Anticoagulant effects of an antidiabetic drug on monocytes in vitro

Introduction

Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties.

Methods

We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry).

Results

Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs.

Conclusions

Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.     

Differential ability of tissue factor antibody clones on detection of tissue factor in blood cells and microparticles

Introduction

Tissue factor (TF), the primary initiator of coagulation in vivo, plays a major role in both thrombosis and hemostasis. The expression of TF in monocytes is well documented, but its presence in other blood cells has been disputed, possibly due to methodological variations among different studies.

Materials and methods

We studied TF expression on platelets, monocytes, lymphocytes and microparticles (MPs) by flow cytometry (FCM) with five commercially available mouse anti-human TF antibodies (HTF-1, TF9-10H10, CLB/TF-5, VIC7 and VD8). The ability of different TF antibodies to inhibit cell surface TF activity was explored by incubating LPS-stimulated monocytes and MPs derived from LPS-stimulated monocytes (MMPs) with TF antibodies followed by measuring TF activity.

Results

HTF-1 detected TF only on LPS-stimulated monocytes, whereas, TF9-10H10 and VD8 detected TF associated with MPs and MMPs in addition to LPS stimulated monocytes. Surprisingly, CLB/TF-5 and VIC7 detected TF on platelets, monocytes even under unstimulated conditions, in addition to MPs and MMPs. CLB/TF-5 also detected TF on unstimulated lymphocytes. Inhibitory studies showed that at a final concentration of 10 μg/mL, HTF-1, CLB/TF-5 and VD8 inhibited monocyte TF activity by 81-84% and MMP TF activity by 92-96%; whereas TF9-10H10 had no inhibitory effect on TF activity in monocytes and MMPs.

Conclusions

Our results suggest non-specific binding by the CLB/TF-5 and VIC7 antibodies in a FCM test system and explain at least some of the reports on TF presence in blood cells, particularly TF associated with platelets and MPs. TF9-10H10 and VD8 are more suitable to detect TF on MPs by FCM.     

Circulating monocytes mirror the imbalance in TF and TFPI expression in carotid atherosclerotic plaques with lipid-rich and calcified morphology

Background

Thrombogenicity of atherosclerotic plaque largely depends on plaque morphology and their content of tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The relationship between morphological composition of plaque (lipid-rich or calcified) and expression of TF and TFPI in circulating blood monocytes and within the plaques is not characterized.

Objective

To investigate whether lipid-rich (echolucent) or calcified (echogenic) morphology of carotid atherosclerotic plaques is associated with differences in TF and TFPI expression in circulating blood monocytes and within carotid atherosclerotic plaques.

Methods

We studied levels of monocyte TF and TFPI mRNA and protein expression and association with traditional risk factors for atherosclerosis in asymptomatic subjects with echolucent (n = 20) or echogenic (n = 20) carotid plaques, or controls without carotid atherosclerosis (n = 20) determined by ultrasonography. Sections of calcified or lipid-rich carotid plaques obtained from symptomatic patients were assessed for TF and TFPI antigen expression.

Results

TF and TFPI surface presentation, surface TF/TFPI ratio, and TF activity were higher in monocytes obtained from subjects with echolucent than with echogenic plaques or controls without carotid atherosclerosis. Multiple regression analyses revealed inverse association between serum apoA1 and monocyte surface TF antigen expression (p = 0.007), and positive association between serum apoB and monocyte surface TFPI expression (p = 0.028). Sections from lipid-rich carotid plaques contained 2.5-fold more TF and 1.5-fold more TFPI antigens relative to calcified lesions, also yielding a higher TF/TFPI ratio.

Conclusions

Our findings indicate that circulating monocytes of asymptomatic individuals with echolucent lipid-rich carotid atherosclerosis express an imbalance between TF and TFPI expression cohering with changes found within advanced carotid atherosclerotic plaques obtained from symptomatic patients.     

Porphyromonas gingivalis infection and prothrombotic effects in human aortic smooth muscle cells

Introduction

Accumulating evidence has demonstrated an association between periodontal infectious agents, such as Porphyromonas gingivalis, and vascular disease. Tissue factor (TF) and its specific tissue factor pathway inhibitor (TFPI) are produced by vascular cells and are important regulators of the coagulation cascade.

Materials and Methods

To assess the role of P. gingivalis in atherothrombosis, we infected primary human aortic smooth muscle cells (HASMC) with either P. gingivalis 381, its non-invasive mutant DPG3, or heat-killed P. gingivalis 381. Levels and activity of TF and TFPI were measured 8 and 24 hours after infection in cell extracts and cell culture supernatants.

Results

P. gingivalis 381 did not affect total TF antigen or TF activity in HASMC, but it significantly suppressed TFPI levels and activity compared to uninfected control cells, and those infected with the non-invasive mutant strain or the heat-killed bacteria. Further, P. gingivalis' LPS (up to a concentration of 5 µg/ml) failed to induce prothrombotic effects in HASMC, suggesting a significant role for the ability of whole viable bacteria to invade this cell type.

Conclusion

These data demonstrate for the first time that infection with a periodontal pathogen induces a prothrombotic response in HASMC.     

Activation of coagulation in amniotic fluid during normal human pregnancy

Introduction

Amniotic fluid (AF) is an important medium for fetal development which exhibits high procoagulant activities; however, the role of these procoagulants during pregnancy has not been elucidated and might be associated with pregnancy complications. The current study aimed to evaluate factor X (FX) activation and its association with tissue factor (TF), tissue factor pathway inhibitor (TFPI) and coagulation activation markers in AF during normal human pregnancy.

Methods

Activation of FX and concentration of TF, free TFPI, D-dimer and prothrombin fragments (F1 + 2) were evaluated in AF samples obtained for chromosome analysis from 91 women with normal pregnancy: 65 samples were taken from patients at 16-20 weeks of gestation, 9 samples were drawn at 21-30 weeks and 17 samples−after 30 weeks of gestation.

Results

Activation of FX in AF significantly increased during normal pregnancy (from 65 ± 41 to 205 ± 80 equivalent RVV ng/mg total protein, P < 0.0001). TF and TFPI levels in AF also rose with gestational age. In contrast, the AF concentration of D-dimer and F1 + 2, markers of coagulation activation significantly decreased when expressed per mg total protein. Levels of free TFPI correlated with TF (r = 0.5, P < 0.001), and were 8-fold higher than those of TF during pregnancy.

Conclusion

High levels of TFPI might be associated with the inhibition of procoagulant activity in amniotic fluid during normal pregnancy, which may account for the rarity of clinical amniotic fluid embolism.     

Anti-phospholipid antibodies contribute to arteriosclerosis in patients with systemic lupus erythematosus through induction of tissue factor expression and cytokine production from peripheral blood mononuclear cells

Introduction

In systemic lupus erythematosus (SLE) patients, the prevalence of arteriosclerosis obliterans (ASO) is high despite a lack of common risk factors for ASO. The main objective of this study was to investigate a possible direct role of anti-phospholipid antibodies (aPLs), which are frequently detected in SLE patients, in the pathogenesis of ASO.

Materials and Methods

We examined tissue factor (TF) expression on the monocyte surface by flow cytometric analysis in 89 SLE patients with or without ASO and/or aPLs and studied the in vitro effect of purified IgG fractions from plasma of SLE patients or normal healthy volunteers (aPLs(+) IgG, n = 8; aPLs(−) IgG, n = 6; Normal IgG, n = 6) on the expression of TF and production of TNF-α and IL-1β in healthy peripheral blood mononuclear cells (PBMCs) or isolated monocytes.

Results

We confirmed that high expression of monocyte TF was strongly associated with the prevalence of ASO and the presence of aPLs. Treatments of PBMCs with aPLs(−) IgG or normal IgG did not significantly increase expression of TF, TNF-α, and IL-1β messenger RNA (mRNA) and the production of TNF-α and IL-1β. However, stimulation of PBMCs with aPLs(+) IgG caused significant increase in expression of TF, TNF-α, and IL-1β mRNA. Moreover, aPLs(+) IgG stimulated PBMCs and significantly enhanced the production of TNF-α and IL-1β.

Conclusion

These results suggest that IgG-aPLs cause persistently high TF expression and inflammatory cytokine production by interacting with peripheral blood monocytes and lymphocytes, which may be an important mechanism in the pathogenesis of ASO peculiar to SLE patients.     

Carbon monoxide (CO)-releasing molecule-derived CO regulates tissue factor and plasminogen activator inhibitor type 1 in human endothelial cells

Introduction

Heme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects.

Materials and Methods

HUVECs were pretreated with 50 μM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively.

Results

Pretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs.

Conclusions

CORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions.     

Increased heparanase level and procoagulant activity in orthopedic surgery patients receiving prophylactic dose of enoxaparin

Background

Orthopedic hip and knee surgeries are followed by a hypercoagulable state. Heparanase is implicated in inflammation, coagulation activation and angiogenesis. Recently, heparanase was shown to directly interact with tissue factor (TF) and to enhance the generation of factor Xa (Nadir et al., Haematologica, 2010). In addition, an assay assessing heparanase procoagulant activity has been lately developed (Nadir et al., Thromb Res, 2011). In the present study heparanase level and procoagulant activity in patients undergoing orthopedic surgery were assessed.

Methods

The study group included 50 orthopedic patients. 31 patients underwent hip surgery and 19 had knee operation. 15 individuals suffered from traumatic hip fractures and 35 had osteoarthrosis of hip or knee joints. All patients received prophylactic dose of enoxaparin starting 6-8 hours post operation and lasting for 5 weeks. Plasma samples were drawn preoperatively and at 1 hour, 1 week and 1 month post operation. Samples were tested for heparanase levels by ELISA and TF/heparanase complex activity, TF activity, heparanase procoagulant activity, factor Xa and thrombin levels using chromogenic substrates.

Results

Heparanase levels were significantly higher 1 hour and 1 week post operatively compared to preoperative levels (p < 0.05, p < 0.005, respectively). The most dramatic changes were observed in heparanase procoagulant activity reaching a 2 fold increase 1 week postoperatively and 1.7 fold increase 1 month after surgery (p < 0.0001, p < 0.0001, respectively). Levels of factor Xa and thrombin did not significantly change.

Conclusions

Heparanase is involved in coagulation activation of orthopedic surgery patients. Heparanase procoagulant activity is highest 1 week postoperatively and remains high 1 month after operation. Considering extending prophylactic anticoagulant therapy or evaluating heparanase procoagulant activity may potentially prevent late thrombotic events.     

Myeloid cell tissue factor does not contribute to venous thrombogenesis in an electrolytic injury model

Introduction

Tissue factor (TF) is a potent initiator of the extrinsic coagulation cascade. The role and source of TF in venous thrombotic disease is not clearly defined. Our study objective was to identify the contribution of myeloid cell TF to venous thrombogenesis in mice.

Materials and methods

The mouse electrolytic inferior vena cava model was used to induce thrombosis. The following groups of mice were used (1) TF^flox/floxLysMCre⁺ mice that have reduced TF expression in myeloid cells, (2) TF^flox/floxLysMCre^- littermate controls, (3) Wild type mice given a monoclonal anti-mouse TF antibody (1H1) to inhibit TF activity, and (4) Wild type mice given rat IgG. Evaluations at baseline, day 2, and day 6 post thrombosis included thrombus weight, vein wall inflammatory cell migration, vein wall TF mRNA, and plasma D-dimer levels.

Results

Inhibition of TF significantly decreased thrombus weight 2 days post venous thrombosis. In contrast, TF^flox/floxLysMCre⁺ had no change in thrombus weight when compared to littermate controls. The absence of myeloid cell TF did not affect infiltration of neutrophils or monocytes into the vein wall. TF mRNA expression in the vein wall decreased at 2 days but then returned to baseline levels by 6 days post thrombosis. D-dimer levels peaked at 2 days post thrombosis in mice with or without myeloid cell TF.

Conclusions

TF is important in the formation of venous thrombi in the macrovasculature. However, TF expression by myeloid cells does not significantly contribute to venous thrombogenesis in this model.     

Microparticles in newborn cord blood: slight elevation after normal delivery

Introduction

Microparticles formed during delivery may add to the well functioning hemostasis, but also to hypercoaguability in the newborn.We wanted to investigate whether microparticles in newborn cord plasma differ from those in adult plasma in terms of concentration, procoagulant activity, and effect on thrombin generation.

Materials and Methods

Three different techniques were used to analyze microparticles. To enumerate and characterize microparticles, flow cytometry and ELISA, based on the prothrombinase reaction, were used. The effect of microparticles derived tissue factor on thrombin generation was measured indirectly by Calibrated Automated Thrombography in newborn cord and adult platelet free plasma.

Results

The flow cytometric measurements of microparticles showed no significantly increased microparticle concentration in newborn cord compared with adult plasma. By the use of ELISA a significantly increased procoagulant activity of microparticles was found in newborn cord plasma as compared to adult plasma. Initiation of thrombin generation by adding phospholipids alone suggested a higher microparticle activity in newborn cord plasma than in adult plasma.

Conclusions

Our results show a higher impact of microparticles on the hemostatic system in newborn cord plasma than in adult plasma in terms of activity, but not concentration. Calibrated Automated Thrombography and ELISA suggest an increased microparticle activity in newborn cord plasma, but comparable results in microparticle number as determined by flow cytometry argue against strong platelet activation during birth.     

Anthracycline treatment of the human monocytic leukemia cell line THP-1 increases phosphatidylserine exposure and tissue factor activity

Introduction

Cancer associated thrombosis is a well-recognized phenomenon that results in considerable patient morbidity and mortality. Malignancy conveys an increased risk for thrombosis and chemotherapy further elevates this risk. The pathophysiological mechanisms underlying this process remain poorly defined.

Materials and Methods

A human acute monocytic leukemia cell line (THP-1) was treated with commonly used anthracycline chemotherapeutics at concentrations similar to those found in the plasma of cancer patients. Cells were analyzed for tissue factor (TF) mRNA, protein, and activity. Microparticle (MP) TF activity was also measured. Phosphatidylserine (PS) exposure on cells and MPs was analyzed by flow cytometry. PS levels on MPs was also evaluated in an annexin V capture assay.

Results

Anthracycline treatment of THP-1 cells resulted in a concentration-dependent increase in cellular TF activity without a change in TF protein, which was associated with increased PS exposure on the cell surface and apoptosis. The increase in TF activity was abolished by annexin V or lactadherin indicating that PS exposure was required. Anthracycline treatment of THP-1 cells also increased the number of TF-positive MPs.

Conclusion

Treatment of THP-1 cells with anthracyclines induces apoptosis and increases cellular TF activity. The increased activity required an increase in exposure of PS. Additionally, anthracyclines increase the release of TF-positive MPs from THP-1 cells. We propose that the increase in cellular TF activity in circulating leukemic cells, combined with increased numbers of TF-positive MPs, may contribute to thrombosis in cancer patients receiving chemotherapy.     

Daunorubicin induces procoagulant response through phosphatidylserine exposure in red blood cells

Introduction

Cytotoxic chemotherapy induces or worsens hemostatic disorders in patients with acute myeloid leukemia. Procoagulant release and tissue factor expression on tumor cells are considered to contribute to chemotherapeutic agents-associated coagulopathy. The role of red blood cells during this process has not been determined; although they lack tissue factor, they may contribute suitable membranes for the amplification phase of blood coagulation. The present study aims to evaluate the possible impact of daunorubicin on phosphatidylserine exposure and consequent procoagulant property of red blood cells.

Materials and methods

Red blood cells from acute myeloid leukemia patients and healthy donors were treated with daunorubicin as well as all-trans-retinoic acid, arsenic trioxide or etoposide for 0-48 h. Procoagulant activity was assessed by measurement of a clotting time and by purified coagulation complex assays. Lactadherin was used as a probe for phosphatidylserine.

Results

Daunorubicin treatment increased procoagulant activity of red blood cells regardless of the cell origin. Moreover, coagulation complexes assays supported daunorubicin-evoked procoagulant response. The procoagulant property of red blood cells was not affected by the other three agents. The modulating effect of procoagulant activity was concomitant with and dependent on level of phosphatidylserine on the outer surface. Blockade of phosphatidylserine with lactadherin inhibited over 90% of tenase generation and prothrombinase activity and prolonged the coagulation time.

Conclusions

We conclude that daunorubicin interacts with red blood cells in a manner that increases phosphatidylserine exposure and consequent procoagulant activity. Lactadherin is an efficient anticoagulant of this process.     

An assay to evaluate heparanase procoagulant activity

Background

Heparanase that was cloned from and is abundant in the placenta is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is directly involved in the regulation of the hemostatic system. Heparanase was shown to form a complex and enhance tissue factor (TF) activity, resulting in increased factor Xa production (Nadir et al. Haematologica, 2010). The present work suggests a novel assay to evaluate heparanase procoagulant activity.

Methods

Heparanase procoagulant activity was studied using purified proteins of heparanase, TF, factor VIIa and factor X. The assay was verified in 55 plasma samples and compared to heparanase and tissue factor pathway inhibitor (TFPI) levels by ELISA and factor Xa, thrombin levels and antithrombin activity by chromogenic substrates. Thirty five samples were of third-trimester pregnant women (weeks 36–41) who were in labor or came for appointed elective cesarean section and 20 control samples were of non-pregnant healthy women.

Results

heparanase procoagulant activity assay was shown to differentiate heparanase procoagulant effect from TF activity, in purified proteins. Heparanase procoagulant activity was significantly higher in the plasma of pregnant women compared to non-pregnant (p < 0.005). Heparanase relative contribution to the TF / heparanase complex activity was significantly higher in the plasma of pregnant women compared to non-pregnant (29% increase, p < 0.0001). Differences in heparanase procoagulant activity were more prominent than changes in heparanase levels by ELISA, TF activity, factor Xa, thrombin and free TFPI levels.

Conclusions

Heparanase procoagulant activity can be determined by the suggested assay. The assay revealed a significant contribution of heparanase to the procoagulant state in late third-trimester pregnancy and at delivery.     

The relationship between plasma and placental tissue factor, and tissue factor pathway inhibitors in severe pre-eclampsia patients

Objective

To investigate the mechanism underlying the hypercoagulable state in severe pre-eclampsia.

Methods

Plasma tissue factor (TF) and tissue factor pathway inhibitor (TFPI) expression from pre-eclampsia patients and healthy pregnant controls were determined by ELISA. Placental TF and TFPI gene and protein expression were detected by quantitative RT-PCR, immunohistochemistry, and Western analysis.

Results

The plasma TF level in the pre-eclampsia group was significantly higher than the control group (p < 0.01), and surprisingly, the plasma TFPI-1 and TFPI-2 in the pre-eclampsia group were significantly lower (p < 0.01). Placental TF gene and protein expression levels in the pre-eclampsia group was significantly higher than the control group, while TFPI-1 and TFPI-2 levels were significantly lower (p < 0.05). Lastly, a significant correlation was found between plasma and placental TF protein levels in the pre-eclampsia group (p < 0.01).

Conclusion

Higher expression and/or release of TF from the placenta may contribute towards a pathological hypercoagulable state in pre-eclampsia patients.     

The Influence of Direct Thrombin Inhibitors on the Formation of Platelet-leukocyte Aggregates and Tissue Factor Expression

Introduction

High concentrations of platelet-monocyte aggregates (PMAs) have been found in patients with myocardial infarction (MI). Oral direct thrombin inhibitors (DTIs) are under evaluation as long-term antithrombotic treatment. The aim was to evaluate whether DTIs affect the formation of platelet-leukocyte aggregates, TF expression and procoagulant microparticles (MPs).

Material and Methods

DTIs were added to an experimental whole blood model before platelet activation with thrombin or ADP. The concentrations of PMAs, platelet-granulocyte aggregates (PGAs), the amount of platelets bound per leukocyte and MPs were investigated by flow cytometry. TF mRNA and activity were recorded in all settings. TF activity was evaluated in a MI population treated with or without an oral DTI.

Results

In vitro, thrombin and ADP increased the formation of PMAs and PGAs as well as TF mRNA expression. DTIs reduced the amount platelets bound to monocytes (p = 0.02) and to granulocytes (p = 0.001) upon thrombin stimulation together with a reduction of TF mRNA. In contrast, the ADP-induced formation of PMAs, PGAs and TF mRNA was not affected by the DTIs. Both thrombin and ADP stimulation increased the amount of TF-expressing MPs, which was effectively inhibited by the DTIs (p = 0.02-0.002). In the MI population, the DTI reduced the TF activity (p < 0.001).

Conclusion

DTIs modulate the formation of PMAs, PGAs and the TF production therein. Together with a reduction of procoagulant MPs, these results may contribute to the clinical benefit found of oral DTIs. Targeting different mechanisms in platelet and coagulation activation may be of importance due to the lack of effect of DTIs on ADP-induced platelet-leukocyte aggregates and TF production.     

Plasma factor VII: a potential marker of pre-eclampsia

Introduction

Normal pregnancy is associated with a local hypercoagulable state that becomes more profound in certain obstetric complications such pre-eclampsia (P-EC). Current literature on the levels of individual haemostatic factors in women with P-EC is limited and results are inconsistent. In this study we provide detailed investigation on the tissue factor (TF)-dependent pathway in women with P-EC.

Materials and Methods

Enzyme-linked immunosorbent assays (ELISA) were used to measure plasma factor (F) FVII, FVIIa, TF and tissue factor pathway inhibitor (TFPI) in healthy non-pregnant women (n = 22), normal pregnant women (n = 15), and women with P-EC (n = 20). All subjects were age matched. In addition, pregnant women were matched for gestational age, parity and were all at the third trimester.

Results

Plasma FVII levels were significantly higher in women with P-EC compared to the healthy non-pregnant (P < 0.001) or the normal pregnant groups (P < 0.001). No such significant trends were observed for plasma FVIIa, TF or TFPI levels. Plasma FVII levels can distinguish women with P-EC from healthy non-pregnant women or normal pregnant women at the third trimester, with high sensitivity (90%), specificity (80%), positive and negative predictive values (86%).

Conclusions

Plasma FVII levels are significantly elevated in women with P-EC, in the absence of comparable changes in other TF-dependent pathway factors (FVIIa, TF and TFPI). We propose the use of plasma FVII as a marker for P-EC.     

Circulating tissue factor positive microparticles in patients with acute recurrent deep venous thrombosis

Introduction

Circulating tissue factor positive microparticles (MPTF) were reported in a wide range of diseases 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 therefore quantified MPTF in patients with acute recurrent DVT to determine whether MPTF levels could be used to predict recurrent DVT.

Materials and Methods

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 Kit.

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 (P < 0.01, respectively). The platelet and endothelial cell derived MPTF in recurrent DVT were significantly increased relative to other MPTF (P < 0.05), although there was no difference between initial DVT patients and normal individuals. We demonstrated elevated procoagulant activity of platelet-free plasma in DVT patients relative 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.