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Emily A. Andreae, Debra J. Warejcka, Sally S. Twining; Analysis of Thrombin Activity and Cyr61 Regulation in a Rat Cornea Organ Culture System. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2025.
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Thrombin stimulation increases Cyr61 (CCN1) gene and protein expression in cultured corneal cells. Cyr61 is an extracellular matrix-associated protein that promotes migration and proliferation of various cell types and stimulates angiogenesis. Our goal is to determine whether prothrombin is activated and whether thrombin upregulates Cyr61 synthesis and stimulates Cyr61 cleavage in a rat cornea organ culture system.
Corneas were removed from Sprague-Dawley rats and placed in organ culture. The rat lens was used as a base to maintain corneal shape. Epithelial scrape wounds were made in one group of corneas prior to organ culture. Serum-free medium was added drop-wise to the corneal surface every 24 hours. In a set of wounded and unwounded corneas, serum-free medium spiked with hirudin, a specific inhibitor of thrombin, was added to the corneal surface. Conditioned medium was collected for all experiments and the proteins separated by SDS-PAGE, transblotted, and probed for Cyr61 and antithrombin III.
Prothrombin activation to thrombin was observed in response to wounding through detection of a two-fold increase in levels of antithrombin III-thrombin complexes. Cyr61 protein was also increased approximately three-fold in the wounded corneas. The rate of extracellular Cyr61 degradation appeared to increase in response to injury. Hirudin inhibited the increase in Cyr61 secretion and proteolytic degradation in wounded corneas which indicates that thrombin is involved in these effects.
Prothrombin is activated to thrombin upon corneal injury, and thrombin upregulates Cyr61 protein expression in response to corneal injury. These results support the involvement of thrombin generated upon corneal injury in the regulation of synthesis and processing of proteins such as Cyr61.
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