Tissue Factor Microparticles Research Articles

Sars-Cov-2 Spike Protein Triggers Inflammasome-Mediated Tissue Factor Activity

Introduction: An exaggerated inflammatory response both locally and systemically mediated by innate immune signaling is a near universal feature of severe COVID-19 illness and has been associated with multi-organ damage and thrombosis. As part of an antiviral response, activation and formation of the intracellular NLRP3 inflammasome leads to expression of proinflammatory IL-1 family cytokines. In macrophages, the NLRP3 inflammasome is linked to the release of tissue factor (TF) bearing microvesicles. Multiple studies provide evidence of NLRP3 inflammasome activation in COVID-19 including increased IL-1β in monocytes, sera, and bronchoalveolar lavage fluid from COVID-19 patients. Patient monocytes bear features of inflammasome activation, which correlate with disease severity (Potere et al. Cardiovascular Research 2023). Given the central role of thromboinflammation in COVID-19, we explored the ability of various coronavirus spike proteins to increase TF activity in a monocytic cell line. Mechanistically, we sought to explore the role of the NLRP3 inflammasome in SARS-CoV-2 spike protein triggered tissue factor generation. Methods: The SARS-CoV-2 α variant S1 subunit (S1α) and Omicron variant BA.1.1.529 S1 (S1O) recombinant proteins were obtained from Raybiotech and OC43, a non-COVID respiratory human coronavirus, recombinant spike protein (OC43 S) from Sino Biological. THP-1 cells at a concentration of 2x10 6/mL were incubated with spike proteins at 37°C in RPMI, supplemented with 10% fetal bovine serum. A specific NLRP3 inflammasome inhibitor (MCC950) was incubated with select samples for 1 hour following addition of the spike protein. The cells were harvested, subjected to RNA extraction and cDNA synthesis by reverse transcription. TF, NLRP3, caspase (CASP)-1, and IL-1β relative mRNA expression was quantified using RT-PCR and normalized to GADPH expression. For quantifying microparticle TF activity, cells were incubated with the spike proteins for 6 hours then microparticles were isolated from the supernatant after centrifugation at 20,000 g for 30 minutes at 4°C, washed and TF activity was measured using a two-stage chromogenic assay kit (Abcam). A monoclonal anti-TF antibody (Santa Cruz) was used to confirm specificity for TF generation. Experiments were performed in triplicate. Data is presented as mean ± standard deviation. The student t test was used to assess the difference between unpaired groups with a value of p <.05 considered statistically significant. Results: Our previous data showed that S1α, but not S1O or OC43 S, leads to increased TF mRNA expression in THP-1 cells in a dose dependent manner. Here we show TF microparticle activity was increased with S1α (20 µg/mL) as compared to incubation of THP-1 cells with S1O or OC43 spike proteins (20 µg/mL), which were comparable to the activity of cells alone ( Figure 1). After treatment with S1α (20 µg/mL) for 1 hour, we found increased expression of TF (10-fold), NLRP3 (2-fold), CASP-1 (3-fold), and IL-1β (3.5-fold). Treatment with S1O and OC43 S did not lead to a significant change in relative mRNA expression of TF, NLRP3, CASP-1, and IL-1β. Following incubation with the S1α spike protein for 1 hour, cells were re-plated in fresh media containing 10 µM MCC950. Treatment with MCC950 significantly reduced expression of TF, NLRP3, CASP-1, and IL-1β ( Figure 2). Conclusion: We confirmed that TF expression induced by the S1α spike protein leads to an increase in TF activity, as assessed by microparticle generation. The NLRP3 inflammasome is a mediator of coagulopathy induced by the spike protein, and TF, CASP-1, and IL-1β expression are abrogated after specific NLRP3 inflammasome inhibition. TF activity and inflammation induced by the coronavirus spike proteins in vitro correlates with the respective clinical phenotype and disease severity of the variants. This suggests that the effects of SARS-CoV-2 on TF and inflammasome activation derive in part from the spike protein, and these assays may be useful in predicting the effects of spike protein variants.

Intradialytic cycling does not exacerbate microparticles or circulating markers of systemic inflammation in haemodialysis patients

PurposePatients receiving haemodialysis (HD) display elevated circulating microparticle (MP) concentration, tissue factor (TF) expression and markers of systemic inflammation, though regular intradialytic cycling (IDC) may have a therapeutic effect. This study investigated the impact of regular, moderate-intensity IDC on circulating MPs and inflammatory markers in unit-based HD patients.MethodsPatients were cluster-randomised to intervention (n = 20, age: 51.4 ± 18.1 years, body mass: 77.6 ± 18.3 kg, mean ± SD) or no-exercise control (n = 20, 56.8 ± 14.0 years, 80.5 ± 26.5 kg). Intervention participants completed 30 min of moderate intensity (rating of perceived exertion [RPE] of 12–14) IDC, thrice weekly for 6 months. Pre-dialysis venous blood samples were obtained at 0, 3 and 6 months. Circulating MP phenotypes, cytokines, chemokine and MP TF expression were quantified using flow cytometry and cytometric bead array assays.ResultsDespite high exercise compliance (82%), no IDC-dependent effects were observed for any MP, cytokine or chemokine measure (p ≥ 0.051, ηρ2 ≤ 0.399) other than TNF-α (p = 0.001, ηρ2 = 0.186), though no significance was revealed upon post hoc analysis.ConclusionSix months of regular, moderate-intensity IDC had no effect on MPs, cytokines or chemokines. This suggests that the exercise did not exacerbate thrombotic or inflammatory status, though further functional assays are required to confirm this.Trial registrationISRCTN1129707, prospectively registered on 05/03/2015.

Air Bubbles Activate Complement and Trigger Hemostasis and C3-Dependent Cytokine Release Ex Vivo in Human Whole Blood.

Venous air embolism, which may complicate medical and surgical procedures, activates complement and triggers thromboinflammation. In lepirudin-anticoagulated human whole blood, we examined the effect of air bubbles on complement and its role in thromboinflammation. Whole blood from 16 donors was incubated with air bubbles without or with inhibitors of C3, C5, C5aR1, or CD14. Complement activation, hemostasis, and cytokine release were measured using ELISA and quantitative PCR. Compared with no air, incubating blood with air bubbles increased, on average, C3a 6.5-fold, C3bc 6-fold, C3bBbP 3.7-fold, C5a 4.6-fold, terminal complement complex sC5b9 3.6-fold, prothrombin fragments 1+2 (PTF1+2) 25-fold, tissue factor mRNA (TF-mRNA) 26-fold, microparticle tissue factor 6.1-fold, β-thromboglobulin 26-fold (all p < 0.05), and 25 cytokines 11-fold (range, 1.5-78-fold; all p < 0.0001). C3 inhibition attenuated complement and reduced PTF1+2 2-fold, TF-mRNA 5.4-fold, microparticle tissue factor 2-fold, and the 25 cytokines 2.7-fold (range, 1.4-4.9-fold; all p < 0.05). C5 inhibition reduced PTF1+2 2-fold and TF-mRNA 12-fold (all p < 0.05). C5 or CD14 inhibition alone reduced three cytokines, including IL-1β (p = 0.02 and p = 0.03). Combined C3 and CD14 inhibition reduced all cytokines 3.9-fold (range, 1.3-9.5-fold; p < 0.003) and was most pronounced for IL-1β (3.2- versus 6.4-fold), IL-6 (2.5- versus 9.3-fold), IL-8 (4.9- versus 8.6-fold), and IFN-γ (5- versus 9.5-fold). Antifoam activated complement and was avoided. PTF1+2 was generated in whole blood but not in plasma. In summary, air bubbles activated complement and triggered a C3-driven thromboinflammation. C3 inhibition reduced all mediators, whereas C5 inhibition reduced only TF-mRNA. Combined C5 and CD14 inhibition reduced IL-1β release. These data have implications for future mechanistic studies and possible pharmacological interventions in patients with air embolism.

Inflammatory Biomarkers in the Short-Term Prognosis of Venous Thromboembolism: A Narrative Review.

The relationship between inflammation and venous thrombosis is not well understood. An inflammatory response may be both the cause and consequence of venous thromboembolism (VTE). In fact, several risk factors of VTE modulate thrombosis through inflammatory markers. Acute pulmonary embolism (PE) is burdened by a remarkable mortality rate, up to 34% in severely ill patients presenting with hemodynamic instability. Initial mortality risk stratification is based on hemodynamic instability. Patients with a situation of hemodynamic stability require immediate further risk assessment based on clinical, imaging, and circulating biomarkers, as well as the presence of comorbidities. Some inflammatory biomarkers have shown potential usefulness in the risk stratification of patients with VTE, especially acute PE. C-reactive protein on admission is associated with 30-day mortality and bleeding in VTE patients. P-selectin is associated with right ventricle dysfunction in PE patients and might be associated with VTE recurrences and the extension of thrombosis. Tissue factor microparticles are associated with VTE recurrence in cancer-associated thrombosis. Other inflammatory biomarkers present scarce evidence (inflammatory cytokines, erythrocyte sedimentation rate, fibrinogen, leukocyte count). In this manuscript, we will review the prognostic role of different inflammatory biomarkers available both for clinical practice and research in VTE patients.

Doxorubicin Enhances Procoagulant Activity of Endothelial Cells after Exposure to Tumour Microparticles on Microfluidic Devices

The majority of cancer patients undergoing chemotherapy have a significantly increased risk of venous thromboembolism via a mechanism not yet fully elucidated but which most probably involves tumour microparticles (MP) combined with damaged/activated endothelium. Tumour cell lines (ES-2 and U87) were cultured as 3D spheroids and transferred to biochips connected through to a second chip precultured with an endothelial cell layer (human umbilical vein endothelial cells [HUVECs]). Media were introduced with and without doxorubicin (DOX) to the spheroids in parallel chips under constant flow conditions. Media samples collected pre- and post-flow through the biochip were analysed for tissue factor microparticles (TFMP) and procoagulant activity (PCA). HUVECs were also harvested and tested for PCA at a constant cell number. TFMP levels in media decreased after passing over HUVECs in both conditions over time and this was accompanied by a reduction in PCA (indicated by a slower coagulation time) of the media. The relationship between PCA and TFMP was correlated (r = −0.85) and consistent across experiments. Harvested HUVECs displayed increased PCA when exposed to tumour spheroid media containing TFMP, which was increased further after the addition of DOX, suggesting that the TFMP in the media had bound to HUVEC cell surfaces. The enhanced PCA of HUVECs associated with the DOX treatment was attributed to a loss of viability of these cells rather than additional MP binding. The data suggest that tumour MP interact with HUVECs through ligand-receptor binding. The model described is a robust and reproducible method to investigate cytotoxic agents on tumour spheroids and subsequent downstream interaction with endothelial cells.

Biomarkers of Hemostatic Activation and Inflammation Are Associated with Altered Coagulation Parameters in Sepsis Patients

Background : Sepsis is characterized by a simultaneous activation of inflammation and hemostasis in response to microbial infection. This systemic inflammatory response is due to the release of pro-inflammatory cytokines, pro-coagulants and adhesion molecules from immune cells and/or damaged endothelial tissue. Simultaneous activation of coagulation and fibrinolysis leads to consumption coagulopathy and severe vascular dysfunction. Profiling of biomarkers of hemostatic activation and inflammation along with the measurement of coagulation parameters has provided useful data in the understanding of the pathogenesis of sepsis. This study was designed to profile biomarkers of hemostatic activation, inflammation and endothelial dysfunction along with the measurement of coagulation parameters in a defined clinically confirmed sepsis population in conjunction with an IRB approved clinical trial. Materials &amp; Methods: Citrated blood samples were collected from sepsis patients with suspected or confirmed infection, and organ dysfunction as defined by a SOFA ³ baseline. Plasma samples from septic shock patients were collected in citrated tubes within 72 hours of ICU admission under an IRB approved protocol in conjunction with an ongoing trial at the University of Utah and Veteran's Affair FFC Health Care System VAMC. Normal controls were comprised of commercially available 25 male and 25 female citrated plasma samples (George King Biomedical, Overland Park, Kansas City). Such biomarkers as CRP, PAI-1, D-Dimer, vWF and microparticle tissue factor complex (MP-TF) were measured using a commercially available sandwich ELISA methods. Nitric oxide (NO) levels were measured using a commercially available Griess reaction based colorimetric method. PT/INR, aPTT and fibrinogen measurements were based on clot based assays. All results were compiled as mean ± SD and SEM. Correlation analysis was carried out to determine relevance between different parameters. Results: Most of the biomarkers of hemostatic activation and inflammation were elevated in patients with sepsis as shown on table 1a, CRP (66 fold) and D-Dimer (23 fold) showed the most pronounced increase in comparison to the control. Other parameters also showed increase levels including MP-TF (5.3 fold), PAI-1 (3.5 fold), vWF (3.1 fold) and NO (3.0 fold). Clotting parameters such as PT/INR (2.0 fold), aPTT (2.5 fold) and fibrinogen (2.0 fold) were also significantly elevated in the sepsis patients. These differences were significant (p value ≤0.0009) for all of the parameters except for NO (p value 0.0937) and fibrinogen (p value 0.4694). As shown on table 1b, there was no correlation between various biomarkers and fibrinogen in the sepsis patients. Summary &amp; Conclusion: In comparison to the control group, the sepsis patients showed wide variations in all of the parameters investigated in this study. The marked prolongation of PT and aPTT are suggestive of both the extrinsic and intrinsic pathway defects and consumption of clotting factors. The aPTT data showed wider scatter in comparison to PT data. The fibrinogen levels were also elevated and nearly 1/3 of the patients showed &gt;1000 mg/dL levels. The markedly higher level of CRP in the sepsis group are indicative of severe inflammatory response. Marked elevation of D-Dimer is indicative of endogenous fibrin formation and its consumption consistent with activation of secondary fibrinolysis. MP-TF, vWF and PAI-1 were also increased in the sepsis patients suggesting marked endothelial dysfunction. This is consistent with increased NO levels which may be due to induction of iNOS in the endothelial lining of sepsis patients. These results further underscore the multifactorial pathophysiology of sepsis which results in the dysregulation of hemostasis, upregulation of inflammatory responses and generalized endothelialopathy. Profiling of the biomarkers included in this study and coagulation parameters may be helpful in the risk stratification and clinical management of patients with sepsis and related disorders. Disclosures No relevant conflicts of interest to declare.

Coagulation System Disorders and Thrombosis Prophylaxis During Laparoscopic Fundoplications.

The aim of this study was to assess and recommend the optimal deep vein thrombosis (DVT) prophylaxis regimen during and after laparoscopic fundoplication according to the blood coagulation disorders and the rate of DVT in 2 patient groups, receiving different DVT prophylaxis regimens. This was a prospective randomized, single-center clinical study. The study population, 121 patients, were divided into 2 groups: group I received low-molecular-weight heparin 12 hours before the operation; group II received low-molecular-weight heparin only 1 hour before the laparoscopic fundoplication. Both groups received intermittent pneumatic compression during the entire procedure. Bilateral Doppler ultrasound to exclude DVT was performed before the surgery. Venous phase computed tomographic images were acquired from the ankle to the iliac tubercles on the third postoperative day to determine the presence and location of DVT. Hypercoagulation state was assessed by measuring the prothrombin fragment F1+2 (F1+2), the thrombin-antithrombin complex (TAT), and tissue factor microparticles activity (MP-TF) in plasma. The hypocoagulation effect was evaluated by measuring plasma free tissue factor pathway inhibitor (fTFPI). F1+2, TAT, and MP-TF indexes increased significantly, whereas fTFPI levels decreased significantly during and after laparoscopic fundoplication, when molecular-weight heparin was administered 12 hours before the operation. Computed tomography venography revealed peroneal vein thrombosis in 2 group I patients on the third postoperative day. Total postsurgical DVT frequency was 1.65%: 3.6% in group I, with no DVT in group II. Molecular-weight heparin and intraoperative intermittent pneumatic compression controls the hypercoagulation effect more efficiently when it is administered 1 hour before surgery: it causes significant reduction of F1+2, TAT, and MP-TF indexes and significant increases of fTFPI levels during and after laparoscopic fundoplication.

High Tissue Factor Microparticle Level in Major Thalassemic Patients with Pulmonary Hypertension

Objective: To analyze the correlation between tissue factor microparticles (TF-MP) levels and pulmonary hypertension (PH) in adult thalassemic patients. Methods: This study was conducted from September to October 2018, using secondary and primary data. The secondary data consisted of the PH parameter, which was retrieved from a 2017 previous study entitled ‘Clinical Characteristic and Complication due to Iron Overload in Thalassaemic Patients‘in 2017 while the primary data were the TF-MP, which were obtained from the analysis of frozen serum of the same population using ELISA method. The mean pulmonary arterial pressure (mPAP) values were obtained from echocardiography results and PH was defined as mPAP &gt;25 mmHg. Results: Seven (16.7%) major thalassemic patients experienced PH. The median values of TF-MP levels were higher among major thalassemic patients with PH when compared to the non-PH patients (1569 vs 11.5 pg/dL; p=0.023). No significant difference was observed in the median TF-MP levels between subjects with splenectomy and subjects without splenectomy (11.6 vs 12.3 pg/dL; p=0.44). There was also no difference in mPAP values between subjects with splenectomy and subjects without splenectomy (18.0 vs 17.0 mmHg; p=0.663). When the median TF-MP levels among major thalassemic patients were analyzed in terms of correlation with transfusion level, no statistically significant difference was seen between subjects who received sufficient transfusions (≥180 mL/kgbb/year) and those who received insufficient transfusions (&lt;180 mL/kgbb/year) (r= 0.138; p=0.390). Conclusions: There is a positive correlation between the TF-MP levels and PH in adult major thalassemic subjects.

Cholesterol Crystals Induce Coagulation Activation through Complement-Dependent Expression of Monocytic Tissue Factor.

Cholesterol crystals (CC) are strong activators of complement and could potentially be involved in thromboinflammation through complement-coagulation cross-talk. To explore the coagulation-inducing potential of CC, we performed studies in lepirudin-based human whole blood and plasma models. In addition, immunohistological examinations of brain thrombi and vulnerable plaque material from patients with advanced carotid atherosclerosis were performed using polarization filter reflected light microscopy to identify CC. In whole blood, CC exposure induced a time- and concentration-dependent generation of prothrombin fragment 1+2 (PTF1.2), tissue factor (TF) mRNA synthesis, and monocyte TF expression. Blocking Abs against TF abolished CC-mediated coagulation, thus indicating involvement of the TF-dependent pathway. Blockade of FXII by corn trypsin inhibitor had a significant inhibitory effect on CC-induced PTF1.2 in platelet-free plasma, although the overall activation potential was low. CC exposure did not induce platelet aggregation, TF microparticle induction, or TF on granulocytes or eosinophils. Inhibition of complement C3 by CP40 (compstatin), C5 by eculizumab, or C5aR1 by PMX53 blocked CC-induced PTF1.2 by 90% and reduced TF+ monocytes from 18-20 to 1-2%. The physiologic relevance was supported by birefringent CC structures adjacent to monocytes (CD14), TF, and activated complement iC3b and C5b-9 in a human brain thrombus. Furthermore, monocyte influx and TF induction in close proximity to CC-rich regions with activated complement were found in a vulnerable plaque. In conclusion, CC could be active, releasable contributors to thrombosis by inducing monocyte TF secondary to complement C5aR1 signaling.

Elevated Microparticle Tissue Factor Activity Differentiates Patients With Venous Thromboembolism in Anti-neutrophil Cytoplasmic Autoantibody Vasculitis

IntroductionVenous thromboembolism (VTE) is a life-threatening complication of anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis whose mechanism remains incompletely elucidated. We tested the hypothesis that elevated microparticle tissue factor activity (MPTFa) or anti-plasminogen antibodies (anti-Plg) may identify patients at risk for VTE.MethodsIn this prospective study, patients were enrolled during active disease and followed longitudinally. Twelve patients who experienced a VTE (VTEpos) were compared with patients without VTE (VTEneg, n = 29) and healthy controls (HC, n = 70). MPTFa, anti-Plg, interleukin-6, high-sensitivity C-reactive protein (hs-CRP), D-dimer, serum creatinine, and serum albumin were assessed. Fisher’s exact tests and Wilcoxon tests compared categorical and continuous variables, respectively. Cox regression for time to VTE or last follow-up was performed.ResultsVTEpos patients had higher MPTFa (peak median = 14.0, interquartile range = 4.3–36.6) than HC (0, 0–3.5) and VTEneg patients (0, 0–1.4). In time-to-event analysis, MPTFa was associated with VTE when measured during both active disease (hazard ratio [HR]; 95% confidence interval [CI]: 1.04; 1.01–1.08) and remission (1.4; 1.11–1.77). Anti-Plg during remission was also associated with VTE (1.17; 1.03–1.33). Each g/dl decrease of serum albumin was associated with a 4-fold increase in VTE risk (4.4; 1.5–12.9). Adjusting for estimated glomerular filtration rate (eGFR), anti-Plg during remission remained significantly associated with VTE.ConclusionElevated MPTFa and increased anti-Plg in remission are strong indicators of VTE independent of renal function. Association of anti-Plg during remission with VTE implies hypercoagulability even during disease quiescence. Hypoalbuminemia strongly portends VTE risk, which is a novel finding in ANCA vasculitis. A thrombotic signature would allow improved management of patients to minimize VTE risk and complications of anticoagulation.

Elevated Microparticle Tissue Factor Activity Is Associated With Carotid Artery Plaque in HIV-Infected Women.

Expression of tissue factor (TF) on the surface of activated monocytes may trigger thrombosis, leading to clotting risk, inflammation, and atherosclerosis. TF-positive microparticles (MP-TF) represent a functionally active form of TF that may be promulgated by long-term HIV infection. We hypothesized that greater MP-TF activity is associated with carotid artery plaque in HIV+ women. In a case-control study nested within the Women's Interagency HIV Study (WIHS), eligible HIV+ participants underwent B-mode carotid artery ultrasound at 2 study visits occurring 7 years apart. Cases were defined by the presence of at least 1 carotid artery plaque assessed at either visit. Cases were matched 1:2 to controls who were found not to have carotid artery plaques. Conditional logistic regression estimated the association of MP-TF activity with the presence of carotid artery plaque, adjusting for demographic and behavioral characteristics, HIV-related factors, cardiometabolic risk factors, and serum inflammation biomarkers (high-sensitivity C-reactive protein, IL-6, sCD14, sCD163, Gal-3, and Gal-3BP). Elevated MP-TF activity (>0.537 pg/mL) was found to be significantly associated with greater odds of plaque (adjusted odds ratio 3.86, 95% confidence interval: 1.06 to 14.07, P = 0.04). The association was attenuated after further adjustment for IL-6 but was unaffected by adjustment for other biomarkers including those denoting monocyte activation. Our findings suggest a link among HIV infection, innate immune system perturbation, coagulation, and atherosclerosis.

Microparticle Responses to Aerobic Exercise and Meal Consumption in Healthy Men.

Microparticles (MP) are shed extracellular vesicles that express the prothrombotic tissue factor (TF). Aerobic exercise may reduce MP count and TF expression. This study investigated the impact of acute running or rest followed by standardized meal consumption on MP phenotypes and TF expression. Fifteen males (age, 22.9 ± 3.3 yr; body mass, 81.9 ± 11.4 kg; V˙O2max, 54.9 ± 6.5 mL·kg·min; mean ± SD) completed 1 h of running (70% V˙O2max) or rest at 9:00 AM and consumed a standardized meal (1170 kcal, 43% CHO, 17% PRO, 40% fat) at 10:45 AM. Venous blood samples were taken at 9:00 AM, 10:00 AM, and 11:30 AM. The MP concentration, diameter, phenotypes, and TF expression were assessed using nanoparticle tracking analysis and flow cytometry. Nanoparticle tracking analysis identified no changes in MP concentration or diameter in response to time or trial. Flow cytometry revealed total MP count increased from 9:00 AM to 10:00 AM (1.62 ± 2.28 to 1.74 ± 2.61 × 10 L, P = 0.016, effect size (η) = 0.105), but was unaffected by trial. TF platelet-derived MP % reduced from 9:00 AM to 10:00 AM (44.0% ± 21.2% to 21.5% ± 9.3%, P = 0.001, η = 0.582) after exercise only (control, 36.8% ± 18.2% to 34.9% ± 11.9%; P = 0.972). TF neutrophil-derived MP percentage reduced from 9:00 AM to 11:30 AM (42.3% ± 17.2% to 25.1% ± 14.9%; P = 0.048, η = 0.801) in the exercise trial only (control, 28.5% ± 15.7% to 32.2% ± 9.6%; P = 0.508). Running induced a significant reduction in %TF platelet and neutrophil MP, suggesting a transient reduction in cardiovascular risk via reduced TF-stimulated thrombosis. This requires further investigation over longer periods in cardiovascular disease populations.

Procoagulant Activity of Platelets and Tissue Factor Microparticles in Behçets Disease

Background: Microparticles are found in all body fluids including peripheral blood. They play an important role in blood clot formation and increased cell aggregation. Aim of this work: Was to evaluate the level of circulating platelet microparticles (PMP) and tissue factor microparticles (TFMP) in Behcet`s disease and to assess the relation of these procoagulant markers and Behcet’s disease activity. Patients and methods: This study included 100 persons divided to three groups; Behcet’s disease (BD) group (40) patients, systemic lupus (SLE) group (40) patients as a comparative and control group (20) persons. Platelet-free plasma was ultra-centrifuged. PMPs and TFMPs were identified and enumerated with flow cytometry using anti CD61PE and CD142Percp antibodies. D-dimer was measured by ELISA. Results: There was statistically significant increase of PMP (CD61), tissue factor MP (CD142), CD61-MFI and CD142-MFI in Behcet`s group in comparison to Control group (p˂0.001). There was statistically significant increase of PMP and tissue factor MP in Behcet`s when compared to Lupus group (p˂0.001), while CD61/CD142, CD61- MFI and CD142-MFI revealed no significant difference. D-dimer was significantly increased during Behcet`s and SLE disease activity (p=0.036 & p=0.021 respectively). Active Behcet`s disease had significant increase of both CD61 and tissue factor expressing PMP (CD61/CD142) when compared to patients with inactive state (p˂0.001 & p=0.004 respectively), while CD142 showed no significant difference (p=0.38). In Behcet`s group, there was significant positive correlation between CD61 and each of CD61/CD142 and D-dimer (p=0.001). Conclusion: PMP and TF MP are significantly elevated in BD in comparison to SLE. PMP, PMP expressing TF and D-dimer are markers of disease activity in both BD and SLE, while ESR and CRP are not. PMP is positively correlated to TF expressing PMP and D-dimer in BD denoting enhanced prothrombotic state. PMP, PMP expressing TF and D-dimer are characters of ANCA positive BD.

Procoagulant Activity of Platelets and Tissue Factor Microparticles in Behçets Disease

Procoagulant Activity of Platelets and Tissue Factor Microparticles in Behçets Disease

The Release of Monocyte-Derived Tissue Factor-Positive Microparticles Contributes to a Hypercoagulable State in Idiopathic Membranous Nephropathy.

Aim: Idiopathic membranous nephropathy (IMN) is an immune-mediated inflammatory disease characterized by a high risk of thromboembolic complications. Microparticles (MPs), a type of extracellular vesicles, have procoagulant properties, especially when they display tissue factor (TF). This study aimed to investigate whether circulating TF-positive MPs contributed to the hypercoagulable state in patients with IMN.Methods: Twenty adult IMN patients and fourteen healthy subjects were included in the study. The basic indexes of a routine biochemical examination and coagulative function were determined. The plasma levels of MPs were detected by flow cytometry, and TF activity of MPs was examined using an assay kit. The plasma levels of lipopolysaccharide (LPS) were measured by an enzyme-linked immunosorbent assay.Results: Total circulating MPs were not increased in patients with IMN compared with healthy controls. Circulating CD14+/TF+MPs were significantly increased in IMN patients, but this achieved significance was not observed in CD41+/TF+MPs between the two groups. Interestingly, the circulating TF-positive MPs were increased significantly. Plasma MPs TF assays revealed high procoagulant activity, which was positively associated with the D-dimer level in IMN. In addition, circulating LPS in IMN patients were significantly higher than those in the controls. Furthermore, after two hours' incubation with healthy whole blood, LPS enhanced the release of circulating TF-positive MPs and the TF activity of MPs.Conclusion: Increased circulating LPS may mediate the release of monocyte-derived TF-positive MPs, which further contributes to the hypercoagulable state in IMN patients. These findings provide an additional mechanism by which patients with IMN have a higher risk of thromboembolic complication.

Matrix Signaling Subsequent to a Myocardial Infarction: A Proteomic Profile of Tissue Factor Microparticles

This study investigated the release and proteomic profile of tissue factor microparticles (TFMPs) prospectively (up to 6 months) following a myocardial infarction (MI) in a chronic porcine model to establish their utility in tracking cellular level activities that predict physiologic outcomes. Our animal groups (n= 6 to 8 each) consisted of control, noninfarcted (negative control); infarcted only (positive control); and infarcted animals treated with cardiac resynchronization therapy (CRT) and a β-blocker (BB) (metoprolol succinate). The authors found different protein profiles in TFMPs between the control, infarcted only group, and the CRT+ BB treated group with predictive impact on the outward phenotype of pathological remodeling after an MI within and between groups. This novel approach of monitoring cellular level activities by profiling the content of TFMPshas the potential of addressing a shortfall of the current crop of cardiac biomarkers, which is the inability to capture composite molecular changes associated with chronic maladaptive signaling in a spatial and temporal manner.

Clinical significance of circulating microparticles in Ph- myeloproliferative neoplasms.

Microparticles (MPs) are small membrane vesicles that are classified into subcategories based on their origin, such as platelet-derived MPs (PMPs), endothelial MPs (EMPs), red blood cell MPs (RMPs) and tissue factor MPs (TF + MPs). Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPN) are disorders characterized by abnormal haematopoiesis, thrombosis and the JAK2V617F mutation. MPs are biomarkers for procoagulant state in cancer patients, but their relevance in patients with Ph-MPN was unclear. The present study aimed to measure MP variation in MPN patients and evaluate association with the JAK2V617F mutation and with thrombosis and splenomegaly. In total, 92 patients with MPN were enrolled in the present study, including 60 with essential thrombocythaemia (ET), 20 with polycythaemia vera (PV), and 12 with primary myelofibrosis (PMF). RMPs, PMPs, TF + MPs and EMPs were measured by flow cytometry. The levels of RMPs, PMPs, EMPs and TF + MPs in patients with Ph-MPN were all found to be significantly increased compared with controls (P<0.05). Additionally, the levels of all four types of MPs in the PMF group were significantly increased compared with the PV group (P<0.05), and the level of RMPs in the PMF group was significantly increased compared with the ET group (P<0.05). MP levels were increased in the Ph-MPN patients with thrombosis compared with patients without thrombosis (P<0.05). MP levels were increased in Ph-MPN patients with splenomegaly compared with patients without splenomegaly (P<0.05). The level of PMPs in patients with the JAK2V617F mutation was increased compared with patients without the mutation (P<0.05). In conclusion, the present study showed that MPs are associated with Ph-MPN pathogenesis, and may promote thrombosis.

Procoagulant effects of lung cancer chemotherapy: impact on microparticles and cell-free DNA.

Lung cancer is the second leading type of cancer, with venous thromboembolism being the second leading cause of death. Studies have shown increased levels of microparticles and cell-free DNA (CFDNA) in cancer patients, which can activate coagulation through extrinsic and intrinsic pathways, respectively. However, the impact of lung cancer chemotherapy on microparticle and/or CFDNA generation is not completely understood. The aim of the study was to study the effects of platinum-based chemotherapeutic agents on generation of procoagulant microparticles and CFDNA in vitro and in vivo. Microparticles were isolated from chemotherapy-treated monocytes, human umbilical vein endothelial cells, or cancer cells. Tissue factor (TF) and phosphatidylserine levels were characterized and thrombin/factor Xa generation assays were used to determine microparticle procoagulant activity. CFDNA levels were isolated from cell supernatants and plasma. A murine xenograft model of human lung carcinoma was used to study the procoagulant effects of TF microparticles and CFDNA in vivo. In vitro, platinum-based chemotherapy induced TF/phosphatidylserine microparticle shedding from A549 and A427 lung cancers cells, which enhanced thrombin generation in plasma in a FVII-dependent manner. CFDNA levels were increased in supernatants of chemotherapy-treated neutrophils and plasma of chemotherapy-treated mice. TF microparticles were elevated in plasma of chemotherapy-treated tumour-bearing mice. Plasma CFDNA levels are increased in chemotherapy-treated tumour-free mice and correlate with increased thrombin generation. In tumour-bearing mice, chemotherapy increases plasma levels of CFDNA and TF/phosphatidylserine microparticles. Platinum-based chemotherapy induces the shedding of TF/phosphatidylserine microparticles from tumour cells and the release of CFDNA from host neutrophils.

Investigation for role of tissue factor and blood coagulation system in severe acute pancreatitis and associated liver injury

This study aims to investigate the molecular mechanisms underlying the pathogenesis of severe acute pancreatitis (SAP) and SAP-associated liver injury, we performed an association analysis of the functions of tissue factor (TF) and blood coagulation system in both SAP patients and mouse SAP model. Our results showed that serum TF and tissue factor-microparticle (TF-MP) levels were highly up-regulated in both SAP patients and SAP mouse model, which was accompanied by the dysfunction of blood coagulation system. Besides, TF expression was also highly up-regulated in the Kupffer cells (KCs) of SAP mouse model. After inhibiting KCs in SAP mouse model, the amelioration of blood coagulation system functions was associated with the decrease in serum TF and TF-MPs levels, and the reduction of SAP-associated liver injury was associated with the decrease of TF expression in KCs. In conclusion, the dis-regulated TF expression and associated dysfunction of blood coagulation system are critical factors for the pathogenesis of SAP and SAP-associated liver injury. TF may serve as a potential and effective target for treating SAP and SAP-associated liver injury.

Increased Level of Thrombotic Biomarkers in Patients with Atrial Fibrillation Despite Traditional and New Anticoagulant Therapy.

The aim of this study was to examine the effect of the traditional oral anticoagulant, warfarin (W), and new anticoagulants, apixaban (A) and rivaroxaban (R), on the level of thrombotic biomarkers in patients with atrial fibrillation (AF). Circulating plasma levels of von Willebrand factor (vWF), prothrombin fragment 1.2 (F1.2), microparticle tissue factor (MP-TF), and plasminogen activator inhibitor (PAI-1) were analyzed as potential markers of clot formation in 30 patients with AF prior to ablation surgery. Patients with AF were divided into 2 groups based on their usage (n = 21) and nonusage (n = 9) of any oral anticoagulant. Furthermore, those on anticoagulants were divided based on their use of newer (R and A, 16) or traditional (W, 4) anticoagulants. A statistical increase (P < .05) in the levels of vWF, MP-TF, and PAI-1 were seen in anticoagulated patients with AF, whereas F1.2 and PAI-1 were increased in nonanticoagulated patients with AF compared to normal. There was no statistical difference (P > .05) in levels of any thrombotic biomarker between patients treated with the traditional anticoagulant, W, and those treated with new anticoagulants, R and A. Our data suggest that, despite the use of traditional or newer anticoagulants, prothrombotic biomarkers are still generated at increased levels in patients with AF. Further studies to confirm these findings are warranted.