Development and application of global assays of hyper‐ and hypofibrinolysis

Abstract

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. AbstractNumerous methods for evaluation of global fibrinolytic activity in whole blood or plasma have been proposed, with the majority based on tissue-type plasminogen activator (t-PA) addition to initiate fibrinolysis. We propose that such an approach is useful to reveal hypofibrinolysis, but t-PA concentrations should be kept to a minimum. In this paper, we describe a low-concentration t-PA plasma turbidity assay to evaluate several congenital factor deficiencies, including plasminogen activator inhibitor-1 (PAI-1) and plasminogen deficiency, as well as hemophilia A and B. In addition, we demonstrate a threshold dependency on endogenous PAI-1 levels. To assess endogenous hyperfibrinolysis, we suggest that assays that avoid t-PA addition are preferable, with assays based on euglobulin fractionation remaining a viable choice.We describe a euglobulin fraction clot lysis time (ECLT) assay with spectrophotometric readout and other modifications, and evaluate it as a tool to measure hyperfibrinolysis in inherited clotting factor deficiency states. We demonstrate that the ECLT is predominantly driven by residual amounts of PAI-1, t-PA, and α 2 -antiplasmin.These assays should be further evaluated for the detection of hypo-or hyperfibrinolysis in acquired thrombotic or hemorrhagic disorders. K E Y W O R D Seuglobulin clot lysis time, fibrinolysis, hemophilia, plasminogen, plasminogen activator inhibitor 1 Essentials• Clot lysis of the euglobulin fraction of plasma reliably assesses endogenous hyperfibrinolysis.• A modified version of the euglobulin clot lysis time (ECLT) is described herein.• This version of ECLT is sensitive to low plasminogen activator inhibitor-1 and high tissue-type plasminogen activator (t-PA) levels.• Turbidimetric assays of clot lysis using low amounts of t-PA are sensitive to hypofibrinolysis.

Functional Fibrinolysis Assays Reveal Different Mechanisms underlying Plasminogen Dysfunction in Ligneous Conjunctivitis

“…by urokinase. In agreement with the results of the cell-based ECLT assay, the K19E/other group had a significantly decreased urokinase-induced plasmin formation rate compared with controls (26 [22][23][24][25][26][27][28] vs. 41 [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] mDO/min À1 , p < 0.05; ►Fig. 4C).…”

“…Similar results have been recently found in LC patients with inherited PLG deficiency using a turbidimetric t-PA resistance assay and a t-PA plasmin generation assay. 25,26 These results suggest that t-PA-based functional assays could provide a reliable tool to predict clinical expression in PLG deficiency. In contrast, the fact that some LC patients had normal urokinase-induced PLG activity indicate that urokinase-dependent assays might not be adapted for such purposes.…”

Impaired Fibrinolysis in Patients with Isolated Aortic Stenosis is Associated with Enhanced Oxidative Stress

“…This methodological difference might justify why oxidative stress has a crucial effect on impaired fibrinolysis in AS despite the various demographic and laboratory determinants of prolonged lysis time between these assays (Siudut et al manuscript in review). The assay by Pieters et al that was introduced in 2019 and supported by the International Society on Thrombosis and Hemostasis Subcommittee has been used in a few studies [ 23 , 25 , 26 ] and is known to be more sensitive to endogenous PAI-1 levels, especially in both hypofibrinolytic and/or hyperfibrinolytic conditions [ 27 ]. On the other hand, the Lys50 assay has been previously applied in studies investigating fibrinolytic capacity in patients with metabolic syndrome, including diabetes mellitus [ 24 ].…”

Assessing plasminogen activation potential with global fibrinolytic assays

“…ECLT cannot assess the main regulatory function of the second step (fibrin degradation) of fibrinolysis in which α2AP and TAFI play essential roles. Though trace amounts of α2AP remain in the euglobulin fraction and show negative correlation with ECLT, ECLT does not show meaningful correlation with plasma α2AP level . TAFI’s effect is also not detected by ECLT when soluble thrombomodulin is not supplemented .…”