Fibrinogen Attentuates Thrombin Decay In a Concentration-Dependent Manner
Authors: Kremers Romy , Wagenvoord Rob , Hemker Coenraad 
Published: October 2019

Abstract

Introduction
Clotting of blood(-plasma) is caused by the first traces of thrombin that appear. The bulk of thrombin emerges in the clot and is subsequently inactivated. Fibrin(-ogen) has a stimulatory effect on the amount of thrombin activity observed. This can be due to stimulation of prothrombin conversion, to attenuation of thrombin inhibition or both. Thrombin is known to bind reversibly to specific sites on the E domain of fibrin and to remain active when bound. We tested the hypothesis that this binding makes thrombin less accessible to inhibitors and thus fosters the presence of active thrombin. Thrombin decay is caused by serpins, mainly antithrombin (AT), and by α2Macroglobulin (α2M).

Aim
In this study we aim to determine the effect of fibrin(-ogen) on the rate of thrombin decay and on the contribution of α2M to total decay.

Methods
Thrombin generation (TG) in normal pool plasma in the presence or absence of fibrinogen was triggered with 5 pM tissue factor (TF) and measured by calibrated automated thrombinography (CAT). Thrombin decay was measured by triggering thrombin generation with high (50 pM) tissue factor (TF) causing all prothrombin to be converted within 3 minutes, so that the part of the curve after 3 min is governed by thrombin decay alone. Total thrombin decay rate was calculated as the pseudo first order decay constant of that part of the curve. The α2M-dependent thrombin decay rate was measured as the pseudo first order constant with which the amidolytically active α2M-thrombin complex forms.
AT and α2M levels were determined by functional assays that were developed in house. Fibrinogen was measured by the von Clauss method. Defibrination was done by adding reptilase and winding out the formed fibrin. Anonymized plasma samples remaining from routine analyses were obtained from the clinical routine laboratory (n=77), including samples from patients in which a shift in the ratio of AT and α2M could be expected (liver cirrhosis, nephrotic syndrome). Pearson’s correlation analysis was used to establish the relationships between thrombin decay rates and inhibitor concentrations, and the influence of plasma fibrinogen concentration on these reactions.

Results
Thrombin generation and thrombin decay were measured in normal pooled plasma, defibrinated normal pooled plasma and defibrinated normal pooled plasma spiked with 1, 2 or 3 g/L purified human fibrinogen. The removal of fibrinogen from plasma decreases thrombin generation by 40% (p<0.001) and the addition of purified human fibrinogen to defibrinated plasma concentration-dependently restores TG.
In addition, plasma defibrination causes the rate of total thrombin decay to increase significantly from 0.36 min-1 to 0.47 min-1 (p<0.001). This effect can be reversed as well by the addition of purified human fibrinogen. Thrombin decay by α2M is inhibited by fibrinogen in a concentration-dependent manner (36% at 3 g/L fibrinogen) (p<0.001). No significant concentration dependence was found for AT-dependent decay.
In the patient samples fibrinogen levels varied between 1.20 and 4.79 g/L with a mean value of 2.79 g/L (± 0.62 g/L). AT concentrations ranged from 0.58 µM to 3.08 µM and α2M levels from 1.85 µM to 7.73 µM. Both total and AT-dependent thrombin decay rates were significantly correlated with the plasma AT levels (p<0.001) and α2M-dependent thrombin decay was significantly correlated with the plasma α2M concentration (p<0.001). Plasma fibrinogen levels were significantly and inversely correlated with the rate of thrombin decay by α2M, but not AT (p=0.001).

Conclusion
The presence of fibrin(-ogen) decreases the decay rate of thrombin in a concentration-dependent manner, primarily by its influence on α2M-dependent thrombin inactivation. This causes thrombin generation to increase. Together, these results suggest that elevated fibrinogen levels may predispose individuals to thrombosis by protecting thrombin.

Disclosures:
No relevant conflicts of interest to declare.