Urokinase binding to bovine corneal endothelial cells

Prior research has shown that thrombin binds to bovine corneal endothelial cells and forms a covalent complex with a secreted protein. However, the role of other proteases in these interactions remained unclear. It was already known that urokinase is involved in clot dissolution and may interact with cell surfaces. This gap motivated an investigation into whether urokinase also binds to corneal endothelial cells. No prior work had resolved the binding specificity of urokinase in this context. The study aimed to clarify if urokinase interacts with these cells and how this might differ from thrombin binding. This uncertainty drove the need to examine binding mechanisms and affinity. The findings may help distinguish urokinase's role from thrombin's in ocular physiology.

The aim of this study was to determine whether urokinase binds to bovine corneal endothelial cells and to compare its binding properties to thrombin. The specific problem addressed was the lack of clarity about urokinase's interaction with these cells. The motivation stemmed from prior findings on thrombin binding and the need to understand if urokinase might play a similar or distinct role. The researchers sought to test if urokinase binding is specific and how it compares to thrombin's affinity. They also wanted to determine if the cells release a binding partner for urokinase. The study aimed to assess the molecular weight of any complexes formed. The goal was to establish whether urokinase binding is physiologically relevant in the eye. This could inform models of protease regulation in the anterior chamber.

The researchers used radiolabeled [125I]-urokinase to assess binding to bovine corneal endothelial cells. They tested the effect of inactivating urokinase with diisoprophylfluorophosphate to determine if the serine active site is essential for binding. They pre-incubated cells with unlabeled urokinase to observe changes in binding site availability. Sodium dodecyl sulfate gel electrophoresis was used to analyze complex formation between urokinase and cell proteins. Thrombin was used as a competitor to compare binding affinities. The molecular weight of the complex was measured as 73,300 daltons. The cells were also tested for their ability to produce a plasminogen activator. These methods allowed the team to assess binding specificity, affinity, and physiological relevance.

The study found that [125I]-urokinase binds rapidly to bovine corneal endothelial cells. Inactivation of urokinase with diisoprophylfluorophosphate prevented binding, indicating the serine active site is necessary. Pre-incubation with unlabeled urokinase increased binding site release into the media and boosted cell binding by at least 3.5-fold. A 73,300 dalton complex formed between urokinase and a cell protein. Thrombin competed with urokinase for binding but had at least a 10-fold lower affinity. The cells produced a plasminogen activator matching urokinase's molecular weight. These findings suggest urokinase binds more strongly than thrombin under physiological conditions. Binding may regulate urokinase activity in the anterior chamber of the eye.

The authors propose that urokinase binds to bovine corneal endothelial cells more effectively than thrombin. They suggest that the serine active site is essential for this interaction. The release of binding sites into the media may reflect a regulatory mechanism. The formation of a 73,300 dalton complex indicates a specific interaction with a cell protein. The higher affinity of urokinase compared to thrombin suggests it is the dominant protease binding under normal conditions. The cells' production of a plasminogen activator matching urokinase's size supports this idea. Binding may limit extracellular proteolytic activity in the eye's anterior chamber. These findings imply a role for urokinase in regulating protease activity in ocular tissues.