Circulating tissue factor positive microparticles in patients with acute recurrent deep
Runyi Yea,1, Caisheng Yea,1, Yongbo Huangb, Longshan Liuc, Shenming Wanga,⁎
aDepartment of Vascular Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
bZhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
cDepartment of Surgical Laboratory, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
a b s t r a c ta r t i c l ei n f o
Received 15 July 2011
Received in revised form 24 September 2011
Accepted 17 October 2011
Available online 17 November 2011
Deep venous thrombosis
Introduction: Circulating tissue factor positive microparticles (MPTF) were reported in a wide range of dis-
eases with thrombotic tendency. Though D-dimer assay had a high negative predictive value for deep venous
thrombosis (DVT) recurrence, there are currently no reliable positive predictors for recurrent DVT. We there-
fore quantified MPTF in patients with acute recurrent DVT to determine whether MPTF levels could be used
to predict recurrent DVT.
MaterialsandMethods:Microparticles (MPs) were isolated from plasma of initial DVT patients (n=25), recurrent
DVT patients (n=25) and sex- and age-matched healthy individuals (n=25), stained with annexin V, cell-
specific monoclonal antibodies (MoAbs) and a MoAb directed against tissue factor (TF), and analyzed by flow
cytometry. We also determined the plasma procoagulant activity with a Human TF Chromogenic Activity Assay
Results: We found total MPTF to be elevated in recurrent DVT patients versus normal individuals (P=0.001). The
number of monocyte-derived MPTF in both initial and recurrent DVT was higher than in normal individuals
(Pb0.01, respectively). The platelet and endothelial cell derived MPTF in recurrent DVT were significantly in-
creased relative to other MPTF (Pb0.05), although there was no difference between initial DVT patients and nor-
to normal individuals, and a positive correlation with MPTF.
Conclusions: The elevated MPTF could be a potentially predictor for DVT recurrence. Further studies are needed
to validate its sensitivity and specificity.
© 2011 Elsevier Ltd. All rights reserved.
Deep venous thrombosis (DVT) of the lower limbs is a common
disease with serious complications, including pulmonary thrombo-
embolism and post-thrombotic syndrome (PTS). After stopping anti-
coagulant treatment, individuals who have had a first episode of
DVT are at an increased risk of new events. In patients who received
standard anticoagulation for 3 to 6 months after an incident event,
the cumulative rate of recurrence is 21% to 23% at 4 to 5 years, and
as high as 40% at 10 years after diagnosis [1,2]. About 5% of recurrent
episodes are fatal, recurrent DVT markedly increases the risk of PTS
and recurrent pulmonary embolism . Although oral anticoagulant
treatment is highly effective, its use is hampered by its drawbacks
such as frequent laboratory monitoring, dose adjustment and an in-
creased risk of major hemorrhage. Consequently, identification of
risk factors for recurrent DVT is important and could help to decide
whether to stop or to continue anticoagulant therapy. Risk factors as-
sociated with increased recurrence were idiopathic or unprovoked
DVT, proximal DVT, cancer, male gender, oral contraceptive use, and
shorter duration of anticoagulation .
The objective test of D-dimer assay after vitamin K antagonist dis-
continuation had a high negative predictive value for DVT recurrence
and could be used to guide the optimal duration of anticoagulation for
patients with a first unprovoked DVT . However, there are currently
no reliable positive predictors for recurrent DVT. The discovery of vari-
ous tissue factor positive microparticles (MPTF) and their procoagulant
activity, makes them a potentially predictor alternative.
Giesen and colleagues  first demonstrated the existence of func-
tionally intact microparticles (MPs) containing tissue factor in the
blood of normal individuals. Tissue factor (TF) is an important initiator
of the extrinsic pathway of blood coagulation, and can be detected in
in the form of MPs, which are small membrane fragments released from
Thrombosis Research 130 (2012) 253–258
Abbreviations: DVT, Deep venous thrombosis; PTS, post-thrombotic syndrome;
MPs, microparticles; MPTF, tissue factor positive microparticles; TF, tissue factor; PFP,
platelet-free plasma; MoAbs, monoclonal antibodies.
of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, China.
Fax: +86 20 87335856.
E-mail address: email@example.com (S. Wang).
1These two authors contributed equally to this paper.
0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
Contents lists available at SciVerse ScienceDirect
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size, with dimensions of 0.1–1 μm, and with a composition dependent
upon the stimulus and cellular origin . MPTF may originate from leu-
kocytes, endothelial cells, platelets or erythrocytes , and thepropor-
tion of different cellular origins may correlate with the underlying
disease. Cardiovascular disease, diabetes, cancer, sickle cell anemia,
gulability may be due in part to elevated levels of circulating MPTF,
predict recurrent DVT.
Materials and methods
The ethics committee of the First Affiliated Hospital of Sun Yat-sen
University approved the study, and written informed consent was
obtained from all subjects. A test sample including 25 patients with
acute initial DVT (group A) and 25 patients with recurrent DVT
(group B) of the lower limbs were compared with 25 healthy individ-
uals (group C) over the same time period from January 2009 to Janu-
ary 2011. We selected acute DVT patients confirmed by Doppler
ultrasound and that had not received any anticoagulant or thrombo-
lytic therapy before admission. The criterion for initial DVT was that
venous thrombosis occurred in the absence of an antecedent major
clinical risk factor such as surgery, trauma, active cancer, immobility,
pregnancy or the puerperium of a thrombophilic blood abnormality.
Recurrent DVT was defined as another episode of venous thrombosis
after a first episode of unprovoked DVT with standard anticoagulation
for 3 to 6 months and the withdrawal of oral anticoagulants at least
for 3 months. Healthy individuals were unrelated to patients and
were also not related to each other. They were spouses or acquain-
tances of patients, hospital staff, or friends or relatives of hospital
staff. The control group was matched for sex and age to the patient
groups. We excluded subjects with any of the clinical risk factors
mentioned above or a family history of thrombosis. We recorded de-
mographic data and past medical history for each subject, and Dopp-
ler ultrasound results and the clinical state for DVT patients. Blood
count and D-dimer tests were completed before plasma specimen
collection. All the DVT patients also have thrombophilia factors tests
including protein C and protein S.
Reagents and assays
MPs and MPTF were detected by flow cytometry using cell-specific
monoclonal antibodies (MoAbs) . The MoAbs stained for monocytes
(CD14), platelets (CD41a), endothelial cells (CD144), and erythrocytes
(CD235a). Isotype control antibodies, respectively, were Mouse IgG2a, κ,
reagents were labeled with Phycoerythrin (PE) and obtained from BD-
ine (PS) was also obtained from BD-Pharmingen. Fluorescein isothiocya-
nate (FITC)–labeled anti-human Tissue Factor MoAb (4507CJ) was
obtained from American Diagnostica Inc (Greenwich, CT), and isotype
control mouse immunoglobulin G1 (IgG1), labeled with carboxyfluores-
cein(CFS) (11711) was obtained from R&D Systems (Minneapolis, MN).
Fluorescent microbeads of 0.1 μm (S37204) and 1 μm (S37498)
were used for gating experiments and 7 μm nonfluorescent beads
for enumeration of MPs were obtained from Molecular Probes.
The AssaySense Human Tissue Factor Chromogenic Activity Assay
Kit (CT1002b, AssayPro) was used to determine plasma Tissue Factor
TF/FVIIa to convert factor X (FX) to factor Xa. The amidolytic activity of
the TF/FVIIa complex is quantitated by the amount of FXa produced
using a highly specific FXa substrate, which releases a yellow para-
nitroaniline (pNA) chromophore. The change in absorbance of pNA at
405 nm is directly proportional to TF enzymatic activity.
Specimen collection and preliminary processing
Blood samples were drawn into buffered citrate using a 21-gauge
in 30 minutes using a 2-step centrifugation procedure (1500 g for
10 minutes at 20 °C to make platelet-rich plasma, followed by 13000 g
for 10 minutes at 20 °C). The PFP was immediately stored at −80 °C.
Preparation of microparticles
MPs were isolated as previously reported . Briefly, wash buffer
(10 mMHEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic
acid) , 140 mM NaCl, 4.5 mM KCl, 1% bovine serum albumin [BSA],
0.1% Na azide, 2.5 mM CaCl2 , pH 7.4) was filtered using a 0.2 μm filter
before washing the PFP. Washed PFP was ultracentrifuged at
100,000 g for 120 minutes at 20 °C. Finally, MPs were resuspended
in the remainder wash buffer by gentle vortexing and pipetting, and
used immediately for flow cytometry.
Flow cytometric detection of microparticles
We used a modification of standard methods  to detect the mi-
croparticles with a FACS Calibur flow cytometer, and CellQuest pro
software (Becton Dickinson). MPs were quantified by spiking a
known quantity of 7 μm beads (23500 beads/25 μl) into each sample.
Acquisition was stopped after 5000 beads and counted in R1 or the
beads gate (Fig. 1-A). We defined MPs as the events falling in the R2
or MPs gate (based on size, Fig. 1-A) and the annexin V-positive
gate (Fig. 1-B). To determine the cellular origin of MPTF, samples
were triple labeled with Cy5-labeled annexin V, a cell type–specific
PE-labeled MoAb, and a FITC-labeled MAb against TF (Fig. 1-C). Positive
triple labeled events were defined as cellular MPTF. Finally, total MPs
and MPTF (per milliliter PFP) was calculated as reported  with the
actual dilution factor.
Procoagulant activity of PFP assay
We detected PFP procoagulant activity on the basis of the Assay-
Sense Human TF Chromogenic Activity Assay Kit (CT1002b, Assay-
Pro). PFP and all reagents were brought to room temperature, and
70 μl freshly prepared Assay Mix added (Assay Diluent 50 μl, FVII
10 μl, FX 10 μl) to each well of the 96-well plate. To this was added
10 μl of TF standards or samples per well with gentle mixing before
incubating at 37 °C for 30 minutes. Next, 20 μl of FXa substrate was
added to each well and absorbance at 405 nm by the Multifunction
Microplate Reader (BioTek Instruments, USA) was recorded every
2 minutes for 12 minutes. Finally, a standard curve was generated
by regression analysis using 4-parameters, from which the unknown
sample concentration was determined.
Summary statistics were expressed as medians and Inter-Quartile
Ranges (IQR) since the results were not normally distributed. The me-
dian value is the 50th percentile. The 25th and 75th percentiles of the
data specify the values covered by the IQR. The Kruskal-Wallis test
was used for an overall comparison between the groups. Significance
determined by the Kruskal-Wallis test enabled subsequent intergroup
comparisons using the Mann–Whitney U test; Pb0.05 (2-tailed) was
considered significant. Bivariate correlations were determined using
Spearman's Rho test. Statistical analysis was performed using SPSS ver-
sion 16.0 for Windows (Chicago, IL, USA).
R. Ye et al. / Thrombosis Research 130 (2012) 253–258
Basic characteristics of the subjects
Demographic, thrombosis clinical data, and medical test results
are shown in Table 1. There were no significant statistical differences
with respect to sex, average age, body mass index (BMI) and blood
count, and no differences in the locations of the thrombosis between
the initial DVT and recurrent DVT group. In the recurrent DVT group,
the first thrombosis occurred in the ipsilateral limbs in 13 of 25 cases.
D-dimer was elevated in DVT groups relative to controls. The heredi-
tary thrombophilia factors including protein C and protein S were all
in the normal range.
Total MPs were elevated among recurrent DVT patients
Enumeration revealed normal individuals (group C) to have total
MPs of 223.6 (182.3-299.4)×103/ml PFP [Median (IRQ)]. In compari-
son, total MPs were elevated for patients with DVT in group A [256.2
(217.5-330.1)×103/ml] and in group B [340.1 (205.7-450.1)×103/
ml]. Overall, the group showed a significant difference (P=0.025,
Table 2), and pairwise comparisons indicated recurrent DVT patients
(group B) had significantly elevated total MPs (P=0.012, versus nor-
mal individuals, Fig. 2-A). However, there was no difference in group
A vs. group B or group A vs. group C (P=0.054 and P=0.327
Number and cell origin of MPs
The levels of MPs from different cells are shown in Table 2. We
found no difference in erythrocyte origin (P=0.197) or in platlet or-
igin (P=0.062), but a significant difference in monocyte cell origin
(P=0.003, group B vs. group C, Fig. 3-A) and an higher level of endo-
thelial cell MPs in recurrent DVT patients than initial DVT patients
and normal individuals (Fig. 3-A).
Total MPTF were elevated among recurrent DVT patients
Normal individuals (group C) had total MPTF of 36.2 (19.7-
47.3)×103/ml PFP (Median (IRQ)). Total MPTF were elevated for pa-
tients with DVT in group A [36.1 (31.1-73.1)×103/ml] and group B
[97.7(34.2-116.5)×103/ml]. The overall group showed a significant
difference (P=0.001, Table 2), and pairwise comparisons showed re-
current DVT patients (group B) had a significantly elevated total
MPTF (P=0.008, and P=0.001, versus group A and group C respec-
tively, Fig. 2-B). However, there was no difference in group A vs.
group C (P=0.114).
Number and cell origin of MPTF
The levels of MPTF from different cells from four different sources
are shown (Table 2, Fig. 3-B). Monocyte derived MPTF were signifi-
cantly elevated in recurrent DVT and initial DVT patients versus
healthy controls, and a higher level for group B than group A. Platelet
and endothelial cell derived MPTF from group B were significantly in-
creased versus group A and group C, with no difference between
group A and group C. Erythrocyte derived MPTF were the same levels
in three groups.
Procoagulant activity of PFP in normal individuals and DVT patients
PFP procoagulant activity was estimated by measuring TF sample
concentration. The median procoagulant activity of normal individ-
uals was 45.95 pM (40.05-82.35 pM), but was significantly elevated
in group A [93.4 pM (82.25-99.98 pM)], and group B [99.35 pM
(77.73-113.9 pM)]. There was no difference between group A and
group B. (Fig. 4).
Fig. 1. Flow cytometric analysis and quantification of MPs. (A) Bead size is reflected by forward scatter (FSC) and side scatter (SSC). The R1 or bead gate included 7-μm beads for
enumerating MPs. The R2 or MP gate included 1.0 μm beads shown in the upper right corner, and contains all microparticles of 1.0 μm or less. (B) The R3 gate shows phosphati-
dylserine (PS)–positive MPs in PFP by Annexin V–Cy5 labeling on the y-axis, in relation to SSC on the x-axis. (C) The cellular origin of MPTF are shown in the upper right quadrant.
Shown is representative triple labeled sample for annexin V (not shown), TF-FITC on the x-axis, and CD144- PE (endothelial cells) on the y-axis.
Demographic and clinic data of the study subjects.
GROUP A GROUP BGROUP C P*
Smoking status, n(%)
FIB, g/L, mean±SD
Protein C deficiency
Protein S deficiency
* N.S., not statistically significant (pb0.05).
BMI , body mass index. FIB, Fibrinogen. SD, standard deviation.
R. Ye et al. / Thrombosis Research 130 (2012) 253–258
Procoagulant activity of PFP and MPs
The bivariate correlations between the procoagulant activity of
PFP and MPs are shown in Table 3. The data indicated that MPTF
was positively correlated with PFP procoagulant activity.
Recent evidence indicates that MPs formation is a highly organized
process, leading to the shedding of distinct domains of the cell
membrane . MPs behave as vectors of bioactive molecules able
to disseminate biological information in the vascular compartment.
MPs contribute to hemostatic and inflammatory responses, vascular
remodeling and angiogenesis, cancer, and apoptosis, well-known
processes involved in atherothrombosis [16–18]. Due to the expres-
sion of both membrane phosphatidylserine, a procoagulant phos-
pholipid necessary for the assembly of the blood clotting enzyme
complex, and functional tissue factor, the major initiator of the co-
agulation cascade, MPs are catalytic procoagulant surfaces involved
in thrombogenesis [8,19]. Decreased numbers of MPs are associated
with bleeding syndromes, such as Scott Syndrome  and Casta-
man Syndrome . However, increased numbers of circulating MPs
cy, including acute myocardial infarction, antiphospholipid syndrome,
preeclampsia, rheumatoid arthritis, thrombotic thrombocytopenic pur-
pura, vasculitis, heparin-induced thrombocytopenia (HIT), and parox-
ysmal nocturnal hemoglobinuria (PNH) [22,23].
In our study, both the total number of MPs and the number of TF
positive MPs are increased in the acute recurrent DVT patients rela-
tive to healthy individuals, but no difference between initial DVT
and healthy individuals. Cihan Ay et al. had found no difference of
Flow cytometry results for MPs and MPTF levels among the three study groups.
Cell OriginGroup AGroup BGroup C Z-value*P*
Median(IQR) Median(IQR) Median(IQR)
Results are shown as medians and inter-quartile ranges (IQR). EC: Endothelial cells, RBC: Red Blood cells. * Kruskal-Wallis test.
GROUP A GROUP BGROUP C
GROUP AGROUP B
Fig. 2. Total blood MPs and MPTF. Data are expressed as number per milliliter of
platelet-free plasma and shown as medians and IQR. (A) Total MP. (B) Total MPTF.
Error bars represent IQR. *P=0.001, **P=0.001 compared with group C.
MonocytePlatelets Endothelial RBC
Monocyte PlateletsEndothelial RBC
GROUP A GROUP BGROUP C
Fig. 3. Cell origin of MPs and MPTF. Data are expressed as number per milliliter of PFP
and shown as medians IQR. (A) Cell origin of MPs. (B) Cell origin of MPTF. Error bars
represent IQR. *Pb0.05, compared with group C in Monocytes and Endothelial cell
MPs respectively; **Pb0.01, compared with group C in Monocytes; ΔPb0.05, compared
with other groups in Platelets; #P=0.006 compared with other groups in Endothelial.
R. Ye et al. / Thrombosis Research 130 (2012) 253–258
circulating procoagulant MPs between patients in the chronic stage of
recurrent DVT and healthy controls . This suggests that the MPs
may play a role in the hypercoagulable state of acute recurrent DVT
but not for chronic DVT.
Circulating MPs are derived from blood cells and the vascular wall,
and the cellular origin of elevated MPs is variable based on the dis-
ease. Atherosclerotic plaques contain highly MPTF of mainly mono-
cytic and lymphocytic origin . Lung cancer patients had a higher
concentration of monocyte and platelet derived MPs than normal
subjects , and sickle blood contains elevated numbers of TF posi-
tive MPs derived from endothelial cells and monocytes . In our
study, both MPs and MPTF derived from endothelial cells in recurrent
DVT patients were elevated relative to other group, but no difference
between normal individuals and initial DVT patients, suggesting acti-
vation or damage of vascular endothelial cells after the first episode of
venous thromboembolism. However, more important is the observa-
tion that both initial and recurrent DVT patients had a higher level of
monocyte derived MPTF than normal individuals. In addition, platelet
derived MPTF from recurrent DVT patients was higher than initial
DVT patients. Taken together, this suggests that monocyte derived
MPTF is the main difference between DVT and non-DVT patients,
and that platelet and endothelial cell derived MPTF are the main dif-
ference between recurrent and inital DVT patients. Some studies have
shown that both monocyte and platelet contained TF mRNA , and
that cancer patients exhibit an increased number of monocyte and
platelet derived MPTF [12,27,28]. Increased monocyte TF activity is
associated with higher risk of acute coronary syndrome  and dis-
seminated intravascular coagulation (DIC) in endotoxemia and sepsis
. Cardiac myocyte-specific overexpression of TF can restore he-
mostasis in the hearts of low TF mice . Thus, monocyte derived
MPTF may play an important role in thrombosis and could be a pre-
dictor for recurrent DVT. Although there is limited prospective
study data demonstrating that MPTF levels can be considered a true
marker of thrombotic risk, an increase in monocyte associated TF ac-
tivity after total knee arthroplasty suggests monocyte MPTF is a po-
tential risk factor for DVT .
Some animal models experiments indicated that circulating MPTF
was an important mediator of thrombotic risk in disease states
[32–34]. Other models explained that MPTF was recruited to a throm-
bus and enhance its growth through the interaction of P-selectin gly-
coprotein ligand 1 (PSGL-1) on the MPs with P-selectin expressed on
the surface of platelets [35,36]. Cancer patients with acute DVT have
higher levels of MPTF activity than normal individuals, cancer pa-
tients without DVT, and subjects with idiopathic DVT . Our data
show elevated TF activity of PFP in DVT patients and a positive corre-
lation with MPTF. So the increased levels of MPTF may indicate an un-
derlying prothrombotic state and levels of MPTF may be a useful
determinant of thrombotic episodes.
Something of our study need to be addressed. We performed our
specimen collection from unprovoked DVT patients and healthy indi-
viduals without medications to avoid any influence of trigger factors
such as malignancy, surgery and pregnancy on circulating MPs, as
these condition are associated with the presumed mechanisms for
MPs generation. We used a MPTF assay from several ways . The
methodological variables that need to be considered and the com-
plexity of our detection methods may limit use of MPTF as a predictor
for recurrent DVT in the clinic.
In summary, we have demonstrated that MPTF levels are signifi-
cantly elevated in recurrent DVT patients from monocyte, platelet
and endothelial cell. We believe that the levels of MPTF could help
to predict the risk of DVT recurrence, and combining the results of
D-dimer we could decide whether or when anticoagulant therapy
can be safely withdrawn. Further studies are needed to validate
their sensitivity and specificity.
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Fig. 4. A box-whisker pot that shows the TF activity (pM) of the PFP from DVT patients
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