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

Area of Science:

• Neuroscience
• Biochemistry
• Pharmacology

Background:

• Coagulation factor X (FX) and its activated form (FXa) are implicated in neuroinflammation.
• Detecting FXa in neural tissues is challenging due to low levels compared to plasma.
• A sensitive and specific method is needed to quantify FXa activity in neural samples.

Purpose of the Study:

• To develop and validate a sensitive method for measuring FXa activity in neural tissues.
• To investigate the regulation of FXa activity in brain slices and glioma cell lines.
• To differentiate between basal, activated, and secreted FXa activity.

Main Methods:

• Utilized a fluorogenic substrate assay with specific inhibitors for FXa quantification.
• Validated the assay in human plasma and subsequently applied it to mouse brain slices and human, rat, and mouse glioma cell lines.
• Employed FXa inhibitors (apixaban) and activators (Russell's viper venom X - RVVX), alongside FX and FXa, to probe FXa activity.

Main Results:

• The developed assay successfully measured FXa activity in plasma and neural samples.
• RVVX significantly increased FXa activity in human plasma and mouse brain slices.
• Apixaban differentially inhibited FXa activity in mouse brain slices (secreted vs. slice-associated) and glioma cells (secreted).
• Lipopolysaccharide modulated RVVX-induced FXa activity and thrombin activity in glioma cells.
• FXa activity in neural tissue requires specific activators for detection.

Conclusions:

• A sensitive fluorescence-based method was established for detecting subtle changes in FXa activity within neural tissue.
• Brain slice-associated FXa activity exhibits distinct responses to activators and inhibitors compared to secreted FXa activity, highlighting complex regulation.
• Specific experimental conditions, including the use of activators, are crucial for detecting FXa activity in neural environments.