The Hypofibrinolysis State Associated with the Dysfibrinogenemia Dusart is Mainly Related to the Altered Fibrin Clot Structure

Abstract

Introduction

The fibrinogen Dusart (p.Aα573Arg > Cys) is a dysfibrinogenemia associated with an increased risk of thrombosis. The aim of this study was to identify factors that could explain the hypofibrinolytic state associated with the Dusart mutation.

Methods

The fibrin α2-antiplasmin (α2-AP) incorporation was quantified by a homemade enzyme-linked immunosorbent assay. The fibrin formation and lysis were studied by turbidity at 405 nm, and the global fibrinolytic capacity (GFC) with the Lysis timer device. The plasmin generation was assessed through an automated method. The clot growth was examined using Thrombodynamics. The clot structure was evaluated by measuring the permeation constant and scanning electron microscopy (SEM).

Results

The plasma levels of D-dimer, PAI-1, FXIII, PAP, and α2-AP were within the normal range, as was α2-AP incorporation into fibrin. By turbidity the patient's clots were almost transparent, and very resistant to fibrinolysis. The patient's GFC was 51 minutes compared with 45 minutes in control. The patient's endogenous plasmin potential and the peak plasmin were increased. The Thrombodynamics analysis revealed an increased lag time and a decreased initial velocity of patient's clot growth. The fibrin clot structure was characterized by a strong reduction in clot's flow (small pores size), and very thin fibers.

Conclusion

The patient's procoagulant phenotype appears primarily driven by the formation of abnormally compact fibrin networks, leading to impaired perfusion and resistance to fibrinolysis, independent of any imbalance in fibrinolytic activators or inhibitors. These findings highlight the critical role of the fibrin clot structure in the thrombotic risk of this variant.

Keywords

dysfibrinogenemia - thrombosis - hypofibrinolysis - plasmin generation - Thrombodynamics

Introduction

Fibrinogen is one of the most abundant protein in the blood with an average concentration of 2 to 4 g/L. Fibrinogen is composed of six polypeptide chains (Aα, Bβ, γ)₂ arranged as a dimer with a 2-fold axis of symmetry passing through the center and perpendicular to the long axis of the molecule.[1] Following partial degradation by thrombin, which removes the N-terminal fibrinopeptides A and B of the α and β chains, fibrinogen spontaneously polymerizes to form a three-dimensional network. Fibrin polymerizes to form a diverse range of clots with varying structural and biological properties, depending on the conditions of formation. Correlations have been established between these clot properties and numerous pathophysiological conditions.[2] The fibrin clot structure can be characterized by fibrin fiber thickness, fiber length, fiber density, degree of branching, and porosity. Two distinct clot structure phenotypes have been identified, with the potential to either facilitate bleeding or thrombosis.[3] A hemorrhagic clot is distinguished by a reduction in fibrin density and stiffness, the presence of thick fibers, and large pores. In contrast, a thrombotic clot is distinguished by elevated fibrin density and stiffness, thin fibers, and small pores. These clot structures have functional consequences, including increased or decreased susceptibility to fibrinolysis and resistance to deformation.[3] In general, the rate of lysis appears to be faster for clots composed of thicker fibers than for clots composed of thinner fibers. However, this is dependent on other biophysical properties of the clot and interactions with cells.[2] [4]

Congenital fibrinogen disorders (CFDs) constitute a heterogeneous group of rare inherited abnormalities of hemostasis encompassing both quantitative and qualitative fibrinogen deficiencies.[5] Among the qualitative defects, dysfibrinogenemia is defined by decreased functional and normal antigenic fibrinogen levels.[6] [7] Most patients with dysfibrinogenemia are asymptomatic at the time of diagnosis. However, a significant number of patients do report a bleeding tendency or have experienced a thrombotic event.[8] A few dysfibrinogenemic variants are strongly associated with a thrombotic phenotype, which is referred to as thrombotic-related dysfibrinogenemia. The mutations causing thrombotic-related dysfibrinogenemia are predominantly found in the C-terminal domain of the Aα chain and the N-terminal segment of the Bβ chain. These include fibrinogen Dusart, AαArg573Cys or AαArg554Cys (numbering without the signal peptide), also known as Paris V or Chapel Hill III, fibrinogen Caracas V (AαSer551Cys), fibrinogen Ijmuiden (BβArg14Cys), fibrinogen New York I (deletion Bβ39–102), fibrinogen Nijmegen (BβArg44Cys), fibrinogen Naples (BβAla98Thr) at homozygous state, and fibrinogen Melun (γAsp390Val).[9] [10] [11] Patients who are carriers of one of these fibrinogen mutations typically experience thrombotic events at a young age and have a first-degree familial thrombotic history (relatives with the same genotype) without any other hereditary thrombophilia.[12] Two mechanisms have been proposed to explain the procoagulant state: (i) Defective binding of thrombin to abnormal fibrin, which results in elevated thrombin levels in the circulation, as observed in the cases of fibrinogens Naples and New York I; and (ii) defective tissue plasminogen activator (t-PA)-mediated fibrinolysis (in fibrinogens New York I, Nijmegen, and Dusart) due to impaired plasminogen binding on fibrin or abnormal fibrin clot structure (increased fibrin density, thin fibers, and small pores) or increased clot stiffness.[8] [13] [14] [15]

The objective of this study was to further investigate coagulation and fibrinolysis in a patient with the Dusart variant.

Materials and Methods

The experiments were conducted using as a control a commercial pooled normal plasma CRYOcheck, from Precision BioLogic with a mean fibrinogen activity of 2.7 g/L. For the experiments related to the incorporation of α2-antiplasmin by FXIII, three controls with normal prothrombin, activated partial thromboplastin, and thrombin times and normal fibrinogen were randomly selected in the laboratory of hemostasis. Furthermore, since the patient was undergoing treatment with a vitamin K antagonist at the time of the collection of plasma samples, a matched control was used from a patient with a 2.29 INR (anticoagulated patient) only for plasmin generation assay, and plasmin generation normal control parameters were determined from 35 individual normal plasmas from CRYOcheck. The patient provided written informed consent for this study, in accordance with the Declaration of Helsinki.

Laboratory Studies

Functional fibrinogen (Clauss method, Thrombin reagent, Siemens, Germany) and INR were measured in a Sysmex CN6000. Fibrinogen antigen was measured by a latex immunoassay (Liaphen Fibrinogen, Hyphen BioMed, France) on a BCS XP coagulometer (Siemens, Germany), D-dimer with Vidas (BioMérieux, France), PAI 1 with Zymutest antigen (Hyphen BioMed, France), plasmin-antiplasmin (PAP) complex with Technozym (Technoclone, GmbH, Austria), and FXIII-A₂B₂ antigen levels by an in-house sandwich ELISA.[16]

α2-Antiplasmin Binding to the Fibrin Clot

The amount of α2-AP incorporated into plasma fibrin clot was determined by an in-house sandwich ELISA.[17] The assay measures all plasmatic forms of α2-AP and it is not influenced by the presence of plasmin–antiplasmin complexes. Plasma samples were clotted by adding 2 IU/mL thrombin (CoaChrom, Maria Enzersdorf, Austria) and 20 mM CaCl₂ (final). After incubation at 37°C for 30 minutes serum was separated by centrifugation (16,100 g, 5 minutes). α2-AP antigen levels were measured from the plasma and serum samples and the extent of α2-AP incorporation into fibrin clot was calculated using the following formula:

α2-AP incorporation (%) = (plasma α2-AP [mg/L] − serum α2-AP [mg/L])/plasma α2-AP (mg/L) × 100

Turbidity

Turbidity was performed essentially as described.[18] Plasma was diluted 1:4 with Tris- buffered saline (TBS, 50 mM Tris, 0.1 M NaCl pH 7.4) for fibrin formation or with TBS supplemented with tissue type plasminogen activator (tPA) (Technoclone, GmbH, Austria) for fibrinolysis. Coagulation was triggered by adding human thrombin (Merck, KGAa, Germany) and CaCl₂. The final plasma dilution was 1:6, thrombin 0.1 units/mL, CaCl₂ 5 mM, and tPA 90 ng/mL. Optical density (OD) was recorded each 14 s during 1 h at 405 nm in a BioTek Instruments ELx800 series (Witec AG, Sursee, Switzerland). Samples were run each in triplicates. The fibrin kinetic parameters for fibrin formation were the lag time (LT, time to reach 10 mOD, min), the slope (first derivative of the curve, OD/min), and the MaxAbs (mOD), and for fibrin degradation the T50 (min), defined as the time elapsed between 50% of MaxAbs in the ascending side of the curve and 50% MaxAbs in the descending side. An open access, online software was used to calculate T50[19] and GraphPad Prism 8.01 was utilized for the calculation of the slopes of the polymerization curves. For better illustration due to the dramatic difference between the patient's OD and that of control, the ODs were normalized by dividing by their respective MaxAbs.

Global Fibrinolytic Capacity

The global fibrinolytic capacity (GFC) was measured with the Lysis Timer (LT) device, using R1 and R2 reagents (HYPHEN BioMed, Neuville sur Oise, France), following the manufacturer protocol detailed in Amiral et al.[20] Briefly, a mixture of tPA (100 ng/mL) and silica (100 μL) was added to 100 μL of plasma, and incubated for 1 minute at 37°C. Then 100 μL of thrombin (4 NIH/mL) and calcium was added, and the progression of the reaction was measured until the complete dissolution of the plasma clot at 940 nm. The GFC (min) is measured from the primary derivatives of the light transmittance variation curve calculated by the software of the LT. GFC ranges provided by manufacturer are: hyperfibrinolysis, 15.3 (0.0) min; normal, 37.9 (2.5) min; hypofibrinolysis, 87.7 (6.4) min. Commercial control plasmas (HYPHEN BioMed, Neuville sur Oise, France) were also tested. Experiments were done by duplicate.

Plasmin Generation

The plasmin generation (PG) was quantified using the PG kit provided by Synapse Research Institute (Maastricht, Netherlands), which relies on an automated method based on the cleavage of a plasmin-specific fluorogenic substrate.[21] Given the paucity of publications in the literature using this kit, and with the objective to establishing an in-house range, we tested 35 individual control plasmas from CRYOCheck.

Briefly, 15 μL of plasma was added to each well in quadruplicate: two wells for calibrator (α2-macroglobulin–plasmin complex, 310 nM) and two wells for the reaction (endogenous PG). Then 35 μL of calibrator solution was added to the calibrator wells and 35 μL of trigger solution to the reaction wells and warmed for 10 minutes at 37°C. In each well 10 μL of the PG substrate (fluorogenic substrate and CaCl₂) was dispensed. Final concentrations were tissue factor 0.5 pmol/L, phospholipids 4 μmol/L, rtPA 0.31 μg/mL, CaCl₂ 16.6 mmol/L, and fluorogenic substrate 0.5 mmol/L. Reactions were monitored every 20 seconds during 40 minutes with a fluorometer (Fluoroskan Ascent, Thrombinoscope, France) equipped with a dispenser and 390/460 filter set (excitation/emission). The parameters lag time (min; time the plasmin concentration reached 6 nmol/L), the time to peak (min; TtPeak), the velocity (nM/min; peak/[TtPeak-lag time]), the peak (nM), and the endogenous plasmin potential (EPP; nM*min) were measured using the same software for thrombin generation.[22]

Thrombodynamics Analyser T2-T

Thrombodynamics Analyser T2-T (HemaCore LLC, Moscow, Russia) records and analyzes spatiotemporal dynamics of thrombin generation and fibrin formation simultaneously. The reagents (Thrombodynamics kit) and protocol were provided by Hemacore LLC (Moscow, Russia). Briefly, 120 μL plasma was transferred to reagent I, which contains a corn trypsin inhibitor to inhibit the contact pathway of coagulation and also a fluorogenic substrate for thrombin, gently mixed, and 5 μL of reagent PLS (suspension of phospholipid vesicles) was added. The tube was incubated for 15 minutes at 37°C. Then 120 μL was transferred to the tube that contains reagent II (lyophilized solution of CaCl₂) and carefully mixed avoiding foaming. The solution was immediately transferred to the channel of the measurement cuvette (two channels per cuvette). Then an activating insert (plastic comb with tissue factor immobilized on its bottom end face) was introduced in the channel, closing the thermostat cap, and the start bottom was pressed for recording. The following parameters were analyzed: lag time, initial and stationary rates of clot growth, clot size at 30 minutes, and clot density. The lag time (Tlag; min) is the time between contact of activator with plasma and start of clot growth; the initial rate of clot growth (Vi; μm/min) is the slope of the curve on a clot versus time graph during the first 2 to 6 minutes of cloth growth.[23] Stationary rate of clot growth (Vst; μm/min) is measured as a slope of the curve on a clot size versus time graph within the interval 15 to 25 minutes after clot growth begins,[23] clot size (μm) after 30 minutes and clot density (a.u.).

Permeation

The flow measurements through preformed plasma fibrin clots were done essentially as described with some modifications.[24] Fibrin clots were made using the same protocol used for the fibrin polymerization with thrombin. Then 40 μL of the solution was immediately transferred to the well of a six-chamber Ibidi slide (Ibidi, GmbH, München, Germany), and left for 2 h in a humidity chamber at room temperature. After 2 h the wells on both side of the channel were filled with 50 μL of TBS, and in one side a 2.5-mL syringe without the plunger was carefully plugged inside the well and filled until a height of 4 cm. Clots were washed for 1 h with TBS and the flowed buffer at the opposite end discarded; then the buffer flowed through the channel was collected at regular time intervals of 10 minutes for control (5 measurements) and 4 h for the patient (1 measurement/clot), weighed and flow rate calculated (g/min). Experiments were run in triplicates. The permeation or Darcy constant (Ks) was calculated as described[25]:

Ks = (J × L × η)/(A × P)

J: flow, g/s; L: length, 1.7 cm; A: area, 0.38 cm × 0.04 cm; P = Pressure, 4,998 dyna /cm²; η: viscosity, 0.01 Poise.

Scanning Electron Microscopy

Plasma samples (100 μL) were clotted with thrombin (1 U/mL and 20 mM CaCl₂, final concentrations), carefully mixed, and immediately transferred into a pre-etched plastic serologic tip, sealed at the bottom with parafilm M, and placed for 2 h in a humidity chamber at 37°C. Clots were processed essentially as described for SEM imaging[26] in a Sigma 300 VP FE-SEM (Field-emission SEM) from Zeiss (Oberkochen. Germany). Duplicates of each sample were made. Fibrin fiber diameters were measured manually using Image J 1.49v (Fiji, National Institute of Health, Bethesda, Maryland, USA).

Statistics

Results are presented as mean ± standard deviation (SD) or median (95% CI of the median), after assessing normality by Shapiro-Wilk test. Mann Whitney test was used for non-normally distributed data, using GraphPad Prism version 8.01.

Results

Clinical History

The Dusart mutation (Aα 573 Arg > Cys) has been identified in three generations of the patient's family: the father and paternal uncle, who experienced recurrent venous thrombosis, and the brother, who is currently undergoing treatment with a vitamin K antagonist for primary prevention. The patient did not experience thrombotic events while receiving a vitamin K antagonist (Fluindione) for primary prevention. The patient's son, aged 17, suffered a severe right sylvian ischemic stroke resistant to fibrinolysis (rtPA) with unsuccessful thrombectomy. This resulted in the need for decompression craniectomy, which was complicated by the subsequent development of spasticity and epilepsy. As observed in other families with Dusart dysfibrinogenemia, fibrinogen levels measured by the Clauss method were only slightly decreased (and on some occasions even within the normal range) while consistently lower than the antigen fibrinogen level. Anticoagulation was monitored by INR on a STAR analyzer with an electromagnetic detection system. However, the measurement of INR with an optical detection system was not possible, likely due to the abnormal structure of the fibrin clot. Recently, the patient was trained to use a point-of-care CoaguChek XS (Roche) for self-testing of INR.

Functional and Structural Studies

The results of the fibrinogen workup and coagulation assays performed on the patient are presented in [Table 1]. The patient had decreased functional fibrinogen relative to antigen concentration, consistent with dysfibrinogenemia. The levels of D-dimers, PAI-1, α2-antiplasmin (α2-AP), and FXIII (FXIII-A2B2) antigen in the patient's plasma were within the normal range. Furthermore, the incorporation of α2-AP into the clot was also within the normal range ([Table 2] and [Supplemental Table S1]).

Table 1 Coagulation assays

	Patient (Aα p.Arg573Cys)	Normal range
Fg functional (g/L)	1.44	2–4
Fg antigen (g/L)	2.99	2–4
INR	2.19	2–3
D-Dimer (ng/mL)	58.23	<500
PAI 1 (ng/mL)	13.78	0–50
PAP complex (ng/mL)	195	0–514

Abbreviations: INR, international normalized ratio; PAP, plasmin–antiplasmin complex.

Table 2 Summary of α2-antiplasmin and FXIII determination in plasma samples

Sample	α2-AP plasma (mg/L)	α2-AP serum (mg/L)	α2-AP – fibrin clot (mg/L)	α2-AP – fibrin clot (%)	Plasma FXIII (mg/L)
Control 1	75.06	32.26	42.80	57.02	24.85
Control 2	72.32	39.12	33.20	45.90	24.77
Control 3	67.28	40.33	26.95	39.38	22.69
Patient	68.44	32.12	36.32	53.07	24.05
Reference range	48.0–84.7	–	–	–	14-28

Abbreviation: α2AP, α2-antiplasmin.

Note: –, not available.

The kinetic of fibrin polymerization triggered with thrombin is shown in [Fig. 1A] (left). The patient had a very decreased MaxAbs (mean ± SD, mOD) of 9 ± 1 (control: 222 ± 4) and slope (mean ± SD, [OD x s] 10⁻⁴) of 0.6 ± 0.0 (control 6.0 ± 0.3), while the lag time (min) was almost similar to control at 3.6 ± 0.3 (control 2.9 ± 0.1). The patient's curves looked better when they were normalized by dividing the OD by the MaxAbs ([Fig. 1B], left). A significant decrease in fibrinolysis was observed in the patient compared with the control. The T50% value was 8.8 (0.2) min for the control, but no lysis was observed after 60 minutes in the patient ([Fig. 1A, B]; right). When fibrinolysis was recorded in the lysis timer device, the patient's GFC was 51.1 (1.2) min compared with 44.8 (1.2) min in control.

Fig. 1 Fibrin turbidity. (A) Fibrin formation (left) and degradation (right). (B) Fibrin formation and degradation after normalization. The curves represent one experiment in triplicate. (•) Control (▪) Patient. Values represent the mean (SD). Graphs were prepared using GraphPad Prism version 8.01.

Plasmin generation results are shown in [Fig. 2] and [Supplemental Table S2]. The patient with Dusart had a prolonged lag time and ttPeak, a normal EPP, and a higher peak compared with the control group (P2.5^th to P97.5^th values). When compared with an anticoagulated patient with similar INR, it showed similarities with regard to the lag time and VelIndex, but the anticoagulated patient had a lower EPP, below the P2.5^th of the control group.

Fig. 2 Plasmin generation. The graphs show the different parameters of plasmin generation of the patient and a matched control with similar INR. The dashed lines represent the P2.5^th and P97.5^th values from a normal control group from CRYOcheck (n = 35). Graphs were prepared using GraphPad Prism version 8.01.

The results of the Thrombodynamics (TD) analysis are summarized in [Table 3]. The patient's fibrin kinetic showed an increased lag time and decreased initial velocity of clot growth, along with a reduction in clot size and density when compared with the control at 10 minutes ([Fig. 3], arrows point to the clot size in control and patient) and 30 minutes. However, at the 1-h interval, the patient's clot size was similar to that of the control ([Fig. 3]; [Videos 1] and [2] are available in the supplemental material), despite the anticoagulation. The patient's clot demonstrated linear growth, while the control presented a parabolic trend.

Table 3 Summary of Thrombodynamics

Fibrin dynamics	Control	Patient
Rate of clot growth (μm/min)	32.8 (2.9)	31.4 (5.7)
Tlag (min)	1.4 (0.0)	2.3 (0.0)
Vi (μm/min)	65.9 (2.1)	44.2 (0.5)
Vst (μm/min)	32.8 (2.9)	31.4 (5.7)
Clot size at 30 min (μm)	1,391 (74)	1,072 (79)
Clot density (a.u.)	20,667 (385)	15,045 (1380)

Notes: Fibrin dynamics: a.u., arbitrary units; Tlag, lag time; Vi, initial rate of growth; Vst, stationary rate of clot growth.

Results are reported as mean (SD).

Fig. 3 Thrombodynamics: dynamic fibrin formation. (A) Control. (B) Patient. Left: Clot size variation versus time. Right: Visual clot density. The arrows point to the thickness of the clot at 10 minutes. The curves and clots images were exported from Thrombodynamics and edited with Gimp 2.10.34 software.

Download Video

Video 1 Control's fibrin: spaciotemporal control clot formation visualized by Thrombodynamics.

Download Video

Video 2 Patient's fibrin: spaciotemporal patient clot formation visualized by Thrombodynamics.

Notably, this technique enabled the visualization of patient's clots that were otherwise difficult to detect at 405 nm.

Finally, the clot structure was evaluated indirectly through the measurement of the permeation constant and by imaging. The patient's Ks was 7.3 (0.3) × 10⁻¹⁰ cm², which was two orders of magnitude lower than the control value of 6.6 (0.3) × 10⁻⁸ cm². As indicated in [Fig. 4A], the fibrin fibers' size distribution (Tukey representation) was not normal with a median (95% CI of the median) of 106 (102–114) nm for control and 57 (55–60) nm for patient (p ≤ 0.0001). SEM images revealed that the patient's plasma clots were composed of highly branched, curved, and thinner fibers in comparison to the control (n = 300, 59 ± 6 nm versus 106 ± 12 nm) with the presence of very small pores ([Fig. 4]).

Fig. 4 Plasma clots' fibrin fiber diameter (Ø) distribution and scanning electron microscopy images (SEM). (A) Plasma fibrin fibers distribution. (B) SEM images. The upper panel represents the clot structure of the control and the lower panel the patient clot. Scale bar 1 μm for both micrographs. Gimp 2.10.34 software was used to combine the graph from GraphPad Prism and scanning electron microscopy image.

Discussion

We present a new case of a patient carrier of the Dusart fibrinogen variant, demonstrating that the abnormal fibrin clot structure leads to hypofibrinolysis and a procoagulant state.

The Aα573 Arg > Cys mutation has been identified in six apparently unrelated families.[10] [11] Fibrinogen Dusart was initially reported in 1983,[27] with three of the five family members who carried the mutation exhibiting thromboembolic disease. Shen et al reported a 36-year-old patient with recurrent splanchnic thromboses.[28] Ramanathan et al described a Scandinavian family with five patients experiencing venous (deep venous thrombosis, pulmonary embolisms, or cerebral vein thrombosis) or arterial (cerebral) thrombosis at young age.[29] In the family with the Aα573 Arg > Cys mutation reported by Tarumi et al,[11] venous thromboembolism was observed in 6 of the 11 family members. The association of the fibrinogen Aα573 Arg > Cys mutation with thrombosis has been supported by the absence of other inherited or acquired factors predisposing to thrombosis.

Extensive imaging and functional analyses have been conducted to characterize the hypofibrinolysis observed in the Dusart variant. Similarly to our observation, Collet et al described that the Dusart clot structure had a 175× decreased permeation constant, a 11× diminished fibrin fiber diameter[14] and in addition a 6× increased rigidity.[15] Furthermore, Mosesson et al reported a 2× faster γ-chain crosslinking.[30] Overall, these modifications in the fibrin clot network resulted in a reduction of plasminogen binding to fibrin and defective plasminogen activation by tissue plasminogen activator.

We tested the fibrinolysis by two methods, the GFC (Lysis Timer) and the clot lysis time (T50). Our findings confirmed that the patient had a marked hypofibrinolysis by both the methods. When assessed with the GFC, the patient's fibrin degradation occurred, on average, 6 minutes later than that observed in the control group. Conversely, when assessed by the clot lysis time, the patient's clots exhibited a degradation rate of only 31%, whereas the control demonstrated complete degradation. The enhanced sensitivity of the clot lysis time assay in comparison to the GFC may be attributed to the tPA brand, and the differing endpoint outcome measures (time to half-clot degradation versus maximum fibrinolysis speed). Second, we investigated the plasmin generation (PG) which aims to evaluate the endogenous capacity of fibrinolysis.[31] The patient's prolonged lag time and increased time to peak were likely associated with the fibrin clot structure and anticoagulation, as the matched, anticoagulated control without fibrinogen disorder not only exhibited a ttPeak within the normal range but also a prolonged lag time. The increased plasmin peak despite hypofibrinolysis is consistent with the concept that PG reflects enzymatic potential, while effective fibrinolysis depends on the accessibility of fibrin-binding sites. In the compact Dusart network, the high density of fibrin fibers likely restricts plasmin diffusion and substrate accessibility, despite adequate enzyme generation. In a recent large study of patients with bleeding disorder of unknown cause, reduced peak plasmin levels were observed, also likely related to modified fibrin clot structure in that disease.[32] These differences in the PG profile between patients with bleeding and thrombotic phenotypes highlight the potential role of fibrin clot properties in defining the clinical presentation.

To further elucidate the fibrinolysis process in Dusart syndrome, we initially investigated whether cross-linking of α2-AP to fibrin could be enhanced, thereby retarding the lysis of fibrin. It has been demonstrated that FXIIIa cross-links α2-AP to the fibrin(ogen) Aα chain at Lys322, thereby localizing α2-AP into the clot-slowing down fibrinolysis.[33] [34] In our study, no differences were observed in comparison to the control group, indicating that the hypofibrinolysis was not a consequence of an imbalance between activators and inhibitors of fibrinolysis. Overall, these observations suggested that the hypofibrinolysis associated to this fibrinogen variant was related exclusively to the altered clot structure. It would also have been of interest to assess thrombin-activatable fibrinolysis inhibitor (TAFI) activity, as TAFIa can modulate plasmin generation by removing C-terminal lysine residues from fibrin, thereby limiting plasminogen binding. However, due to the limited plasma volume available, this measurement could not be performed. Nonetheless, the increased PG and normal α₂-AP incorporation observed in our patient both indicate that lysine residues on fibrin were preserved and functionally accessible, making a TAFI-related mechanism unlikely.

In conclusion, the present study has demonstrated that hypofibrinolysis in Dusart dysfibrinogenemia is a consequence of the abnormal fibrin clot structure, rather than a result of altered plasmin generation or fibrinolytic factors levels. Given the procoagulant state induced by the hypofibrinolysis, it is recommended to start anticoagulation at the earliest opportunity for patients who are carriers of the Dusart variant.

Conflict of Interest

Éva Katona reports all support for the present manuscript from “OTKA Bridging Fund from the University of Debrecen” and Alessandro Casini reports grants or contracts from few/some entities from “NovoNordisk research grant” and consulting fees from “LFB, SOBI.”

Acknowledgments

We want to thank Synapse Research Institute for providing the kits necessary for plasmin generation measurements, and to the patient for the blood donation that made this study possible.

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Publication History

Received: 06 August 2025

Accepted: 05 November 2025

Article published online:
20 January 2026