Relationship between Endothelial Tissue Factor and Thrombogenesis under Blood Flow Conditions

1995 ◽  
Vol 74 (02) ◽  
pp. 778-783 ◽  
Author(s):  
Armelle Diquélou ◽  
Dominique Dupouy ◽  
Dominique Gaspin ◽  
Jacques Constans ◽  
Pierre Sié ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Tissue Factor ◽  
Thrombus Formation ◽  
Luminal Surface ◽  
Fibrin Deposition ◽  
Flow Conditions ◽  
The Impact ◽  
The Relationship ◽  
Endothelial Tissue

SummaryWe have evaluated the relationship between the level of tissue factor (TF) expression by stimulated endothelial cells and thrombus formation under blood flow conditions. Cultures of human umbilical venous endothelial cells (HUVECs) were treated in order to express different levels of TF activity. They were stimulated for 4 h with either I) lipopolysaccharides (LPS, 10 µg/ml), II) recombinant interleukin Iß (IL1ß, 50 Ul/ml) or III) simultaneously with LPS and IL1ß (LPS + IL1ß). TF activity was low on confluent HUVECs or on the corresponding extracellular-matrix (ECM prepared by exposure of HUVECs to 0.1 N NH4OH). In contrast, it was high when HUVECs were stimulated with LPS or IL1ß, and significantly higher (p <0.05) with LPS+IL1ß. The TF activity associated with the stimulated ECM was 2-fold higher (p <0.05) than that expressed on the luminal surface of the stimulated HUVECs, irrespective of the agonist or combination of agonists used.These surfaces were exposed to non-anticoagulated human blood at a venous (50 s-1) and an arterial (650 s-1) wall shear rate in parallel-plate perfusion chambers for 5 min. Thrombus formation was morphologically quantified by measuring the deposition of platelets and fibrin. Fibrin deposition was also immunologically quantified. Fibrin deposition was related to the level of TF expression. Non-stimulated HUVECs and corresponding ECMs were not thrombogenic. The luminal surface of HUVECs stimulated with LPS or IL1ß alone expressed low levels of TF activity and was a poor inducer of platelet deposition and fibrin deposition (<15%) at 50 s-1. In contrast, fibrin deposition increased to 80% when the cells were stimulated with LPS and IL1ß simultaneously. This fibrin deposition was comparable to that found on the corresponding ECM, despite a two-fold lower TF activity. However, at 650 s-1, platelet and fibrin deposition on HUVECs stimulated with LPS + IL1ß were significantly lower than that observed on the corresponding ECM. In all circumstances, the thrombogenicity was TF-dependent, since fibrin deposition was totally blocked by anti-TF antibodies. Thus, it appears that the level of TF activity expressed on endothelial cells governs thrombus formation. However, the impact of TF expression on thrombus formation is also affected by the blood flow.

Collagen Induced Thrombus Formation at the Apex of Eccentric Stenoses - A Time Course Study with Non-Anticoagulated Human Blood

1996 ◽  
Vol 75 (04) ◽  
pp. 685-692 ◽  
Author(s):  
R Marius Barstad ◽  
Peter Kierulf ◽  
Kjell S Sakariassen
Keyword(s):  
Blood Flow ◽  
Human Blood ◽  
Time Course ◽  
Thrombus Formation ◽  
Shear Stresses ◽  
Fibrin Deposition ◽  
Luminal Narrowing ◽  
Perfusion Time ◽  
Platelet Thrombus ◽  
The Impact

SummaryAtherosclerotic plaque rupture may trigger the formation of a mural thrombus. This thrombus formation is apparently affected by very high and complex shear conditions introduced by the luminal narrowing (stenosis) of the atheroma. To study the impact of such blood flow behaviour on thrombus formation we employed a model system where collagen-induced thrombogenesis is studied at the apex of well-defined eccentric stenoses.Thrombus formation in non-anticoagulated human blood drawn directly from an antecubital vein over the collagen coated stenosis apex for periods of 0.5, 1, 3 or 5 min was quantified by morphometry. The stenoses reduced the cross-sectional area of the blood flow channel by 60, 80 and 89%, which corresponded to apex wall shear rates of 2600, 10,500 and 32,000 s−1, respectively. Platelet-collagen adhesion decreased by increasing shear at the stenosis apex. The corresponding adhesion rates were highest at 1 min, then they gradually decreased upon prolongation of the perfusion time. The platelet thrombus volume increased in concert with increasing shear rate up to 10,500 s−1, whereas, at 32,000 s−1, the volume was decreased. The corresponding growth rates and rates of thrombus occlusion at the apex levelled off at 3 min. Significant fibrin deposition was not observed before 3 min, and was most pronounced at 10,500 and 32,000 s−1. The plasma levels of fibrinopeptide A and P-thromboglobulin increased in concert with increasing shear and perfusion time, particularly at the two highest shear conditions.Thus, hallmarks of thrombus formation at these stenoses with increasing shear are decreased platelet-collagen adhesion, and increased platelet-platelet interaction and fibrin deposition. A fibrin tail downstream to the collagen-attached platelet thrombus is regularly observed when thrombus occlusion exceeds 40%. However, the reduced thrombus growth at the most occlusive stenosis (89%) is presumably due to the high shear stresses which may reduce the rate of platelet incorporation into the thrombus and/or tear off thrombus fragments.

Platelets, circulating tissue factor, and fibrin colocalize in ex vivo thrombi: real-time fluorescence images of thrombus formation and propagation under defined flow conditions

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2787-2792 ◽  
Author(s):  
Viji Balasubramanian ◽  
Eric Grabowski ◽  
Alessandra Bini ◽  
Yale Nemerson
Keyword(s):  
Real Time ◽  
Tissue Factor ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
High Sensitivity ◽  
Flow Chamber ◽  
Flow Conditions ◽  
Coated Glass ◽  
Relative Contribution ◽  
Fluorescent Markers

Although it is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of tissue factor (TF) to blood, there is still little agreement about the mechanisms of thrombus propagation and the identities of the molecular species participating in this process. In this study, we characterized the thrombotic process in real-time and under defined flow conditions to determine the relative contribution and spatial distribution of 3 components of the thrombi: circulating or blood-borne TF (cTF), fibrin, and platelets. For this purpose, we used high-sensitivity, multicolor immunofluorescence microscopy coupled with a laminar flow chamber. Freshly drawn blood, labeled with mepacrine (marker for platelets and white cells), anti-hTF1Alexa.568 (marker for tissue factor), and anti-T2G1Cy­5 (marker for fibrin) was perfused over collagen-coated glass slides at wall shear rates of 100 and 650 s−1. A motorized filter cube selector facilitated imaging every 5 seconds at 1 of 3 different wavelengths, corresponding to optimal wavelengths for the 3 markers above. Real-time video recordings obtained during each of 10 discrete experiments show rapid deposition of platelets and fibrin onto collagen-coated glass. Overlay images of fluorescent markers corresponding to platelets, fibrin, and cTF clearly demonstrate colocalization of these 3 components in growing thrombi. These data further support our earlier observations that, in addition to TF present in the vessel wall, there is a pool of TF in circulating blood that contributes to the propagation of thrombosis at a site of vascular injury.

scholarly journals The functional expression of tissue factor by fibroblasts and endothelial cells under flow conditions

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3265-3270 ◽  
Author(s):  
EF Grabowski ◽  
DB Zuckerman ◽  
Y Nemerson
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Cell Types ◽  
Shear Stresses ◽  
Factor X ◽  
Flow Conditions ◽  
Production Values

Abstract The expression of tissue factor (TF) by a variety of vascular cell types under physiologic flow conditions is critical to factor X activation and in vivo clotting. Therefore, in a parallel-plate flow chamber (volume 40 microL) we mounted monolayers of human embryonic fibroblasts (FBs) or interleukin-1 alpha (IL-1 alpha) (5 U/mL x 4 hours)-stimulated human umbilical vein endothelial cells (ECs). Inflow buffer contained 10 nmol/L factor VIIa, 100 nmol/L factor X, and 2.0 mmol/L CaCl. With FBs, production of factor Xa (product of outflow concentration of factor Xa-and flow rate) increased 200-fold over the range of shear stress from 0 to 2.7 dynes/cm2. Production values (mean +/- SE (N)) were 7.93 +/- 0.024 (6), 312 +/- 7.3 (6), 688 +/- 33.1 (8), 1,033 +/- 119 (6), and 1,601 +/- 183 (7) fmol/cm2.minute at shear stresses of 0, 0.27, 0.68, 1.35, and 2.7 dynes/cm2, respectively. Further experiments at 0.68 dynes/cm2 indicated that factor Xa production increased with factor X concentration over the range from 3 to 100 nmol/L, but changed little from 300 to 1,000 nmol/L. With ECs, production was 0.13 +/- 0.86 (6), 8.17 +/- 1.65 (13), and 1.66 +/- 1.66 (5) fmol/cm2.minute at 0, 0.68, and 2.7 dynes/cm2, respectively. However, in the presence of an antibody directed against tissue factor pathway inhibitor (TFPI) production with ECs was augmented to 16.46 +/- 0.80 (8), 149.8 +/- 18.6 (8), and 48.9 +/- 10.3 (10), respectively, at these same shear stresses. Control experiments with factor VIIa, factor X, or both absent confirm for both cell types the specificity of the reaction for the TF pathway. Similarly, specificity for TF itself is shown by the virtual absence of factor Xa generation in the presence of the monoclonal antibody HTF1–7B8 directed against human TF. We conclude that ECs, even when activated, are normally unable to generate significant quantities of factor Xa in the presence of factors X and VIIa. However, significant quantities of factor Xa are possible in the presence of an inhibitor of TFPI. On the other hand, production of factor Xa by fibroblasts is markedly augmented by shear stress, yet independent of the availability of substrate factor X above an inflow concentration of 100 nmol/L. The latter suggests a direct effect of flow on the fibroblast monolayers, not substrate limitation by convective diffusion.

CD40 Engagement on Endothelial Cells Promotes Tissue Factor-dependent Procoagulant Activity

1998 ◽  
Vol 79 (05) ◽  
pp. 1025-1028 ◽  
Author(s):  
Ling Zhou ◽  
Patrick Stordeur ◽  
Aurore de Lavareille ◽  
Kris Thielemans ◽  
Paul Capel ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tissue Factor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
The Impact

SummaryThe CD40 molecule expressed on endothelial cells has been shown to transduce activation signals resulting in upregulation of adhesion molecules. Herein, we studied the impact of CD40 engagement on the induction of tissue factor (TF)-dependent procoagulant activity (PCA) at the surface of human umbilical vein endothelial cells (HUVECs). First, we found that co-incubation of HUVECs with 3T6 fibroblasts transfected with the CD40L gene (3T6-CD40L) resulted in a clear induction of PCA which was not observed with control untransfected fibroblasts. The specificity of this finding was established by inhibition experiments using monoclonal antibodies (mAbs) blocking CD40 or CD40L. PCA induced by CD40 ligation was TF-related as it was not observed in factor VII-deficient plasma and was associated with the accumulation of TF mRNA. To investigate the role of CD40/CD40L interactions in the induction of endothelial cell PCA by lymphocytes, interferon (IFN)-γ-stimulated EC were incubated with T cells in the absence or presence of anti-CD40 or anti-CD40L mAb. The 60-70% inhibition of PCA induced by these mAbs but not their isotype-matched control indicated that the CD40 pathway is involved in the induction of PCA resulting from interactions between activated HUVECs and T cells. We conclude that activation signals elicited by CD40 engagement on endothelial cells result in the induction of TF-dependent PCA. The CD40/CD40L pathway might therefore be involved in the development of prothrombic states during diseases associated with endothelial cell and T cell activation.

Endothelium but Not Platelet-Derived Protein Disulfide Isomerase Is Required for Fibrin Generation during Thrombus Formation in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 691-691 ◽  
Author(s):  
Reema Jasuja ◽  
Jaehyung Cho ◽  
Bruce Furie ◽  
Barbara Furie
Keyword(s):  
Endothelial Cells ◽  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Fibrin Deposition ◽  
Wild Type ◽  
Disulfide Isomerase ◽  
Laser Injury ◽  
Injury Model ◽  
Protein Disulfide

Abstract We have previously reported that protein disulfide isomerase is required in wild-type mice for platelet thrombus formation and fibrin generation in an in vivo laser injury model of thrombosis (Cho et al. J. Clin. Invest., 2008; 118:1123–31). Fibrin deposition after laser injury to the vessel wall in Par4−/− mice, lacking the G protein-coupled platelet thrombin receptor, is independent of platelets or requires minimal platelet activation or accumulation (Vandendries et al. Proc. Natl. Acad. Sci., 2007; 104:288–92). However, protein disulfide isomerase inhibitors have a dramatic effect on fibrin accumulation in Par4− mice, suggesting that these inhibitors may function by a platelet independent mechanism. Here, we compare the contributions of endothelium and platelet-derived protein disulfide isomerase to fibrin generation in the mouse laser injury model of thrombosis. In vitro studies using cultured human umbilical vein endothelial cells and human aortic endothelial cells show that protein disulfide isomerase can be secreted rapidly into the culture medium from these cells upon thrombin stimulation. Using intravital microscopy, we observe that protein disulfide isomerase is not detectable on the vessel wall prior to laser injury but can be detected on the injured cremaster arteriolar wall and in the developing thrombus very rapidly after laser induced injury in the live mouse. The median integrated fluorescence intensity for protein disulfide isomerase in wild-type mice was compared to wild-type mice injected with 10ug/g mouse of Integrilin, an inhibitor of platelet activation and platelet thrombus formation, and thus, an inhibitor of the contribution of platelet derived protein disulfide isomerase to thrombus formation. Protein disulfide isomerase expression was similar in both treated and untreated animals upto 30 seconds post-laser injury. After 30 seconds, the expression of protein disulfide isomerase in integrilin treated mice was significantly decreased compared to that in untreated mice, indicating that the initial protein disulfide isomerase was derived from the endothelium and later additional protein disulfide isomerase was derived from the platelets following their accumulation in the developing thrombus. Fibrin deposition, a measure of thrombin generation was comparable in wild-type mice that had been treated with Integrilin or treated with a control buffer, suggesting that endothelial-derived protein disulfide isomerase was sufficient for fibrin generation. The rate and amount of fibrin generation was indistinguishable in both groups. Furthermore, inhibition of the protein disulfide isomerase with the function blocking monoclonal antibody RL-90 (3ug/g mouse) eliminated any fibrin deposition in wild-type mice that had been treated with Integrilin. Taken together, these data indicate that endothelium-derived protein disulfide isomerase is necessary to support fibrin deposition in vivo in our laser injury model of thrombus formation. The initial protein disulfide isomerase expressed at the site of injury is derived from endothelial cells but platelets activated at the site of thrombus formation contribute, amplify and sustain protein disulfide isomerase expression.

scholarly journals Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation

Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2133-2143 ◽  
Author(s):  
Roxane Darbousset ◽  
Grace M. Thomas ◽  
Soraya Mezouar ◽  
Corinne Frère ◽  
Rénaté Bonier ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Thrombus Formation ◽  
Coagulation Cascade ◽  
Factor Xii ◽  
Long Time ◽  
Platelet Accumulation

AbstractFor a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1–ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

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