A novel simultaneous clot‐fibrinolysis waveform analysis for assessing fibrin formation and clot lysis in haemorrhagic disorders

Abstract

Simultaneous evaluation of coagulation and fibrinolysis facilitates an overall understanding of normal and pathological haemostasis. We established an assay for assessing clot formation and fibrinolysis simultaneously using clot waveform analysis by the trigger of a mixture of activated partial thromboplastin time reagent and an optimized concentration of tissue-type plasminogen activator (0Á63 lg/ml) to examine the temporal reactions in a short monitoring time (<500 s). The interplay between clot formation and fibrinolysis was confirmed by analysing the effects of argatroban, tranexamic acid and thrombomodulin. Fibrinogen levels positively correlated with coagulation and fibrinolytic potential and initial fibrin clot formation was independent of plasminogen concentration. Plasminogen activator inhibitor-1deficient (-def) and a2-antiplasmin-def plasmas demonstrated different characteristic hyper-fibrinolytic patterns. For the specificity of individual clotting factor-def plasmas, factor (F)VIII-def and FIX-def plasmas in particular demonstrated shortened fibrinolysis lag-times (FLT) and enhanced endogenous fibrinolysis potential in addition to decreased maximum coagulation velocity, possibly reflecting the fragile formation of fibrin clots. Tranexamic acid depressed fibrinolysis to a similar extent in FVIII-def and FIX-def plasmas. We concluded that the clot-fibrinolysis waveform analysis technique could sensitively monitor both sides of fibrin clot formation and fibrinolysis, and could provide an easy-to-use assay to help clarify the underlying pathogenesis of bleeding disorders in routine clinical practice.

Hemostatic function in hyperfibrinolytic disseminated intravascular coagulation

“…A modification of the conventional clot waveform technique to include analysis of fibrinolysis has been recently established by our group . The novel CFWA provides comprehensive data not only on fibrin clot formation but also on fibrinolytic potential, to simultaneously quantify clot fibrinolysis.…”

“…A modification of the conventional clot waveform technique to include analysis of fibrinolysis has been recently established by our group. 17 The novel CFWA provides comprehensive data not only on fibrin clot formation but also on fibrinolytic potential, to simultaneously quantify clot fibrinolysis. In outline, fibrin formation and fibrinolytic reactions were initiated by the addition of a mixture of 50 lL normal plasma, 50 lL APTT reagent (APTT-SLA â ; Sysmex, Kobe, Japan) and 50 lL 100 mmol/L CaCl 2 containing t-PA (f.c., 0.63 lg/mL).…”

“…The marked shortening of the P-LT observed at onset strongly supported the diagnosis of enhanced fibrinolysis associated with DIC, and hence simultaneous assessment of coagulation and fibrinolytic potential using CFWA could be informative in these circumstances. 17 We adopted this technique, therefore, in the current investigations. Figure 2b shows the time-dependent changes in fibrin clot formation and fibrinolysis detected on CFWA in the present young patient at diagnosis and at day +1.…”

Application of clot‐fibrinolysis waveform analysis to assessment of in vitro effects of direct oral anticoagulants on fibrinolysis

“…Recombinant human t-PA (ProSpec) was solved in H 2 O at 100 µg/mL as recommended by the manufacturer and as performed in a previous study. 9 The stock solutions of t-PA were also aliquoted and stored at −80°C. DOACs were diluted in series and applied to spiking the CRYOcheck Pooled Normal Plasma (Precision BioLogic) with concentrations ranging from 0 to 800 ng/mL.…”

“…While the recent reports focus on application of CFWA to assessment of hemorrhagic disorders, CFWA is also expected to aid in investigating whether and how anticoagulants impact fibrinolysis. 9,10 The present study aims to evaluate the usability of CFWA for assessment of in vitro effects of DOACs on fibrinolysis.…”