scholarly journals Flow-Cytometry Evaluation of Cellular Surface FVIII in Blood Cells and Its Correlation with FVIII Activity and Coagulation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4236-4236
Author(s):  
Anjud Al-Mohannadi ◽  
Muhammad Elnaggar ◽  
Hani Bibawi ◽  
Che-Ann Lachica ◽  
Igor Pavlovski ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Protein Detection ◽  
Coagulation Cascade ◽  
Binding Motif ◽  
Healthy Controls ◽  
Bleeding Diathesis ◽  
Surface Binding ◽  
Clinical Scenarios

Abstract FVIII deficiency or inactivity leads to coagulation malfunction known as Hemophilia A (HA). FVIII overactivity on the contrary is commonly linked to a hypercoagulability state. In steady state, FVIII remains quiescent by forming a non-covalent complex with vWF, and upon coagulation cascade activation, thrombin liberates active FVIII (FVIIIa) from vWF. FVIIIa binds to the plasma membrane of the platelets and other blood cells via its C2 domain phospholipids binding motif to exert its coagulative role. Recently, we developed a reliable Flow-Cytometry (FC) platform for FVIII protein detection (Elnaggar M. et al, 2020). The assay is suitable to measure both intracellular and extracellular FVIII. When applied to human blood it showed that by overexpressing FVIII through a lentiviral-vector-based transduction, a high proportion of circulating FVIII is bound on leukocytes membranes. Leukocyte abundance is reported to be highly associated with thrombotic events but with no clear mechanistic explanation. Therefore, we sought to evaluate the percentage of FVIIIa bound on different leukocyte subsets (T cells, B cells, monocytes and granulocytes) in different clinical scenarios, including HA and other abnormal coagulation states and to correlate it to the plasmatic levels of FVIII. The analysis was conducted on 31 pediatric/young-adult subjects (median age 9y), including patients diagnosed with HA (n=8), patients with coagulopathies (von Willebrand Disease, menorrhagia and unexplained bleedings (n=11) and 1 patient with known high levels of FVIII), and patients without coagulopathies/healthy controls (n=11) referred to Hematology/Oncology clinic at Sidra Medicine-Qatar. Fresh blood samples were analyzed with a Sysmex XN1000 Hematologic Counter for complete blood count and with a BD-Symphony FC for the following surface markers: CD3/CD4/CD8/CD14/CD16/CD19/CD33/Live/Dead and for FVIII protein A2 domain (Ab clone GMA8024), after light-chain Zenon-labeling, with proper IgG controls, as described in the publication above. In parallel, plasma FVIII measurements were performed for clinical diagnostic purposes, and results were correlated. Expectedly, HA patients showed the lowest FVIII percentage in plasma (p<0.0001), while leukocytes' surface FVIII was generally low (on average <1%), trended to a lower rate in patients with coagulopathies and HA compared to healthy-controls and was significantly higher in the patient with hypercoagulability (p=0.0007). Surprisingly, among leukocytes' subpopulations, screened on patients with bleeding diathesis and HA subjects vs healthy-controls, only CD8-surface bound FVIIIa was significantly lower compared to healthy-controls (p=0.015 healthy-controls vs HA, and p=0,0093 healthy-controls vs bleeding diathesis). These results raise the possibility of a CD8-cell mediated controlled binding mechanism regulating FVIII availability in case of biological need of the protein, as bleeding. We did not find any significant correlation between leukocyte-surface bound FVIII and plasma FVIII level, and this further indicates that the differences in FVIIIa leukocyte surface binding are not directly proportional to FVIII availability in the circulation, rather actively regulated by a biological still unknown mechanism. While the study is still ongoing and recruiting other subjects with hypercoagulation state, we showed in this preliminary analysis that our in-house FC platform for FVIII protein detection screens in depth FVIII protein adherence to blood cells, may shed light on coagulation novel mechanisms, and potentially serve as a diagnostic and prognostic tool. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Role of erythrocytes and platelets in the hypercoagulable status in polycythemia vera through phosphatidylserine exposure and microparticle generation

2013 ◽  
Vol 109 (06) ◽  
pp. 1025-1032 ◽  
Author(s):  
Chunyan Gao ◽  
Xue Yang ◽  
Jianan Li ◽  
Wei Wang ◽  
Jinxiao Hou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Cells ◽  
Significant Inhibition ◽  
Polycythaemia Vera ◽  
Factor V ◽  
Healthy Controls ◽  
Clotting Time ◽  
Thrombotic Risk ◽  
Few Data

SummaryThe development of thrombosis in polycythaemia vera (PV) involves multifactorial processes including pathological activation of blood cells. Release of microparticles (MPs) by activated cells in diseases is associated with thrombotic risk, but relatively few data are available in PV. The aim of the present study was to investigate the increase in MP release and exposure of phosphatidylserine (PS) on the outer membrane of MP-origin cells in patients with PV, and to analyse their procoagulant activity (PCA). PS-positive MPs and cells were detected by flow cytometry, while PCA was assessed with clotting time and purified coagulation complex assays. We found that PV patients had elevated circulating lactadherin+ MPs, which mostly originating from erythrocytes, platelets, granulocytes, and endothelial cells, as well as increased PS exposing erythrocytes/platelets as compared to secondary polycythaemia patients or healthy controls. These PS-bearing MPs and cells were highly procoagulant. Moreover, lactadherin competed factor V and VIII to PS and inhibited about 90% of the detected PCA in a dose-response manner while anti-TF antibody did no significant inhibition. Treatment with hydroxyurea is associated with a decrease in PS exposure and lactadherin+ MP release of erythrocytes/platelets. Our data demonstrate that PV patients are characterised by increased circulating procoagulant MPs and PS exposing erythrocytes/platelets, which could contribute to the hypercoagulable state in these patients.

scholarly journals Expression Pattern of CD55 and CD59 on Red Blood Cells in Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4815-4815
Author(s):  
Salah A. Al Humood ◽  
Lama A. Al-Faris ◽  
Monera Al-Rukhayes
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Healthy Controls ◽  
Cell Disease ◽  
Altered Expression ◽  
Cd59 Expression

Abstract Background: Altered expression of glycosylphosphatidylinositol (GPI)-anchored proteins, might result in increased susceptibility of red blood cells (RBCs) to complement-mediated lysis. Limited information is available on the pattern of CD55 and CD59 expression on RBCs of sickle cell disease (SCD) patients. Methods: Flow cytometric analysis was performed on RBCs from 71 adult SCD patients and 53 healthy controls, using the commercial REDQUANT kit. Results: CD59 deficiency was significantly higher among SCD patients than among healthy controls. The proportions of CD55-deficient and CD59-deficient RBCs from SCD patients were significantly higher when compared with those from healthy controls (0.17 vs. 0.09 and 2.1 vs. 1.2, respectively). The MFI of CD55 and CD59 expression on RBCs in SCD was significantly reduced when compared to the expression healthy controls (5.2 vs. 6.4 and 19.4 vs 20.3, respectively). The pattern of CD55 and CD59 expression was not correlated with anemia, biomarkers of hemolysis, erythropoietin level or other pro-inflammatory markers. Conclusions: There is an altered pattern of CD55 and CD59 expression on RBCs of SCD Patients; however, it does not seem to play a causal role in the pathophysiology of anemia, and is unlikely to be influenced by the level of erythropoietin or other inflammatory mediators. Disclosures No relevant conflicts of interest to declare.

Procoagulant Profile of Platelets from Immune Thrombocytopenia Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1370-1370
Author(s):  
María Teresa Álvarez Román ◽  
Raul Justo Sanz ◽  
Elena Monzon Manzano ◽  
Monica Martín Salces ◽  
Ihosvany Fernandez Bello ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Compensatory Mechanism ◽  
Healthy Controls ◽  
Hepes Buffer ◽  
Prothrombinase Complex ◽  
Fibrinogen Receptor

Abstract Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder in which both increased platelet destruction and insufficient platelet production are involved. Patients can have a range of bleeding manifestations from none to severe at a similar platelet count. In some cases, patients have fewer bleeding symptoms than expected considering the low platelet count that they might have. Objective: The aim of this study was to determine the procoagulant profile of platelets from ITP patients in order to determine whether any of their features may explain this observation. Methods: Twenty-five patients with chronic ITP [(68±100)x109 platelets/L, mean age: 59.6 ± 16.1 years old, 56% female)] and thirty-five healthy controls [(256±36)x109 platelets/L, mean age: 41.6 ± 13.5 years old, 51% female) were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP). To obtain washed platelets, the top two-thirds volumes of PRP were collected and centrifuged (650 g for 10 min at 23°C) after the addition of acid-citrate-dextrose (ACD, 1:10) and the pellet was resuspended in an equal volume of HEPES buffer. Platelet activation was determined by flow cytometry through binding of FITC-PAC1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 micromol/L thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland) or 20 micromol/L ADP. Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions. To characterize platelet ability to bind coagulation factors, washed platelets (1x108/mL) were activated with 100 micromol/L TRAP and then incubated with FVa and/or FXa (5nM each, 10 min, ambient temperature). After fixation with 2% paraformaldehyde to cross-link the platelet-bound factors Va and Xa, platelets were washed two times with Hepes Buffer. Non-specific binding sites were blocked with 8% bovine serum albumin (30 min, room temperature). Following centrifugation, platelets were first incubated with anti-CD41-PE, anti-FVa and/or anti-FXa and then with a secondary FITC-goat anti-mouse IgG and stored at 4°C until flow cytometry analyses. Results: Platelets from ITP patients showed a basal expression of activated fibrinogen receptor similar to controls and a reduced ability for being activated by agonists (% of positive platelets for TRAP-induced PAC1 binding: 60±20 % in controls and 35±23 % in ITP, p<0.01; ADP-induced PAC1 binding: 63±14 % in controls and 50±23 % in ITP, p<0.05). Diminished responses to activation were not due to a reduction in surface expression of fibrinogen receptor in platelets from ITP patients. Platelets from ITP patients expressed more PS than controls under basal conditions [mean fluorescence (MF) for FITC-annexin V binding was: 336±128 in controls, 588±25 in ITP, p<0.05]. Since the PS is the anchor site of the prothrombinase complex, we studied the binding of FVa and FXa at baseline and after activating platelets with TRAP. The binding of these factors in both conditions was higher in the group of patients with ITP (MF for basal FVa binding: 41.4±14.4 in controls, 58.1±24 in ITP, p <0.02; MF for TRAP-induced FVa binding: 44.1±11.4 in controls, 81.4±38 in ITP, p<0.001; MF for basal FXa binding: 45.7±18.4 in controls, 58.1±24 in ITP, p <0.005; MF for TRAP-induced FXa binding: 46.1±16.4 in controls, 72.0±24 in ITP, p<0.05). The lower the platelet count the higher increase in PS exposure (Spearman r =-0,518, p <0.001) and the union of FVa (Spearman r = -0.8571, p <0.001) and FXa (Spearman r = -0.7455, p<0.05). Conclusions: Platelets from ITP patients, despite having less capacity of activation by agonist stimulation, have an increased procoagulant surface with greater ability to bind prothrombinase complex (FXaVa) than those from healthy controls. This feature might be a procoagulant compensatory mechanism that could reduce the risk of bleeding in patients with ITP. This work was supported by a grants from the FIS-FEDER, PI12/01831 and PI15/01457 Disclosures No relevant conflicts of interest to declare.

scholarly journals Asparaginase Immune Complexes Detectable after Asparaginase-Induced Hypersensitivities Activate Basophils Via FcγRIII

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5210-5210
Author(s):  
Sanjay Rathod ◽  
Fred D Finkelman ◽  
Christian Fernandez
Keyword(s):  
Flow Cytometry ◽  
Immune Complexes ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Peripheral Blood Cells ◽  
Dependent Manner ◽  
Igg Antibodies

Abstract Background: Asparaginase (ASNase) is an important chemotherapeutic agent for the treatment of acute lymphoblastic leukemia (ALL). However, the development of anti-ASNase IgG antibodies are frequent, can neutralize the enzyme activity of ASNase, and can increase the risk of ALL relapse. Nevertheless, the role of anti-ASNase IgG antibodies on the onset of ASNase-induced hypersensitivity reactions is unknown, but likely require the formation of ASNase immune complexes (ICs) to activate cells involved in hypersensitivities due to the low binding affinity of the Fcγ receptor for the IgG immunoglobulin. Objective: To detect plasma ASNase ICs after the onset of ASNase hypersensitivities and to demonstrate that binding of ASNase ICs to peripheral blood cells, and the subsequent immune cell activation, is FcγRIII-dependent. Methods: Mice were sensitized to native E.coli ASNase using aluminum hydroxide adjuvant. ICs prepared using the plasma of sensitized mice were precipitated using polyethylene glycol to remove free ASNase and anti-ASNase IgG. ICs were detected by flow cytometry using APC labeled ASNase and protein G beads, characterized by dynamic light scattering and TEM, and the binding properties of ASNase ICs or free ASNase were determined by flow cytometry using non-sensitized and sensitized peripheral murine blood cells and the anti-IgE and/or anti-FcγRIIβ/III blocking mAb. Activation of basophils, which can occur in an IgE- or FcγRIII-dependent manner, was determined ex vivo by measuring changes in CD200R1 or CD200R3 expression after incubating peripheral blood cells with ASNase or ASNase ICs. Results: ASNase ICs were detected after the onset of ASNase hypersensitivities in mice. ASNase ICs formed ex vivo at high anti-ASNase IgG1 concentrations, bound to naïve peripheral blood cells (e.g., neutrophils macrophage/monocytes, and basophils), and activated basophils in an FcγRIII-dependent manner. In contrast, basophil binding and activation by free ASNase was IgE- and not FcγRIII-dependent. Conclusion: Our data indicate that ASNase sensitized mice form ICs after drug administration and can activate basophils via the FcγRIII receptor in mice, whereas the binding and activation by free ASNase occurs via cell-associated IgE. Our study suggests that ASNase ICs can contribute to the onset and severity of ASNase hypersensitivity. Disclosures No relevant conflicts of interest to declare.

Circulating Microparticles and Microparticle Procoagulant Activity in Patients with Antiphospholipid Antibodies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5244-5244
Author(s):  
Karen A Breen ◽  
Katy Sanchez ◽  
Nicola Kirkman ◽  
Kiran Parmar ◽  
Beverley J Hunt
Keyword(s):  
Flow Cytometry ◽  
Antiphospholipid Antibodies ◽  
Conflicts Of Interest ◽  
Procoagulant Activity ◽  
P Value ◽  
Healthy Controls ◽  
Endothelial Microparticles ◽  
Significant Difference ◽  
Thrombotic Complications ◽  
Platelet Microparticles

Abstract Abstract 5244 Background: Microparticles are membrane fragments from cells and microparticles derived from platelets, monocytes and endothelial cells have been implicated in prothrombotic states. Their procoagulant activity is thought to be through exposure of procoagulant phospholipids. Elevated microparticle levels have been found in artherosclerosis, inflammatory states and pregnancy loss. More recently, elevated levels of both endothelial and platelet microparticles have been implicated in APS and thrombosis and in APS and recurrent miscarriage. Microparticles may play a role in the pathogenesis of APS. Aims: The aim of this study was to assess levels of circulating endothelial and platelet microparticles and to measure microparticle procoagulant activity in patients with aPL. Materials & Methods: We obtained samples from 69 patients (69 females, median age 45 (range 19–73) years) who had PAPS according to International Consensus statement criteria, or had persistent aPL without associated complications. 18 healthy controls (18 females, median age 37.5 (range 20–58) years) were recruited and were not known to have antiphospholipid antibodies. Patients with PAPS included 38 with thrombotic complications, 12 with obstetric complications and 19 with isolated aPL. 37/69 patients with thrombotic complications of APS were receiving vitamin K antagonists and 9/19 of those with isolated aPL were receiving aspirin. Blood was drawn by flawless venepuncture into tubes containing citrate. Samples were centrifuged at 3000 rpm for 15 min at 4°C and stored at −80°C until use. A functional ELISA assay of microparticle procoagulant activity (based on prothrombinase activity stimulated by microparticle phospholipid exposure on binding to annexin V immobilised on the ELISA plates) was performed on platelet depleted plasma samples according to manufacturer's protocol (Hyphen Biomed, UK). Intra-assay CV was 3–8%. Endothelial and platelet derived microparticles were detected by flow cytometry, specific surface markers were used to identify platelet microparticles (CD41&CD61, Beckman Coulter, UK) and endothelial microparticles (CD51&CD105, Beckman Coulter, UK). Samples were thawed at room temperature and incubated with CD41-PC, CD61-PC, CD51-FITC and CD105-PE. Enumeration beads were added prior to analysis by flow cytometry for quantification of microparticle levels. The unpaired t-test was used to compare platelet microparticle levels between groups and a p-value of <0.05 was considered statistically significant. Results: Results are shown below in table 1 and are represented as medians and 95% confidence intervals. Microparticle procoagulant activity was lower in patients with aPL/PAPS but did not reach statistical difference(p=0.05). No significant difference was observed in platelet microparticle levels(CD41, CD61) between patients with aPL/PAPS. Endothelial microparticle(CD51) levels were significantly higher in patients with aPL/PAPS compared to healthy controls(p=0.02) but endothelial microparticles(CD105) were not significantly different(p=0.08). No statistical differences were seen in procoagulant activity, platelet or endothelial microparticle levels between patients with aPL according to complication(thrombotic APS, obstetric APS or isolated aPL). Conclusions: Endothelial microparticles (CD51) were elevated but no differences were observed in endothelial microparticles (CD105), platelet microparticles or microparticle procoagulant activity in patients with aPL/PAPS. Most of these patients were receiving anticoagulants and antiplatelet agents but this has not been demonstrated to have an effect on microparticle activity. There are currently a variety of methods to quantify microparticles and as standardisation of methods improve, so too will understanding of relevance of results from this clinical study. Disclosures: No relevant conflicts of interest to declare.

Soluble Endothelial Protein C Receptor (sEPCR) Is Increased in Cirrhotic Patients. A Possible Role in Procoagulant Imbalance?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1080-1080
Author(s):  
Thomas Sinegre ◽  
Cedric Duron ◽  
Ahmed Bouras ◽  
Arnaud Serres ◽  
Marc G Berger ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Coagulation Cascade ◽  
Soluble Form ◽  
Healthy Controls ◽  
Endothelial Protein C Receptor ◽  
Potential Marker ◽  
Post Test ◽  
Cirrhotic Patients

Abstract Context: Cirrhosis impacts the coagulation cascade since the first stage of the liver disease. For years, a procoagulant imbalance, related to the severity of cirrhosis, has been demonstrated. This procoagulant imbalance is detected when coagulation assays are sensibilised to the protein C pathway. A resistance to thrombomodulin (TM) is consistently found in thrombin generation assays. TM and endothelial protein C receptor (EPCR) are two transmembrane proteins involved in the anticoagulant protein C pathway. A soluble form of EPCR (sEPCR) is release of the endothelial surface upon cleavage by mettaloprotease. Soluble EPCR have a procoagulant and proinflammatory effects: bound to PC or activated PC (APC), PC is unavailable for activation by thrombin-TM complex and APC is unable to inactivate FVa and FVIIIa. The aim of this study was to evaluate the plasma levels of sEPCR in non-complicated cirrhotic patients as a potential marker for procoagulant imbalance, compared to healthy controls. Materials and Methods: Patients prospectively included were confirmed cirrhotic patients (prothrombin time <70% and/or liver dysmorphia and/or fibroscan > 20 kPa and/or histology and/or association of portal hypertension and liver failure). Patients were free of hepatocellular carcinoma and were not anticoagulated. Patients with on-going infection or inflammatory complication were excluded. None of them had a thromboembolic event or a familial history of thromboembolism. Controls were free of anticoagulation, coagulation disorders and without oral contraceptives. All ethical requirments were obtained and patients and controls gave their written informed consent. Kruskal-Wallis test and Dunn post test were used for statistical analysis. Results are expressed as mean (Q1 - Q3) values of sEPCR. Results: Fifty two cirrhotic patients (24 Child-Pugh A, 21 Child-Pugh B and 7 Child-Pugh C) and 32 healthy controls were prospectively included in our hospital. The sEPCR values for healthy controls were 100 ng/mL (81 - 135 ng/mL). Comparatively to controls, Child-Pugh A patients and Child-Pugh B patients had statistically significant higher values of sEPCR : 169 ng/mL (117-225 ng/mL, p < 0.01) and 176 (135 - 230 ng/mL, p < 0.001) respectively (Figure 1). Compared to healthy controls, Child-Pugh C patients had an increased level of sEPCR : 174 ng/mL (78 - 254 ng/mL, not significant but probably underpowered). These data are in lines with the thrombin generation profiles showing a resistance to TM in cirrhotic patients increasing from Child-Pugh A to C. No patient have developed thrombosis during the follow-up period. Conclusion: Patients with Child-Pugh A and B cirrhosis had significantly higher values of sEPCR compared to healthy controls (p < 0.01 for both). The putative role of sEPCR as a marker of the procoagulant imbalance associated to cirrhosis remains to be accurately evaluated. Figure 1. sEPCR levels in plasma of cirrhotic patients. Figure 1. sEPCR levels in plasma of cirrhotic patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Procoagulant Activity of Blood and Endothelial Cells via Phosphatidylserine Exposure and Microparticle Delivery in Patients with Diabetic Retinopathy

2018 ◽  
Vol 45 (6) ◽  
pp. 2411-2420 ◽  
Author(s):  
Ying Su ◽  
Jingli Chen ◽  
Zengxiang Dong ◽  
Yan Zhang ◽  
Ruishuang Ma ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Diabetic Retinopathy ◽  
Confocal Microscopy ◽  
Blood Cells ◽  
Procoagulant Activity ◽  
Diabetic Patients ◽  
Healthy Controls ◽  
Clotting Time ◽  
Coagulation Time

Background/Aims: The mechanisms for thrombosis in diabetic retinopathy (DR) are complex and need to be further elucidated. The purpose of this study was to test phosphatidylserine (PS) exposure on microparticles (MPs) and MP-origin cells from the circulation and to analyze cell-/MP-associated procoagulant activity (PCA) in DR patients. Methods: PS-positive MPs and cells from healthy controls (n = 20) and diabetic patients (n = 60) were analyzed by flow cytometry and confocal microscopy. Clotting time and purified coagulation complex assays were used to measure PCA. Results: PS exposure on platelets and monocytes was higher in proliferative DR (PDR) patients than in non-PDR patients or controls. The highest levels of MPs (derived from platelets [30%], erythrocytes [13%], leukocytes [28%], and endothelial cells [10%]) were found in patients with PDR. In addition, PS exposure on blood cells and shed MPs in DR patients led to significantly increased FXa and FIIa generation, fibrin formation, and markedly shortened coagulation time. Moreover, lactadherin reduced 70% of PCA by blocking PS, while an anti-tissue factor antibody had a smaller effect. Conclusion: Our results confirmed that PCA in DR patients may be partly ascribed to PS exposure and MP release from blood and endothelial cells. Lactadherin may act as an efficient anticoagulant factor in this process.

scholarly journals Increased Phosphatidylserine-Exposing Microparticles and Their Originating Cells Are Associated with the Coagulation Process in Patients with Oral Squamous Cell Carcinoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1222-1222
Author(s):  
Yingmiao Liu ◽  
Cong Zhang ◽  
Yan Kou ◽  
Lili Zou ◽  
Hui Liang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Squamous Cell Carcinoma ◽  
Endothelial Cells ◽  
Cell Carcinoma ◽  
Binding Sites ◽  
Blood Cells ◽  
Plasma Levels ◽  
Stage Iv ◽  
Radical Resection ◽  
Healthy Controls

Abstract Introduction:Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck area, and the incidence remains high.Despite advances in diagnosis and treatment, the poor prognosis of OSCC is characterized by a high rate of local recurrence and the overall five-year survival rate remains at approximately 50%. Therefore, the mechanisms underlying the development of OSCC still need to be clarified. Patients with cancer tend to develop a hypercoagulable state which predisposes them to thromboembolic events. Cancer increases the risk of venous thrombosis several fold with varying degree of relative risks (range 4-7). A recent study has reported that microparticles (MPs) increased procoagulant activity (PCA) in OSCC. MPs are small membrane vesicles of 0.1-1 µm containing negatively charged, procoagulant phosphatidylserine (PS), which plays an important role in thrombosis. The definitive role of PS in the hypercoagulable state in patients with OSCC remains unclear. Our objectives were to measure the PS exposure on MPs, blood cells, and endothelium, and to evaluate their PCA in different stages of OSCC. Methods: OSCC patients (n = 57) and healthy controls (n = 26) were included in our study. Blood samples were obtained from controls and OSCC patients within 1 day before surgery and 2-week after surgery. Human umbilical vein endothelial cells (HUVECs) were incubated in growth media containing 20% of pool serum obtained from either OSCC patients or healthy donors at room temperature for 24 h, respectively. Exposed PS was analyzed with flow cytometry and confocal microscopy. Lactadherin was used to quantify PS exposure on MPs and their original cells. PCA of MPs and these cells was evaluated using clotting time, purified coagulation complex, and fibrin formation assays. Meanwhile, the inflammation-related cytokines were detected by enzyme-linked immunosorbent assay. Results: Using flow cytometry, plasma levels of PS+ blood cells and MPs in OSCC patients with stage III/IV were significantly higher than those in stage I/II patients or healthy controls (all P < 0.05). However, we only found a significant difference between stage I or II and controls (P < 0.05) in total PS+ MPs and PMPs. Similarly, we found that the endothelial cells (ECs) treated with OSCC serum in vitro exposed more PS than those with healthy serum. Moreover, in OSCC patients with stage IV, MPs primarily originated from platelets (53.9 ± 3.2%) followed by leukocytes (21.8 ± 2.1%, including MPs from neutrophils, monocytes and lymphocytes), erythrocytes (6.3 ± 0.6%) and ECs (6.9 ± 0.8%). Additionally, PS+ blood cells, MPs and OSCC serum-cultured ECs markedly promoted shortened coagulation time and significantly increased FXa/thrombin/fibrin generation in stage IV or III OSCC patients compared with controls (all P < 0.05). Interestingly, confocal imaging of MPs or OSCC serum-treated ECs showed binding sites for FVa and FXa to form prothrombinase. Furthermore, blockade of PS on MPs/blood cells/ECs with lactadherin inhibited PCA by approximately 80%. Most importantly, we found treatment with radical resection significantly reduced the amount of PS+ blood cells, ECs and MPs, and prolonged the clotting times of those cells and MPs compared with presurgery patients. Lastly, the correlation analysis revealed that the plasma levels of interleukin 6, interleukin 8 and tumor necrosis factor α were positively correlated with the levels of total PS+ MPs and PS+ platelets in OSCC patients. Conclusions: Our results suggested that PS+ blood cells and MPs play a prominent role in inducing the hypercoagulable and prothrombotic state especially in advanced OSCC. Interestingly, we found treatment with radical resection could attenuate this effect. Moreover, the released inflammatory cytokines may contribute to PS exposure on platelets and MPs and the increased procoagulant activity in patients with OSCC. Notably, our findings also show that PS provides binding sites for FXa and prothrombinase complexes and promotes thrombin formation. Therefore, directly targeting FXa and prothrombinase complexes might decrease thrombotic risk OSCC patients. This study shows that future research should focus on the application of PS inhibitors as a novel therapeutic strategy in OSCC patients when coagulation is abnormally enhanced. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tim-3 associated with apoptotic NK cells and disease activity in SLE

2021 ◽  
Vol 19 ◽  
pp. 205873922110005
Author(s):  
Di Zhao ◽  
Xiao Yang ◽  
Jie Zhang ◽  
Yi Zhang
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
T Cell ◽  
Disease Activity ◽  
Nk Cells ◽  
Lupus Erythematosus ◽  
Healthy Controls ◽  
Systemic Lupus ◽  
Potential Target

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) has been found to play important roles in systemic lupus erythematosus (SLE), however, whether Tim-3 is involved in apoptosis of NK cells in SLE remains unknown. The proportion of CD3−CD56+ NK cells and the percentage of AnnexinV+ NK cells were analyzed by flow cytometry in SLE patients and healthy controls. Tim-3 expression on NK cells was also evaluated by flow cytometry. We firstly observed a decreased proportion of NK cells and an increased proportion of apoptotic NK cells in SLE patients. The proportion of apoptotic NK cells was positively correlated with anti-dsDNA and SLEDAI. Tim-3 expression on NK cells was up-regulated in SLE patients. Further analysis showed that Tim-3 expression on NK cells was negatively correlated with the proportion of apoptotic NK cells, anti-dsDNA and SLEDAI, while positively correlated with the proportion of NK cells. The present results suggest that Tim-3 might play roles in SLE by regulating the apoptosis of NK cells and Tim-3 might serve as a potential target for the treatment of SLE.

scholarly journals POS0776 CORRELATION OF PERIPHERAL CD4+GRANZB+CTLS WITH DISEASE SEVERITY IN PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 641.3-642
Author(s):  
Q. Wang ◽  
L. Gu ◽  
M. Zhang
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Sjögren’S Syndrome ◽  
Mhc Class Ii ◽  
Sjögren's Syndrome ◽  
Sjogren’S Syndrome ◽  
Sjogren's Syndrome ◽  
Healthy Controls ◽  
Extraglandular Manifestations ◽  
Sjögren‘S Syndrome

Background:Sjögren’s syndrome (SS) is a chronic autoimmune disorder. The major histopathologic lesion of it is a focal lymphocytic infiltrate around ductal and acinar epithelial cells, which include a majority of CD4+T. Several studies have shown that the epithelial cells in SS present diverse phenomena, such as MHC class II overexpression. CD4+T cells with cytotoxic activity (CD4 CTL) have been detected in various immune responses. They are characterized by their ability to secrete perforin and granzyme B to kill the target cells in an MHC class II-restricted fashion.Objectives:So this study was to investigate the correlation of peripheral CD4+GranzB+CTLs with disease severity and organ involvement in patients with primary Sjögren’s syndrome.Methods:We recruited 116 pSS patients and 46 healthy controls using flow cytometry to examine proportion of CD4+GranzB+CTLs in their peripheral blood, and immunofluorescence to test the expression of CD4+GranzB+CTLs in labial gland. The correlations of CD4+GranzB+CTLs and the relevant clinical data were analyzed.Results:We analyzed the percentage of CD4+GranzB+cytotoxic T cells in peripheral blood mononuclear cells (PBMCs) by flow cytometry. Frequency of peripheral CD4+GranzB+CTLs were measured in 116 patients with pSS and 46 healthy controls matched for age and sex. The percentage of CD4+GranzB+CTLs were significantly up-regulated in pSS patients than healthy controls (7.1%±4.9% vs 3.1%±1.9%, p <0.0001) and positive correlation with ESSDAI in pSS patients(r = 0.6332, p<0.001). The percentage of CD4+GranzB+CTLs were markedly higher in pSS patients with extraglandular manifestations. Moreover, CD4+GranzB+CTLs were observed in the lymphocytic foci and periductal areas of the LSGs and were elevated with increased foci index (FI). After excluding the other risk factors associated with pSS, CD4+GranzB+CTLs were still related to ESSDIA and extraglandular manifestations independently(p<0.05). ROC curve analysis indicated that the area under the curve (AUC) of CD4+GranzB+CTLs was 0.796 to predict the activity of pSS, and 0.851 to presume extraglandular manifestations. The best diagnostic cut-off point was 4.865 for pSS patients.Conclusion:In this study, We provide new evidence indicating involvement of CD4+GranzB+CTLs over activation in the disease pathophysiology of pSS, which may serve as a new biomarker to evaluate the activity and severity of pSS.References:[1]Takeuchi A, Saito T. Front Immunol. (2017) 23:194.[2]Brown DM, et al. Front Immunol. (2016) 9:93.[3]Polihronis M, et al. Clin Exp Immunol. (1998) 114:485-90.[4]Xanthou G, et al. Clin Exp Immunol. (1999) 118:154-63.[5]Maehara T, et al. Ann Rheum Dis. (2017) 76:377-385.[6]Goules AV, et al. Clin Immunol. (2017) 182:30-40.[7]Hashimoto K, et al. Proc Natl Acad Sci U S A. (2019) 116:24242-24251.[8]Croia C, et al. Arthritis Rheumatol. (2014) 66:2545-57.[9]Schmidt D,et al. J Clin Invest. (1996) 97:2027–37.[10]Pandya JM, et al. Arthritis Rheum. (2010) 62:3457–66.[11]Moosig F, et al. Clin Exp Immunol. (1998) 114:113–8.[12]Peeters LM, et al. Front Immunol. (2017) 20:1160.Table 1.Multivariate analysis of CD4+GranzB+CTLs influenced by pSS-related factorsregression coefficientstandard errort-statisticsp value95%CICD8+GranzB+CTLs(%)0.1440.0334.3346.9E-50.077, 0.211ESSDAI0.2560.1222.0950.0410.011, 0.502extraglandular manifestations2.6121.2682.0590.0450.065, 5.158Figure 1.Receiver operating characteristic (ROC) curve of the frequency of CD4+GranzB+CTLs to predict ESSDAI and extraglandular manifestations responseDisclosure of Interests:None declared

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