Calibrating Thrombin Generation in Different Samples: Less Effort with a Less Efficient Substrate

Abstract

Abstract:The technique of thrombin generation has been established as a promising tool for the diagnosis, risk estimation, and perhaps prognosis of coagulation insufficiencies. Without further experimentation an indication can be obtained if a sample donor carries the risk for haemophilia or thrombophilia, either congenital or acquired. A comparison of two different thrombin generation assays is presented, demonstrating that a currently not commercially available test allows an easier and cheaper calibration than its commercial counterpart. This is achieved by employment of a less efficient but highly specific peptide substrate for thrombin and the involvement of low amounts of analyte (plasma samples).

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

“…Plasma concentration: The plasma concentration in commercial fluorogenic TGA varies between 67 % and 40 % [33], but also 25 % plasma has been successfully used [40]. Here 25 % plasma was selected, because optimum FSAP activation was seen at this dilution (Figure 1 A), similar to reports that in TGA the highest AUC values were seen at 20 % to 30 % plasma [41].…”

Factor VIII assay mimicking in vivo coagulation conditions

“…Attempts to get even closer to the in vivo situation by performing analogous experiments according to our TGT were less successful. In these experiments, FIXa was replaced by 1 p m tissue factor and the FXa specific substrate Glut‐GR‐AMC was exchanged against the thrombin specific substrate B‐VR‐AMC .…”

“…We employed thrombin generation test (TGT) as a global test to demonstrate that the ‘peripheral’ inhibitors, CTI and BPTI do not exhibit noticeable interfere with the events leading to coagulation and beyond. This test was performed with CTI/BPTI plasma from four donors with different coagulation characteristics: One represents a normal donor, one was carrier of the FV‐Leiden mutation, causing activated protein C (APC) resistance, another donor has prothrombin overload due to an as yet unknown defect (this donor had a history of two thrombotic events, but G20210A mutation had been excluded by sequencing), and a fourth donor was carrier of FV‐Leiden mutation and was under phenprocoumon therapy (INR ~ 2.5) at the time of blood donation.…”

“…Thrombin generation test ( TGT ) in corn trypsin inhibitor/bovine pancreatic trypsin inhibitor ( CTI / BPTI )‐plasma. TGT was performed as described in CTI / BPTI ‐plasma with 1 p m tissue factor as trigger. Thrombomodulin (6 n m ) was used to identify disturbances in the APC ‐pathway.…”

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres

“…The materials as well as the methods used for this study are described in the Supporting Information. The authors have cited additional references within the Supporting Information [29,39,40,43,[59][60][61][62][63][64][65][66][67][68][69][70][71][72]. Supplementary Figures of the protein similarity calculation (Figure S1), fluorometric inhibition assays (Figures S2 and S3), absorption spectra (Figure S4), stability studies (Figure S5), NMR-spectra and HPLC-chromatograms (Figures S6a-S21c) and Table S1 of the computation of physicochemical parameters can be accessed in the supporting information.…”