Characterizing Modulators of Protease-activated Receptors with a Calcium Mobilization Assay Using a Plate Reader.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.

A thrombin-PAR1/2 feedback loop amplifies thromboinflammatory endothelial responses to the viral RNA analogue poly(I:C).

Activation of blood coagulation and endothelial inflammation are hallmarks of respiratory infections with RNA viruses that contribute significantly to the morbidity and mortality of patients with severe disease. We investigated how signaling by coagulation proteases affects the quality and extent of the response to the TLR3-ligand poly(I:C) in human endothelial cells. Genome-wide RNA profiling documented additive and synergistic effects of thrombin and poly(I:C) on the expression level of many genes.