scholarly journals Impact of Epicatechin on the Procoagulant Activities of Microparticles

Nutrients ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2935
Author(s):  
Thomas Sinegre ◽  
Dragan Milenkovic ◽  
Céline Bourgne ◽  
Dorian Teissandier ◽  
Yosra Nasri ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Flow Cytometry ◽  
Tissue Factor ◽  
Healthy Subjects ◽  
Thrombin Generation ◽  
Plasma Samples ◽  
Flow Cytometry Analysis ◽  
Cardioprotective Effects ◽  
The Impact ◽  
Platelet Microparticles

Microparticles play a role in cardiovascular disease pathology. The flavanol-like epicatechin is increasingly considered due to its cardioprotective effects. The aim of this study was to investigate the impact of epicatechin on microparticle generation, phenotype and procoagulant properties. Plasma samples from 15 healthy subjects were incubated with increasing concentrations of epicatechin (1 to 100 μM). Then, the expression of glycoprotein IIb, phosphatidylserine (PS), glycoprotein Ib (GPIb) and P-selectin was assessed by flow cytometry analysis after (or not) platelet stimulation. Microparticle procoagulant activity was determined using ZymuphenTM MP and ZymuphenTM MP-TF for phospholipid and tissue factor content, and with thrombin generation (TG) assays for procoagulant function. Platelet microparticles that express GPIb (/µL) decreased from 20,743 ± 24,985 (vehicle) to 14,939 ± 14,333 (p = 0.6), 21,366 ± 16,949 (p = 0.9) and 15,425 ± 9953 (p < 0.05) in samples incubated with 1, 10 and 100 µM epicatechin, respectively. Microparticle concentration (nM PS) decreased from 5.6 ± 2.0 (vehicle) to 5.1 ± 2.2 (p = 0.5), 4.5 ± 1.5 (p < 0.05) and 4.7 ± 2.0 (p < 0.05) in samples incubated with 1, 10 and 100µM epicatechin, respectively. Epicatechin had no impact on tissue factor-positive microparticle concentration. Epicatechin decreased TG (endogenous thrombin potential, nM.min) from 586 ± 302 to 509 ± 226 (p = 0.3), 512 ± 270 (p = 0.3) and 445 ± 283 (p < 0.05). These findings indicate that epicatechin affects microparticle release, phenotype and procoagulant properties.

The Impact of Factor IX Level on Thrombin Generation and Bleeding During Warfarin Anticoagulation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 541-541
Author(s):  
Yesim Dargaud ◽  
Maureane Hoffman ◽  
Claude Negrier ◽  
Leana Lefrapper ◽  
Dougald M. Monroe
Keyword(s):  
Tissue Factor ◽  
Vitamin K ◽  
Intracranial Haemorrhage ◽  
Thrombin Generation ◽  
Factor Ix ◽  
Target Range ◽  
Significant Difference ◽  
Generation Capacity ◽  
The Impact

Abstract Abstract 541 Bleeding occurs in from 10 – 16% of warfarin-treated patients. Having a PT-INR in the target range is associated with better outcomes. However, even patients with an INR in the target range of 2–3 can suffer bleeding, suggesting that INR does not perfectly reflect the therapeutic effect of warfarin. The goal of our studies was to determine whether the level of specific coagulation factors could predict the risk of bleeding while the INR was in the target range. We modeled warfarin anticoagulation in our previously published in vitro cell based-model by adjusting the levels of vitamin K-dependent factors to those of patients with an INR of 2–3. We then examined the effect of variations in the level of FIX. The cogulation reactions were initiated by monocyte-expressed tissue factor (assayed at 1pM). Variation in FIX had a marked effect on thrombin generation. However, in plasma with the same levels of factors, as expected, variations in FIX had no effect on the PT-INR. Thus, we hypothesized that a subject with a lower FIX level than average may have a lower level of thrombin generation than is indicated by the INR. The INR might, therefore, underestimate the level of anticoagulation in such a subject. If s/he is maintained in the “therapeutic range” as measured by the INR, s/he will actually be over-anticoagulated and prone to hemorrhage. A prospective, single centre clinical study has been carried out to test this hypothesis in warfarinized patients. Between October 2010 and June 2011, 312 consecutive patients admitted to the emergency department of Edouard Herriot Hospital in Lyon, with an INR between 1.8 and 3.2, were included in the study after obtaining informed consent. Twenty six patients were admitted for a bleeding episode, 18 for recurrent thrombosis and 268 for other medical reasons. Patients presenting with bleeding, 17 males and 9 females, were aged 74±14 years old compared to the rest of the patients aged 76±14. Among the 26 bleeders, 7 had a spontaneous intracranial haemorrhage, 2 had a trauma-induced intracranial haemorrhage, 12 presented a gastrointestinal bleeding and 5 exhibited muscle hematomas, severe epistaxis or urinary tract bleeding. PT-INR and vitamin K-dependent factor levels were determined in all patients. Thrombin generation capacity in platelet poor plasma was measured using Calibrated Automated Thrombin generation assay (Thrombinoscope bv, Maastricht, The Netherlands), with tissue factor 1pM and phospholipids PC:PS:PE 4μM. No statistically significant difference was observed in the PT-INR of bleeding patients (INR=2.4±0.4) and those having a thrombosis (INR=2.5±0.5) or patients admitted for other reasons (INR=2.6±0.2). Plasma prothrombin and factor × levels were also similar in all three groups. However, a statistically lower plasma factor IX activity was observed in bleeders (p=0.01, Mann Whitney test) compared to other groups, 47.6±20 IU/dL vs. 63±33 IU/dL. In all the warfarinized subjects with an INR between 1.8 and 3.2, no correlation was found between thrombin generation capacity and PT-INR results (p=0.85, Spearman correlation test). However, a statistically significant correlation was observed between thrombin generation capacity and factor IX levels (p=0.0002). In patients, presenting with warfarin-related haemorrhage, the endogenous thrombin potential (ETP) was significantly lower at 340±335 nM.min (p=0.05) then that of warfarinized subjects who did not suffer bleeding (ETP 406±215 nM.min). These data support our hypothesis based on our in vitro results and show that patients who bleed when their PT-INR is in the target range 2 – 3 might have defective thrombin generation related to a lower level of factor IX than expected. Thus, our results suggest that the appropriate target INR level might not be the same for all patients. Those with factor IX levels that differ significantly from the mean of the population might be managed best by selecting a target INR that is based on the level of thrombin generation. Of course, a “target range” for parameters of thrombin generation during warfarin therapy would need to be developed if the assay were to be used for this purpose. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Correlation of Circulating Microparticles with Coagulation and Fibrinolysis Is Healthy Individuals

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4975-4975
Author(s):  
Venencia Albert ◽  
Arulselvi Subramanian ◽  
Hara Prasad Pati
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Plasma Concentrations ◽  
Annexin V ◽  
Coagulation System ◽  
Low Grade ◽  
Healthy Individuals ◽  
Circulating Microparticles ◽  
The Impact

Abstract Introduction Microparticles (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 & Methods Flow 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. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Rate of Prothrombin Conversion Is Increased in Antiphospholipid Syndrome and Is Insensitive to Thrombomodulin

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 718-718
Author(s):  
Romy Kremers ◽  
Stéphane Zuily ◽  
Hilde Kelchtermans ◽  
Tessa Peters ◽  
Saartje Bloemen ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Antiphospholipid Syndrome ◽  
Conversion Rate ◽  
Healthy Subjects ◽  
Thrombin Generation ◽  
Lag Time ◽  
Peak Height ◽  
Thrombin Inhibitors ◽  
Time To Peak ◽  
Glycoprotein I

Abstract Background: The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies directed mainly against prothrombin and β2-glycoprotein I. The syndrome is associated with an increased risk of thrombosis. The global hemostatic state in a patient can be tested by measuring thrombin generation (TG). Recently, we developed a method to study the main pro- and anticoagulant processes at the basis of TG, called the thrombin dynamics method. Aim: In this study we investigated the dynamics of thrombin generation in healthy subjects and APS patients. Materials and methods: Healthy subjects (n=129) and antiphospholipid syndrome (APS) patients (n=31) were included in the study. Sixty-eight percent of the APS patients were lupus anticoagulant positive, anti-cardiolipin antibodies were detected in 84% of the patients, and 52% presented with anti-β2-glycoprotein I antibodies. Patients on anticoagulant therapy were excluded from the study. Thrombin generation was measured at 1 pM tissue factor (TF) and activated protein C (APC) system sensitivity was tested by measuring TG in the presence and absence of 20 nM thrombomodulin (TM). Results: Thrombin generation was measured in platelet poor plasma at 1 pM tissue factor. The lag time and time-to-peak were significantly prolonged in APS patients compared to healthy subjects (lag time: 3.30 ± 0.59 vs. 6.69 ± 4.26 min, p<0.001; time-to-peak: 8.33 ± 1.29 vs. 10.76 ± 4.51 min, p<0.001). The peak height was significantly higher in APS patients (240 ± 84 vs. 214 ± 58 nM, p<0.05) and the velocity index was elevated in APS patients (134 ± 66 vs. 70 ± 32 nM/min, p<0.001) compared to healthy subjects. The ETP values were comparable between healthy subjects and APS patients (1260 ± 235 vs. 1176 ± 362 nM*min). The pro- and anticoagulant processes underlying thrombin generation were studied separately. The total amount of prothrombin converted during thrombin generation (PCtot) did not differ between healthy subjects and patients (1234 vs. 1165 nM). However, the maximum rate of prothrombin conversion (PCmax) was significantly elevated in APS patients (291 vs 425 nM/min; p<0.001). The amount of thrombin-antithrombin (T-AT) complexes formed was comparable between patients and controls (1169 vs. 1098 nM), and the thrombin decay capacity (TDC) was comparable as well (0.675 vs. 0.674 min-1). These results are in line with the finding that the plasma levels of the main thrombin inhibitors are unchanged in APS patients. Antithrombin levels are on average 2.31 ± 0.44 μM in healthy subjects and 2.36 ± 0.56 μM in APS patients, and the mean α2-macroglobulin levels were 3.22 ± 0.77 μM in healthy subjects and 3.23 ± 1.11 μM in patients. Thrombomodulin reduced the ETP by 45% in healthy subjects, but had significantly less effect in APS patients (10%). The addition of TM decreased total prothrombin conversion by 40% and the maximum prothrombin conversion rate by 50% in healthy subjects. In patients, TM only slightly reduced total prothrombin conversion (8%) and the maximum prothrombin conversion rate (7%). Discussion: The thrombin generation results indicate a predisposition to thrombosis in APS patients, as the TG parameters peak height and the velocity index are increased. Examination of the underlying pro- and anticoagulant processes of prothrombin conversion and thrombin inactivation revealed that although the same amount of prothrombin is converted in patients, the maximum activity of the prothrombinase complex is higher, indicating that patients can generate thrombin faster. In addition, APS patients have a dysfunctional APC system, as prothrombin conversion and thrombin generation could be only slightly inhibited by the addition of thrombomodulin. Disclosures No relevant conflicts of interest to declare.

Single-cell RNA-sequencing reveals profound changes in circulating immune cells in patients with heart failure

2020 ◽  
Author(s):  
Wesley T Abplanalp ◽  
David John ◽  
Sebastian Cremer ◽  
Birgit Assmus ◽  
Lena Dorsheimer ◽  
...  
Keyword(s):  
Heart Failure ◽  
Flow Cytometry ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cells ◽  
Healthy Subjects ◽  
Patients With Heart Failure ◽  
Single Cell Rna Sequencing ◽  
The Impact ◽  
Classical Monocytes

Abstract Aims Identification of signatures of immune cells at single-cell level may provide novel insights into changes of immune-related disorders. Therefore, we used single-cell RNA-sequencing to determine the impact of heart failure on circulating immune cells. Methods and results We demonstrate a significant change in monocyte to T-cell ratio in patients with heart failure, compared to healthy subjects, which were validated by flow cytometry analysis. Subclustering of monocytes and stratification of the clusters according to relative CD14 and FCGR3A (CD16) expression allowed annotation of classical, intermediate, and non-classical monocytes. Heart failure had a specific impact on the gene expression patterns in these subpopulations. Metabolically active genes such as FABP5 were highly enriched in classical monocytes of heart failure patients, whereas β-catenin expression was significantly higher in intermediate monocytes. The selective regulation of signatures in the monocyte subpopulations was validated by classical and multifactor dimensionality reduction flow cytometry analyses. Conclusion Together this study shows that circulating cells derived from patients with heart failure have altered phenotypes. These data provide a rich source for identification of signatures of immune cells in heart failure compared to healthy subjects. The observed increase in FABP5 and signatures of Wnt signalling may contribute to enhanced monocyte activation.

Tissue Factor-Bearing Microparticles Derived from Tumor Cells: Impact on Coagulation Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1757-1757 ◽  
Author(s):  
Monica Davila ◽  
Ali Amirkhosravi ◽  
Enriqueta Coll ◽  
Robles D. Liza ◽  
John Francis
Keyword(s):  
Flow Cytometry ◽  
Tissue Factor ◽  
Tumor Cells ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Annexin V ◽  
Cancer Tissue ◽  
Cancer Associated Thrombosis ◽  
Mcf 7

Abstract Thromboembolic disease is a frequent complication in cancer. Tissue factor (TF), shown to be involved in tumor growth and metastasis, is also considered to play a central role in the pathogenesis of cancer-associated thrombosis. Circulating TF-bearing microparticles (TF+ MPs) have been found in the plasma of patients with different malignancies and are thought to contribute to their hypercoagulable state. Although numerous studies have focused on TF+ MPs derived from blood cells, there is no information available on the pathological relevance of MPs originating from tumor cells. We conducted a study to detect, enumerate and characterize the procoagulant activity (PCA) of MPs released specifically from tumor cells. MPs from high (MDA-231) and low (MCF-7) TF-expressing human breast carcinoma cells were generated ex vivo in whole blood or in buffer under stirring conditions for 45 minutes. The numbers (MPs/ml) of total and TF-expressing tumor-derived particles (TMPs) in cell-free plasmas were measured by flow cytometry using FITC-labeled annexin V and a PE-labeled monoclonal anti-TF antibody respectively. The PCA of TMPs was measured by a one stage clotting assay and a highly sensitive fluorogenic thrombin generation assay. In order to evaluate the PCA of circulating TMPs, we injected 2x106 TF+ MPs derived from MDA-231 cells into mice via the tail vein. Human TF antigen and activity were measured in cell-free mouse plasmas at various intervals (5–420 min) after injections by ELISA and clotting assay, respectively. MPs less than 1μm in diameter were released from tumor cells in both buffer and whole blood by stirring. TMPs positive for TF consisted of approximately 40% of the annexin V+ MPs, and such particles derived from as low as 1x105 MDA-231 cells could be enumerated reliably (2.5x104 MPs/105 cells). By ultracentrifugation of cell-free plasmas, we confirmed that TF antigen was associated entirely with the MP fraction. TMPs derived from as few as 450 MDA-231 cells shortened plasma recalcification times from 525 ± 114 to 265 ± 148 (P<0.01), and significantly accelerated thrombin generation as evidenced by a 3 fold shortening in lag time, and a 2 fold increase in the rate of thrombin formation and thrombin concentration. No PCA was detected with MCF-7-derived TMPs. The PCA of TMPs was inhibited completely by a blocking anti-TF monoclonal antibody, but not by saturating concentrations of annexin V (an inhibitor of phospholipid PCA) or corn trypsin inhibitor (an inhibitor of the intrinsic pathway). Five minutes following the injection of TMPs into mice, appreciable levels of human TF antigen and activity were detected in cell-free plasmas. Both TF activity and antigen decreased over time and were detectable no longer than 30 minutes after injection, indicating a rapid clearance of circulating TMPs in vivo. In contrast, when TMPs were incubated in human whole blood ex vivo for various intervals, TF activity remained unchanged in cell-free plasmas for at least 5 hrs and TF antigen was not detected by flow cytometry on any blood corpuscles, including platelets, at all intervals. However, when whole blood containing TMPs was clotted by recalcification, no TF activity could be detected in the serum, indicating the incorporation of TMPs in formed clots. In summary, MPs bearing active TF are released readily from tumor cells and can significantly activate coagulation even at very low concentrations. These results provide new insights towards the potential contribution of TMPs to the pathogenesis of cancer-associated thrombosis.

scholarly journals Levels of Circulating Alternatively Spliced Tissue Factor (asTF) in the Plasma of Patients with Pancreatic Ductal Adenocarcinoma (PDAC) May Help Predict Aggressive Tumor Phenotype

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1486-1486
Author(s):  
Dusten Unruh ◽  
Farah Sagin ◽  
Mariette Adam ◽  
Patrick Van Dreden ◽  
Barry J Woodhams ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Emergency Room ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Tissue Factor ◽  
Healthy Subjects ◽  
Ductal Adenocarcinoma ◽  
Control Group ◽  
Chi Square ◽  
Exact Test ◽  
Alternatively Spliced

Abstract Tissue Factor (TF) present in blood cells and plasma is referred to as blood-borne or circulating TF. TF has been implicated in the pathogenesis of several chronic disease states, most notably cardiovascular disease/thrombosis, diabetes, and cancer. Full-length TF is an integral membrane protein while alternatively spliced TF can be secreted in a free form and features a unique C-terminal domain enabling its selective detection in bio-specimens. Recently, asTF was shown to circulate in the blood of metastatic breast cancer patients at concentrations exceeding 1 ng/mL (Kocaturk et al, PNAS 2013), and it promoted tumor growth and spread in an orthotopic model of pancreatic ductal adenocarcinoma (PDAC, Unruh et al, Int J Cancer, 2014). asTF protein acts as a cell agonist driving angiogenesis, cancer cell proliferation, and monocyte recruitment via integrin binding. It is not known whether circulating asTF may contribute to or serve as a biomarker in patients suffering from cardiovascular disease, diabetes, and/or solid cancers including PDAC. We evaluated circulating asTF in healthy subjects and individuals with ongoing acute coronary syndrome (ACS); diabetes mellitus (DM); ongoing ACS+DM; and PDAC. Samples of platelet poor plasma from 204 subjects were obtained from University of Cincinnati Cancer Institute’s Tumor Bank and Diagnostica Stago collections, blood specimens drawn from emergency room visitors at four medical centers in the US, and George King Bio-Medical, Inc. ACS was defined by positive troponin levels; DM was self-identified. Blood was drawn into tubes containing heparin (ASC, DM, ACS+DM), acid citrate dextrose (PDAC), or sodium citrate (healthy subjects), centrifuged at 3000 rpm for 15 min at 4°C, and stored at -80°C until use. Blinded asTF ELISA was performed on plasma samples as per the prototype-tailored procedure (Diagnostica Stago). Samples with asTF concentrations ≥0.2 ng/mL were deemed positive. asTF concentrations are presented as mean±SD. Kruskal-Wallis one-way analysis of variance was used to compare differences in concentration levels between the cohorts; Chi-Square and/or Fisher’s exact test were used to compare proportions. asTF protein was detectable in the plasma of 3/19 (15.8%) subjects in the healthy cohort (CORE Set 50, George King Bio-Medical); 7/38 (18.4%) in the no ACS/no DM cohort (emergency room visitors’ control group); 2/40 (5%) in the DM cohort; 5/39 (12.8%) in the ACS cohort; 4/25 (16.0%) in the ACS/DM cohort; and 20/43 (46.5%) in the PDAC cohort; the proportion of PDAC patients positive for asTF was significantly higher compared to that in all other cohorts (p<0.01, Chi-Square test). The mean asTF concentrations in the cohorts were as follows: PDAC, 0.403±0.912 ng/mL; healthy subjects, 0.169±0.596 ng/mL; emergency room visitors’ control group, 0.159±0.357 ng/mL; ACS, 0.0925±0.258 ng/mL; DM, 0.0423±0.19 ng/mL; ACS+DM, 0.208±0.642 ng/mL; the differences between mean asTF levels in the cohorts did not reach significance. Next, we evaluated asTF’s potential as a biomarker to help detect a more aggressive PDAC phenotype. Among the 43 patients with PDAC, 36 were initially deemed resectable and 7 unresectable due to the presence of metastatic disease as determined by diagnostic screening; following exploratory laparoscopic surgery, 11 out of 36 patients initially deemed resectable were deemed unresectable due to the presence of metastatic disease. When the entire PDAC cohort was split into bona fide resectable (25) and unresectable (18) sub-cohorts, positivity for asTF was significantly more prevalent in the unresectable sub-cohort irrespective of the results of initial evaluation and/or pre-operative CA19-9 levels (asTF ≥0.2 ng/mL: 13 unresectable and 7 resectable patients; asTF<0.2 ng/mL: 5 unresectable and 18 resectable patients, p=0.0059, Fisher’s exact test). We here report that asTF at levels ≥0.2 ng/mL occurs more frequently in the plasma of patients with PDAC compared to healthy subjects and/or individuals with ACS, DM, and ACS/DM. Further, PDAC patients whose plasma asTF levels were equal to or exceeded 0.2 ng/mL had a significantly lower chance to qualify for tumor resection, irrespective of initial pre-surgical diagnostic evaluation. asTF may thus comprise a novel marker of aggressive PDAC phenotype with potential utility in patient stratification, warranting prospective evaluation of larger PDAC patient cohorts. Disclosures No relevant conflicts of interest to declare.

Microparticle-Associated Tissue Factor: A Molecular Link Between Coagulation Activation, Inflammation and Disease Progression in Early-Stage Prostate Cancer?

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3813-3813
Author(s):  
Katja Haubold ◽  
Michael Rink ◽  
Brigitte Spath ◽  
Ali Amirkhosravi ◽  
John L. Francis ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Monoclonal Antibody ◽  
Disease Progression ◽  
Tissue Factor ◽  
Tumor Cells ◽  
Thrombin Generation ◽  
Early Stage ◽  
Plasma Samples ◽  
Coagulation Activation ◽  
D Dimer

Abstract Activation of coagulation and inflammation is a characteristic finding in patients with advanced malignancies, including prostate cancer. Tissue factor (TF), a molecule involved in hemostasis, thrombosis and pro-inflammatory signaling pathways, is over-expressed on tumor cells and cells of the tumor microenvironment (i.e. endothelial cells, fibroblasts and tissue macrophages). Moreover, the enhanced release of TF into plasma in association with sub-cellular membrane vesicles, so-called plasma microparticles (MPs), has recently been associated with key events in molecular oncogenesis and cancer progression. In this study, we measured TF-specific procoagulant activity (PCA) of plasma MPs in 58 consecutive patients with clinically localized prostate cancer (mean age, 64±5 years) to explore its potential as a prognostic marker in this tumor entity. MPs were isolated from pre-operative plasma samples by sequential high-speed centrifugation for 1 h at 16,100 × g. TF-specific PCA of plasma MPs was quantified using a highly sensitive one-stage clotting assay in the presence and absence of inhibitory TF monoclonal antibody and calibration of clotting times against serial dilutions (1:10–1:105) of lipidated recombinant human full-length TF (rhTF1–263), showing a linear correlation in a log-log plot with R2>0.99. The lower detection limit of this assay for rhTF1–263 (33 kDa) was <5 pg/ml (<150 fM), and the intra- and inter-assay coefficients of variation were 7.3% and 5.4%, respectively. Total numbers of TF-positive MPs were measured by single-color flow cytometry using PE-conjugated TF monoclonal antibody (HTF-1) and microspheres for size calibration (1 μm) and sample flow standardization. TF antigen was quantified in plasma by ELISA. Calibrated automated thrombography (CAT) was used to monitor thrombin generation in platelet-free plasma samples over a 2-h period without the addition of exogenous TF or phospholipids. Intravascular coagulation activation was assessed by measuring plasma D-dimer. All assay systems were validated using MPs spontaneously shed from prostate cancer cell lines (PC-3, LNCaP and DU145) or from whole blood monocytes after challenge with endotoxin. Based on plasma fibrinogen and C-reactive protein levels, patients were stratified into those with (n=26) and those without (n=32) laboratory evidence of an acute-phase reaction (APR). Compared to healthy male controls (n=20), patients had significantly increased levels of both D-dimer (0.46±0.19 vs. 0.21±0.05 mg/l) and TF-specific PCA of plasma MPs (563±301 vs. 292±74 U/ml) (P<0.001). Among patients, laboratory evidence of an APR was associated with a significant increase in MP-associated TF PCA (699±351 vs. 452±196 U/ml) (P=0.001). Overall, we found a significant correlation between MP-associated TF PCA and plasma D-Dimer (P=0.015), suggesting that plasma MPs contributed to in-vivo coagulation activation in a TF-dependent manner. CAT also revealed significantly increased thrombin generation in patient compared to control plasmas, as indicated by a shortening in lag phase (25±4 vs. 29±5 min) and an increase in both peak thrombin generation (184±76 vs. 127±71 nM) and the endogenous thrombin potential, defined as the area under the thrombin generation curve (3576±509 vs. 2980±562 nM*min) (P<0.01). Importantly, TF-specific PCA of plasma MPs correlated neither with absolute numbers of TF-positive MPs nor with plasma TF antigen, suggesting that a substantial and variable fraction of the total plasma TF pool circulated as an inactive variant. Interestingly, systemic levels of IL-8, an inflammatory cytokine involved in TF/FVIIa-dependent, PAR-2-mediated pro-migratory signaling pathways in tumor cells and shown to be of biological relevance in advanced, hormone-refractory prostate cancer, were elevated in patients compared to controls (9±11 vs. 4±6 pg/ml) (P<0.01). In summary, our findings suggest that TF-specific PCA of plasma MPs contributes to intravascular coagulation activation in patients with early-stage prostate cancer and may represent an important molecular link between hypercoagulability, inflammation and disease progression. The above-described assay for the quantification of MP-associated TF PCA could thus be of prognostic value in the risk stratification of patients with localized prostate cancer with respect to thromboembolic complications and/or tumor recurrence.

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