scholarly journals Activation of prothrombin in the presence of human umbilical-vein endothelial cells

1992 ◽  
Vol 281 (3) ◽  
pp. 661-664 ◽  
Author(s):  
P Schoen ◽  
C Reutelingsperger ◽  
T Lindhout
Keyword(s):  
Endothelial Cells ◽  
Time Delay ◽  
Umbilical Vein ◽  
Factor Xa ◽  
Fluid Phase ◽  
Human Umbilical Vein ◽  
Factor Va ◽  
Umbilical Vein Endothelial Cells ◽  
Prothrombinase Activity

Addition of Factor Xa, Factor Va and prothrombin to immobilized cultured human umbilical-vein endothelial cells resulted after a time delay in thrombin formation. The prothrombin-converting (prothrombinase) activity, however, was not associated with the cell surface. Rather, perturbation by thrombin, either formed in situ or exogenously added, induced a procoagulant phospholipid surface in the fluid phase, which, in the presence of Factor Xa and Factor Va, enabled the assembly of prothrombinase.

scholarly journals Enhancement by human umbilical vein endothelial cells of factor Xa- catalyzed activation of factor VII

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 791-796
Author(s):  
LV Rao ◽  
SI Rapaport ◽  
M Lorenzi
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Vascular Endothelial Cell ◽  
Factor Xa ◽  
Reaction Rates ◽  
Factor Vii ◽  
Activate Factor ◽  
Human Umbilical Vein ◽  
Factor Va ◽  
Umbilical Vein Endothelial Cells

In blood coagulation on endothelium, an unperturbed vascular endothelial cell surface apparently provides activity equivalent to the phospholipid needed for generation of factor Xa and thrombin in soluble systems. Phospholipid in soluble systems also markedly enhances the ability of factor Xa to activate factor VII; therefore we investigated the influence of an unperturbed monolayer of human umbilical vein endothelial cells (HUVEC) upon factor VII activation. HUVEC were found to augment factor Xa-catalyzed activation of factor VII. This appeared to result from the binding of trace amounts of factor Xa to the cells. Adding active site-inhibited factor Xa to reaction mixtures, but not factor X, abolished the enhanced activation. Adding either anti-factor V antibodies or exogenous factor Va had no effect upon reaction rates. Thus factor Va does not function as a cofactor for the reaction. In further experiments the effect upon activation of factor VII and prothrombin was studied by varying the order of addition of factor Xa and factor Va to supernatants of HUVEC monolayers. Evidence was obtained that HUVEC, unlike platelets, possess a functional factor Xa binding site that is independent of factor Va. Since phospholipid is the only known cofactor for factor Xa/Ca2+-induced activation of factor VII, the demonstration of enhanced activation of factor VII in the presence of unperturbed cultured HUVEC supports a hypothesis that the functional equivalent of procoagulant phospholipid is available on the luminal surface of vascular endothelium in vivo.

scholarly journals Enhancement by human umbilical vein endothelial cells of factor Xa- catalyzed activation of factor VII

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 791-796 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport ◽  
M Lorenzi
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Vascular Endothelial Cell ◽  
Factor Xa ◽  
Reaction Rates ◽  
Factor Vii ◽  
Activate Factor ◽  
Human Umbilical Vein ◽  
Factor Va ◽  
Umbilical Vein Endothelial Cells

Abstract In blood coagulation on endothelium, an unperturbed vascular endothelial cell surface apparently provides activity equivalent to the phospholipid needed for generation of factor Xa and thrombin in soluble systems. Phospholipid in soluble systems also markedly enhances the ability of factor Xa to activate factor VII; therefore we investigated the influence of an unperturbed monolayer of human umbilical vein endothelial cells (HUVEC) upon factor VII activation. HUVEC were found to augment factor Xa-catalyzed activation of factor VII. This appeared to result from the binding of trace amounts of factor Xa to the cells. Adding active site-inhibited factor Xa to reaction mixtures, but not factor X, abolished the enhanced activation. Adding either anti-factor V antibodies or exogenous factor Va had no effect upon reaction rates. Thus factor Va does not function as a cofactor for the reaction. In further experiments the effect upon activation of factor VII and prothrombin was studied by varying the order of addition of factor Xa and factor Va to supernatants of HUVEC monolayers. Evidence was obtained that HUVEC, unlike platelets, possess a functional factor Xa binding site that is independent of factor Va. Since phospholipid is the only known cofactor for factor Xa/Ca2+-induced activation of factor VII, the demonstration of enhanced activation of factor VII in the presence of unperturbed cultured HUVEC supports a hypothesis that the functional equivalent of procoagulant phospholipid is available on the luminal surface of vascular endothelium in vivo.

Studies of the Factor Xa-Dependent Inhibitor of Factor VIIa/Tissue Factor (Extrinsic Pathway Inhibitor) from Cell Supernates of Cultured Human Umbilical Vein Endothelial Cells

1989 ◽  
Vol 61 (01) ◽  
pp. 101-105 ◽  
Author(s):  
Bonnie J Warn-Cramer ◽  
Fanny E Almus ◽  
Samuel I Rapaport
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Phorbol Ester ◽  
Umbilical Vein ◽  
Primary Cultures ◽  
Factor Xa ◽  
Extrinsic Pathway ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VHa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with 125I-factor Xa as the ligand.

Stressed Endothelial Cells Compartmentalize the Prothrombinase Complex on Filopodia and Other Convex Membrane Features near the Cell Margin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 372-372
Author(s):  
Jialan Shi ◽  
Dessislava N. Nikova ◽  
Gary E. Gilbert
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Factor Xa ◽  
Annexin V ◽  
Complex Assembly ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Tnf Α ◽  
Prothrombinase Activity

Abstract Abstract 372 The dependence of procoagulant activity on phosphatidylserine (PS) has been recognized for at least four decades but the location of physiologically relevant membranes with PS exposure remains uncertain. PS is exposed on apoptotic cells and cell microparticles but in vitro and in vivo studies have failed to demonstrate a clear relationship of microparticles or apoptotic cells to fibrin deposition. Exposure of endothelial cells to stimulants or toxins leads to retraction of cell margins, mounding of the central cell, and extension of filopodia. We have also found that cell stress also leads to limited, focal PS exposure. Furthermore, we found that binding sites for lactadherin, a PS-binding protein that shares homology with factor VIII and factor V, are concentrated on convex surfaces such as filopodia. In this study we ask whether the limited, focal PS exposure on stressed human umbilical vein endothelial cells is sufficient to support prothrombinase complex assembly and whether the prothrombinase complex assembly is restricted to the convex membrane features that bind lactadherin. We allowed Human Umbilical Vein Endothelial Cells (HUVEC) to grow to confluent monolayers prior to exposure to TNF-α, 10 ng/ml, for 5–24 hours. PS exposure was detected by simultaneous staining using 10 nM lactadherin–Alexa 488 and annexin V–Cy 3.18, both exhibiting high affinity for PS. Stressed cells withdrew from their prior borders, leaving residual fibrils connected to original attachment points. In addition, they extended filopodia that were up to several cell diameters in length. Confocal microscopy demonstrated focal staining of filopodia, fibrils and cell margins with lactadherin and patches near the nucleus with annexin A5. We asked whether the selective binding might be determined by the membrane topology. To mimic the curvature of a cell membrane we prepared nano-fabricated silica substrates with ridge radii of 10 nm. The AFM topographic and fluorescent images of synthetic membrane bilayers supported by the substrates showed that, over a PS content of 4–15%, lactadherin preferentially binds to the convex nano-ridges with a ridge: valley staining ratio >80:1, while annexin V selectively binds the concave areas of the nano-trenches with a ridge. Combined fluorescence/AFM imaging of TNF-α treated HUVEC's, demonstrated that the new thin filaments staining with lactadherin had radii of curvature of approx. 12 nm, similar to the ridges of our synthetic bilayers. We asked whether factor Va and factor Xa share preference for convex surfaces, analogous to lactadherin. Supported membranes of 4% PS had preferential ridge staining by factor Va-fluorescein-maleimide with a ridge/valley ratio > 10/1. Co-staining with factor Va and factor Xa-EGRck-biotin (complexed to Alexa 647-steptavidin) indicated that factor Va enhanced binding of factor Xa to ridges, thus the prothrombinase complex has highly preferential binding to convex ridges. TNF-α-treated endothelial cells bound factor Va, like lactadherin, selectively on filopodia and fibrils near the retracted edges of endothelial cells. Factor Xa also localized to these features in the presence of factor Va, indicating prothrombinase complex assembly. Stressed endothelial cells exhibited at least 8-fold higher support for thrombin production and prothrombinase activity. Prothrombinase activity was efficiently inhibited by lactadherin, demonstrating that the lactadherin-binding sites were the functional sites for prothrombinase activity. Together, these data indicate that stressed endothelial cells can support the prothrombinase complex and that prothrombinase activity is compartmentalized near the periphery of the cell and in the intracellular area through binding sites on highly convex membrane features with exposed PS. We have hypothesized that this compartment of procoagulant activity is relatively protected from anti-coagulant proteins that are localized elsewhere on the stimulated/stressed endothelial cell. Disclosures: No relevant conflicts of interest to declare.

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

2009 ◽  
Vol 394 (2) ◽  
pp. 217-222 ◽  
Author(s):  
Jin-Young Park ◽  
Se-Hui Jung ◽  
Jae-Wan Jung ◽  
Mi-Hye Kwon ◽  
Je-Ok Yoo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Tissue Transglutaminase ◽  
Human Umbilical Vein ◽  
Transglutaminase Activity ◽  
Umbilical Vein Endothelial Cells

scholarly journals Protein C Activation and Factor Va Inactivation on Human Umbilical Vein Endothelial Cells

1997 ◽  
Vol 17 (11) ◽  
pp. 2765-2775 ◽  
Author(s):  
Matthew F. Hockin ◽  
Michael Kalafatis ◽  
Marie Shatos ◽  
Kenneth G. Mann
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Factor Va ◽  
Umbilical Vein Endothelial Cells

scholarly journals Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells

Blood ◽  
2012 ◽  
Vol 119 (10) ◽  
pp. 2325-2334 ◽  
Author(s):  
Rui Xie ◽  
Chunyan Gao ◽  
Wen Li ◽  
Jiuxin Zhu ◽  
Valerie Novakovic ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Acute Promyelocytic Leukemia ◽  
Fibrinolytic Activity ◽  
Umbilical Vein ◽  
Factor Xa ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Prothrombinase Activity

Abstract The coagulopathy of acute promyelocytic leukemia (APL) is mainly related to procoagulant substances and fibrinolytic activators of APL blasts, but the fate of these leukemic cells is unknown. The aim of this study was to investigate the removal of APL blasts by macrophages and endothelial cells in vitro and consequent procoagulant and fibrinolytic activity of APL cells. We found that human umbilical vein endothelial cells as well as THP-1 and monocyte-derived macrophages bound, engulfed, and subsequently degraded immortalized APL cell line NB4 and primary APL cells. Lactadherin promoted phagocytosis of APL cells in a time-dependent fashion. Furthermore, factor Xa and prothrombinase activity of phosphatidylserine-exposed target APL cells was time-dependently decreased after incubation with phagocytes (THP-1–derived macrophages or HUVECs). Thrombin production on target APL cells was reduced by 40%-45% after 2 hours of coincubation with phagocytes and 80% by a combination of lactadherin and phagocytes. Moreover, plasmin generation of target APL cells was inhibited 30% by 2 hours of phagocytosis and ∼ 50% by lactadherin-mediated engulfment. These results suggest that engulfment by macrophages and endothelial cells reduce procoagulant and fibrinolytic activity of APL blasts. Lactadherin and phagocytosis could cooperatively ameliorate the clotting disorders in APL.

Human umbilical vein endothelial cells express high affinity receptors for factor Xa

1997 ◽  
Vol 172 (1) ◽  
pp. 36-43 ◽  
Author(s):  
Françoise Bono ◽  
Jean-Pascal Herault ◽  
Corinne Avril ◽  
Paul Schaeffer ◽  
Jean-Claude Lormeau ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Factor Xa ◽  
Human Umbilical Vein ◽  
High Affinity ◽  
Umbilical Vein Endothelial Cells
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