Microparticle-associated tissue factor activity in patients with acute unprovoked deep vein thrombosis and during the course of one year

doi:10.1016/j.thromres.2014.07.041

Thrombosis Research

Volume 134, Issue 5, November 2014, Pages 1093-1096

https://doi.org/10.1016/j.thromres.2014.07.041 Get rights and content

Highlights

•
Elevated tissue factor (TF)-bearing microparticles (MPs) have been found in different prothrombotic conditions
•
We hypothesized that MP-associated TF activity may contribute to the pathogenesis of unprovoked deep vein thrombosis (DVT).
•
MP-TF activity did not differ between patients with acute DVT and healthy controls
•
No changes in MP-TF activity were found in four follow-up measurements during one year

Abstract

Background

Tissue factor (TF) is the main in-vivo initiator of blood coagulation. Microparticles (MPs) are small procoagulant membrane vesicles. Elevated TF-bearing MPs have been found in different prothrombotic conditions and MP-associated TF activity may contribute to the pathogenesis of unprovoked deep vein thrombosis (DVT).

Objective

To determine MP-TF activity levels at diagnosis of DVT and at four additional time points during the course of one year in a well-defined group of patients with unprovoked DVT of the lower limb.

Patients/Methods

In this study, 41 patients with acute unilateral symptomatic and unprovoked DVT of the lower limb were included and followed for 1 year. Venous blood samples for determination of MP-TF activity were drawn at diagnosis of acute DVT, and 1-, 3-, 6-, and 12 months later. In addition, 10 young and healthy control subjects were included.

Results

The median MP-TF activity was 0.06 pg/mL (25th-75th percentile: 0.0-0.53) in patients with acute DVT and 0.18 pg/mL (0.07-0.33) in healthy controls, and did not differ significantly (p = 0.35). No significant changes in MP-TF activity were found in the follow-up measurements. MP-TF activity did also not differ significantly between patients with proximal- or distal DVT and between those with- or without residual DVT after 6 months.

Conclusions

MP-TF activity is low at the acute event in patients with unprovoked DVT of the lower limb and remains unchanged during the course of the disease. Our data do not support the hypothesis that TF-bearing MPs play a determining role in the pathogenesis of unprovoked DVT.

Introduction

More than 150 years ago Virchow published his thoughts on the pathogenesis of deep vein thrombosis (DVT), centered on the triad of stasis, changes in the vessel wall and hypercoagulability [1]. To date, it is well established that DVT most frequently occurs on the surface of activated but largely intact endothelium [2]. It was demonstrated that particularly the valve pockets of the large deep veins of the lower limbs are prone to thrombosis, due to irregular patterns of blood flow and hypoxia-induced endothelial activation [3]. Increasing evidence also indicates a central role of prothrombotic changes in the blood, referred to as thrombophilia, in the pathogenesis of unprovoked DVT [4]. However, most of the to date established contributors to thrombophilia, like the presence of heterozygous factor V Leiden- or prothrombin mutation, have been shown to increase the propensity of developing venous thromboembolism (VTE) only moderately [5].

Tissue factor (TF) is a transmembrane receptor and the main in-vivo initiator of blood coagulation. It was hypothesized that DVT is triggered by increased amounts of active circulating TF, which is bound to small procoagulant membrane vesicles, so-called microparticles (MPs). Indeed, elevated levels of MP-associated TF activity were found in different prothrombotic conditions with significant activation of monocytes like cancer-related VTE [6], [7], [8], disseminated intravascular coagulation [9], [10] and sickle cell disease [11]. Since MPs are known to fuse with activated platelets and neutrophils, TF-bearing MPs may also contribute to diseases like antiphospholipid syndrome [12] and the formation of neutrophil extracellular traps [13].

Data on the course of plasma MP-TF activity after acute unprovoked DVT are missing, so far. We therefore sought to determine MP-TF activity levels at the time of diagnosis and at four different time points in the following year in a well- defined group of patients with unprovoked DVT of the lower limb. We also investigated whether MP-TF activity levels differ between patients with acute proximal- or distal DVT, and between those with- or without residual DVT after 6 months. In addition, we determined TF antigen levels, and investigated whether they correlate with MP-TF activity levels.

Section snippets

Study population

In this prospective longitudinal study 41 patients with acute unilateral symptomatic DVT of the lower limb were included and followed for 1 year. All patients suffered acute unprovoked DVT, which was defined as thrombosis without a triggering event such as surgery, trauma causing immobilization, pregnancy, delivery, or malignancy. All patients were diagnosed at the outpatient department of the Division of Angiology of the Medical University of Vienna, Austria. DVT was confirmed in all patients

Patients’ characteristics

The median age of 41 patients with acute unprovoked DVT of the lower limb was 51 years (25th-75th percentile: 39-64 years). Twenty-one (51.2%) patients were females. The median BMI was 29 kg/m² (25th-75th percentile: 25-31 kg/m²) and 11 (27%) patients were active smokers. Four (9.8%) patients had ileofemoral-, 17 (41.5%) had femoropoliteal- and 20 (48.7%) had distal DVT. Seven (17.1%) patients had pulmonary embolism (PE) in addition to DVT (Table 1).

MP-TF activity and unprovoked DVT

The median MP-TF activity was 0.06 pg/mL

Discussion

In this longitudinal study we determined MP-TF activity levels in a well-defined group of patients with unprovoked DVT of the lower limb at the acute event and at four additional time points during the following year. MP-TF activity was neither elevated during acute DVT nor at any of the four following measurement time points. In subgroup analyses MP-TF activity was not significantly higher in patients with proximal DVT or residual DVT after 6 months compared to those without. MP-TF activity did

Addendum

Contribution: T. Gremmel, J. Thaler, C. Ay, R. Koppensteiner, I. Pabinger were responsible for study concept and design; J. Thaler and T. Gremmel acquired the data; J. Thaler, T. Gremmel, C. Ay, R. Koppensteiner, I. Pabinger analyzed and interpreted the data; J. Thaler and C. Ay drafted the manuscript; T. Gremmel, R. Koppensteiner, I. Pabinger reviewed the manuscript for important intellectual content; J. Thaler provided statistical analysis; R. Koppensteiner and I. Pabinger provided

Funding Sources

This study was supported by a grant from the Austrian Science Fund (FWF): Special Research Program "Inflammation and Thrombosis", SFB-F54.

Disclosure of conflict of interests

The authors declare no competing financial interests.

References (31)

M.E. Tesselaar et al.
Microparticle-associated tissue factor activity: a link between cancer and thrombosis?
J Thromb Haemost
(2007)
D.A. Manly et al.
Increased microparticle tissue factor activity in cancer patients with Venous Thromboembolism
Thromb Res
(2010)
M.E. Tesselaar et al.
Microparticle-associated tissue factor activity in cancer patients with and without thrombosis
J Thromb Haemost
(2009)
J. Thaler et al.
Clinical evidence for a link between microparticle-associated tissue factor activity and overt disseminated intravascular coagulation in patients with acute myelocytic leukemia
Thromb Res
(2014)
N.S. Key et al.
Whole blood tissue factor procoagulant activity is elevated in patients with sickle cell disease
Blood
(1998)
A.A. Khorana et al.
Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer
J Thromb Haemost
(2008)
S. Butenas et al.
Tissue factor activity in whole blood
Blood
(2005)
C. Ay et al.
Circulating procoagulant microparticles in patients with venous thromboembolism
Thromb Res
(2009)
P. Garcia Rodriguez et al.
Plasma levels of microparticle-associated tissue factor activity in patients with clinically suspected pulmonary embolism
Thromb Res
(2010)
J. Thaler et al.
Microparticle-associated tissue factor activity, venous thromboembolism and mortality in pancreatic, gastric, colorectal and brain cancer patients
J Thromb Haemost
(2012)

J. Thaler et al.

Microparticle-associated tissue factor activity in patients with metastatic pancreatic cancer and its effect on fibrin clot formation

Transl Res

(2014)

R.D. Lee et al.

Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity

Thromb Res

(2012)

Y. Yuana et al.

Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles

J Thromb Haemost

(2010)

T.W. Wakefield et al.

Mechanisms of venous thrombosis and resolution

Arterioscler Thromb Vasc Biol

(2008)

N. Mackman

New insights into the mechanisms of venous thrombosis

J Clin Invest

(2012)

Cited by (19)

Circulating levels of tissue factor and the risk of thrombosis associated with antiphospholipid syndrome
2018, Thrombosis Research
Citation Excerpt :
Despite TF having been associated with thrombosis related to many chronic diseases, the role of TF in unprovoked VTE has not been confirmed. A recent clinical trial showed that patients with unprovoked VTE presented similar levels of circulating TF as healthy controls [24]. Our results point to the same direction of a lack of association between circulating TF and unprovoked VTE, suggesting that the profile of hypercoagulability in APS-related thrombosis is different from that observed in unprovoked VTE, which could ultimately explain the clinical differences between these thrombotic diseases.
The mechanisms behind the severe hypercoagulable state in antiphospholipid syndrome (APS) have not yet been fully elucidated. Knowledge on the etiology of thrombosis in APS is needed to improve treatment. We performed a case control study to evaluate the association of the levels of circulating tissue factor (TF) with thrombotic APS and unprovoked venous thromboembolism (VTE), as compared with controls without a history of thrombosis. Study participants were selected in the same geographic area. Linear regression was used to evaluate possible determinants of TF levels among controls and logistic regression was used to evaluate the association between TF, unprovoked VTE and t-APS. TF levels were grouped into three categories based on: below 50th percentile [reference], between 50-75th percentiles [second category] and 75th percentile [third category]. Two hundred and eighty participants were included in the study; 51 patients with unprovoked VTE, 111 patients with t-APS and 118 control individuals. The levels of TF were not associated with an increased risk of unprovoked VTE, as compared with controls. The adjusted odds ratio for t-APS was 2.62 (95%CI 1.03 to 6.62) with TF levels between 50-75th percentiles and 8.62 (95%CI 3.76 to 19.80) with TF levels above the 75th percentile, as compared with the reference category (below the 50th percentile). In the subgroup analysis, higher levels of TF were associated with both arterial and venous thrombosis in APS and with both primary and secondary APS. Circulating TF is associated with thrombotic complications related to APS, but not with the risk of unprovoked VTE.
Deferiprone inhibits iron overload-induced tissue factor bearing endothelial microparticle generation by inhibition oxidative stress induced mitochondrial injury, and apoptosis
2018, Toxicology and Applied Pharmacology
Citation Excerpt :
TF activates factor VII and then triggers the initiation of the intrinsic coagulation pathway, leading to thrombus formation. TF-bearing MPs are extremely procoagulant and elevated MPs within blood circulation are associated with increased risk of thrombosis (Thaler et al., 2014). In this study, we aim to: 1) explore the possible underlying mechanisms of iron-induced EMP release; 2) investigate the contents of iron overload-induced EMPs, and 3) find the protective chelator with the most potential against iron-induced oxidative stress, EMP generation and apoptosis of endothelial cells.
Iron overload-induced cardiovascular toxicity is one of the most common causes of morbidity and mortality in beta-thalassemia major patients. We have previously shown that iron overload-induced systemic arterial changes characterized by endothelial dysfunction are associated with increased endothelial microparticle (EMP) release. In this study, we further demonstrate how EMP release is associated with iron-induced mitochondrial injury and apoptosis of endothelial cells. Iron increased the production of reactive oxygen species (ROS) and calcium influx into mitochondria [Ca^2 +]_m. Iron also disturbed mitochondrial respiration function and eventually led to loss of mitochondrial membrane potential (ΔΨ_m). A significant increase in apoptotic cells and EMPs were found under iron treatment. EMPs contained tissue factor (TF), which has potential clinical impact on thromboembolic phenomenon. Then, we investigated the salvaging effect of deferiprone (L1) on endothelial cell damage and EMP release. We found that L1 could inhibit iron-induced ROS generation, and decrease mitochondrial damage with the resultant effect of less endothelial cell apoptosis and EMP release. L1 could protect endothelial cells from iron-induced toxic effects and minimize EMP release, which could be potentially helpful in a subgroup of thalassemia patients who have increased thromboembolic complications.
Using the laboratory to predict thrombosis in dogs: An achievable goal?
2016, Veterinary Journal
Citation Excerpt :
In a retrospective case–control study, elevated MP levels were an independent risk factor for unprovoked VTE (Bucciarelli et al., 2012). However, MP-TF activity is not consistently elevated, even in patients with existing VTE compared with healthy controls (Thaler et al., 2014), potentially reflecting a greater relevance of TF-MPs to the pathogenesis of cancer-associated VTE rather than unprovoked VTE (Zwicker et al., 2009). Only a few groups have assessed MPs in canine thrombotic disease.
Thrombosis is a major cause of mortality and morbidity in humans and dogs; however, anti-thrombotic drugs carry a risk of bleeding and increase the cost of patient care. The ability to identify individuals at high risk of thrombosis would allow targeting of anti-coagulant therapy at those most likely to derive a net benefit. Significant advances have been made towards predicting thrombotic risk in humans using laboratory tests individually and as part of risk prediction models. Assays that have shown potential in humans include D-dimers, activated partial thromboplastin time and viscoelastic testing, all of which are available to veterinarians. This review discusses the extent to which these assays are likely to predict thrombosis in dogs, and introduces new research techniques which may have future clinical value.
Role of thromboxane-dependent platelet activation in venous thrombosis: Aspirin effects in mouse model
2016, Pharmacological Research
Citation Excerpt :
The increased levels of TXB-M and the higher circulating levels of DNA, as surrogate marker of NETs, have been reported in pathological conditions including VTE and/or DVT [18⿿20]. As regards to TF, its relevance in venous thrombus formation is mostly accepted [6,8], but contradicting results have been reported concerning its levels in DVT [21⿿24]. Our data are in agreement with Ramacciotti et al. [25] that stated an increased TF activity on MPs increased in mice that developed venous thrombi after IVC ligation, suggesting that MP-TF activity might represent an early circulating marker of venous thrombosis.
Recent trials suggest that Aspirin (ASA) reduces the incidence of venous thromboembolism in human. However, the molecular mechanisms underlying this effect are still unclear. In this study we assessed the effects of ASA in venous thrombosis mouse model induced by inferior vena cava (IVC) ligation and we investigated the mechanisms responsible for this effect.
ASA (3 mg/kg daily for 2 days) treatment decreased the thrombus size, the amounts of tissue factor activity in plasma microvesicles (TF-MP) and the levels of 2,3-dinor Thromboxane B₂ (TXB-M) in urine compared to control mice. Interestingly, the thrombus size positively correlated with both TF-MP activity and TXB-M. In addition, positive correlation was observed between TF-MP activity and TXB-M. A reduced number of neutrophils and monocytes, and of TF-positive cells accompanied to a lower amount of fibrin and neutrophil extracellular traps (NETs) were also found in thrombi of ASA-treated mice. Similar results were obtained when mice were treated 24 h before IVC ligation with SQ29548 (1 mg/kg), a selective thromboxane receptor antagonist. In addition, transfusion of platelets in SQ29548 treated-mice excluded the likelihood of a redundant role of platelet-TP receptor in this context.
Finally, incubation of macrophages and neutrophils with SQ29548 prevented TF activity and/or NETs formation induced by supernatant of activated platelets or by IBOP, a selective thromboxane analogue.
In conclusion, ASA, suppressing TXA₂, prevents macrophages and neutrophils activation and markedly reduces thrombus size with a mechanism most likely dependent of the inhibition of TF activity and NETs formation. These results provide a new link between platelet-produced thromboxane and the occurrence of venous thrombosis.
Measurement of microparticle tissue factor activity in clinical samples: A summary of two tissue factor-dependent FXa generation assays
2016, Thrombosis Research
Citation Excerpt :
This has been discussed extensively in a recent review [12]. For example, there are several centrifugation conditions reported for preparing platelet-poor-plasma (PPP) [15,16,19–26]. Other studies used various conditions to prepare platelet-free-plasma (PFP) [17,27–30].
Thrombosis is a leading cause of morbidity and mortality. Detection of a prothrombotic state using biomarkers would be of great benefit to identify patients at risk of thrombosis that would benefit from thromboprophylaxis. Tissue factor (TF) is a highly procoagulant protein that under normal conditions is not present in the blood. However, increased levels of TF in the blood in the form of microparticles (MPs) (also called extracellular vesicles) are observed under various pathological conditions. In this review, we will discuss studies that have measured MP-TF activity in a variety of diseases using two similar FXa generation assay. One of the most robust signals for MP-TF activity (16–26 fold higher than healthy controls) is observed in pancreatic cancer patients with venous thromboembolism. In this case, the TF + MPs appear to be derived from the cancer cells. Surprisingly, cirrhosis and acute liver injury are associated with 17-fold and 38-fold increases in MP-TF activity, respectively. Based on mouse models, we speculate that the TF + MPs are derived from hepatocytes. More modest increases are observed in patients with urinary tract infections (6-fold) and in a human endotoxemia model (9-fold) where monocytes are the likely source of the TF + MPs. Finally, there is no increase in MP-TF activity in the majority of cardiovascular disease patients. These studies indicate that MP-TF activity may be a useful biomarker to identify patients with particular diseases that have an increased risk of thrombosis.
Extracellular vesicles in venous thromboembolism and pulmonary hypertension
2023, Journal of Nanobiotechnology

View all citing articles on Scopus

¹: Thomas Gremmel and Cihan Ay equally contributed to senior authorship.

View full text

Regular articleMicroparticle-associated tissue factor activity in patients with acute unprovoked deep vein thrombosis and during the course of one year

Highlights

Abstract

Background

Objective

Patients/Methods

Results

Conclusions

Introduction

Section snippets

Study population

Patients’ characteristics

MP-TF activity and unprovoked DVT

Discussion

Addendum

Funding Sources

Disclosure of conflict of interests

J Thromb Haemost

Thromb Res

J Thromb Haemost

Thromb Res

Blood

J Thromb Haemost

Blood

Thromb Res

Thromb Res

J Thromb Haemost

Transl Res

Thromb Res

J Thromb Haemost

Mechanisms of venous thrombosis and resolution

Arterioscler Thromb Vasc Biol

New insights into the mechanisms of venous thrombosis

J Clin Invest

Regular article
Microparticle-associated tissue factor activity in patients with acute unprovoked deep vein thrombosis and during the course of one year