scholarly journals The Exposure of Phosphatidylserine Influences Procoagulant Activity in Retinal Vein Occlusion by Microparticles, Blood Cells, and Endothelium

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Ying Su ◽  
Xueqing Deng ◽  
Ruishuang Ma ◽  
Zengxiang Dong ◽  
Feng Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Retinal Vein Occlusion ◽  
Blood Cells ◽  
Coagulation Factors ◽  
Procoagulant Activity ◽  
Anticoagulant Effect ◽  
Clotting Time ◽  
Coagulation Time ◽  
Vein Occlusion

The pathogenesis of hypercoagulability in retinal vein occlusion (RVO) is largely unknown. Whether the exposure of phosphatidylserine (PS) and microparticle (MPs) release will affect procoagulant activity (PCA) in RVO needs to be investigated.Objectives. To evaluate PS expression, circulating MPs, and the corresponding PCA in RVO patients. Twenty-five RVO patients were compared with 25 controls. PS-positive cells were detected by flow cytometry. Cell-specific MPs were measured by lactadherin for PS and relevant CD antibody. We explored PCA with coagulation time, purified coagulation complex assays, and fibrin production assays. In RVO, MPs from platelets, erythrocytes, leukocyte, and endothelial cells were increased and the exposure of PS was elevated significantly when compared with controls. In addition, we showed that circulating MPs in RVO patients were mostly derived from platelets, representing about 60–70% of all MPs, followed by erythrocytes and leukocytes. Moreover, PS exposure, ECs, and MPs in RVO lead to shortened clotting time with upregulation of FXa and thrombin formation obviously. Importantly, ECs treated with RVO serum which bounded FVa and FXa explicitly suggested the damage of retinal vein endothelial cells. Furthermore, lactadherin can inhibit the combination between PS and coagulation factors by approximately 70% and then exert an anticoagulant effect. In summary, circulating MPs and exposed PS from different cells may contribute to the increased PCA in patients with RVO. Lactadherin can be used for PS detection and an anticoagulant agent.

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.

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.

Daunorubicin induces procoagulant activity of cultured endothelial cells through phosphatidylserine exposure and microparticles release

2010 ◽  
Vol 104 (12) ◽  
pp. 1235-1241 ◽  
Author(s):  
Huibo Li ◽  
Fenglin Cao ◽  
Yanhua Su ◽  
Shengjin Fan ◽  
Yinghua Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Chemotherapeutic Agents ◽  
Significant Inhibition ◽  
Procoagulant Activity ◽  
Clotting Time ◽  
Increased Risk ◽  
Umbilical Vein Endothelial Cells ◽  
The Impact ◽  
Prothrombotic Effect

SummaryAdministration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 μM) for 24 hours. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 μM of daunorubicin or more. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process.

Lactadherin as a Probe for Phosphatidylserine Exposure and as An Anticoagulant for the Procoagulant Activity in the Study of Stored Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3150-3150
Author(s):  
Jin Zhou ◽  
Jinxiao Hou ◽  
Wen Li ◽  
Xiaoqian Zhang ◽  
Yueyue Fu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Progressive Increase ◽  
Procoagulant Activity ◽  
Platelet Concentrates ◽  
Time Analysis ◽  
Coagulation Time ◽  
Enzymatic Assays

Abstract Abstract 3150 Poster Board III-87 Background Phosphatidylserine (PS) can support coagulant reactions. However, it is uncertain how the location and extent of PS exposure to the membranes of stored platelets affect such reactions. We compared annexin V with lactadherin as a way of detecting how of PS exposure influences the procoagulant properties of stored platelets in platelet concentrates (PCs). Method PS exposure and the relevant procoagulant activity (PCA) of platelets in 5 different PCs were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. PS exposure was separately measured using annexin V and lactadherin, respectively. Results Exposure of PS to stored platelets promoted thrombin formation. A progressive increase in PS exposure was detected by flow cytometry. Moreover, using lactadherin, we identifed higher levels of PS exposure on the platelets and platelet-derived microparticles (PMPs) compared to detection using annexin V. The percentage of PS-positive cells was 0.02 % by annexin V versus 0.3 % by lactadherin on day 0, 7.5 % by annexin V versus 12.3 % by lactadherin on day 5, and 29 % by annexin V versus 44.3 % by lactadherin on day 9. Rare microparticles (MPs) were released from fresh platelets, and, the number of PMPs increased approximately 2-fold on day 5 and further progressively increased. Using lactadherin and platelets in the earlier stage of storage, confocal microscopy revealed earlier and localized PS exposure based on plasma membrane staining. For later storage platelets, increased levels of PS-positive platelets and PMPs were clearly detected by both annexin V and lactadherin. Thirty-two nM lactadherin or annexin V prolonged coagulation time 2.4 fold versus 2 fold. The productions of thrombin and intrinsic/extrinsic factor Xase were approximately inhibited 85 % and 60 % by lactadherin and annexin V, respectively. Conclusion PS exposure was localized to the cellular rims, blebbing vesicles and thin elongated filopodia-like areas on banked platelets. Furthermore, lactadherin provides a more accurate measurement of PS exposure and the relevant with PCA, which is an important factor to consider for transfusion medicine. Our findings of elevated PS-positive platelets and PMPs indicate that platelets should not be stored for extended periods of time. Disclosures No relevant conflicts of interest to declare.

Daunorubicin Induces Procoagulant Activity of Cultured Endothelial Cells through Phosphatidylserine Exposure and Microparticles Release

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5185-5185
Author(s):  
Yueyue Fu ◽  
Huibo Li ◽  
Wen Li ◽  
Xiushuai Dong ◽  
Jinxiao Hou ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Conflicts Of Interest ◽  
Umbilical Vein ◽  
Chemotherapeutic Agents ◽  
Significant Inhibition ◽  
Procoagulant Activity ◽  
Clotting Time ◽  
Increased Risk ◽  
Umbilical Vein Endothelial Cells ◽  
The Impact

Abstract Abstract 5185 Administration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 μ M) for 24 h. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 μ M of daunorubicin or over. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 380-388 ◽  
Author(s):  
EM Bevers ◽  
T Wiedmer ◽  
P Comfurius ◽  
SJ Shattil ◽  
HJ Weiss ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Calcium Ionophore ◽  
Coagulation Factors ◽  
Calcium Ionophore A23187 ◽  
Bleeding Disorder ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor Va ◽  
Resealed Ghosts

The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.

scholarly journals Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 380-388 ◽  
Author(s):  
EM Bevers ◽  
T Wiedmer ◽  
P Comfurius ◽  
SJ Shattil ◽  
HJ Weiss ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Calcium Ionophore ◽  
Coagulation Factors ◽  
Calcium Ionophore A23187 ◽  
Bleeding Disorder ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor Va ◽  
Resealed Ghosts

Abstract The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.

EFFECTS OF INTRAVENOUS INJECTIONS OF THE ANTICOAGULANT FRACTION OF INCUBATED FIBRINOGEN ON BLOOD COAGULATION

1960 ◽  
Vol 38 (1) ◽  
pp. 909-918 ◽  
Author(s):  
D. C. Triantaphyllopoulos
Keyword(s):  
Prothrombin Time ◽  
Bleeding Time ◽  
Jugular Vein ◽  
Coagulation Factors ◽  
External Jugular Vein ◽  
Anticoagulant Effect ◽  
Clotting Time ◽  
Intravenous Injections ◽  
Fibrinogen Content ◽  
Ammonium Sulphate Saturation

AFIF (Anticoagulant Fraction of Incubated Fibrinogen precipitated between 25 and 50% ammonium sulphate saturation) prepared from Armour's incubated bovine fibrinogen was injected in a dosage of 46–106 mg (mean: 66.5 mg) tyrosine per kilogram body weight in the external jugular vein of 10 rabbits. Blood samples were withdrawn at [Formula: see text]- or 1-hour intervals until the aspirated blood began showing signs of coagulation. The anticoagulant effect was manifested immediately and lasted for [Formula: see text] to [Formula: see text] hours in the animal. Although the blood aspirated during this time remained unclotted even after 24 hours, the surgical wound in the neck of the animal did not bleed and autopsies revealed no sign of internal haemorrhage. The bleeding time, however, was found prolonged when tested by cutting the marginal vein of the ear. The level of the coagulation factors was determined both in oxalated specimens and in specimens with no anticoagulant added. Both kinds of sample showed: (1) clottable fibrinogen content normal, thus excluding fibrinolysis as the cause of the anticoagulant effect; (2) thrombin clotting time of infinity; (3) one-stage prothrombin time longer than 60 seconds; (4) no correction of the infinite thrombin clotting time of oxalated specimens following the addition of 0.25 mg protamine per ml. This makes unlikely any appreciable release of heparin by the animal. However, oxalated and non-oxalated specimens differed in the following respects: (1) prothrombin time determined after adsorption and mixing of the eluate with adsorbed plasma was found to be normal for the oxalated blood but variably increased for the non-oxalated specimen (10–34.5 seconds); (2) the plasma precursors of plasma thromboplastin were normal in oxalated but very low in native specimens. The serum precursors of plasma thromboplastin were normal.

scholarly journals Prevention of Heme-Induced Human Endothelial Cell Activation By Hemopexin in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 8-8
Author(s):  
Jacqueline Adam ◽  
Thomas Gentinetta ◽  
Svetlana Diditchenko ◽  
Alexander Schaub ◽  
Gregory J Kato ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Sickle Cell Disease ◽  
Blood Cells ◽  
Cell Activation ◽  
Cell Disease ◽  
Endothelial Cell Activation ◽  
Free Heme

Hemoglobin (Hb) is one of the most abundant proteins in the human body. When red blood cells rupture, cell-free Hb may initiate adverse pathophysiological reactions. Pathophysiology triggered by cell-free Hb plays an important role in modifying the phenotype of sickle cell disease (SCD). SCD is caused by a single nucleotide mutation of the β-globin gene resulting in Hemoglobin-S (HbS) instead of the normal HbA found in healthy individuals. Polymerization of HbS shortens the lifespan of sickle red blood cells and promotes intra- and extravascular hemolysis. In cell-free Hb ferrous Hb (Fe2+) is oxidized into ferric Hb (Fe3+) promoting the dissociation and transfer of heme into lipid compartments where it triggers lipid peroxidation and generation of cytotoxic and pro-inflammatory reaction products. These processes promote endothelial cell activation and damage. The endogenous plasma protein hemopexin exhibits the highest binding affinity for heme and binds heme in a 1:1 binding ratio. Heme bound to hemopexin is rendered relatively non-reactive and is delivered safely to hepatocytes for endocytosis and degradation. To investigate the endothelial-protective function of hemopexin based on its ability to scavenge heme, we exposed human umbilical vein endothelial cells (HUVEC) in vitro to heme(NaOH) in the presence or absence of different hemopexin doses. As a read-out, different markers for endothelial cell activation were analyzed by either flow cytometry or multiplexed particle-based flow cytometry (Luminex). Briefly, confluent HUVEC were preincubated with hemopexin at different concentrations for 5 min before stimulation with heme(NaOH) for 25 min. Following stimulation cells were analyzed by flow cytometry for expression of membrane bound P-Selectin, a robust marker of endothelial cell activation. Alternatively, heme(NaOH) stimulation of hemopexin-preincubated HUVEC was conducted for 16 h and cell culture supernatants were analyzed by Luminex for three additional well-characterized plasma markers of endothelial cell activation: pro-inflammatory cytokine IL-8, cell adhesion molecule VCAM-1 and glycoprotein Von Willebrand factor (vWF). In the absence of hemopexin, heme(NaOH) consistently induced robust cell surface expression of P-Selectin and elevated levels of soluble IL-8, VCAM-1 and vWF. However, hemopexin completely blocked the stimulatory potential of heme as HUVEC exposed to pre-formed heme:hemopexin complexes showed unchanged P-Selectin expression levels compared to negative control samples. We found that hemopexin reduced heme(NaOH)-mediated P-selectin expression on HUVEC in a dose-dependent fashion. Once an equimolar ratio between heme and hemopexin was reached, P-selectin expression was abolished as shown in figure 1. In addition to P-Selectin, hemopexin also had a strong effect to reduce the heme-induced expression of IL-8, VCAM-1 and vWF to background levels. Thus, the presented data underlines on the one hand the stimulatory capacity of heme(NaOH) on endothelial cells and demonstrates on the other hand the potential of hemopexin to efficiently neutralize free heme. In a stoichiometric fashion, hemopexin potently prevents the pro-inflammatory effect of heme on endothelial cells. Hence, our study suggests a protective role of hemopexin for endothelial cells exposed to elevated levels of cell-free heme due to intravascular hemolysis. Additional experiments are required to elucidate the effect of hemopexin on the endothelium in more detail. Combined with our other lines of data, our results further support the investigation of hemopexin as a potential therapeutic agent in the treatment of sickle cell disease. Disclosures Adam: CSL Behring AG: Current Employment. Gentinetta:CSL Behring: Current Employment. Diditchenko:CSL Behring AG: Current Employment. Schaub:CSL Behring AG: Current Employment. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment.

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