The hemostatic system stops bleeding at the site of vascular injury and maintains the integrity of blood vessels through clot formation. This process is regulated by complex interactions between coagulation factors, platelets and other blood cells, endothelial cells and von Willebrand factor. Coagulation is an important process in hemostasis, responsible for the formation of a blood clot. The central enzyme in coagulation is thrombin, which converts soluble fibrinogen into insoluble fibrin fibers.
Thrombin generation is a global hemostatic assay, which describes the kinetics of the formation and inactivation of thrombin in clotting plasma or whole blood. Whole blood thrombin generation allows to assess the contribution of blood cells to coagulation.
Whole blood thrombin generation is based on the Calibrated Automated Thrombogram (CAT) assay in plasma. It is based on the use of a fluorogenic thrombin substrate and a thrombin calibrator. Whole blood is mixed with a fluorogenic thrombin substrate and added to either TG calibrator (α2-Macroglobulin-thrombin) or TG trigger (tissue factor and CaCl2). The raw thrombin generation curves are measured by the CAT machine and a dedicated spreadsheet is used to calculate thrombin generation parameters from these curves. The main parameters are the lag time, the endogenous thrombin potential (ETP; area under the thrombin generation curve), the peak height and the time to peak.
The use of α2-Macroglobulin-thrombin as calibrator, allows to correct for inner filter effect and substrate consumption, donor-to-donor variability in color of the blood and instrumental differences.
We developed a dedicated spreadsheet to determine the thrombin generation parameters from the raw fluorescent traces. These parameters include: the lag time (the time needed to form the first traces of thrombin), the ETP (represents the total amount of active thrombin formed during thrombin generation), the peak height (the maximal amount of thrombin formed) and the time to peak (time needed to reach maximum thrombin activity).
