Endotheliopathy in septic conditions: mechanistic insight into intravascular coagulation

AbstractEndothelial cells play a key role in maintaining intravascular patency through their anticoagulant properties. They provide a favorable environment for plasma anticoagulant proteins, including antithrombin, tissue factor pathway inhibitor, and protein C. Under septic conditions, however, the anticoagulant properties of endothelial cells are compromised. Rather, activated/injured endothelial cells can provide a scaffold for intravascular coagulation. For example, the expression of tissue factor, an important initiator of the coagulation pathway, is induced on the surface of activated endothelial cells. Phosphatidylserine, a high-affinity scaffold for gamma-carboxyglutamate domain containing coagulation factors, including FII, FVII, FIX, and FX, is externalized to the outer leaflet of the plasma membrane of injured endothelial cells. Hemodilution decreases not only coagulation factors but also plasma anticoagulant proteins, resulting in unleashed activation of coagulation on the surface of activated/injured endothelial cells. The aberrant activation of coagulation can be suppressed in part by the supplementation of recombinant antithrombin and recombinant thrombomodulin. This review aims to overview the physiological and pathological functions of endothelial cells along with proof-of-concept in vitro studies. The pathophysiology of COVID-19-associated thrombosis is also discussed.

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Association of Tissue Factor Pathway Inhibitor With Human Umbilical Vein Endothelial Cells

Blood ◽

10.1182/blood.v90.9.3568 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3568-3578 ◽

Cited By ~ 20

Author(s):

John-Bjarne Hansen ◽

Randi Olsen ◽

Paul Webster

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Umbilical Vein ◽

Coagulation Factors ◽

Coagulation System ◽

Human Umbilical Vein ◽

Tissue Factor Pathway ◽

Umbilical Vein Endothelial Cells

AbstractTissue factor pathway inhibitor (TFPI) is a serine protease inhibitor of the extrinsic coagulation system, synthesized in endothelial cells, which has recently been shown to play an important role in the regulation of activated coagulation factors at the endothelial cell surface. In the present study we investigated the subcellular localization and metabolism of TFPI in human umbilical vein endothelial cells (HUVEC). Immunocytochemical labeling of HUVEC with anti-TFPI showed specific labeling associated with the cell surface and with many intracellular organelles including the Golgi complex. Further characterization of these organelles was performed by colocalizing the anti-TFPI with 3-(2,4-dinitroanilino)′-amino-N-methyldipropylamine (DAMP; to demonstrate low pH), mannose phosphate receptor (endosomes), and LAMP 1 (late endocytic compartments). TFPI also colocalized with antibodies to the human transferrin receptor, a marker for early endocytic, recycling compartment. Endogenous TFPI colocalized with biotin in intracellular vesicles during endocytosis after biotinylation of the cell surface, which indicated that TFPI was being co-internalized with the surface biotin. The binding of exogenously added 125I-TFPI increased linearly to HUVEC over the concentration range of 0 to 32 nmol/L without saturation, the binding was not affected by up to a thousand-fold molar excess of unlabeled TFPI, and heparin inhibited the binding dose dependently. An intact C-terminal domain was important for the interaction between TFPI and the cell surface of HUVEC, because less than 10% of a C-terminal truncated form of TFPI (TFPI1-161 ) was bound after addition of equimolar concentrations of full-length TFPI. Exogenously added 125I-TFPI was not degraded in HUVEC during 4 hours at 37°C. The presence of TFPI in endocytic and recycling compartments support the hypothesis that endogenous, membrane-anchored TFPI could be internalized for subsequent recycling back to the cell surface.

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Tissue factor pathway inhibitor (TFPI) interferes with endothelial cell migration by inhibition of both the Erk pathway and focal adhesion proteins

Thrombosis and Haemostasis ◽

10.1160/th07-10-0623 ◽

2008 ◽

Vol 99 (03) ◽

pp. 576-585 ◽

Cited By ~ 19

Author(s):

Mathieu Provençal ◽

Marisol Michaud ◽

Édith Beaulieu ◽

David Ratel ◽

Georges-Étienne Rivard ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Tissue Factor ◽

Focal Adhesion ◽

Tissue Factor Pathway Inhibitor ◽

Endothelial Cell Migration ◽

Cell Functions ◽

Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that is mainly known for its inhibition of tissue factor-mediated coagulation. In addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. In this work we demonstrate that at concentrations within the physiological range,TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro. These effects were specific to endothelial cells since no inhibitory effect was observed on the migration of tumor (glio- blastoma) cells. Inhibition of endothelial cell migration was correlated with a concomitant loss in cell adhesion,suggesting an alteration of focal adhesion complex integrity. Accordingly,we observed thatTFPI inhibited the phosphorylation of focal adhesion kinase and paxillin,two key proteins involved in the scaffolding of these complexes, and that this effect was specific to endothelial cells. These results suggest that TFPI influences the angiogenic process via a non-haemostatic pathway, by downregulating the migratory mechanisms of endothelial cells.

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Platelet effects on tissue factor and fibrinolytic inhibition of cultured human fibroblasts and vascular cells

Blood ◽

10.1182/blood.v60.1.140.bloodjournal601140 ◽

1982 ◽

Vol 60 (1) ◽

pp. 140-147

Author(s):

PE Smariga ◽

JR Maynard

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Human Fibroblasts ◽

Cellular Tissue ◽

Cultured Human Fibroblasts ◽

Platelet Concentration ◽

The Media ◽

Low Levels ◽

Coagulation Pathway

Platelets stimulate tissue factor, the initiator of the extrinsic coagulation pathway, and increase fibrinolytic inhibition in fibroblasts grown in vitro. Cellular tissue factor increases an average of 2.8-fold over the control levels after a 6-hr incubation with platelets, and no activity is present in the media. Fibrinolytic inhibition is stimulated in both the fibroblasts and their media in the presence of platelets and accumulates throughout a 24-hr incubation. Neither leukocytes nor erythrocytes stimulate these changes. Both tissue factor and fibrinolytic inhibition increases are dependent on platelet concentration and are blocked by inhibitors of RNA or protein synthesis. Control smooth muscle cells have higher tissue factor and fibrinolytic inhibition than fibroblasts, but their response to the presence of platelets is similar. Confluent monolayers of endothelial cells have very low levels of tissue factor that are not altered by the presence of platelets. However, the ability of endothelial cells to inhibit fibrinolysis is enhanced by the presence of platelets. The fraction that stimulates tissue factor and fibrinolytic inhibition is distinct from platelet-derived growth factor and from the fraction that enhances leukocyte tissue factor. It is associated with an insoluble, nonmitogenic fraction that is not inactivated by phospholipase C, or diisopropylfluorophosphate, nor is it chloroform:methanol extractable. Platelets are a physiologic modulator for both cellular tissue factor and the fibrinolytic system in vitro.

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Effect of Intradermal Tumor Necrosis Factor-α-induced Inflammation on Coagulation Factors in Dermal Vessel Endothelium

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615693 ◽

2001 ◽

Vol 85 (02) ◽

pp. 362-367 ◽

Cited By ~ 23

Author(s):

Christoph Kopp ◽

Ingrid Simonitsch ◽

Bernd Jilma ◽

Burkhard Jansen ◽

Markus Exner ◽

...

Keyword(s):

Tumor Necrosis Factor ◽

Tissue Factor ◽

Tumor Necrosis ◽

Tissue Factor Pathway Inhibitor ◽

Coagulation Factors ◽

Tissue Factor Pathway ◽

Factor Α ◽

Necrosis Factor

SummaryInflammatory mediators were shown to exert procoagulant effects on cultured human endothelial cells (EC). In the present study the effect of intradermal application of tumor necrosis factor- (TNF-) on the expression of factors involved in regulation of coagulation at the EC surface, i. e. tissue factor (TF), thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI) was studied in humans in vivo. The endothelial expression of these factors was evaluated immunohistochemically in biopsies taken after intradermal application of 5000 U TNF- in 8 healthy volunteers. After 6 and 22 h biopsies were taken from the injection sites. At TNF- injected sites typical inflammatory changes, e. g. EC upregulation of adhesion molecules and accumulation of leukocytes were detected. In parallel we could document EC expression of TF, downregulation of TM and depletion of tissue factor pathway inhibitor (TFPI) in inflamed areas. Early depletion of endothelial I B at the site of inflammation after application of TNF- points to an activation of the NF- B pathway. Our data suggest that, as shown in in vitro experiments, TNF- activates the NF- B pathway and induces specific procoagulant changes of EC due to expression of TF, down-regulation of TM and depletion of TFPI in vivo in humans. This procoagulant shift in the haemostatic balance on the cell surface, caused by TNF- -induced inflammation, is likely to contribute to thrombosis associated with tissue inflammation in humans.

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Localization of Tissue Factor Pathway Inhibitor In Endothelial Cells: Pitfalls and Challenges

Microscopy and Microanalysis ◽

10.1017/s1431927600018948 ◽

1999 ◽

Vol 5 (S2) ◽

pp. 1124-1125

Author(s):

R. Olsen ◽

J.-B. Hansen ◽

P. Webster

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Umbilical Vein ◽

Light And Electron Microscopy ◽

Coagulation Factors ◽

Endocytic Pathway ◽

Coagulation System ◽

Tissue Factor Pathway ◽

Umbilical Vein Endothelial Cells

Tissue Factor Pathway Inhibitor (TFPI) is a potent inhibitor of the extrinsic coagulation system, synthesized in endothelial cells. Membrane bound TFPI has recently been shown to play an important role in the degradation of activated coagulation factors in the endothelium. Comparison of subcellular TFPI localization patterns and TFPI production between Human Umbilical Vein Endothelial Cells (HUVEC) and an immortalized cell line (ECV 304) are presented here, as are the essential protocol modifications that we used to obtain labeling results by both light- and electron microscopy. We show that ECV 304 cells are similar to HUVEC in that TFPI is present in the Golgi complex (fig. 1) as well as in the endocytic pathway of ECV cells. Additionally, they secrete higher levels of TFPI into the culture medium than HUVEC. A colocalization of anti-TFPI with endocytosed BSA-gold, as well as with accumulated DAMP, demonstrated the presence of TFPI in the endocytic pathway.

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Bemiparin and Fluid Flow Modulate the Expression, Activity and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1616761 ◽

2001 ◽

Vol 86 (12) ◽

pp. 1547-1554 ◽

Cited By ~ 13

Author(s):

Andrew Westmuckett ◽

Vijay Kakkar ◽

Tsutomu Hamuro ◽

Florea Lupu ◽

Cristina Lupu

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Cell Surface ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Arterial Flow ◽

Immunogold Electron Microscopy ◽

Tissue Factor Pathway ◽

Static Conditions

SummaryWe investigated the localisation, gene expression, and activity of tissue factor pathway inhibitor (TFPI) in endothelial cells (EC) grown in static conditions or under shear stress, in the presence of unfractionated heparin (UFH) and two low-molecular-weight heparins (LMWHs), dalteparin and bemiparin (a second generation of LMWHs). All three preparations induced increased release, cellular redistribution, and enhanced activity of TFPI on the cell surface in static EC. In EC grown under shear stress (0.27, 4.1 and 19 dyne/cm2) and incubated with each heparin for 24 h, the release of TFPI was significantly correlated with the level of flow for bemiparin and dalteparin, but not for UFH. For all three levels of flow tested, bemiparin induced the highest secretion and increase of both cellular TFPI and cell surface activity of the inhibitor. The expression of TFPI mRNA, determined by Northern blotting, was specifically modulated by heparins. All three preparations increased the expression of TFPI by 60 to 120% in EC under minimal flow, but only bemiparin enhanced TFPI mRNA in EC under the arterial flow. Immunogold electron microscopy revealed that EC exhibited strong cellular labelling for TFPI when grown under arterial flow in the presence of bemiparin. We conclude that in EC subjected to shear stress in vitro bemiparin is more efficient than UFH or dalteparin in modulating the expression, release and activity of TFPI. We therefore suggest that bemiparin may be superior over the conventional heparins in maintaining the anticoagulant properties of the endothelium.

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Platelet effects on tissue factor and fibrinolytic inhibition of cultured human fibroblasts and vascular cells

Blood ◽

10.1182/blood.v60.1.140.140 ◽

1982 ◽

Vol 60 (1) ◽

pp. 140-147 ◽

Cited By ~ 31

Author(s):

PE Smariga ◽

JR Maynard

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Human Fibroblasts ◽

Cellular Tissue ◽

Cultured Human Fibroblasts ◽

Platelet Concentration ◽

The Media ◽

Low Levels ◽

Coagulation Pathway

Abstract Platelets stimulate tissue factor, the initiator of the extrinsic coagulation pathway, and increase fibrinolytic inhibition in fibroblasts grown in vitro. Cellular tissue factor increases an average of 2.8-fold over the control levels after a 6-hr incubation with platelets, and no activity is present in the media. Fibrinolytic inhibition is stimulated in both the fibroblasts and their media in the presence of platelets and accumulates throughout a 24-hr incubation. Neither leukocytes nor erythrocytes stimulate these changes. Both tissue factor and fibrinolytic inhibition increases are dependent on platelet concentration and are blocked by inhibitors of RNA or protein synthesis. Control smooth muscle cells have higher tissue factor and fibrinolytic inhibition than fibroblasts, but their response to the presence of platelets is similar. Confluent monolayers of endothelial cells have very low levels of tissue factor that are not altered by the presence of platelets. However, the ability of endothelial cells to inhibit fibrinolysis is enhanced by the presence of platelets. The fraction that stimulates tissue factor and fibrinolytic inhibition is distinct from platelet-derived growth factor and from the fraction that enhances leukocyte tissue factor. It is associated with an insoluble, nonmitogenic fraction that is not inactivated by phospholipase C, or diisopropylfluorophosphate, nor is it chloroform:methanol extractable. Platelets are a physiologic modulator for both cellular tissue factor and the fibrinolytic system in vitro.

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Emicizumab, A Bispecific Antibody to Factors IX/IXa and X/Xa, Does Not Interfere with Antithrombin or TFPI Activity In Vitro

TH Open ◽

10.1055/s-0038-1636538 ◽

2018 ◽

Vol 02 (01) ◽

pp. e96-e103 ◽

Cited By ~ 7

Author(s):

Mariko Noguchi-Sasaki ◽

Tetsuhiro Soeda ◽

Atsunori Ueyama ◽

Atsushi Muto ◽

Michinori Hirata ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Bispecific Antibody ◽

Coagulation Factors ◽

Enzymatic Assays ◽

Monospecific Antibody ◽

Tissue Factor Pathway ◽

Cofactor Activity

AbstractEmicizumab is a humanized bispecific antibody that binds simultaneously to factor (F) IXa and FX replacing the cofactor function of FVIIIa. Because emicizumab recognizes FIX/FIXa and FX/FXa, a question may arise whether emicizumab competes with antithrombin (AT) and/or tissue factor pathway inhibitor (TFPI), thereby enhancing overall hemostatic potential by blocking their antihemostatic effects. To address this question, we performed enzymatic assays using purified coagulation factors to confirm whether emicizumab interferes with the action of AT on FIXa or FXa, or with the action of TFPI on FXa. In those assays, we found no interference of emicizumab on the actions of AT and TFPI. We next assessed emicizumab's influences on the anticoagulation actions of AT or TFPI in thrombin generation assays triggered with FXIa or tissue factor (TF) in AT-depleted or TFPI-depleted plasma supplemented with AT or TFPI in vitro. In those assays, we employed anti-FIXa and anti-FX monospecific one-armed antibodies derived from emicizumab instead of emicizumab itself so as to prevent emicizumab's FVIIIa cofactor activity from boosting thrombin generation. Consequently, we found that neither anti-FIXa, anti-FX monospecific antibody, nor the mixture of the two interfered with the anticoagulation actions of AT or TFPI in plasma. Although emicizumab can bind to FIXa and FXa, our results showed no interference of emicizumab with the action of AT or TFPI on FIXa or FXa. This indicates that the presence of emicizumab is irrelevant to the action of AT and TFPI, and thus should not alter the coagulant/anticoagulant balance related to AT and TFPI.

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