Evaluation of the Impact of Metabolite M10 on FXIa and on the Plasma Clotting Time

Potential inhibitory activities of M10 on human FXIa or on the clotting process in human plasma after contact activation were investigated in enzymatic assays in buffer and in the aPTT assay in human plasma, respectively, as described previously [20]. In brief, different solutions of M10 in DMSO were prepared to yield final assay concentrations up to 50 µM. For the buffer assay, a solution of M10 in DMSO (1 µL), a buffer solution (pH 7.4) consisting of 50 mM Tris/HCl, 100 mM NaCl, 5 mM CaCl₂, and 0.1% bovine serum albumin (20 μL), a solution of human FXIa (20 μL), and a solution containing a fluorogenic peptidic FXIa substrate (20 μL) were mixed. The fluorescence (excitation wavelength: 360 nm; emission wavelength: 460 nm) was measured over time to determine the enzymatic activity at different M10 concentrations and thereby calculate the half-maximal inhibitory concentration (IC₅₀). For the clotting-time determination, 49 µL of human plasma was incubated for 3 minutes with different concentrations of M10 in 1 µL DMSO. To this mixture, 50 µL of a kaolin/cephalin suspension (C.K. Prest, Diagnostica Stago, France) was added. After 3 minutes the clotting process was initiated with 50 µL of a calcium dichloride solution. The clotting time was determined in an automated coagulometer (AMAX 200, Trinity Biotech Plc, Ireland) and compared with the clotting time without M10.