Human endothelial cells and fibroblasts express and produce the coagulation proteins necessary for thrombin generation

AbstractIn a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV–FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin–antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.

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Human Endothelial Cells And Fibroblasts Express And Produce The Coagulation Proteins Necessary For Thrombin Generation

10.21203/rs.3.rs-784474/v1 ◽

2021 ◽

Author(s):

Clay Cohen ◽

Nancy Turner ◽

Joel Moake

Keyword(s):

Endothelial Cells ◽

Thrombin Generation ◽

Coagulation Factors ◽

Cell Types ◽

Human Endothelial Cells ◽

Surface Binding ◽

Prothrombinase Complex ◽

Coagulation Proteins ◽

Exogenous Addition ◽

Activate Cell

Abstract In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV-FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin-antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and g-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.

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HiPS-Endothelial Cells Acquire Cardiac Endothelial Phenotype in Co-culture With hiPS-Cardiomyocytes

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.715093 ◽

2021 ◽

Vol 9 ◽

Author(s):

Emmi Helle ◽

Minna Ampuja ◽

Alexandra Dainis ◽

Laura Antola ◽

Elina Temmes ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Animal Studies ◽

Cardiac Development ◽

Cell Types ◽

Human Endothelial Cells ◽

Bidirectional Communication ◽

Induced Pluripotent ◽

And Function ◽

Cell Crosstalk

Cell-cell interactions are crucial for organ development and function. In the heart, endothelial cells engage in bidirectional communication with cardiomyocytes regulating cardiac development and growth. We aimed to elucidate the organotypic development of cardiac endothelial cells and cardiomyocyte and endothelial cell crosstalk using human induced pluripotent stem cells (hiPSC). Single-cell RNA sequencing was performed with hiPSC-derived cardiomyocytes (hiPS-CMs) and endothelial cells (hiPS-ECs) in mono- and co-culture. The presence of hiPS-CMs led to increased expression of transcripts related to vascular development and maturation, cardiac development, as well as cardiac endothelial cell and endocardium-specific genes in hiPS-ECs. Interestingly, co-culture induced the expression of cardiomyocyte myofibrillar genes and MYL7 and MYL4 protein expression was detected in hiPS-ECs. Major regulators of BMP- and Notch-signaling pathways were induced in both cell types in co-culture. These results reflect the findings from animal studies and extend them to human endothelial cells, demonstrating the importance of EC-CM interactions during development.

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EXPRESSION OF PLATELET ALLOANTIGENS ON HUMAN ENDOTHELIAL CELLS AND HEL CELLS

10.1055/s-0038-1642813 ◽

1987 ◽

Author(s):

Y Kawai ◽

R R Montgomery ◽

K Furihata ◽

T J Kunicki

Keyword(s):

Endothelial Cells ◽

Protein A ◽

Cell Types ◽

Membrane Glycoproteins ◽

Human Endothelial Cells ◽

Immunoblot Assay ◽

Sds Page ◽

Hel Cells ◽

Erythroleukemia Cell ◽

Different Cell Types

Analogs of platelet membrane glycoproteins IIb and IIIa (GPIIb-IIIa) have been shown to be synthesized and expressed by human endothelial cells (HEC), a human erythroleukemia cell line (HEL) and various other cells. Previous studies from our laboratory demonstrated that the platelet alloantigen P1A1, is expressed on HEC GPIIIa. Other alloantigen systems, namely, Pen and Bak, are known to be localized on platelet GPIIIa and GPIIb, respectively. Utilizing additional antibodies from patients with PTP specific for Pena, Baka, and Bakb allo-antigens, and isoantibodies (iso-ab) from a patient with Glanzmann's Thrombasthenia (GT), we have studied cultured HEC and HEL cells for expression of epitopes recognized by these antibodies. HEC and HEL cells were meta-bolically labeled with 35S-methionine and lysed in 0.5% TX-100 in the presence of 5mM EDTA. Soluble antigens were immunoprecipitated with these antibodies coupled to Protein A-Sepharose and subjected to SDS-PAGE and fluorography. Anti-Pena and the GT iso-ab reacted with the GPIIb-IIIa complex from both HEC and HEL lysates, but anti-Baka and anti-Bakb failed to immunoprecipitate GPIIb-IIIa analogs from either HEC or HEL. In an immunoblot assay, the GT iso-ab bound to GPIIIa of both HEC and HEL. Anti-Pena failed to react with SDS-denatured proteins. HEL GPIIIa migrates slightly faster than HEC GPIIIa and slightly slower than platelet GPIIIa. These results indicate that the epitopes of platelet GPIIIa recognized by alloantibodies and isoantibodies are shared by GPIIIa analogs of HEC and HEL. GPIIb-associated alloantigens are not expressed by HEC and HEL GPIIb analogs, an observation that is consistent with the decreased structural homology between GPIIb analogs derived from different cell types.

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Atherogenic Lipoproteins Support Assembly of the Prothrombinase Complex and Thrombin Generation: Modulation by Oxidation and Vitamin E

Blood ◽

10.1182/blood.v91.2.508 ◽

1998 ◽

Vol 91 (2) ◽

pp. 508-515 ◽

Cited By ~ 64

Author(s):

Simin Rota ◽

Nicola A. McWilliam ◽

Trevor P. Baglin ◽

Christopher D. Byrne

Keyword(s):

Endothelial Cells ◽

Vitamin E ◽

Thrombin Generation ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Very Low Density Lipoprotein ◽

Low Density ◽

Oxidized Low Density Lipoprotein ◽

Lipoprotein Oxidation ◽

Prothrombinase Complex

AbstractThe importance of lipoproteins in the etiology of atherosclerosis is well established. Evidence is now accumulating to implicate thrombin in the pathogenesis of atherosclerosis. We have investigated whether atherogenic lipoproteins can support thrombin generation. In the absence of platelets or endothelial cells, both very low-density lipoprotein (VLDL) and oxidized low-density lipoprotein (LDL) support assembly of the prothrombinase complex and generation of thrombin. Thrombin generation (per μg of apolipoprotein) supported by VLDL was 19.4-fold greater than that supported by high-density lipoprotein (HDL), P < .00001, and 11.7-fold greater than that supported by LDL, P < .00001. Oxidation of LDL increased lipoprotein-supported thrombin generation 12-fold compared to unmodified LDL, P < .0001. We have shown that the phenomenon of lipoprotein-supported thrombin generation is mediated predominantly by specific phospholipids and is enhanced by oxidation of these phospholipids. The addition of vitamin E (α-tocopherol) markedly reduced the increase in thrombin generation observed after oxidation of LDL (822 ± 57 v 138 ± 47 nmol/L;P < .0001). These effects suggest that lipoproteins are important in the production of thrombin and that vitamin E may confer protection from the detrimental effects of lipoprotein oxidation by limiting thrombin formation. These results suggest that atherogenic lipoproteins are linked to the development of atherosclerosis in part by their capacity to support thrombin generation.

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Attachment of cultured human endothelial cells is promoted by specific association with S protein (vitronectin) as well as with the ternary S protein-thrombin-antithrombin III complex

Blood ◽

10.1182/blood.v71.6.1581.1581 ◽

1988 ◽

Vol 71 (6) ◽

pp. 1581-1589 ◽

Cited By ~ 45

Author(s):

KT Preissner ◽

E Anders ◽

J Grulich-Henn ◽

G Muller-Berghaus

Keyword(s):

Endothelial Cells ◽

Antithrombin Iii ◽

Cell Attachment ◽

Umbilical Vein ◽

Cell Types ◽

Human Endothelial Cells ◽

S Protein ◽

Major Vessel ◽

Specific Association ◽

Dose Dependent

Abstract The interaction of the multifunctional S protein (vitronectin) with cultured human endothelial cells of macrovascular and microvascular origin was investigated. Purified S protein, coated on polystyrene Petri dishes, induced dose-dependent and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVECs) as well as human omental tissue microvascular endothelial cells (HOTMECs) at 37 degrees C. Not only isolated S protein, but also the ternary S protein- thrombin-antithrombin III (STAT) complex promoted attachment of approximately 90% of the cells within 2 hours at an S protein concentration of 0.13 mumol/L. Inhibition of attachment in these experiments was achieved by the addition of the cell-attachment pentapeptide Gly-Arg-Gly-Asp-Ser and by monospecific antibodies against S protein, whereas nonrelated peptides or antibodies against fibronectin, fibrinogen, or von Willebrand factor (vWF) were ineffective. Direct binding of S protein to HUVECs and HOTMECs was studied with cells in suspension at a density of 1 x 10(6) cells/mL and was maximal after 120 minutes. S protein bound to both cell types in a dose-dependent fashion with an estimated dissociation constant Kd = 0.2 mumol/L. At a 200-fold to 500-fold molar excess of unlabeled S protein, greater than 80% of bound radiolabeled S protein was displaceable, whereas binding was reduced to 30% to 50% by addition of the pentapeptide, the STAT complex, or by physiologic concentrations of fibrinogen or vWF as well as Fab fragments of anti(human S protein)IgG, but not by Fab rabbit IgG. These findings present evidence for the specific association of S protein with endothelial cells ultimately leading to attachment and spreading of cells. Moreover, a novel function for the ternary STAT complex, which induced endothelial cell attachment and spreading virtually identical to free S protein, is described. These data further suggest a possible role for S protein during coagulation as major vessel wall-related adhesive protein at sites of vascular injury.

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Degradation of Sulphated Glycosaminoglycans at the Surface of Cultured Human Endothelial Cells by a Platelet Heparitinase

10.1055/s-0039-1682699 ◽

1977 ◽

Author(s):

C. Busch ◽

B. Glimelius ◽

Å Wastesson ◽

B. Westermark

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Incubation Medium ◽

Chondroitin Sulphate ◽

Coagulation Factors ◽

Platelet Lysate ◽

Human Endothelial Cells ◽

Endothelial Cell Surface ◽

Sulphated Glycosaminoglycans

The non-thrombogenic property of the endothelial cell surface is a prerequisite for maintainance of blood circulation. The nature of this property is poorly understood. Recent advances in culturing techniques of endothelial cells in vitro may facilitate studies of the surface biochemistry. Human endothelial cells (EC) isolated from umbilical veins were shown to synthesize and secrete sulphated glycosaminoglycans (GAG). The recent finding of a platelet enzyme capable of degrading heparin sulphate (HS) raised the question:Can platelet lysate or a purified heparitinase detach and degrade endothelial HS? EC cultured in the presence of 35S-sulphate, produce 35S-labelled GAG which was isolated from the incubation medium from a cell associated trypsin labile pool and from a cellular pool not liberated by trypsin. After 48 hours of incorporation about 95% of 35S-GAG was found in the medium fraction, 5% in the trypsin fraction and negligible amounts in the cell fraction. In the trypsin pool (“surface fraction”) heparin sulphate comprised about 85%, while the remaining 15% consisted of chondroitin sulphate and/or dermatan sulphate. Incubation of 35S-labelled EC with platelet lysate or a partially purified preparation of the enzyme from the same source caused a marked release of cell-surface associated HS to the incubation medium as oligosaccharides. These effects could be ascribed to heparitinase activity and may alter the properties of the EC-surface and influence the interaction between these cells on one hand and blood cells or plasma components, e.g., coagulation factors on the other.

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CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2 and are differentially expressed in lung and kidney epithelial and endothelial cells

10.1101/2020.06.22.165803 ◽

2020 ◽

Cited By ~ 13

Author(s):

Razie Amraei ◽

Wenqing Yin ◽

Marc A. Napoleon ◽

Ellen L. Suder ◽

Jacob Berrigan ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular System ◽

Virus Entry ◽

Antiviral Drug ◽

Cell Types ◽

Host Cells ◽

Human Endothelial Cells ◽

High Importance ◽

Lung And Kidney ◽

Low Expression

AbstractAs the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development.SignificanceUnderstanding the interactions between SARS-CoV-2 with host cells is of high importance. ACE2 is recognized as a major entry receptor, but SARS-CoV-2 may also employ alternative receptors for cell entry and these may hold the key to infection in tissues, where ACE2 has a low expression level or is absent. We identify CD209L/L-SIGN and CD209/DC-SIGN as receptors for SARS-CoV-2. We show that CD209L is N-glycosylated and this modification modulates the binding of CD209L with spike protein. CD209L interacts with ACE2, suggesting that CD209L and ACE2 could function as co-receptors for SARS-CoV-2 entry and infection. Human endothelial cells are permissive to SARS-CoV-2 infection. We show that interfering with CD209L activity in endothelial cells by knockdown or with introduction of soluble CD209L inhibits virus entry, suggesting a novel target for development of antiviral drugs.

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Expression of the Drug-Dependent Antigen for Quinine-Dependent Antiplatelet Antibodies on GP III a but not that on GPI b, II b or IX on Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645209 ◽

1990 ◽

Vol 63 (02) ◽

pp. 279-281 ◽

Cited By ~ 8

Author(s):

Sharron L Pfueller ◽

Robyn A Bilston ◽

Stephen Jane ◽

Jannine Gibson

Keyword(s):

Endothelial Cells ◽

Cell Types ◽

Dependent Manner ◽

Igg Antibodies ◽

Human Endothelial Cells ◽

Thrombocytopenic Purpura ◽

Antiplatelet Antibodies ◽

Igg Binding ◽

Drug Dependent ◽

Surface Glycoproteins

SummaryIn quinine- and quinidine-induced thrombocytopenic purpura IgG antibodies are known to react in a drug-dependent manner with different combinations of surface glycoproteins (GP) Ib , IIb, IIIa and IX. Because endothelial cells share a number of properties of platelets, including the presence of GP Ilia and GPIb-like proteins, we have compared these two cell types for their quinine-dependent IgG binding abilities. By immunoblotting of endothelial cells, quinine-dependent IgG binding from four patient sera was observed only to a 93 kDa component corresponding to GP Ilia. Strong IgG binding independent of the drug was found at 170–180 kDa. Thus endothelial cells express the GP Ilia quinine-dependent epitope on platelet GP IIIa , but not those on other platelet glycoproteins.

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Covalently linking heparin to antithrombin enhances prothrombinase inhibition on activated platelets

Thrombosis and Haemostasis ◽

10.1160/th12-10-0766 ◽

2013 ◽

Vol 109 (06) ◽

pp. 1016-1024 ◽

Cited By ~ 2

Author(s):

Ivan Stevic ◽

Howard H. W. Chan ◽

Ankush Chander ◽

Leslie R. Berry ◽

Anthony K. C. Chan

Keyword(s):

Unfractionated Heparin ◽

Thrombin Generation ◽

Anticoagulant Activity ◽

Coagulation Factors ◽

Enzyme Complex ◽

Prothrombinase Complex ◽

Activated Platelets ◽

Artificial Surfaces

SummaryFactor (F)Xa within the prothrombinase complex is protected from inhibition by unfractionated heparin (UFH), enoxaparin and fondaparinux. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. We have also demonstrated that ATH is superior at inhibiting coagulation factors when assembled on artificial surfaces. The objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit FXa within the prothrombinase complex when the enzyme complex is assembled on the more native platelet system. Discontinuous inhibition assays were performed to determine final k 2-values for inhibition of FXa, FXa within the platelet-prothrombinase, or FXa within prothrombinase devoid of various components. Thrombin generation and plasma clotting was also assayed in the presence of resting/activated platelets ± inhibitors. Protection of FXa was not observed for ATH, whereas a moderate 60% protection was observed for AT+UFH. ATH inhibited platelet-prothrombinase ∼4-fold faster than AT+UFH. Relative to intact prothrombinase, rates for FXa inhibition by AT+UFH in prothrombinase complexes devoid of either prothrombin (II)/activated platelets/FVa were higher. However, inhibition by AT+UFH of prothrombinase devoid of FII yielded slightly lower rates compared to free FXa inhibition. Thrombin generation and plasma clotting was enhanced with activated platelets, while inhibition was better by ATH compared to AT+UFH, thus suggesting an overall enhanced anticoagulant activity of ATH against platelet-bound prothrombinase complexes.

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The Human Cytomegalovirus Ribonucleotide Reductase Homolog UL45 Is Dispensable for Growth in Endothelial Cells, as Determined by a BAC-Cloned Clinical Isolate of Human Cytomegalovirus with Preserved Wild-Type Characteristics

Journal of Virology ◽

10.1128/jvi.76.18.9551-9555.2002 ◽

2002 ◽

Vol 76 (18) ◽

pp. 9551-9555 ◽

Cited By ~ 94

Author(s):

Gabriele Hahn ◽

Hanna Khan ◽

Fausto Baldanti ◽

Ulrich H. Koszinowski ◽

M. Grazia Revello ◽

...

Keyword(s):

Endothelial Cells ◽

Clinical Isolate ◽

Human Cytomegalovirus ◽

Ribonucleotide Reductase ◽

Human Fibroblasts ◽

Artificial Chromosome ◽

Cell Types ◽

Factor X ◽

Wild Type ◽

Human Endothelial Cells

ABSTRACT An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and the ribonucleotide reductase homolog UL45 was deleted. Reconstituted virus RVFIX and RVΔUL45 grew equally well in human fibroblasts and human endothelial cells. Thus, UL45 is dispensable for growth of HCMV in both cell types.

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