Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of plasmin generation by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases.
The plasmin generation assay (PGA) is a calibrated method to monitor plasmin formation and inhibition in plasma. Per sample, at least one PG calibrator well (α2-Macroglobulin-plasmin) and PG trigger well (tissue factor, phospholipids and tissue plasminogen activator) are required. After addition of plasma to these wells, the plate is incubated in the CAT machine, whereafter the PG substrate mix, containing CaCl2, is automatically dispensed. PG curves obtained are expressed in molar concentrations and provide quantitative information on the kinetics of plasmin formation and inhibition during fibrinolysis.
This assay is based on the established approach employed by calibrated automated thrombography, and overcomes several limitations associated with the earlier methods. Firstly, by using the α2-Macroglobulin-plasmin complex as a calibrator, the PG assay not only corrects for substrate exhaustion and the inner filter effect, but also eliminates inhibition of the calibrator by plasmin inhibitors. Secondly, this assay uses the plasmin-specific fluorogenic substrate Boc-Glu-Lys-Lys-AMC, eliminating problems associated with substrate specificity.
Multiple parameters can be obtained from the PG kinetic curve, including lag time (time needed for plasmin concentration to reach 6 nM of the peak concentration), velocity (rate of plasmin formation), peak height, time to peak (ttpeak, time needed to reach maximum plasmin activity), and endogenous plasmin potential (EPP, the overall activity of plasmin formed during fibrinolysis).
