Tissue Factor-bearing Microparticles Research Articles

300 PCSK9 induces a prothrombotic response by activation of NFKB signalling pathway in mononuclear cells

Abstract Aims Strong evidence both experimental and clinical studies point out to an involvement of proprotein convertase subtilisin/kexin 9 (PCSK9), as an important player in hypercholesterolemia and atherosclerosis pathophysiology, identifying it, as a key molecule in the development of new cholesterol-lowering drugs and therapeutic target for atherosclerosis and related diseases. Emerging evidence shown that PCSK9 is straight implicated in inflammatory process where it may directly influence the activity of various cell types through NFkB activation, a key transcription factor involved in the induction of both pro-inflammatory cytokines and Tissue Factor (TF) expression, a major regulator of haemostasis and thrombosis in monocytes. Deductive reasoning makes therefore plausible to investigate a mechanistic link between circulation PCSK9 levels and TF expression, both in monocytes as well as on the surface of microparticles (MPs) generated from the same cells, with NFkB acting as the chain in the link. To investigate the involvement of NFkB signalling pathway in PCSK9-induced TF expression. Methods THP-1 cell line were stimulated with human (h) PCSK9 (5 μg/mL) or pre-incubated with BAY-117082 (BAY, 10−5M) an NFκB inhibitor, CLI-095 (3 × 10−6M), a highly TLR-4 signalling specific inhibitor and LPS-RS(5 μg/mL) a TLR-4 antagonist. TF procoagulant activity (PCA) was assessed by one-stage clotting assay using a STart Max semi-automated coagulation analyser. Concentration of TF-bearing MPs was determined using the Zymuphen MP-activity kit. Results hPCSK9 stimulated TF activity in THP-1 (PCA: from 50 ± 20 to 120 ± 20 ρg/mL, n = 10, P < 0.01). BAY, an NFkB inhibitor (PCA: −71 ± 23%, n = 5, P < 0.01) and CLI-095, a TLR-4 signalling inhibitor, (PCA: −86 ± 26%, n = 3, P < 0.05) and LPS-RS (PCA: −71 ± 23%, n = 5, P < 0.01) down-regulated PCSK9-induced TF activity completely. Furthermore THP-1stimulation with hPCSK9 causes the release of prothrombotic MPs-TF+ (MPs release from: 0.13 ± 0.07 to 0.42 ± 0.1 nM PS, n = 7, P < 0.05; PCA-MP+ from: 14 ± 3 to 44 ± 28 ρg/mL, n = 5, P < 0.05). Conclusions These data confirm the pivotal role of monocytes in the response PCSK9-induced TF-expression as well as the involvement of PCSK9 in inflammatory-thrombotic diseases through a direct stimulation of TF procoagulant activity and by the release of TF-bearing MPs. The possible mechanism of action involves recognition of PCSK9 by TLRs 2 and/or 4, on monocytes membrane surface, leading to activation of the transcription factor NFκB via an intracellular signalling cascade. Further studies will be needed to better understand the regulatory mechanisms underlying this complex set of biological responses that bind PCSK9, and coagulation events.

PAR-4/Ca2+-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes

BackgroundPatients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages.MethodsIn 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages.ResultsWe found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway.ConclusionsThese results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

Determination of Plasma Tissue Factor Antigen and Tissue Factor-Bearing Microparticles Activity in Healthy Subjects

Tissue factor (TF) is a transmembrane glycoprotein that is essential for hemostasis. It binds the coagulation serine protease factor VII/VIIa (FVII/VIIa) to form a bimolecular complex that functions as the primary initiator of coagulation in vivo [1].

Di-(2-Ethylhexyl) Phthalate Promotes Release of Tissue Factor-Bearing Microparticles From Macrophages via the TGFβ1/Smad/PAI-1 Signaling Pathway

BackgroundPlasminogen activator inhibitor type 1 promotes formation of endothelial microparticles with procoagulant activity. However, it remains unclear whether di-(2-ethylhexyl) phthalate, a peroxisome proliferator-activated receptor α agonist, influences microparticle formation. Materials and MethodsThe effect of di-(2-ethylhexyl) phthalate on release of tissue factor-bearing microparticles was investigated using human M1 macrophages. ResultsExposure of M1 macrophages to di-(2-ethylhexyl) phthalate significantly upregulated expression of plasminogen activator inhibitor type 1, whereas incubation of macrophages with small interfering RNA for peroxisome proliferator-activated receptor α attenuated it. Di-(2-ethylhexyl) phthalate significantly increased the tissue factor protein level in culture supernatants of M1 macrophages, but not M2 macrophages. After purification of proteins by centrifugal filtration, western blotting detected 2 high molecular weight bands of tissue factor-bearing microparticles in culture supernatants of M1 macrophages. The upper band showed binding to factor VIIa and tissue factor pathway inhibitor, unlike the lower band. This suggested heterogeneity of the procoagulant activity of tissue factor-bearing microparticles, presumably dependent upon encryption/decryption of tissue factor. Phosphatidylserine contributes to tissue factor decryption, and western blotting revealed that the density of phosphatidylserine was reduced in the upper tissue factor band compared with the lower band. Di-(2-ethylhexyl) phthalate also upregulated transforming growth factor-β1 protein production by M1 macrophages. Moreover, silencing of Smad2, Smad3 or Smad4 attenuated plasminogen activator inhibitor type 1 expression and tissue factor-release from macrophages after di-(2-ethylhexyl) phthalate stimulation. ConclusionsDi-(2-ethylhexyl) phthalate promotes formation of tissue factor-bearing microparticles in human M1 macrophages via the transforming growth factor-β1/Smad/ plasminogen activator inhibitor type 1 signaling pathway.

Tissue factor procoagulant activity in the tumor cell lines and plasma of dogs with various malignant tumors.

Hypercoagulability is a common paraneoplastic complication in dogs with various malignant tumors. Importantly, tissue factor procoagulant activity (TF-PCA) induced by TF-bearing microparticles (TF-MPs) is associated with hypercoagulability in human patients with cancer. However, TF-PCA in tumor cells and the association between circulating TF-MPs and hypercoagulability in dogs with malignant tumors remain poorly understood. Therefore, the present study was conducted to evaluate the TF-PCA in various types of canine tumor cell lines and plasma in dogs with malignant tumors. Mammary gland tumor, hemangiosarcoma, and malignant melanoma cell lines, but not lymphoma cell lines, expressed TF on their surfaces and showed cellular surface and MP-associated TF-PCA. The plasma TF-PCA was elevated in some dogs that naturally developed such tumors. No significant difference was observed in plasma TF-PCA between the disseminated intravascular coagulation (DIC) group (median: 43.40; range: 3.47–85.19; n=5) and non-DIC group (median: 7.73; range: 1.70–16.13; n=12). However, plasma TF-PCA was remarkably elevated in three of five dogs with DIC. To the best of our knowledge, this is the first study to evaluate plasma TF-PCA in dogs with malignant tumors. Further studies must be conducted to determine the cellular origin of TF-MPs and the efficacy of plasma TF-PCA as a biomarker of DIC in dogs with malignant tumors.

Correlation of Circulating Microparticles with Coagulation and Fibrinolysis Is Healthy Individuals

IntroductionMicroparticles (MPs) are small membrane blebs that are released in response to cellular activation or apoptosis from the cell surface by proteolytic cleavage of the cytoskeleton. Previous studies have reported low levels of naturally produced cell-derived microparticle in healthy individuals. This study was conducted to assess whether circulating microparticles induce thrombin generation leading to low grade activation of the coagulation system in healthy individuals.Materials & MethodsFlow cytometry analysis of three phenotypes of Platelet derived (PMP), Endothelial derived (EMP) and TF bearing (TFMP) microparticles was done in in the 20 healthy individuals. Triple gating strategy (i) particle size(<0.5µm) (ii) expression of cell surface markers (PMP, CD42a+; EMP, CD62E+; TF bearing MP(TFMP), CD142+) and (iii) phosphatidylserine(Annexin V+) was used. Plasma concentrations of thrombomodulin (TM), Tissue Factor (TF), Tissue factor pathway inhibitor (TFPI), Protein C (PC), Free Protein S (PS), Thrombin antithrombin (TAT) complexes, soluble fibrin monomer (sFM) was done assess the status of coagulation system. Spearman's rank correlation was done examine the role of cell-derived microparticles as critical effectors of thrombosis.Results: the study group comprised of 75% males of the age group (mean±SD27.3±4.32. Total Microparticles (Annexin V+) enumerated in plasma was 1125±355 (count/µl). 52.8% were TFMP [median (IQR)] [380.3(301.8-710.1) (count/µl)]. Highest number of MP originated from activated platelets [249.1(198.9-404.5) (count/µl)], followed by endothelial cells [140.9(124.9-286.0) (count/µl)]. A statistically significant and inverse correlation of EMP's with TAT complex was observed [12.7(6.8-20.1) ng/ml] (r = -0.46, p = 0.01), PS (93.4±20.6 %) (r = -0.52, p = 0.01) and TM (11.4±4.47 ng/ml) (r = -0.56, p = 0.008). Also, TFMP significantly correlated with TF [3.9(3.0-5.0)] (r = 0.46, p = 0.03) and PC (90.4±17.7) (r=-0.42, p=0.04). No correlation was observed between PMP numbers and coagulation pathway markers.Conclusion: Sub optimal degree of coagulation and natural anticoagulation pathway are occurring in healthy individuals. Microparticles moderately correlated with TF levels. Low EMP's levels correlated with the anticoagulation markers and thrombin generation, suggesting that EMPS may have a role in inhibition of thrombosis. The impact of endothelial vesiculation on basal coagulation should be studied further. DisclosuresNo relevant conflicts of interest to declare.

Changes of microparticle levels before and after daunorubicin-based chemotherapy in patients with acute leukemia

Objective To observe the serum levels of endothelial microparticles (EMP) and tissue factor-bearing microparticles (TF+ MP) in patients with acute leukemia before and after daunorubicin-based chemotherapy. Methods From July 2012 to February 2013, 15 patients with newly diagnosed acute leukemia in Peking Union Medical College Hospital received DA (daunorubicin + cytarabine) regimen or VDCLP (vincristine + daunorubicin + cyclophosphamide + L-asparaginase + prednisone) regimen chemotherapy. There were 8 males and 7 females, and the median age of patients was 44 years old. Eleven patients were acute myeloid leukemia (M0 1 case, M1 1 case, M2 9 cases), and 4 were acute lymphocytic leukemia. The peripheral blood samples were taken before induction chemotherapy and after 3 days of daunorubicin. Levels of EMP and TF+ MP were assessed using flow cytometry. Results The serum EMP and TF+ MP levels were significantly higher after 3-day daunorubicin infusions than those before induction chemotherapy (28.94/μl vs. 10.74/μl, P = 0.001; 64.24/μl vs. 43.80/μl, P = 0.02). Conclusion Daunorubicin-based chemotherapy may cause increased numbers of EMP and TF+ MP in patients with acute leukemia. Key words: Leukemia, acute; Antineoplastic combined chemotherapy protocols; Daunorubicin; Endothelial particles; Tissue factor-bearing microparticles

Pathophysiology of Trousseau’s syndrome

SummaryClinically relevant clotting abnormalities in cancer patients are referred to as Trousseau’s syndrome. While thrombotic complications such as venous thromboembolism are most frequent in every day’s practice, cancer patients may also experience severe bleeding symptoms due to complex systemic coagulopathies, including disseminated intravascular coagulation, haemolytic thrombotic microangiopathy, and hyperfibrinolysis. The pathophysiology of Trousseau’s syndrome involves all aspects of Virchow’s triad, but previous basic research has mainly focused on the cellular and molecular mechanisms underlying blood hypercoagulability in solid cancers and haematological malignancies. In this regard, over-expression of tissue factor (TF), the principal initiator of the extrinsic coagulation pathway, by primary tumour cells and increased shedding of TF-bearing plasma microparticles are critical to both thrombus formation and cancer progression. However, novel findings on intrinsic contact activation in vivo, such as the release of polyphosphates or DNA by activated platelets and neutrophils, respectively, have pointed to additional pathways in the complex pathophysiology of Trousseau’s syndrome.

The Role of Sphingomyelin (SM) in Tissue Factor (TF) Encryption: ATP-Induced Activation of Acid Sphingomyelinase in Macrophages Decrypts Tissue Factor By Hydrolysis of SM in the Outer Leaflet

While tissue factor (TF)-mediated blood coagulation is essential for maintaining hemostasis, the aberrant activation of TF-mediated coagulation is a major determinant of thrombotic occlusions, the precipitating event in acute myocardial infarction, unstable angina, and ischemic stroke. Typically, TF on cell surfaces exists in inactive coagulant status (cryptic TF). Cell injury leads conversion of cryptic TF to coagulant active/prothrombotic TF. Molecular differences between cryptic and procoagulant TF and the mechanisms that are responsible for the conversion from one to the other form are poorly understood and often controversial. A majority of the evidence in the literature suggest that level of anionic phospholipids, such as phosphatidylserine (PS), in the outer leaflet of the plasma membrane plays a critical role in regulating TF procoagulant activity at the cell surface. However, other pathways, such as the thioredoxin system or thiol-disulfide exchange pathways involving protein-disulfide isomerase (PDI), were also shown to contribute to TF activation by inducing structural changes in TF. It is unknown at present whether TF on cell surfaces of naïve cells exists primarily in the cryptic state because of the limited availability of anionic phospholipids at the outer leaflet or phospholipids present in the outer leaflet play an active role in maintaining TF in the cryptic state. In the outer leaflet of mammalian plasma membrane, sphingomyelin (SM) constitutes up to 50% of the total phospholipids present on the cell surface. It is possible that a high SM content in the outer leaflet may be responsible for maintaining TF in its cryptic state at the cell surface in naïve cells, and the hydrolysis of SM on the outer leaflet mediated by factors released in cell injury contributes to TF activation. The present study was carried out to investigate this possibility.First, we tested the potential effect of SM on TF activity in a reconstituted system in which full-length TF was reconstituted into phospholipid vesicles composed of varying molar concentrations of SM with the remainder of the vesicle consisting of phosphatidylcholine (PC). SM, at 35 mol % or higher concentration in the proteoliposome, inhibited TF coagulant activity significantly as measured in factor X activation assay. Ceramide, having a similar sphingosine backbone as of SM, had no inhibitory effect on TF-FVIIa activation of FX. Measurement of FVIIa-TF amidolytic activity showed that SM does not inhibit the amidolytic activity of FVIIa-TF, indicating that SM neither affects FVIIa binding to TF nor TF-FVIIa cleavage of the small substrate peptide. SM also inhibited significantly TF activity of TF reconstituted in PC/PS (94%:6% mol/mol) vesicles. Next, human monocyte-derived macrophages (MDMs) were treated with varying concentrations of bacterial sphingomyelinase (b-SMase) to hydrolyze SM in the outer leaflet. b-SMase treatment increased cell surface TF activity in a dose-dependent manner. SMase treatment also enhanced the release of TF-bearing microparticles (MPs). SMase treatment had no significant effect on cell surface prothrombinase activity or annexin V binding to MDMs, indicating that b-SMase treatment did not increase PS availability at the cell surface under our experimental conditions. Similar to that observed in bone marrow-derived mouse macrophages, ATP (200 µM) stimulation of MDMs increased cell surface TF activity by about 3-fold and triggered the release of TF+ MPs. Immunofluorescence confocal microscopy revealed that ATP stimulation induced in the translocation of acid(a)-SMase from intracellular compartments to the outer leaflet of the plasma membrane. Treatment of MDMs with sphingomyelinase inhibitors, desipramine and imipramine (1 and 5 µM), or silencing a-SMase with siRNA markedly reduced the ATP-induced increased TF activity at the cell surface and TF+ MPs release. Finally, ATP stimulation was shown to increase the hydrolysis of SM in the outer leaflet of MDMs markedly. a-SMase inhibitors or silencing of a-SMase attenuated the ATP-induced SM hydrolysis. In summary, our data indicate that SM plays a critical role in maintaining TF in the cryptic state in resting cells. Activation/translocation of a-SMase to the outer leaflet following the activation of ATP receptor P2X7 leads to hydrolysis of SM and thus relieves the inhibitory effect of SM on TF, leading to TF decryption and the release of TF+ MPs. DisclosuresNo relevant conflicts of interest to declare.

EL-16 - Management of cancer-associated thrombosis

The development of VTE has been known to be one of cautious complications and associated with a worse QOL and survival outcome in cancer patients. Cancer patients have a 4∼7 fold increased risk of VTE compared with general population. Around 10% of the patients with unprovoked VTE have been reported to have hidden cancer. The high incidence of thrombosis is reported in patients with pancreas, ovary, lung, and brain tumor. On the other hand, relatively low risks are seen in patients with mammary or prostate cancer. While the mechanisms on development of VTE associated with cancer progression are actively studied recently, it remains unsolved. One of most attractive molecules is tumor-derived tissue factor-bearing microparticles (TMP) which are produced by cancer cells and spread out in circulating blood. TMP may attach to surface membrane of vascular endothelial cells and leukocytes via the interaction of adhesion molecules, and then blood coagulation system is activated and induces the formation of thrombosis on the phospholipid cell surface membrane. In cancer patients, VTE delays in surgery and administration of chemotherapy and increased risk of bleeding due to anticoagulation, and then results in worse QOL and increased mortality. Therefore, the prediction and adequate prevention of VTE should be essential. In 2008 Khorana et al proposed the predictive model for VTE in cancer patients, which included type of cancer, platelet count ( = >350,000/ml), Hb <10g/dl, WBC >11000/ml and BMI = >35. Recently, D-dimer is indicated to be predictive biomarkers of thrombosis. Several guidelines for prophylaxis and treatment of VTE in cancer patients are presented. Prophylaxis against VTE is generally accepted in cancer patients with high risk Khorana score for DVT. Low molecular weight heparin is mostly used as first line therapy, although it is not currently approved in Japan. The clinical usefulness of direct oral anticoagulants (DOACs) has not yet been concluded.

Haemostatic factors do not account for worse outcomes from ischaemic stroke in patients with higher C-reactive protein concentrations.

Background Although the role of microparticles was recently implicated in stroke pathophysiology, the association between microparticles and inflammation is still not fully understood. The aim of this cohort study of 66 patients was to assess a relation between haemostatic factors, C-reactive protein and clinical outcome of ischaemic stroke. Methods Plasma microparticles procoagulant activity, concentrations of tissue factor-bearing microparticles, tissue factor and tissue factor pathway inhibitor in ischaemic stroke patients were determined with enzyme-linked immunosorbent assays at the time of initial diagnosis, along with serum C-reactive protein concentrations. Patients were divided into two groups depending on their C-reactive protein concentrations (C-reactive protein <3 mg/L; n = 28 vs. C-reactive protein ≥3 mg/L; n = 38). The analysed clinical outcome measures included the National Institutes of Health Stroke Scale and the Barthel Index. Results The two C-reactive protein groups did not differ significantly in terms of microparticles procoagulant activities, tissue factor-bearing microparticles, tissue factor and tissue factor pathway inhibitor concentrations. A significant correlation was observed between tissue factor pathway inhibitor and National Institutes of Health Stroke Scale score at admission ( R = 0.3, P = 0.03). Patients with C-reactive protein ≥3 mg/L presented with significantly higher National Institutes of Health Stroke Scale scores (median, 9.00 vs. 5.50, P = 0.002) and lower Barthel Index scores (median, 20.00 vs. 65.00, P = 0.002) than individuals with C-reactive protein <3 mg/L. The C-reactive protein concentrations correlatedpositively with National Institutes of Health Stroke Scale scores ( R = 0.3, P = 0.02) and inversely with Barthel Index scores ( R = - 0.4, P = 0.002). Conclusions Altogether, these findings imply that haemostatic parameters (microparticles, tissue factor-bearing microparticles, tissue factor, tissue factor pathway inhibitor) do not account for elevated C-reactive protein concentrations in ischaemic stroke patients.

Tissue factor-bearing microparticles and CA19.9: two players in pancreatic cancer-associated thrombosis?

Background:Cancer-related venous thromboembolism (VTE) heralds a poor prognosis, especially in pancreatic adenocarcinoma (PAC). Tissue factor (TF) is implicated as one of the main culprits in PAC-associated VTE and disease progression.Methods:In a prospective cohort study of 79 PAC patients, we measured plasma CA19–9 and microparticle-associated TF activity (MP-TF activity). In addition, we enumerated TF+MPs and MUC1+MPs in plasma (n=55), and studied the expression of TF, MUC1, CD31 and CD68 in tumour tissue (n=44).Results:Plasma MP-TF activity was markedly elevated in PAC patients with VTE compared with those without (median: 1925 vs 113 fM Xa min−1; P<0.001) and correlated with the extent of thromboembolic events, metastatic disease and short survival. Similar results were found for CA19–9. Patients with massively progressing thrombosis and cerebral embolisms despite anticoagulant therapy (n=3) had the highest MP-TF activities (12 118–40 188 fM Xa min−1) and CA19–9 (40 730–197 000 kU l−1). All tumours expressed MUC1 and TF. MP-TF activity did not correlate with intensity of TF expression in adenocarcinoma cells, but corresponded with numbers of TF+ macrophages in the surrounding stroma.Conclusions:Circulating TF+MPs and mucins may concertedly aggravate coagulopathy in PAC. Understanding of underlying mechanisms may result in new treatment strategies for VTE prevention and improvement of survival.

OC-09 - Microparticle-mediated transfer of TF hypercoagulability between cancer cells

Tissue Factor (TF) promotes apoptosis-resistance and facilitates haematogenous metastasis. Cancer cells release TF-bearing microparticles (TF-MP) which have pro-coagulant activity. TF-MPs from cancer cells induce increase in TF expression and procoagulant activity of recipient endothelial cells. Drug resistance microparticles transfer between cancer cells, resulting in induction of recipient cell drug resistance, however transfer of TF-MPs between cancer cells has not been demonstrated. To determine if microparticles isolated from TF expressing cancer cell lines can induced TF-mediated procoagulant activity in cell lines that do not express TF. A7 cells (a melanoma cell line that does not express TF) were transformed to express TF with adenoviral transformation. In addition, the TF-expressing breast cancer cell line MDAMB231 was used. Media was collected from (1) wild-type A7 (A7-WT) cells, which acted as control (2) MDAMB231 cells, (3) A7 cells transformed with 'low' levels of TF-adenovirus and (4) A7 cells transformed with 'high' levels of TF-adenovirus. MPs were isolated from media by centrifugation using standard techniques. Isolated MPs were incubated with wild-type A7 (A7-WT) cells overnight. Pro-coagulant activity of A7-WT cells was assessed using a FXa generation assay. i) A7-WT cells incubated overnight with MDAMB231-derived MPs produced an almost 5 fold increase in procoagulant activity compared to control (Mean 3.5 mOD/min S.E 0.32 vs control 0.68 S.E. 0.05, p<0.01). ii) MPs released from A7-TF cells induced a dose-dependant increase in cell based procoagulant activity when incubated with A7-WTs compared to control:A7-WT cells incubated with control MPs (Control): mean mOD 0.83, S.E. 0.07A7-WT cells incubated with A7-TF derived MPs (low levels of TF-adenovirus): mean mOD 1.27, S.E. 0.06 (p=0.04 vs control)A7-WT cells incubated with A7-TF derived MPs (low levels of TF-adenovirus): mean mOD 2.77, S.E. 0.23 (p<0.01 vs control). These results demonstrate, for the first time, microparticle-mediated transfer of TF procoagulant activity between cancer cells. MPs shed by cancer cells in vivo have been implicated in cancer cell progression and hypercoagulability. The acquisition and spread of of MP-mediated TF activity has implications for cancer cell biology and cancer-induced hypercoagulability.

Assessing Circulating Factor VIIa-Antithrombin Complexes in Acute Ischemic Stroke: A Pilot Study.

The goal of this study was to determine the levels of factor VII (FVII), factor VIIa-antithrombin complexes (FVIIa-AT), total tissue factor (TF), and tissue factor-bearing microparticles (MPs-TF) in patients with acute ischemic stroke. Further, we sought evidence of an association between hemostatic markers, time of blood sampling, type of treatment, and patient outcomes. Venous blood samples were collected from 33 patients on the first day and on the seventh day after stroke diagnosis. Age-matched controls were also included (n = 20). Plasma levels of FVII, FVIIa-AT, total TF, and MPs-TF were measured by enzyme-linked immunosorbent assay. We divided patients into 2 groups: thrombolysis group (n = 13) and nonthrombolysis group (n = 20). Furthermore, evaluation of the National Institutes of Health Stroke Scale and the Barthel Index was performed on the first day and the seventh day. Patients with ischemic stroke showed significantly lower plasma FVII, FVIIa-AT, and total TF levels than controls (median, 112.25% vs 132.05%, P = .004; 107.97 pmol/L vs 154.94 pmol/L, P < .001; 81.74 pg/mL vs 105.71 pg/mL, P < .001, respectively). In contrast, levels of plasma MPs-TF were significantly higher in patients with stroke compared to healthy controls (1.60 pg/mL vs 0.74 pg/mL, P < .001). Additionally, the thrombolysis group had lower FVII levels on the seventh day compared to the first day (median, 109.80% vs 115.74%, P = .04). Factor VII, FVIIa-AT, and total TF are decreased, while MPs-TF are elevated in patients with ischemic stroke. We observed a slight but significant effect of alteplase on FVII plasma levels.

Circulating microparticles and the risk of thrombosis in inherited deficiencies of antithrombin, protein C and protein S.

Many subjects carrying inherited thrombophilic defects will never experience venous thromboembolism (VTE) while other individuals developed recurrent VTE with no known additional risk factors. High levels of circulating microparticles (MP) have been associated with increased risk of VTE in patients with factor V Leiden and prothrombin G20210A mutation, suggesting a possible contribution of MP in the hypercoagulability of mild genetic thrombophilia. The role of MP as additional risk factor of VTE in carriers of natural clotting inhibitors defects (severe thrombophilia) has never been assessed. Plasma levels of annexin V-MP, endothelial-derived MP (EMP), platelet-derived MP (PMP), tissue factor-bearing MP (TF+) and the MP procoagulant activity (PPL) were measured in 132 carriers of natural anticoagulant deficiencies (25 antithrombin, 63 protein C and 64 protein S defect) and in 132 age and gender-matched healthy controls. Carriers of natural anticoagulant deficiencies, overall and separately considered, presented with higher median levels of annexin V-MP, EMP, PMP, TF+MP and PPL activity than healthy controls (p< 0.001, < 0.001, < 0.01, 0.025 and 0.03, respectively). Symptomatic carriers with a previous episode of VTE had significantly higher median levels of annexin-V MP than those without VTE (p=0.027). Carriers with high levels of annexin V-MP, EMP and PMP had an adjusted OR for VTE of 3.36 (95% CI, 1.59 to 7.11), 9.26 (95% CI, 3.55 to 24.1) and 2.72 (95%CI, 1.16 to 6.38), respectively. Elevated levels of circulating MP can play a role in carriers of mild and severe inherited thrombophilia. The clinical implications of this association remain to be defined.

Promotion of hypercoagulability in antineutrophil cytoplasmic antibody-associated vasculitis by C5a-induced tissue factor-expressing microparticles and neutrophil extracellular traps.

Patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) have a high frequency of venous thromboembolic events and a hypercoagulable state. As C5a-primed neutrophils play an important role in the development of AAV, we investigated whether C5a-induced neutrophil tissue factor (TF)-expressing microparticles (MPs) and neutrophil extracellular traps (NETs) might promote hypercoagulability in AAV. TF-expressing MPs were measured by flow cytometry. TF-expressing NETs were assessed by confocal microscopy. Levels of thrombin-antithrombin complexes were determined by enzyme-linked immunosorbent assay. The effect of C5a in sera from AAV patients was evaluated by treating neutrophils with C5a receptor antagonist before incubation with sera from AAV patients with active disease. Treatment of C5a-primed neutrophils with antineutrophil cytoplasmic antibody (ANCA)-positive IgG resulted in the release of TF-bearing MPs and NETs. Neutrophils from healthy donors treated with sera from patients with active AAV released TF-bearing MPs and NETs, which were abolished by treatment with C5a receptor antagonist. Involvement of TF in MP- or NET-dependent thrombin generation was indicated by the findings of antibody neutralization studies. NETs with thrombin-generating capacity were demonstrated by DNase I treatment. C5a-primed neutrophils produce TF-expressing MPs and NETs after stimulation with ANCAs, indicating a mechanism for hypercoagulability in AAV that was not previously recognized.

Postsurgical Inflammation as a Causative Mechanism of Venous Thromboembolism.

Surgery is associated with an increased risk of venous thromboembolic events (VTE) including deep vein thrombosis and pulmonary embolism. Although the current treatment regiments such as mechanical manipulation and administration of pharmacological prophylaxis significantly reduced the incidence of postsurgical VTE, they remain a major cause of postoperative morbidity and mortality worldwide. The pathophysiology of venous thrombosis traditionally emphasizes the series of factors that constitute Virchow triad of factors. However, inflammation can also be a part of this by giving rise to a hypercoagulable state and endothelial damage. The inflammatory response after surgery, which is initiated by a cytokine "storm" and occurs within hours of surgery, creates a prothrombotic environment that is further accentuated by several cellular processes including neutrophil extracellular traps formation, platelet activation, and the generation of tissue factor-bearing microparticles. Although such inflammatory markers are elevated in undergoing surgery, the precise mechanism by which they give rise to venous thrombosis is poorly understood. Here, we discuss the potential mechanisms linking inflammation to thrombosis, and highlight strategies that may minimize surgical inflammation and reduce the incidence of postoperative VTE.

Abstract POSTER-THER-1406: Tissue factor-factor VIIa complex triggers vascular endothelial growth factor-a secretion in epithelial ovarian cancer cell lines via a protease activated receptor-2 dependent mechanism

Abstract Purpose of study: Epithelial ovarian cancer (EOC) is highly sensitive to initial platinum-based chemotherapy, but ultimately re-growth of the tumor occurs in the majority of patients with development of platinum resistance. Identification of: 1) the major mechanisms leading to regrowth, and 2) potential drug targets for maintenance therapy to prevent regrowth/relapse of EOC would potentially have a dramatic impact on patient outcomes. Progression of EOC is often associated with a procoagulant phenotype, characterized by increased tissue factor (TF) expression and enhanced metastasis. TF-factor VIIa (FVIIa) complex on the cell surface activates protease activated receptor-2 (PAR-2) directly, and PAR-1 activation indirectly via triggering local thrombin generation. PAR-2 activation is associated with an induction in growth factor release in renal cell carcinoma. We hypothesize that TF-FVIIa dependent activation of PAR-2 on EOC cells up-regulates expression of angiogenic mediators (VEGF-A) that modify the peritoneal microenvironment, enhancing vascular leakage and ascites formation. Incorporation of anti–VEGF therapy in standard treatment of EOC has been shown to prolong progression-free survival, but the underlying mechanisms are incompletely understood. The objective of this study is to determine the role TF-FVIIa-PAR-2 axis plays in EOC progression. Methods: TF antigen expression in EOC cell lines (CaOV-3, SKOV-3 &amp; OvCAR-3) was determined by western blot (WB) analysis. TF-bearing microparticles were isolated from conditioned media by differential centrifugation (20,800 g) and TF coagulant activity was detected by factor X activation. VEGF-A levels in conditioned media were determined by ELISA. Proliferation of the SKOV-3 and OVCAR-3 cell lines in response to thrombin (PAR-1 agonist) was determined via direct cell count. Results: WB analysis of EOC cell line lysates demonstrated PAR-2 expression (CaOV-3&amp;gt;OvCAR-3&amp;gt;&amp;gt;SKOV-3), and exposure of SKOV-3 and OVCAR-3 cells to 50 nM FVIIa resulted in 3-fold induction of VEGF-A release over 48 hr relative to untreated cells (p&amp;lt;0.05). TF-FVIIa complex was a more potent inducer of VEGF-A when compared to the PAR-2 peptide agonist (SKIGKL-NH2). Pre-incubation with the PAR-2 antagonist ENMD-1068 reduced VEGF-A to basal levels in the presence of TF-FVIIa; further supporting PAR-2 dependent induction of VEGF-A. Pan-expression of PAR-1 was observed in EOC cell lines by WB. Thrombin (0.1-1U/mL), an established PAR-1 agonist, induced dose-dependent proliferation of SKOV-3 and OVCAR-3 cell lines under serum free conditions (p&amp;lt;0.001). Pre-incubation with the PAR-1 antagonist vorapaxar reduced proliferation to basal levels in the presence of thrombin. Conclusion: TF expression and coagulant activity is up regulated in EOC. FVIIa triggers a PAR-2 dependent induction of VEGF-A release from EOC cell lines. Similarly, the PAR-1 agonist thrombin triggered a significant increase in EOC cell proliferation. These results suggest that interaction of EOC with the coagulation system in the tumor microenvironment may enhance both PAR-1 dependent cellular proliferation and PAR-2 dependent release of VEGF, a potent regulator of angiogenesis. Thus, PAR-1 and PAR-2 may represent pharmacologically relevant drug targets to block ovarian cancer progression via inhibition of growth and angiogenesis. Citation Format: Alice Chanakira PhD, John P. Sheehan MD. Tissue factor-factor VIIa complex triggers vascular endothelial growth factor-a secretion in epithelial ovarian cancer cell lines via a protease activated receptor-2 dependent mechanism [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1406.

Origin and levels of circulating microparticles in normal pregnancy: A longitudinal observation in healthy women

Objective. Microparticles (MP) are actively involved in the hypercoagulable state reported both in normal pregnancies and in pregnancy diagnosed with placenta-mediated complications. In this study the origin and the levels of plasma MP as well as MP activity were evaluated in a group of healthy women during the three trimesters of a normal pregnancy. Materials and methods. Seventy-five healthy normotensive pregnant women were enrolled and blood samples were prospectively collected at three different time points corresponding to 1st trimester, 2nd trimester, 3rd trimester of pregnancy. A group of age- matched healthy non-pregnant women acted as controls. Both standard clotting parameters and MP of different origin were measured. MP were identified by size and annexin V- FITC labelling using flow-cytometer. MP subtypes were identified using specific monoclonal antibodies. Procoagulant activity of MP was assessed using the STA® Procoag PPL assay. Results. The levels of total, platelet-, endothelial-, leukocyte-derived and tissue factor-bearing MP, as well as the MP procoagulant activity, in non-complicated pregnancy were higher in the 1st trimester as compared to non-pregnant age-matched women. Regardless of the origin, MP levels gradually increase during pregnancy, with the highest values reached in the 3rd trimester. Conclusions. MP levels gradually increase during normotensive pregnancy. All types of MP including TF+ present with the highest levels in the 3rd trimester. MP convey prothrombotic and proinflammatory antigens already from the first trimester of normal pregnancy. This may contribute to the global hypercoagulable state observed, particularly in the last months of pregnancy, also in healthy women.

Hypercoagulability in overweight and obese subjects who are asymptomatic for thrombotic events.

The role of circulating microparticles (MP) of different origin and tissue factor (TF)-bearing in overweight and obese patients with and without metabolic syndrome is still a matter of debate. In a case-control study, the presence of hypercoagulability was evaluated in overweight and obese patients by measuring MP, thrombin generation (TG) and FVIIa-AT complexes. Twenty overweight patients (body mass index [BMI] range 25-29.9 kg/m²), 20 with I degree (30-34.9 kg/m²), 20 with II degree (35-39.9 kg/m²) and 20 with III degree obesity (≥ 40 kg/m²) were enrolled and compared to 40 age and gender-matched normal weight individuals. A significant increase in median levels of all MP subtypes was observed in the three degrees of obese patients compared to controls. Overweight patients had higher levels of annexin V-MP (AMP), endothelial-derived, leukocyte-derived and TF-bearing MP than controls. Obese patients had a significantly shorter median lag time (p< 0.05), higher median peak thrombin (p< 0.01) and increased median endogenous thrombin potential [ETP] (p< 0.001) compared to controls. Overweight subjects had significantly increased ETP compared to controls (p< 0.05). Both AMP levels and ETP were found to positively correlate with BMI, waist circumference, and inflammatory parameters. No significant increase in FVIIa-AT complex was seen in cases compared to controls. We conclude that obesity is associated with overproduction of procoagulant MP and increase TG. Interestingly, hypercoagulability is found in overweight patients free of metabolic syndrome and increases with the severity of obesity. Assessment of MP and TG may be helpful in the early characterisation of the prothrombotic state in obese patients.