A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis

Abstract

Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were < 9% and 6-25%, respectively. Interplay between coagulation and fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.

Thrombin and plasmin generation in patients with plasminogen or plasminogen activator inhibitor type 1 deficiency

“…17 The NHA is characterized by seven parameters as shown in Figure 1 where. Thrombin generation is initiated with a low tissue factor concentration, and plasmin generation initiated by tPA which is dependent on fibrin formation.…”

“…[8][9][10] PAI-1 downregulates fibrinolysis through inhibition of tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). 17 The impetus to develop the NHA was based upon the need to evaluate overall haemostatic potential in patients utilizing fibrinolytic therapy (eg, tPA, urokinase) where concomitant administration of anticoagulants is required. Reported clinical manifestations include abnormal epistaxis, oral bleeding with injury or dental extractions, haematoma formation, maenorrhagia, haemorrhagic ovarian cysts, haemorrhage in pregnancy and intracranial bleeding with injury.…”

Alterations in markers of coagulation and fibrinolysis in patients with Paroxysmal Nocturnal Hemoglobinuria before and during treatment with eculizumab

“…We have assessed levels of procoagulant microparticles (MP), prothrombin fragment 1+2 (F1+2) and D dimer levels as parameters of thrombin generation and fibrinolysis respectively. To study the interplay between coagulation and fibrinolysis, the Nijmegen Hemostasis Assay (NHA) was used, which allows simultaneous analysis of the plasma potential to generate both thrombin and plasmin [31]. All parameters were correlated with clinical parameters of PNH (LDH, PNH clone size and history of thrombosis).…”

“…The NHA was performed as described previously [31]. This assay simultaneously analyzes coagulation and fibrinolysis in plasma samples upon in vitro activation.…”

A(nother) Test Meant to Fill the Gap between In Vivo and Ex Vivo Hemostasis

“…Other global assays currently under investigation include clot waveform analysis (4 ) and simultaneous thrombin and plasmin generation in platelet-poor plasma (5 ).…”