Does cell-free DNA promote coagulation and inhibit fibrinolysis in patients with unprovoked venous thromboembolism?

doi:10.1016/j.thromres.2019.11.030

Thrombosis Research

Volume 186, February 2020, Pages 13-19

https://doi.org/10.1016/j.thromres.2019.11.030 Get rights and content

Highlights

•
Plasma samples from unprovoked VTE patients are procoagulant and anti-fibrinolytic.
•
CFDNA is elevated in idiopathic VTE patients after stopping warfarin therapy.
•
CFDNA levels do not correlate with coagulation or fibrinolysis parameters.
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Factors in addition to CFDNA may modulate hemostasis in unprovoked VTE patients.

Abstract

Introduction

Cell-free DNA (CFDNA) is the major structural component of neutrophil extracellular traps (NETs). CFDNA contributes to the prothrombotic potential of NETs by promoting thrombin generation and inhibiting fibrinolysis. Patients with venous thromboembolism (VTE) have elevated circulating nucleosomes (i.e. DNA-histone complexes). In this study, we investigated if CFDNA contributes to a procoagulant and an antifibrinolytic state in patients with unprovoked VTE.

Materials and methods

Plasma samples from patients with a first episode of unprovoked VTE were obtained from the D-Dimer Optimal Duration Study (DODS). We measured CFDNA plasma levels in 263 patients while on warfarin and 1-month after stopping. Thrombin generation assays and clot lysis assays were measured in patients after stopping warfarin. Comparisons were made with healthy controls.

Results

CFDNA levels in VTE patients who stopped warfarin (5.53 μg/mL; 95%CI: 5.34–5.72) were higher than during warfarin therapy (3.11 μg/mL; 95%CI: 2.98–3.25; p < .001), and higher than in healthy controls (2.77 μg/mL; 95%CI: 2.42–3.11; p < .001). VTE patients had a procoagulant state as evidenced by a shorter lag time (30.8 min; 95%CI: 29.2–32.4) compared to controls (48.2 min; 95%CI :41.0–55.5; p < .001) and a greater endogenous thrombin potential (2656 nM∗min; 95%CI: 2479–2836) compared to healthy controls (1198 nM ∗ min; 95%CI: 793–1603). There was a higher proportion of clots generated from patient plasma that were resistant to lysis (43.7%) compared to healthy controls (46.3%; p < .05). CFDNA levels were not associated with enhanced thrombin generation or impaired fibrinolysis in VTE patients.

Conclusion

CFDNA levels are elevated in patients with unprovoked VTE but do not correlate with the procoagulant or anti-fibrinolytic properties of patient plasma. This study suggests that additional factors in addition to CFDNA may contribute to the pathogenesis of VTE.

Introduction

Venous thromboembolism (VTE), which manifests as deep vein thrombosis (DVT) and pulmonary embolism (PE), is the most common vascular disease after myocardial infarction and stroke [[1], [2], [3]]. VTE is a multifactorial disease that is the result of a complex interplay between genetic and environmental risk factors [4]. Approximately 25% to 50% of patients have unprovoked VTE, defined as VTE that occurs without an apparent provoking factor [1,5,6]. Unprovoked VTE is associated with a substantial risk of recurrence, with approximately 25% of patients having a recurrence within 5 years of stopping anticoagulant therapy [7]. As a result, many patients with unprovoked VTE continue on long-term anticoagulation [8,9]. However, long term anticoagulant therapy entails a risk of bleeding [9]. An improved understanding of the pathophysiology of thrombosis may lead to new therapeutic strategies that do not increase the risk of bleeding.

In recent years, neutrophils have been shown to contribute to the pathogenesis of VTE [[10], [11], [12]]. Neutrophils are recruited to the vessel wall within the first hours of reduced venous blood flow [12]. Activated neutrophils release neutrophil extracellular traps (NETs) through the process of NETosis, a unique form of neutrophil cell death. NETs consist of cell-free DNA (CFDNA), histones, and granular enzymes (e.g. neutrophil elastase and myeloperoxidase) [13]. In experimental thrombosis models, CFDNA has been shown to provide a scaffold which supports thrombus stability by binding platelets and red blood cells [12]. CFDNA also activates the intrinsic pathway of coagulation and impairs fibrinolysis [[14], [15], [16]]. In a mouse model of flow restriction thrombosis, infusion of DNase I, an enzyme that cleaves CFDNA, protected mice from flow restriction-induced DVT [17]. These findings suggest that CFDNA contributes to the pathogenesis of VTE and may be a target for therapeutic interventions that does not increase the risk of bleeding.

In this study, we tested the hypothesis that circulating CFDNA contributes to a procoagulant state in patients with unprovoked VTE. We measured CFDNA levels in patients with a first episode of unprovoked VTE who were in the D-dimer Optimal Duration Study (DODS) [18]. We determined whether CFDNA is associated with hyper-coagulability and/or impaired fibrinolysis in this population.

Section snippets

Participant eligibility criteria

Patients were eligible for DODS if they were at least 18 years of age with a first episode of unprovoked proximal DVT or PE and were on anticoagulants for 3 to 7 months. Unprovoked VTE was defined as thrombosis not associated with a major risk factor within 3 months, a minor risk factor within 2 months, or cancer within 2 years of diagnosis. Females who had VTE while on estrogens were eligible but classified as having estrogen-associated VTE at baseline (referred to as estrogen-females).

Plasma samples processing

Patient blood samples were collected at enrollment (on warfarin) and 1 month after enrollment (off warfarin). Whole blood from patients and healthy controls was collected via venipuncture into polypropylene tubes containing 3.2% buffered trisodium citrate. Blood was then spun within 2 h of collection at 1700 ×g for 15 min at 20 °C. The plasma was isolated and spun again at 1700 ×g for 5 min at 20 °C. The resultant platelet poor plasma was aliquoted and stored at −80.0 °C.

CFDNA measurement

Thawed platelet poor

Baseline characteristics of patients and healthy controls

Of the 410 patients enrolled in the DODS study, 263 were eligible for inclusion in our analysis (Fig. 1). Baseline characteristics of the VTE patients are shown in Table 1. Mean age was 52, 58% were male, 24% were non-estrogen females, 18% were estrogen females, 59% had presented with DVT alone, 41% presented with PE, 23% had a family history of VTE in a first degree relative, 6% were on an antiplatelet therapy, and 16% were on statins. Of the 35 healthy controls, males and females were equally

Discussion

In this study, we investigated if circulating CFDNA contributes to the procoagulant and/or antifibrinolytic state in patients with unprovoked VTE. We found that VTE patients had evidence of a procoagulant state compared to healthy controls, as reflected by increased thrombin generation with a shortened lag time and increased ETP. VTE patients were also more resistant to fibrinolysis when compared to healthy controls as reflected by an increased proportion of patients that have clots resistant

Conclusion

In conclusion, although plasma samples from unprovoked VTE patients off warfarin exhibit increased coagulability and impaired fibrinolysis compared with plasma samples from healthy volunteers, elevated circulating CFDNA does not directly correlate with the procoagulant and anti-fibrinolytic properties of the plasma. This finding suggests that additional factors may modulate hemostasis in this patient population.

Role of funding source

Funding for this study was provided by the Heart and Stroke Foundation of Ontario, the Canadian Institutes of Health Research, and CanVECTOR. Funding sources did not influence the study design or data analysis/interpretation.

Authorship contributions

S.K.M analyzed the results, made the graphs, and with the help of P.C.L. and V.B. wrote the paper. B.E., D.J.D., and V.B. conducted the experiments. S.K.M., P.C.L., V.B., C.K., and S.P. interpreted the results. C.K., S.P., and N.J.D. extracted clinical data for analysis.

Declaration of competing interest

All authors declare no competing conflicts of interest.

Acknowledgements

This research was supported by grants and awards from the Heart and Stroke Foundation of Ontario (GIA-50), Canadian Institutes for Health Research (MOP-84303; MOP-136878), and CanVECTOR. The authors thank the DODS investigators and the DODS patients, who took part in the clinical study and contributed plasma samples for analysis.

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Full Length ArticleDoes cell-free DNA promote coagulation and inhibit fibrinolysis in patients with unprovoked venous thromboembolism?

Highlights

Abstract

Introduction

Materials and methods

Results

Conclusion

Introduction

Section snippets

Participant eligibility criteria

Plasma samples processing

CFDNA measurement

Baseline characteristics of patients and healthy controls

Discussion

Conclusion

Role of funding source

Authorship contributions

Declaration of competing interest

Acknowledgements

Best Pract. Res. Clin. Haematol.

J. Thromb. Haemost.

Thromb. Res.

J. Thromb. Haemost.

Blood.

The epidemiology of venous thromboembolism

Circulation.

Environmental and genetic risk factors associated with venous thromboembolism

Semin. Thromb. Hemost.

Incidence of idiopathic deep venous thrombosis and secondary thromboembolism among ethnic groups in California

Ann. Intern. Med.

The long-term clinical course of acute deep venous thrombosis

Ann. Intern. Med.

Apixaban for extended treatment of venous thromboembolism

N. Engl. J. Med.

Full Length Article
Does cell-free DNA promote coagulation and inhibit fibrinolysis in patients with unprovoked venous thromboembolism?