Factor Xa Generation Reveals Tissue Factor Fusion Protein Does Not Affect Procoagulant Activity
Mentor 1
Julie Oliver
Mentor 2
Brittany Vanderhoof
Start Date
1-5-2020 12:00 AM
Description
The expression of tissue factor (TF) outside of the vasculature regulates initiation of blood coagulation by recruiting Factor VIIa to convert Factor X into Xa. We hypothesized that TF activity, measured as Factor Xa production, depends on TF expression level and not the presence of an experimental fluorophore tag. To quantify TF expression, cells were transfected with plasmids containing TF, with or without a fluorophore tag. We demonstrated that cell lines expressing TF fusion proteins tagged with mTurquoise (mTQ) or super yellow fluorescent protein 2 (SYFP2) fluorophores, along with lines expressing untagged TF, can be characterized as having high and low expression of TF using indirect staining and flow cytometry analysis. Single transfectants of each fluorophore and double transfectants containing both fluorophores were characterized. Cell lines were matched for total TF expression level, then used in procoagulant activity assays to test whether the fluorophore tags impact TF function. TF activity was quantified with a chromogenic substrate to measure the amount of Factor Xa produced from X. Our results show that Factor Xa generation is similar between lines with similar levels of total TF, whether the proteins are tagged or untagged. This confirms our hypothesis that the fluorophore tag has no significant impact on TF function. While future tests of fluorophore impact on TF signaling functions would be helpful, these results support the broad conclusion that the fluorophore-tagged fusion proteins commonly used in a wide range of biological model systems have normal, unaltered function.
Factor Xa Generation Reveals Tissue Factor Fusion Protein Does Not Affect Procoagulant Activity
The expression of tissue factor (TF) outside of the vasculature regulates initiation of blood coagulation by recruiting Factor VIIa to convert Factor X into Xa. We hypothesized that TF activity, measured as Factor Xa production, depends on TF expression level and not the presence of an experimental fluorophore tag. To quantify TF expression, cells were transfected with plasmids containing TF, with or without a fluorophore tag. We demonstrated that cell lines expressing TF fusion proteins tagged with mTurquoise (mTQ) or super yellow fluorescent protein 2 (SYFP2) fluorophores, along with lines expressing untagged TF, can be characterized as having high and low expression of TF using indirect staining and flow cytometry analysis. Single transfectants of each fluorophore and double transfectants containing both fluorophores were characterized. Cell lines were matched for total TF expression level, then used in procoagulant activity assays to test whether the fluorophore tags impact TF function. TF activity was quantified with a chromogenic substrate to measure the amount of Factor Xa produced from X. Our results show that Factor Xa generation is similar between lines with similar levels of total TF, whether the proteins are tagged or untagged. This confirms our hypothesis that the fluorophore tag has no significant impact on TF function. While future tests of fluorophore impact on TF signaling functions would be helpful, these results support the broad conclusion that the fluorophore-tagged fusion proteins commonly used in a wide range of biological model systems have normal, unaltered function.
