Tissue Factor-bearing Research Articles

Platelets provide robustness of spatial blood coagulation to the variation of initial conditions

Activated platelets provide phospholipid surface and secrete coagulation factors, enhancing blood clotting. We investigated the role of platelets in the regulation of blood coagulation spatial dynamics. We activated blood clotting with tissue factor-bearing (TF) surface in platelet-rich plasma (PRP) or platelet-free plasma (PFP). When blood coagulation was initiated by high TF density, clot growth rate (V) in PRP (2 × 105/μL platelets) was only 15 % greater than in PFP. Spatial distribution of thrombin in PRP had a peak-like shape in the area of the fibrin clot edge, while in PFP thrombin was distributed in the shape of descending plateau. Platelet inhibition with prostaglandin E1 or cytochalasin D made spatial thrombin distribution look like in the case of PFP. Inhibition of blood coagulation by natural endogenous inhibitor heparin was diminished in PRP, while the effect of the exogenous or artificial inhibitors (rivaroxaban, nitrophorin, hirudin) remained undisturbed in the presence of platelets. Ten times decrease of the TF surface density greatly depressed blood coagulation in PFP. In PRP only clotting initiation phase was, while the propagation phase remained intact. Coagulation factor deficiency greatly reduced amount of thrombin and decreased V in PFP rather than in PPR. Thus, platelets were redundant for clotting in normal plasma under physiological conditions but provided robustness of the coagulation system to the changes in initial conditions.

Significance of Tissue Factor-Bearing Microparticle Procoagulation Activity and Antithrombin Ⅲ Detection in Thalassemia Patients

To explore whether the high risk factors possibly leading to hypercoagulative status and thrombosis exist in Thalassemia patients of Guangxi region through detecting plasma tissne factor-bearing microparticles (TF+MP), procoagulatima activity, coagulation and anticoagulation function, fibrinolytic function, endothelial function and platelet count. The TF+MP procoagulation activity was detected by chromogenic saubstract method, the levels of tissue factors (TF), tissue factor pathway inhibitor(TFPI), protein C (PC), protein S (PS), antithrombin Ⅲ(AT-Ⅲ), tissue plasminogen activator (tPA), thrombin-activated fibrinolysis inhibitor (TAFI), soluble E-selectin (sE-sel), intercellular adhesion molecule-1 (ICAM-1) and thrombomodulin (TM) were detected by ELISA in thalassemia group (n=71) and control group (n=20 heathy persons). Compared with control group, the AT-Ⅲ level decreased in β-thalastemia major group (TM) (P<0.05), the AT-Ⅲ level in TM group independeutly posstiody correlated with plt count (r=0.37, P<0.05); the levels of TF and sICAM in α-thalassenia intermediate group (TA) significantly decteased (P<0.05), the procoagulatim activity of TF+MP in β-thalassemia intermediate group (TI) increased sngnificantly (P<0.05)， moreover positively corretated with AT-Ⅲ level (r=0.77, P<0.05). The TF and sICAM-1 levels in normal liver functim group of Thalassemia patients were lower tham those in control group (P<0.01 and P<0.05, respectively), the TF+MP activity between normal and abnormal liver function was significantly different (P<0.05), while there were no significant difference in other correspoding indexes beween thalassemia group and control group as well as between each thalassemia groups. The damage of liver function and reduction of anticoagylation substances exist in patients with β-thalassenia major in Guangxi region, the procoagulation activity of plasma TF+MP in patients with β-thalassemia intermedia abnormally increases. All the above-mentioned factors may increase the risk of high coagulation status or thrombosis is thalassemia patients, the decrease of TF and SICAM-1 levels in patients with α-thalassemia intermedia may be factor against thrombosis.

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.

Postprandial Increase in Blood Plasma Levels of Tissue Factor-Bearing (and Other) Microvesicles Measured by Flow Cytometry: Fact or Artifact?

Tissue factor (TF)–bearing microvesicles (MVs) and exosomes may play a role in hemostasis and thrombosis. MVs may be quantified by flow cytometry (FC)–based detection of phosphatidylserine (PS)-positive submicron particles carrying specific antigens, although interference from lipoproteins complicates this approach. In this study, we evaluated the effect of food intake on blood levels of TF-bearing particles measured by FC and small extracellular vesicles (EVs) measured by a protein microarray–based test termed EV Array. Platelet-free plasma (PFP) was obtained from 20 healthy persons in the fasting state and 75 minutes after consumption of a meal. Postprandial changes in the concentration of PS-positive particles, including subgroups binding labeled antibodies against TF, CD41, CD146, and CD62E, respectively (FC), small EVs (EV Array), and TF antigen and procoagulant phospholipids (PPLs) were measured. Furthermore, we tested the effect on FC results of in vitro addition of lipoproteins to fasting PFP. We found significantly increased plasma concentrations of PS-positive particles and all examined subgroups postprandially, while no changes in small EVs, PPL, or TF antigen levels were found. Levels of all types of particles measured by FC were also elevated by lipoprotein spiking. In conclusion, meal consumption as well as in vitro addition of lipoproteins to fasting plasma induces increased levels of PS-positive particles as measured by FC, including TF-positive subtypes and subtypes exposing other antigens. While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins.

C0282: Tissue Factor-Bearing Microparticles (MPS-TF) in the Cord Blood of Preterm Neonates

C0282: Tissue Factor-Bearing Microparticles (MPS-TF) in the Cord Blood of Preterm Neonates

Angiotensin II induces the generation of procoagulant microparticles by human mononuclear cells via an angiotensin type 2 receptor-mediated pathway

IntroductionMicroparticles are small vesicles shed by cells upon activation and during apoptosis which participate in physiologically relevant phenomena, including blood coagulation. Intracellular calcium mobilization is one of the mechanisms of microparticle generation during cell activation. Because the renin-angiotensin system has been proposed as a link between hypertension and increased thrombotic risk, we investigated whether angiotensin II upregulates the generation of procoagulant microparticles by human mononuclear cells. Materials and MethodsHuman mononuclear cells were exposed to angiotensin II for 15min. Intracellular calcium concentration was assessed by a Fluo 4 based kit. The supernatants were analyzed for both microparticle content, with a commercially available kit based on phosphatidylserine analysis, and microparticle-associated tissue factor, with a one-stage clotting assay. ResultsIntracellular calcium concentration is increased upon exposure of mononuclear cells to angiotensin II. Incubation with angiotensin II stimulates microparticles release; microparticle-associated tissue factor is also upregulated. The effect is inhibited by an angiotensin receptor type 2 antagonist (PD123319) and not by two angiotensin type 1 antagonists (Losartan and Olmesartan). ConclusionsAngiotensin receptor 2-mediated upregulation of tissue factor-bearing, procoagulant microparticle generation represents a novel mechanism linking the renin-angiotensin system to thrombosis.

State-of-the-Art and Current Controversies in the Detection and Quantitation of Tissue Factor-Bearing Microparticles

Abstract SCI-11 Microparticles are submicron vesicles that circulate in blood and are derived from a variety of vascular cells. Microparticles expressing tissue factor have been shown in animal thrombosis models to contribute to thrombus formation in vivo, which has led to the evaluation of tissue factor bearing microparticle populations in a variety of thrombotic disorders including malignancy, sepsis, and coronary thrombosis. Central to the evaluation of tissue factor-bearing microparticle populations in health and disease is the establishment of accurate means for qualitative or quantitative assessment of circulating microparticles. Whilst there is considerable interest in evaluating whether pathologic disturbances in tissue factor-bearing microparticles contribute to thrombosis, analytic techniques for microparticle measurement remain in the validation stage. The method most commonly employed for the detection of tissue factor-bearing microparticles is light-scatter flow cytometry, which relies on the sizing of particles based on light-scatter profile. Light scatter, however, is also influenced by variables independent of particle size, including the wavelength of incident light, particle shape, presence of surface absorptive material, and relative refractive indices of particles and suspension medium. A number of potentially more sensitive methodologies are currently under investigation such as impedance-based flow cytometry, quasi-elastic light scattering based on Brownian movement, and atomic force microscopy. Alternatively, assays have been developed that measure tissue factor antigen or activity following isolation of microparticles from plasma or whole blood but without determination of particle size. Absent a gold standard for the measurement of tissue factor-bearing microparticle populations by which these approaches can be validated, the development of accurate methodology will require inter-assay and intra-assay precision as well as validation of clinical relevance in human studies. Disclosures: No relevant conflicts of interest to declare.

Tissue Factor–Bearing Microparticles and Thrombus Formation

Blood microparticles are vesicular structures with a diameter of 100 to 1000 nm that are present in the blood of normal subjects and in patients with various diseases. These microparticles are derived from cells that circulate in the blood and cells associated with the blood vessel wall. Microparticle membranes retain the protein receptors of their parent cells and may retain RNAs and other cytosolic content. On the basis of surface protein expression, microparticles are known to be derived from platelets, granulocytes, monocytes, endothelial cells, smooth muscle cells, and tumor cells. Only a subpopulation of these microparticles expresses tissue factor.

Tumor-Derived Tissue FactorBearing Microparticles Are Associated With Venous Thromboembolic Events in Malignancy

Despite the strong association between malignant disease and thromboembolic disorders, the molecular and cellular basis of this relationship remains uncertain. We evaluated the hypothesis that tumor-derived tissue factor-bearing microparticles in plasma contribute to cancer-associated thrombosis. We developed impedance-based flow cytometry to detect, quantitate, and size microparticles in platelet-poor plasma. We evaluated the number of tissue factor-bearing microparticles in a cohort of cancer patients of different histologies (N = 96) and conducted a case-control study of 30 cancer patients diagnosed with an acute venous thromboembolic event (VTE) compared with 60 cancer patients of similar age, stage, sex, and diagnosis without known VTE, as well as 22 patients with an idiopathic VTE. Tissue factor-bearing microparticles were detected in patients with advanced malignancy, including two thirds of patients with pancreatic carcinoma. Elevated levels of tissue factor-bearing microparticles were associated VTE in cancer patients (adjusted odds ratio, 3.72; 95% confidence interval, 1.18-11.76; P = 0.01). In cancer patients without VTE, a retrospective analysis revealed a 1-year cumulative incidence of VTE of 34.8% in patients with tissue factor-bearing microparticles versus 0% in those without detectable tissue factor-bearing microparticles (Gray test P = 0.002).The median number of tissue factor-bearing microparticles in the cancer VTE cohort (7.1 x 10(4) microparticles/microL) was significantly greater than both the idiopathic VTE and cancer-no VTE groups (P = 0.002 and P = 0.03, respectively). Pancreatectomy in three patients eliminated or nearly eliminated these microparticles which coexpressed the epithelial tumor antigen, MUC-1. We conclude that tumor-derived tissue factor-bearing microparticles are associated with VTE in cancer patients and may be central to the pathogenesis of cancer-associated thrombosis.

Development of Venous Thromboembolic Events in Cancer Patients with Elevated Levels of Tissue Factor-Bearing Microparticles

Cancer cells shed procoagulant vesicles containing tissue factor, and these tissue factor-bearing microparticles (TFMP) may play a role in thrombus formation in vivo. Using impedance-based flow cytometry to quantify microparticles and a high affinity monoclonal antibody specific for tissue factor, we previously demonstrated the presence of tissue factor-bearing microparticles in platelet-poor plasma in cancer patients. In this case control study, tissue factor-bearing microparticles represented a 4-fold risk factor for venous thromboembolic events (VTE) in cancer patients with acute VTE compared to age, stage, sex, diagnosis-matched controls with cancer but without acute VTE. To further assess the relationship between tissue factor-bearing microparticles and VTE, we performed a retrospective analysis of deep venous thrombosis or pulmonary emboli diagnosed in cancer patients initially enrolled without evidence of VTE. All radiographic reports for the cancer-no VTE group in the 2 years following enrollment were analyzed by a reviewer blinded to microparticle status. Only documented evidence of a new proximal extremity deep vein thrombosis or pulmonary embolism was included in the analysis. The TFMP and no-TFMP groups did not differ significantly for age, sex, active cancer treatment, smoking status, diabetes, or the presence of metastatic disease at time of enrollment. Sixteen of the 60 patients in this group had measurable tissue factor-bearing microparticles, 4 (4/16; 25%) of which subsequently developed radiographic evidence of VTE within 12 months of enrollment. No thrombotic events were recorded among the 44 patients without detectable tissue factor-bearing microparticles within the initial 12 months; however, one patient developed a pulmonary embolism 17 months following enrollment. Identifying death without VTE as a competing risk, the one-year estimate of the rate of VTE in cancer patients with detectable tissue factor-bearing microparticles was 34.8%; among the same group without detectable tissue factor-bearing microparticles, the 1-year rate was 0% (Log Rank p-value=0.002). The presence of tissue factor-bearing microparticles in cancer patients initially thrombosis-free predicted a 7-fold increased risk of thrombosis over cancer patients who were negative for tissue factor-bearing microparticles (OR 7.00, 95% CI 0.85–82.74, P=0.02). These tissue factor-bearing microparticles appear to be derived from the underlying malignancy since samples analyzed from patients with pancreatic cancer demonstrated co-expression of both tissue factor and MUC-1, a transmembrane glycoprotein overexpressed in epithelial malignancies. These data further support the role of tissue factor-bearing microparticles in the pathogenesis of cancer-associated thrombosis and as a biomarker for the prediction of cancer patients at risk of thrombosis. A prospective clinical study, currently being initiated, is required to evaluate this biomarker for the prediction of VTE risk in cancer patients and the utility of thromboprophylaxis in patients with elevated numbers of tissue factor-bearing microparticles.

Tissue Factor–Bearing Microparticles and Cancer

Blood-borne tissue factor (TF)-bearing microparticles have been shown to play an important role in thrombus propagation in experimental models. The pathophysiologic role of these microparticles is being investigated in several prothrombotic conditions including cancer-associated thrombosis. Tumor cells are known to shed TF-bearing microparticles in vitro, and circulating TF-bearing microparticles can be measured in plasma samples from patients with advanced cancer. We are currently using an impedance-based flow cytometer to accurately size and enumerate TF-bearing microparticles to explore the association between cancer thrombosis and elevations in circulating TF-bearing microparticles.

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

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.

Trousseau's Syndrome Revisited: Tissue Factor-Bearing Microparticles in Pancreatic Cancer.

Thromboembolic disease is a major complication of malignant disease, and pancreatic cancer ranks among the malignancies associated with the highest rates of thrombosis. Using laser injury in a mouse model of thrombosis, we previously demonstrated that tissue factor-bearing microparticles circulating in normal plasma accumulate in the developing thrombus through an interaction mediated by P-selectin and PSGL-1. Microparticle tissue factor contributes significantly to fibrin propagation within the thrombus. Based upon this observation, we evaluated the hypothesis that elevated numbers of tissue factor-bearing microparticles might be one of the causes of cancer-associated thrombosis. Microparticles are cell-derived vesicular structures under 1000 nm in diameter. Since the light scattering methodology used in commercial flow cytometers cannot determine particle size when the diameter of the particle (200–1000 nm) is the same order of magnitude as the wavelength of the incident light employed (488 nm), we applied a novel instrumentation to determine the size, size distribution, and concentration of tissue factor-bearing microparticles. An NPE Systems Quanta flow cytometer, using impedance to measure particle size, was extensively modified for this microparticle application. Application of 780 nm fluorescent calibration beads yielded a major population from 740 to 820 nm. Application of 520 nm fluorescent beads yielded a major population from 500 to 540 nm. Using a high affinity antibody to tissue factor, tissue factor-bearing microparticles were measured in platelet-poor plasma derived from normal subjects and subjects with surgically unresectable or metastatic pancreatic cancer. Tissue factor bearing microparticles were detected in significantly more subjects with pancreatic cancer (12 of 17, 70%) than healthy individuals in whom tissue factor-bearing microparticles remained below the level of detection in all but a single individual (1 of 11, P=0.005). By comparison, tissue factor-bearing microparticles were not detected in subjects with advanced stage (III or IV) non-small cell lung cancer (N=9). In subjects with advanced pancreatic cancer, the mean concentration of tissue factor bearing microparticles was 444,000 particles/microliter with a mean diameter ranging between 371 nm to 727 nm. The two patients with pancreatic cancer in this study who suffered a recent lower extremity venous thrombosis had high measurable tissue factor-bearing microparticles levels with a mean concentration of 975,000 particles/microliter. These results indicate that tissue factor-bearing microparticles are present in the plasma of many patients with advanced pancreatic cancer and suggest that tissue factor-bearing microparticles may be central to the pathogenesis of cancer-associated thrombosis.

Factor Xa-A pleuripotential protease.

A current view of vascular thrombosis emphasizes the importance of cellular surface biochemistry and the integrated contribution of platelets, monocytes and endothelial cells that contribute to atherothrombosis. Initiation of coagulation occurs on tissue-factor bearing cells, while amplification (or priming) requires activation of platelets and coagulation proteases. The final phase, propagation is determined by thrombin generation on platelet surfaces [1] (Fig. 1). The cell-based model of vascular thrombosis highlights specific phases or biochemical stages rather than a more traditional view of independent coagulation pathways or cascades. Accordingly, tissue factor is considered the key element for initiation of thrombosis, wherein its ability to complex with factor (f) VIIa and activate f X ultimately causes thrombin generation. Although thrombin is a pivotal enzyme in coagulation and thrombosis, the importance of fXa and its diverse effects on thrombin generation, inflammatory processes, smooth muscle cell proliferation and endothelial cell activation warrants serious consideration as a potential target for pharmacologic intervention.