Procoagulant Activity Of Microparticles Research Articles

Procoagulant activity of red blood cell microparticles in stored packed red blood cell units and its relation to ABO blood grouping

BackgroundThroughout the storage of blood, the red cells undergo alterations known as “storage lesions,” which involve shape changes and the formation of microparticles (MPs). Studies of the formation of red cell microparticles (RMPs) emphasize the prospective application of RMPs as a quality control measure in the preparation and storage of blood components in the future. In the present study, twenty packed RBC units in citrate phosphate dextrose adenine-1 (CPDA1) were collected from volunteers and stored for 35 days. Over 35 days of storage, samples were collected at six distinct time points weekly and evaluated for the presence of RMPs. MPs were separated by the ultracentrifugation method. Electron microscopy was used to characterize the morphology and size of the isolated microparticles, and flow cytometry was performed to determine the percentage of RMPs that expressed glycophorin A (CD235a) and Annexin V antigens. RMPs' procoagulant activity (PCA) was assessed using a plasma recalcification test. RMP concentration in accordance with ABO blood grouping was assessed by using various types of donated blood groups.ResultsRMPs progressively increased over storage. The procoagulant activity (PCA) exhibited a significant increase during storage, as evidenced by a shorter plasma recalcification time (P value = 0.001). A significant negative correlation (P value = 0.001) between plasma recalcification time and Annexin V-positive microparticles, as well as a dual-positive Annexin V/CD235a population, was identified, indicating a strong correlation between the direct quantitative assay by flowcytometry and the functional assay through the PCA.ConclusionRMPs increase on storage with increased PCA. Finding ways to reduce these microparticles in packed RBC units is crucial for reducing the risk of transfusion-related coagulopathy.

Coagulation in Patients with Immune Thrombocytopenia and Other Associated Autoimmune Disorders

Introduction Chronic immune thrombocytopenia (ITP) is characterized by perturbations of immune homeostasis with hyperactivated effector cells as well as defective regulation of the adaptive immune system. It has been reported that, with varying frequency, patients with ITP may also have associated other autoimmune disorders, with circulating autoantibodies such as lupus anticoagulant antibodies (LACs), anti-nuclear antibodies (ANA), anticardiolipin (aCL), anti-beta2-glycoprotein I (anti-beta2GPI), anti-thyroid peroxidase (ATPA), and rheumatoid factor (RF). Objectives To evaluate coagulation in patients with ITP with autoantibodies associated to other autoimmune diseases. Methods This is a prospective study that was approved by the Ethics Committee from Hospital Universitario La Paz. Informed consent was signed before sampling. Adult ITP patients (n=182) without and with associated autoantibodies such as LACs, ANA, aCL, anti-beta2GPI, ATPA, and RF and healthy controls (n=180) were recruited. To evaluate the kinetics of clot formation and fibrinolysis, ROTEM® was performed on fresh platelet rich plasma adjusted to 25x10 9 platelets/L with the subjects own platelet poor plasma (PPP). Recalcification (NATEM) was used to induce coagulation. Clotting time (CT = time from start of measurement until initiation of clotting, in seconds); alpha angle (tangent to the curve at 2 mm amplitude, in degrees, which reflects the rate of fibrin polymerization); maximum clot firmness (MCF = maximum clot firmness, in mm, which reflects the maximum tensile strength of the thrombus); and lysis at 60 min (LI60) were recorded. Thrombin generation was measured in citrated PPP by calibrated automated thrombogram (CAT). To evaluate procoagulant capacity of plasma, coagulation was triggered by proper recalcification and the addition (final concentrations) of 1 pmol/L of recombinant human tissue factor (TF) and 4 µmol/L of phospholipid mixture (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands). Procoagulant activity of microparticles (MPs) associated with their content of either TF or phosphatidylserine (PS) was determined, respectively, with MP reagent (4 µM phospholipids) or PRP reagent (1 pM of recombinant human TF) by CAT. The following parameters were determined: Lag time (LT), or time from the start of the assay until 10 nM thrombin is formed, in min; time-to-peak (ttPeak), or time required to reach the maximum thrombin concentration, in min; peak height (Peak), or maximum thrombin concentration reached, in nM; and endogenous thrombin potential (ETP), or total amount of thrombin generated over time, in nMxmin. Results Fifty of the 182 patients with ITP (27.5%) were positive for autoantibodies associated with other autoimmune diseases, distributed as shown in Table 1. ANA were the most frequent antibodies present in these patients, and 22% had more than one kind of associated antibodies. Patients with ITP without antibodies had a shorter clot formation time (CT) than healthy controls. Presence of associated autoimmune diseases prolongs CT and resulting in values similar to controls. Both groups of patients with ITP had a diminished fibrinolysis (Table 2). Thrombin generation triggered by TF plus phospholipids as well as that dependent on TF content of microparticles (MP-reagent) was impaired in patients with other associated autoimmune disorders as shown by the prolonged LT and the diminution in either ETP or peak of generated thrombin (Table 2). When thrombin generation relayed on phospholipids of MPs, all patients with ITP generated less thrombin. Conclusion Association of other autoimmune diseases with ITP seemed to produce a delay in clot formation and in thrombin generation whereas did not affect the diminished fibrinolysis observed in ITP patients. Work supported by Instituto de Salud Carlos III (ISCIII) (PI19/00772, PI22/01489), co-funded by the European Union.

MPs-ACT, an Assay to Evaluate the Procoagulant Activity of Microparticles.

The procoagulant effect of microparticles (MPs) contributes to hypercoagulability-induced thrombosis. We provide preliminary findings of the MPs-Activated Clotting Time (MPs-ACT) assay to determine the procoagulant activity of MPs. MPs-rich plasma was obtained and recalcified. Changes in plasma viscoelasticity were evaluated and the time to the peak viscoelastic changes was defined as the MPs-ACT. MPs concentration was measured by flow cytometry. Coagulation products produced during plasma clotting were identified by fibrin and fibrinopeptide A. MPs were prepared in vitro and added to standard plasma to simulate pathological samples. In addition, reproducibility and sensitivity were evaluated. We confirmed the linear relationship between MPs-ACT and MP concentrations. Dynamic changes in fibrin production were depicted. We simulated the correlation between MPs-ACT and standard plasma containing MPs prepared in vitro. The reproducibility of high-value and low-value samples was 6.0% and 10.8%, respectively. MPs-ACT sensitively detected hypercoagulable samples from patients with pre-eclampsia, hip fractures, and lung tumors. MPs-ACT largely reflects the procoagulant effect of MPs. MPs-ACT sensitively and rapidly detects hypercoagulability with MPs-rich plasma. It may be promising for the diagnosis of hypercoagulable states induced by MPs.

Haemostatic profile of riboflavin-treated apheresis platelet concentrates.

The haemostatic activity of platelet concentrates (PCs) treated with pathogen reduction technology (PRT) remains a subject of debate. Our aim was to investigate the effect of Mirasol PRT on the haemostatic properties of PCs stored in plasma. Untreated and Mirasol-treated platelets stored in plasma and derived from ten split double-dose apheresis PCs were evaluated in vitro on days 1, 3 and 5 post collection for functionality, microparticle procoagulation activity (MPA), endogenous thrombin potential (ETP), and haemostatic profile using rotational thromboelastometry (ROTEM). P-selectin expression was significantly higher in Mirasol-treated platelets compared with untreated counterparts on days 3 and 5 (p=0.003 and p=0.002, respectively). Clot strength, as shown by EXTEM maximum clot firmness (MCF), was significantly lower in the Mirasol-treated platelets at all time points (days 1, 3, 5) than in untreated platelets (p=0.009, p<0.001, p<0.001, respectively). There was a considerable increase in MPA over time (p<0.001) and this was significantly higher in the Mirasol-treated platelets on day 5 (p=0.015). A notable acceleration of decrease in ETP values was observed for Mirasol-treated PCs over time (p<0.001), with significant differences between PRT-treated and untreated PCs on days 3 and 5 (p=0.038 and p=0.019, respectively). Clot strength attenuation was significantly associated with pH reduction (p<0.001, Spearman's rho: 0.84), increased microparticle procoagulant activity (p<0.001, Spearman's rho: -0.75), and with decreased ETP (p<0.032, Spearman's rho: 0.41). Increased platelet activation induced by PRT treatment leads to a decrease in in vitro haemostatic capacity as seen by reduced clot strength and thrombin generation capacity over time. The clinical relevance of this needs to be investigated.

Effect of Rapid Centrifugation Using High Centrifugal Force on Procoagulant Activity in Plasma Samples.

International guidelines for plasma preparation are not always observed because high-speed centrifugation reduces turnaround times. This study assessed the effect of rapid centrifugation on sample quality and clotting time. Blood samples were obtained from five healthy volunteers. Normal blood samples were spiked with unfractionated heparin to produce abnormal samples. The normal and abnormal coagulation ability samples were centrifuged at 1,500 x g for 15 minutes, 2,000 x g for 10 minutes (both according to international guidelines), or 3,500 x g for 7 minutes (rapid centrifugation). Microparticle procoagulant activity (MP activity), prothrombin time (PT), and activated partial thromboplastin time (APTT) were measured in the supernatant plasma. Rapid centrifugation caused a significant increase in MP activity compared to the two recommended conditions and significantly shortened clotting times, particularly APTT in the abnormal samples. Rapid centrifugation should not be used for routine processing of blood samples for coagulopathy screening and monitoring patients on anticoagulant therapy.

Histophilus somni stimulates bovine monocyte-derived macrophages to release microparticles that increase fibrin clot formation in vitro

Histophilus somni is a Gram-negative coccobacillus that causes diffuse vasculitis and intravascular thrombosis that can lead to multiple organ failure in cattle. Macrophages are important cellular mediators of fibrin deposition and removal at sites of inflammation. It has become evident that macrophages and other cells release microparticles (MPs) that have an array of biological activities, including pro-coagulant activity. We sought to determine whether monocyte-derived macrophages exposed to H. somni in vitro release MPs that activate the clotting cascade in a manner that could lead to thrombus formation. Bovine monocyte-derived macrophages were incubated with H. somni (at a 10:1 ratio) in RPMI with 10% heat inactivated fetal bovine serum for 6 h at 37 °C with 5 % CO2. Membrane-shed MPs were isolated from the conditioned media, washed twice with Ca2+ and Mg2+ free HBSS, and pro-coagulant activity assessed by a one-step plasma clotting assay. We observed greater pro-coagulant activity for MPs from H. somni stimulated macrophages than from unstimulated controls. Microparticle pro-coagulant activity was inhibited by addition of an anti-tissue factor antibody. We also observed co-localization of fluorescein-labeled H. somni cells and annexin V staining as evaluated by confocal microscopy. These results demonstrate that exposure to H. somni cells causes bovine monocyte-derived macrophages to release MPs that contain tissue factor, the first such report for bovine macrophages. We infer that if similar events occur in vivo they could amplify thrombus formation in bovine histophilosis.

Endothelial dysfunction and hypercoagulability in severe sickle-cell acute chest syndrome

RationaleAcute pulmonary hypertension (PH) may develop during sickle-cell acute chest syndrome (ACS), and is associated with an increased mortality. Its mechanisms remain poorly known. We questioned whether there is endothelial dysfunction and hypercoagulability in severe ACS, with and without acute PH.MethodsIn a prospective monocentre cohort follow-up study, all sickle-cell adult patients with ACS admitted to the intensive care unit underwent transthoracic echocardiography and measurement of biomarkers of coagulation, endothelial activation and platelet and erythrocyte activation. Acute PH was defined as a high echocardiographic probability of PH. The biological profiles of sickle-cell patients were analysed at the time of ACS, contrasting with the existence of acute PH, and compared with steady-state and with non-sickle-cell controls (healthy subjects and community-acquired pneumonia).ResultsMost patients (36 patients with 39 ACS episodes; 23 males; median age 27 years) had thoracic pain, dyspnoea and computed tomography scan lung consolidation. Acute PH was diagnosed in seven (19%) patients. Erythrocyte- and platelet-derived microparticles and the pro-coagulant activity of microparticles were higher in ACS patients with acute PH, compared with their counterparts. Compared with healthy controls, ACS patients had higher levels of tissue factor, fibrin monomers, D-dimer, release of pro-coagulant microparticles and erythrocyte- and platelet-derived microparticles. Compared with community-acquired pneumonia patients, ACS patients had increased levels of fibrin monomers and erythrocyte- and platelet-derived microparticles.ConclusionsSevere ACS is characterised by endothelial dysfunction and hypercoagulability, with a marked pro-coagulant profile in cases of associated PH.

Vascular and Hemostatic Alterations Associated with Pulmonary Hypertension in β-Thalassemia Hemoglobin E Patients Receiving Regular Transfusion and Iron Chelation

Background: β-thalassemia hemoglobin E (β-thal/HbE) is currently the commonest β-thalassemia disease worldwide. Pulmonary hypertension (PH) is the most common cardiovascular complication in this genotype, especially in splenectomized patients. Yet the pathogenesis of this major complication remains poorly characterized. Most of the previous reports investigated pathophysiology of PH in β-thal/HbE with inadequate transfusion. In this study we aimed to define the prevalence and comprehensively explore mechanisms of PH in β-thal/HbE patients receiving regular transfusion and iron chelation which were reported to alleviate PH. Our findings will provide deeper insights into pathogenesis and identify targets for management of persistent PH in thalassemia patients after adequate transfusion and chelation.Methods: β-thal/HbE patients who received regular transfusion ≥ 1 unit per month and any forms of iron chelation over 1 year were eligible for the study. Normal healthy subjects were used as controls. Blood sample collection and echocardiographic assessment were performed on the same days. Patients at risk for PH were defined by the tricuspid regurgitant jet velocity (TRV) ≥ 2.5 m/s.Results: There were 68 β-thal/HbE and 38 healthy controls enrolled between May and September 2015. The ages (mean ± SD) were 34.1 ± 12.6, ranging from 19-67 years, and 37.2 ± 8.6 years, ranging from 18-70 years, respectively. Among 68 β-thal/HbE, the average transfusion duration was 8.1 ± 2.2, ranging from 2.2-10.6 years, and the quantity of transfusion was 22.3 ± 9.6 units per year. The pre-transfusion hemoglobin (Hb) level was 8.1 ± 1.3 g/dL. There were 31 (45.6%) splenectomized patients. β-thal/HbE showed markedly increased erythropoiesis (growth differentiation factor, GDF15, and soluble transferrin receptor, sTfR, levels) and iron load (serum ferritin) compared to controls, all p < 0.001. Thrombin antithrombin (TAT) complex and microparticle's procoagulant activity (MPA) were significantly elevated (both p < 0.001), but total tissue factor antigen (TFAg) and microparticle-associated tissue factor activity (MPTFA) were not different from those of controls, suggesting TF-independent hypercoagulability. Serum nitric oxide metabolites (NOx) and prostaglandin E2 (PGE2) levels were significantly lower than those of controls (both p < 0.001), while soluble thrombomodulin (sTM) levels were significantly elevated (p= 0.045). Serum endothelin-1 (ET-1) and soluble p-selectin levels were not different between patients and controls. Echocardiographic profiles demonstrated preserved biventricular systolic functions in β-thal/HbE with a significant increase in TRV (p < 0.001).Among β-thal/HbE, there were 29 (42.6%; 16 non-splenectomized and 13 splenectomized) cases at risk for PH. Duration and amount of transfusion, pre-transfusion Hb, GDF15 and sTfR levels were not statistically different between PH and non-PH subgroups indicating the similar degree of erythropoiesis. Notably, β-thal/HbE with PH had a significant reduction in NOx levels (p < 0.001) but elevations in TAT, ET-1 and sTM levels compared to those without PH (p= 0.046, 0.005 and 0.026, respectively). Additionally, TRV was significantly correlated with NOx, TAT, sTM and ET-1 levels (r = -0.514, p < 0.001; r = 0.281, p= 0.004; r = 0.313, p= 0.001; and r = 0.245, p= 0.012, respectively). Serum ferritin, MPA, MPTFA, TFAg and PGE2 levels were not statistically different between PH and non-PH subgroups. Surprisingly, there were no differences in any biomarkers and echocardiographic profiles between splenectomized and non-splenectomized patients as well as no significant associations between splenectomy statuses and PH (Χ2 = 0.012, p= 0.914).We reviewed another β-thal/HbE cohort with available flow mediated dilatation (FMD) data. 9 β-thal/HbE with PH had significantly lower FMD (3.43%) compared to 18 β-thal/HbE without PH (6.37%) and 27 age-sex matched controls (9.87%; p= 0.025 and p < 0.001, respectively) indicating a greater degree of endothelial dysfunction in β-thal/HbE having PH. In addition, TRV was significantly correlated with FMD (r = -0.474, p < 0.001).Conclusions: PH is still prevalent in β-thal/HbE patients receiving long-term regular transfusion and iron chelation. In this group, PH is not associated with splenectomy statuses but correlated with NO depletion, TF-independent hypercoagulation and endothelial dysfunction. [Display omitted] DisclosuresSutcharitchan:Chulalongkorn University: Consultancy, Employment; Celgene: Research Funding.

Phosphatidylserine on Blood Cells and Microparticles Contributes to the Hyper-Coagulable State in Diabetic Kidney Disease

Introduction: Patients with diabetes mellitus (DM) are considered to be prothrombotic with major disturbances in hemostasis that are associated with an increased risk of venous thromboembolism, especially when microvascular complications of diabetes mellitus occur, such as diabetic kidney disease (DKD). Additionally, microthrombosis in glomerular capillaries could decrease the kidney blood flow and induce renal insufficiency. However, patients with DKD do not receive routine antithrombotic therapy. Therefore, more study is needed on the mechanisms of coagulation in DKD with the aim of identifying therapeutic targets. Recent studies have shown that phosphatidylserine (PS), a membrane constituent, plays an important role in the thrombosis. However, relatively little is known about the precise role of PS in the prothrombotic state of DKD. Our objectives were to study the effect of increased PS exposure on microparticles (MPs) and the outer membrane of MP-origin cells in DKD patients, and to evaluate its effect on procoagulant activity (PCA).Methods: DKD patients (n = 90) were divided into three groups based on urinary albumin excretion rate (UAER), defined as normoalbuminuria (<30 mg/24 h), microalbuminuria (30-299 mg/24 h), or macroalbuminuria (>300 mg/24 h), and compared to healthy controls (n = 30). Lactadherin was used to quantify PS exposure on MPs and their original cells. Healthy blood cells and human umbilical vein endothelial cells (HUVECs) were treated with 25 mM, 5 mM or 2.5 mM glucose and then PS exposure was measured. PCA of MPs and cells was evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. Phosphatidylserine exposure and fibrin strands were observed using confocal microscopy.Results: Usingflow cytometry, we found that PS+ MPs (derived from platelets, erythrocytes, HUVECs, neutrophils, monocytes and lymphocytes) and blood cells (BCs) were significantly higher in patients than in controls. Furthermore, the number of PS+ MPs and BCs in patients with macroalbuminuria was significantly higher than in patients with normoalbuminuria. Similarly, we observed markedly elevated PS exposure on HUVECs cultured with serum from patients with macro-DKD versus serum from patients with micro-DKD or normo-DKD. In addition, circulating PS+ MPs cooperated with PS+ cells, contributing to markedly shortened coagulation time and dramatically increased FXa/thrombin generation and fibrin formation in each DKD group. Confocal microscopy images demonstrated co-localization of fibrin with PS on ECs. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by approximately 80%. Additionally, both hyperglycemia (25 mmol/L) and hypoglycemia (2.5 mmol/L) induced increased PS exposure on cells compared to normoglycemia (5 mmol/L). Linear regression analysis showed that levels of PS+ BCs and MPs were positively correlated with uric acid and proteinuria, but negatively correlated with GFR.Conclusions: Our results suggested that PS+ MPs and blood cells play a prominent role in inducing the hypercoagulable and prothrombotic state in patients with DKD. The occurrence of abnormal glucose during the hypoglycemic therapy is associated with hypercoagulation in DKD. PS+ cells and MPs likely support thrombosis in renal vascular and result in renal dysfunction and disease progression. Our results demonstrate that PS provides binding sites for FXa and prothrombinase complexes and promotes thrombin formation. Thus, directly targeting FXa and prothrombinase complexes might decrease thrombotic and renal disease risk. The results from this study suggest that future research should focus on discovery and deployment of direct PS inhibitors.Disclosure of Interest: Jialan Shi has a patent for the use of lactadherin as a PS probe. DisclosuresNo relevant conflicts of interest to declare.

Tissue Factor-Related Biomarkers and Prediction of Cancer-Associated VTE: Correlative Analyses of the Phacs Trial

Background: Cancer patients at high or low risk for venous thromboembolism (VTE) can be predicted by a validated score (Khorana et al, Blood 2008) but the mechanisms underlying this association are not fully understood. We evaluated the association of VTE-related biomarkers with clinical outcomes in patients enrolled in a randomized trial of outpatient prophylaxis in high-risk patients and a control population of low-risk patients.Methods: Cancer patients with risk score ≥3 starting systemic therapy were randomized to dalteparin or observation for 12 weeks; a low-risk group of controls (score=0) was concurrently enrolled. Analyses for tissue factor (TF) using microparticle procoagulant activity, TF pathway inhibitor (TFPI) using ELISA, D-dimer and factor VIIA were conducted on specimens obtained at enrollment and 4-weekly on study as well as at enrollment in control patients.Results: The study populations comprised 89 randomized high-risk patients and 101 low-risk patients. TF MP-PCA levels were more likely to be detectable in high-risk patients (51.7% vs 32.7% of low-risk patients, p=0.01) and levels were elevated at time of enrollment in high-risk compared to low-risk patients (mean 0.67 vs 0.19 pg/mL in low-risk, p=0.002; median 0.03 vs 0 pg/mL, p=0.001). TF levels were particularly high in the subgroup of high-risk patients with pancreatic cancer (N=38, 57.9% detectable, mean 0.87 pg/mL, median 0.13 pg/mL). D-dimer levels were also significantly elevated in high-risk patients (median 1.23 vs 0.92 mg/mL, p=0.006). Levels of F1.2 and FVIIa were not significantly different between the high- and low-risk groups. Patients randomized to dalteparin had a significant decline in median D-dimer levels (median 1.22 mg/mL for timepoint 1 versus 0.7 mg/mL for timepoint 4, p =0.018) whereas there was no significant change over time for patients randomized to observation (median 1.30 vs 1.33 mg/mL, p=0.43). F1.2 levels also declined over time for patients randomized to dalteparin (median 77.17 to 32.37, p=0.01) but not for patients randomized to observation (median 59.06 to 61.63, p=0.99). In multivariate time-dependent Cox analysis in high-risk patients, VTE was associated with both doubling of TF (HR 1.42 95% CI 1.17-1.84, p=0.0003) and doubling of TFPI (HR 3.21, 95% CI 1.14-9.50, p=0.02).Conclusions: Cancer patients at high-risk for VTE are more likely to have increased TF levels at baseline; increased levels of both TF and TFPI were associated with future VTE. Further investigations into the role of TF and TFPI in cancer-associated thrombosis and their roles as predictive biomarkers are warranted. DisclosuresKhorana:Pfizer: Consultancy, Honoraria; Halozyme: Consultancy, Honoraria; Janssen Scientific Affairs, LLC: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Leo: Consultancy, Honoraria, Research Funding. Francis:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.

Procoagulant Mechanisms in Patients with Myelodysplastic Syndrome

Background: MDS comprise a heterogeneous sort of disorders caused by cytogenetic changes, gene mutations, or both, responsible for altered multilineage morphological patterns that lead to a varying grade of peripheral blood cytopaenias and an increased risk of transformation to acute myeloid leukemia. Thrombocytopaenia is observed in aproximately 35% of MDS patients. Despite it is accepted that haemorrhagic events are more likely at platelet counts below 10,000/μl, in the case of MDS patients, whose platelets had a diminished ability to be activated, bleeding events are lower than expected. So, hemostasis in MDS patients is a complex issue where mechanisms additional to platelet count/function seem to be involved.Aim: This work aimed to identify procoagulant mechanisms in non-bleeders MDS patients that might protect them from bleeding.Methods: This is a prospective, observational and transversal study. Eighty four non-bleeders MDS patients (44% female, mean age of 77±25 years), were included. Sixty healthy subjects (45% female, mean age of 57±22 years) were recruited.Human peripheral blood samples were collected in 3.8% sodium citrate. Blood cell counts were performed with a Coulter Ac.T Diff cell counter (Beckman Coulter, Madrid, Spain). Platelet rich plasma (PRP) and platelet free plasma (PFP) were obtained by centrifugation.Surface exposure of phosphatidylserine (PS) and active caspase-3, -8 or -9 were assessed by flow cytometry measuring the binding of FITC-labeled Annexin V (BD Pharmingen) and with a specific kit for measuring these caspases (Millipore, Madrid, Spain).Prothrombinase complex binding to platelets was determined in quiescent washed platelets (1x108/mL) incubated with factor (F) Xa (5 nmol/L) and FVa (5nmol/L) obtained from Haemtech Inc (Cellsystems, Germany). Following fixation to cross-link platelet-bound FVa and FXa, specific or control monoclonal antibodies (0.1 μmol/L each, Haemtech Inc, Germany) were added. All samples were analized by flow cytometry.PS-associated and tissue factor (TF)-associated procoagulant activity of microparticles (MPs) was determined with the ZYMUPHEN MP-Activity kits (HYPHEN BioMed, Neuvillesur Oise, France).The identification of the MP's cellular origin was determined by flow cytometry, labeling MPs with Annexin-V-FITC and the following specific mAb conjugated with phycoerythrin: anti-CD41 mAb for platelets (Biocytex, Marseille, France); and anti-CD14 mAb for monocytes, anti-CD144 mAb for endothelial cells, anti-CD235 mAb for red cells and anti-CD45 mAb for leukocytes, all from BD Biosciences.The statistical analysis of the experimental data was using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA).Results: Platelets from patients with MDS presented higher levels of activated caspases-3,7, -8 and -9 and this situation was accompanied by a higher PS platelet surface exposure (TABLE 1)We evaluated if increased PS exposure was functional for anchoring prothrombinase complex. Binding of FVa and of FXa on platelet membrane surface was higher in quiescent platelets from MDS patients [FVa (Mean fluorescence), control: 49.7±8.5, MDS patients: 73.9±23.2, p<0.01; FXa (Mean fluorescence), control: 46.4±17.5 , MDS patients: 77.6±12.0, p<0.05)].A significantly higher procoagulant capacity of MPs associated either to PS (control: 8.0±2.5 nanomol/L; MDS: 17.8±3.5 nanomol/L, p<0.01) or TF (control: 0.4±2.5 nanomol/L; MDS: 7.5±2.5 nanomol/L, p<0.001) were observed in the MDS group.To determine the cellular source of MPs, flow cytometry analyses were performed and a significant increase was observed in the amount of monocyte- (events, control: 94±30, MDS patients: 220±29, p<0.05)and red cell-derived MPs (events, control: 142±55, MDS patients: 252±23, p<0.01) in the samples from the patients with MDS. In patients with MDS, TF-MP-associated thrombogenic ability correlated with monocyte percentage (Spearman r=0.5019, p<0.01).Conclusion: MDS patients, even those with normal platelet count, have a reduced platelet function with enhanced apoptosis but do not bleed in many cases. This fact might be due to the increase in tissue factor-containing microparticles and in the enhanced binding of prothrombinase complex to apoptotic platelets which, in turn, augmented procoagulant capacity of these patients.Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresJimenez-Yuste:Roche: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding. Álvarez-Roman:Shire: Consultancy; Novo Nordisk: Consultancy.

Improvement in the cardiovascular profile of patients with morbid obesity following bariatric surgery: Effect on hypercoagulability.

Obesity is an inflammatory state related to vascular endothelium dysfunction. It generates a biological situation of hypercoagulability increasing the risk of thrombosis. This prothrombotic condition could be improved by bariatric surgery.The main objective was to analyze the impact of bariatric surgery on cardiovascular risk factors (CVRF) associated with changes in thrombin generation and procoagulant activity of microparticles (MP).We present a prospective longitudinal study including consecutive patients candidate for bariatric surgery. We performed 3 sequential clinical visits: at inclusion, before surgery after completing the modified fasting phase, and 6 months after surgery. We analyzed CVRF, thrombin generation, and MP activity. The data analysis was performed using a logistic regression model to determine changes over time of hemostatic parameters and body mass index (BMI). McNemar test for binary variables was used to analyze the CVRF.We included 94 patients (66 women), with an average age of 45.7 ± 10.1 years. The mean BMI reduction at the end of the follow-up was 15.5 ± 4.2 kg/m2. We detected a statistically significant improvement in CVRF: hypertension, diabetes mellitus, dyslipidemia, and obstructive sleep apnea, as well as a significant reduction in thrombin generation capacity and procoagulant MP activity.Massive weight loss induced by bariatric surgery improves the cardiovascular profile, associated with a reduction in the hypercoagulable status.

Procoagulant microparticles are associated with arterial disease in patients with systemic lupus erythematosus.

Microparticles (MPs) have been associated with inflammatory and thrombotic disease. High levels of MPs have been identified in patients with systemic lupus erythematosus (SLE) and associated with cardiovascular disease. We analyzed the procoagulant activity of MPs and itscorrelation with arteriosclerosis and arterial thrombosis in SLE patients. Eighty-seven patients with SLE were included: 22 (25.3%) with associated antiphospholipid syndrome (APS), 32 (36.8%) without antiphospholipid antibodies (aPL) and 33 (37.9%) with aPL but without APS. Subclinical arteriosclerosis, defined as the presence and number of plaques, was evaluated by ultrasonography of carotid arteries. Thrombotic events were confirmed by objective methods. The procoagulant activity of MPs was determined by a functional assay with annexin V. Subclinical arteriosclerosis was found in 19 (21.8%) patients. Thirteen episodes of arterial thrombosis and eight of venous thrombosis were recorded. The procoagulant activity of MPs was greater in patients with arterial thrombosis (17.28 ± 8.29nM vs 12.96 ± 7.90nM, p < 0.05). In patients without arterial thrombosis, greater procoagulant activity of MPs was identified in patients with multiple (≥ 2) carotid plaques (17.26 ± 10.63nM vs 12.78 ± 7.15nM, p = 0.04). In the multivariate analysis, the procoagulant activity of MPs was independently associated with multiple (≥ 2) carotid plaques and arterial thrombosis [OR = 1.094 (95%CI 1.010-1.185), p = 0.027 and OR = 1.101 (95%CI 1.025-1.182), p = 0.008; respectively]. In conclusion, the procoagulant activity of MPs is associated with arteriosclerosis burden and arterial thrombosis in patients with SLE.

Impact of Epicatechin on the Procoagulant Activities of 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.

Phosphatidylserine-exposing blood cells, microparticles and neutrophil extracellular traps increase procoagulant activity in patients with pancreatic cancer

Patients with pancreatic cancer (PC) are at increased risk of venous thrombosis, but the precise mechanisms of hypercoagulable state in PC remain unclear. We aimed to identify how phosphatidylserine positive (PS+) blood cells (BCs), PS+ microparticles (MPs) and neutrophil extracellular traps (NETs) regulate procoagulant activity (PCA) in PC, and to assess the relationship between PCA and PC staging. A total of 83 PC patients with different stages of disease were compared to 30 healthy controls, with confocal microscopy and flow cytometry used to assess MP and cellular PS exposure. MP and cell PCA was determined using both fibrin production assays and procoagulant enzyme complex analyses, and coagulation time was further measured. Patients with stage I PC and healthy controls exhibited significantly lower frequencies of PS+ MPs and BCs relative to those with more advanced disease, which may partly due to the increased levels of inflammation cytokines in advanced disease. Functional coagulation assays indicated that PS+ MPs and BCs derived from patients with stage II/III/IV PC directly contribute to elevated FXa, thrombin, and fibrin formation, and to more rapid coagulation relative to healthy control samples. In inhibition assays, lactadherin, which antagonizes PS, led to a roughly 80% inhibition of PCA. We further used isolated NETs to stimulate endothelial cells, revealing that this led to morphological changes including retraction from cell-cell junctions and a more pro-coagulative phenotype, with DNase I and activated protein C treatment reversing these changes. In patients with stage III PC, curative resection surgery significantly reduced PCA, whereas non-curative surgery did not have a marked impact based on studies of pre- and post-operative samples. These results highlight the pathogenic activity of PS+ cells, MPs, and NETs in promoting a prothrombotic environment within individuals suffering from advanced PC. Targeting PS and NETs in these patients may thus be a viable means of preventing pathological 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.

Reduction of plasma procoagulant activity in patients with schizophrenia during pharmacotherapy: thrombodynamic parameters of coagulation before and after treatment

To detect plasma procoagulant activity in patients with schizophrenia at admission to the hospital in a state of exacerbation before (point 1) and after (point 2) pharmacotherapy and evaluate plasma and platelet hemostasis abnormalities. The study included 80 women, aged from 16 to 57 years, median age 28 years, with schizophrenia with continuous, paroxysmal-progressive or paroxysmal course (F20.00, F20.01, F20.02 according to ICD-10). In 42 of 80 patients, depressive disorders in the structure of schizophrenia were observed. The thrombodynamic test (TD) was performed on T-2 Trombodynamis device according to the manufacturer's instructions (Hemacore LLC, Moscow, Russia). Blood for the TD test was taken in admission to the hospital (point 1) and on discharge (point 2). All patients received standard pharmacotherapy according to their condition. For the first time, it was established that in the whole group of patients (n=46) thrombodynamic indicators of the rate of growth of the clot: initial velocity (Vin), stationary velocity (Vst) and adjusted for spontaneous clots velocity (V) and the amount of clot for 30 minutes test TD (ClotSize, CS) were significantly higher compared to normal values. The mean time of occurrence of spontaneous thrombosis (Tsp) was significantly less than 30 min (p<0.0001), indicating rapid, spontaneous thrombosis. Other parameters of TD did not differ significantly from the norm. As a result of treatment, the initial growth rate of the clot from the activator (Vi) decreased from 58,5 μm/min to 54,5 μm/min; V speed from 37,4 μm/min to 33,5 μm/min; CS clot size from 1249 μm to 1219 μm; clot density - from 24 874 units up to 23 658 units. All these changes are significant. Such dynamics of plasma hemostasis clearly indicates a significant decrease in the coagulation activity of the blood plasma of patients as a result of treatment. An increase in the time of appearance of spontaneous clots after treatment (from 23.5 minutes to 30.5 minutes) indicates a decrease in the procoagulant activity of platelet microparticles after treatment, i.e. the reduction of platelet activation as a result of treatment. Our studies have shown for the first time that treatment of patients with antidepressants and antipsychotics reduces the generation of spontaneous clots. The treatment of patients with schizophrenia is accompanied by a decrease in the activity of plasma and platelet hemostasis. This is of great practical importance, since hypercoagulation of spontaneous clots in schizophrenic patients aggravates their chronic inflammatory disorders and affects their resistance to treatment.

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

Thrombodynamic correlates of catatonia severity in children with autism

To study a correlation between the values of thrombodynamics parameters of hypercoagulation measured by the thrombodynamics test and the severity of catatonia in children with infantile psychosis in childhood autism (F84.02). Twenty-four patients (22 boys and 2 girls) aged from 3 to 13 years, were studied. The severity of catatonia was determined by BFCRS. A thrombodynamic test was performed in platelet-free plasma using the analyzer T-2 Thrombodynamics Device (Hemacore LLC, Russia). Thrombodynamic (TD) parameters of clot growth rates from the activator (V, Vi and Vst) were statistically significantly higher than normal values. Similar results were obtained for Clot Size at 30 min (CS, μm): Tlag and D values were within normal limits. The values of Time of appearance of spontaneous clots (Tsp min) were less than the lower limit values for the norm (30 min). Correlation analysis showed that the severity of catatonia is positively correlated with the initial clot growth rate (Vi) (p=0.009) and negatively with Tsp (p=0.002). With an increase in the time of appearance of spontaneous clots (due to a decrease in the procoagulant activity of platelet microparticles in the plasma of patients), the severity of catatonia in children with ASD decreases. The results suggest that normalizing plasma and platelet hemostasis is important for increasing the effectiveness of treatment of patients with ASD with catatonia.