Measuring Neural Factor X Pathway Activity in Rodent Glioma Cell Culture and Brain Slices

Coagulation factor X (FX) plays a significant role in neuroinflammation and is considered a promising target for therapeutic intervention. However, because activated FX (FXa) levels in neural tissues are expected to be significantly lower than in plasma, a more sensitive, direct, and specific method is required for accurate detection. To measure FXa activity with high sensitivity, we used a fluorogenic substrate in combination with a panel of specific inhibitors to prevent nonspecific protease activity that potentially cleaves the substrate, initially validated in human normal and FX-deficient plasma. This approach allowed selective quantification of FXa activity in neural samples. To further characterize the FX pathway, we applied a FXa inhibitor (apixaban), a FX activator (Russell's viper venom X, RVVX), and commercially available FX and FXa to mouse brain slices and to glioma cell lines derived from human, rat, and mouse. FXa activity significantly increased following RVVX application in human plasma and mouse brain and was inhibited in a calcium-dependent manner in human plasma. In the mouse brain, apixaban inhibited only the FXa activity secreted from the slice rather than the slice-associated activity. In glioma cell lines, RVVX application increased cell-bound FXa activity, which was not inhibited by apixaban, while the activity secreted into the medium was elevated and inhibited by apixaban. Exposure to lipopolysaccharide reduced RVVX-induced FXa activity while increasing the thrombin activity in glioma cell lines from mice and rats, respectively. We developed a specific method of measuring basal FXa activity and induced from FX by RVVX, in plasma and secreted from brain slices and astrocyte tumor cells. The results were obtained in tissue with defined experimental conditions, pharmacologically differentiated tools, and biological validation as assessed by thrombin activity measurement. FXa activity in neural tissue is tightly regulated and undetectable without an FX activator. KEY MESSAGES: We developed a sensitive method using fluorescence substrate to measure the fine changes in FXa activity in neural tissue. Brain slice associated FXa activity differs from secreted activity in its response to activators and inhibitors, suggesting the complexity of FXa activity regulation in neural tissue. Specific set of conditions is needed to detect FXa activity in neural tissue.