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.

SummaryPatients with functional deficiency of C1-inhibitor (C1-INH) suffer from recurrent acute attacks (AA) of localized oedema associated with activation of the contact system, complement and fibrinolysis. To unravel further the role of coagulation and fibrinolysis in the pathophysiology of C1-INH deficiency, we performed simultaneous thrombin and plasmin generation measurements in plasma from patients with hereditary angioedema (HAE) due to C1-INH deficiency during AA (n = 23), in remission (R) (n = 20) and in controls (n = 20). During AA thrombin generation after in-vitro activation of plasma was higher than in controls, as demonstrated by shorter thrombin peak-time (P < 0·05), higher thrombin peak-height (P < 0·001) and increased area under the curve (AUC) (P < 0·05). Additionally, elevated levels of prothrombin fragment 1+2 (P < 0·0001) were observed in non-activated plasma from the same patients. In contrast, in activated plasma from patients during AA plasmin generation estimated as plasmin peak-height (P < 0·05) and plasmin potential (P < 0·05) was reduced, but non-activated plasma of the same patients showed elevated plasmin-anti-plasmin (PAP) complexes (P < 0·001). This apparent discrepancy can be reconciled by elevated soluble thrombomodulin (sTM) (P < 0·01) and thrombin activatable fibrinolysis inhibitor (TAFI) in patients during AA providing possible evidence for a regulatory effect on fibrinolysis. Plasminogen activator inhibitor-1 (PAI-1) was reduced in patients during AA indicating, together with the observed reduction of plasmin generation, the consumption of fibrinolytic factors. In conclusion, our results support the involvement of coagulation and fibrinolysis in the pathophysiology of HAE and show the possible application of simultaneous measurement of thrombin and plasmin generation to evaluate different clinical conditions in HAE patients.

Rare bleeding disorders (RBDs) are a heterogeneous group of diseases with varying bleeding tendency, only partially explained by their laboratory phenotype. We analysed the separate groups of RBD abnormalities, and we investigated retrospectively whether the novel haemostasis assay (NHA) was able to differentiate between bleeding tendency. We have performed simultaneous thrombin generation (TG) and plasmin generation (PG) measurements in 41 patients affected with deficiencies in prothrombin, factor (F) V, FVII, FX, FXIII and fibrinogen. Parameters of the NHA were classified based on (major or minor) bleeding tendency. Patients with deficiencies in coagulation propagation (FII, FV and FX) and major type of bleedings had diminished TG (expressed as AUC) below 20% of control. FVII deficient patients only had prolonged thrombin lag-time ratio of 1.6 ± 0.2 (P < 0.05) and normal AUC (92-125%). Afibrinogenemic patients demonstrated PG of 2-29% of normal and appeared to correlate with the type of mutation. Thrombin peak-height (57 ± 16%) was reduced (not significant) in these patients and AUC was comparable to the reference (102 ± 27%). FXIII-deficient plasmas resulted in a reduced thrombin peak-height of 59 ± 13% (P < 0.05) and normal AUC (90 ± 14%). Thrombin peak-height (P < 0.01) and plasmin potential (P < 0.05) were lower in the major bleeders compared with the minor bleeders. These results provided distinct TG and PG curves for each individual abnormality and differentiation of bleeding tendency was observed for thrombin and PG parameters. Prospective studies are warranted to confirm these retrospective results.

No abstract

No abstract