (UM), is not well understood. Herein, we classify the expression of thrombin receptors in five UM cell lines, tissue from a xenograft animal model, and in UM patient samples. We also examine the consequences of thrombin-induced PAR activation in UM cell lines.
Cytospins of five human UM cell lines were immunostained for PAR-1, -3, and -4 expression. In addition, intraocular tumours obtained from an established animal model of UM were also immunostained for PARs. The expression of PAR-1 in human patient samples was also analyzed. Cell lines were exposed to various concentrations of thrombin and changes in both invasion and proliferation were assessed using a Matrigel artificial membrane and a TOX-6 assay kit, respectively. Changes in matrix metalloproteinase (MMP) -2 and -9 levels induced by thrombin were also assessed using a specialized MMP Gelatinase Assay.
All five cell lines expressed PAR-3, two cell lines expressed PAR-1 and one cell line expressed PAR-4. Intraocular tumours from the animal model revealed universal expression of all PARs despite in vitro expression of only PAR-3. Ten of 13 human samples were positive for PAR-1. Widespread increases in MMP-2 and MMP-9 expression were seen in all cell lines using three concentrations of thrombin (10, 1, and 0.1 U/ml) the majority of which were statistically significant (p<0.05). However, thrombin was only capable of increasing the invasive capabilities of two of five cell lines. Thrombin increased proliferation of one of the cell lines - the only cell line with positivity for PAR-1 expression.
The increases in MMP production, invasiveness and proliferation of five human UM cell lines in response to thrombin reveal the oncogenic nature of PAR activation. Evidence that the tumor microenvironment is capable of inducing PAR expression provides further evidence of the importance of PARs in UM progression.
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