Development of a Factor VII Activating Protease (FSAP) generation assay and its application in studying FSAP in venous thrombosis


      • Development of a robust, simple and highly specific FSAP generation assay (FGA)
      • Carriers of the Marburg I-polymorphism can be identified by strongly delayed FSAP generation, low peak FSAP and low FSAP potential
      • Delayed FSAP generation significantly correlated with VT in post-menopausal women after excluding Marburg I carriers


      Human genetic studies based on the Marburg I polymorphism in the factor VII activating protease (FSAP) encoding gene, analysis of FSAP activity in plasma and biochemical characterization of FSAP substrates indicate a possible causal link between FSAP activity and venous thrombosis. We hypothesized that a direct standardized assay to measure FSAP activity in plasma could provide convincing arguments for or against such a potential link. Using Ac-Pro-DTyr-Lys-Arg-AMC as a highly specific and sensitive substrate, histones as a trigger to activate pro-FSAP and plasma-purified active FSAP as a calibrator, we have developed a fluorogenic kinetic assay that reveals the FSAP generating potential in human plasma in real time. This assay is similar to the thrombin generation assay and allows analysis of lag phase, time to peak and velocity, as well as peak FSAP and the endogenous FSAP potential (EFP) of plasma samples. Carriers of the Marburg I polymorphism showed clearly delayed FSAP generation and lower peak FSAP and EFP level. There were no significant differences in all FSAP activity parameters between plasma from patients with a history of venous thrombosis and controls. When excluding Marburg I carriers, which were evenly distributed between groups, delayed FSAP generation significantly correlated with venous thrombosis in postmenopausal women. The novel FSAP activity assay is robust and easy to perform and will be a useful tool for analyzing plasma FSAP activity, also, in other pathophysiological conditions.

      Graphical abstract


      AMC (aminomethyl coumarin), AUC (area under the curve), CTI (corn trypsin inhibitor), EFP (endogenous FSAP potential), FG(A) (FSAP generation (assay)), FSAP (Factor VII Activating Protease), MI-SNP (Marburg I single nucleotide polymorphism), RFU (relative fluorescence unit), TGA (thrombin generation assay), TtPk (time to peak), VT (venous thrombosis)


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