scholarly journals Heterogeneous regulation of constitutive thrombomodulin or inducible tissue-factor activities on the surface of human saphenous-vein endothelial cells in culture following stimulation by interleukin-1, tumour necrosis factor, thrombin or phorbol ester

1991 ◽  
Vol 273 (3) ◽  
pp. 679-684 ◽  
Author(s):  
G Archipoff ◽  
A Beretz ◽  
J M Freyssinet ◽  
C Klein-Soyer ◽  
C Brisson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Saphenous Vein ◽  
Tumour Necrosis ◽  
Protein C ◽  
Interleukin 1 ◽  
Factor Activity ◽  
Human Saphenous Vein ◽  
Tissue Factor Activity ◽  
Necrosis Factor

Thrombomodulin and tissue-factor activities were measured on the surface of confluent human saphenous-vein endothelial cells (HSVEC) cultivated in 96-multiwell plates. Thrombomodulin activity was measured in the presence of purified human thrombin (2.2 nM) and protein C (65 nM). Tissue-factor activity was measured with purified human Factor VII (5 nM) and Factor X (400 nM). Generated activated protein C and Factor Xa released in the supernatant were assayed with chromogenic substrates. Resting cells exhibited significant thrombomodulin activity, but no detectable tissue-factor activity. After 4 h of preincubation with tumour necrosis factor (TNF, 22-2200 pM), interleukin-1 (IL-1, 5.7-570 nM) or phorbol myristate acetate (PMA, 1.61-161 nM) there was an increase in tissue-factor activity and a concomitant decrease in thrombomodulin activity. However, the extent of both responses varied according to the nature of the stimulus. Thrombin (0.44-44 nM) also induced an increase in tissue-factor activity, but had no effect on thrombomodulin activity. Kinetic studies showed that for all stimuli the increase in tissue factor was transient, reaching a maximum after 4-8 h of preincubation with the stimulating agent and returning to normal values after 24 h. IL-1 and TNF induced a time-dependent decrease in thrombomodulin, by respectively 47% and 67% of control values after 24 h. However, PMA induced only a transient down-regulation of thrombomodulin, full activity being recovered after 18 h. Hence this simultaneous assay system, using intact HSVEC and purified human coagulation factors, enabled us to observe that the regulation of thrombin generation could be diversely affected by various substances known to stimulate the endothelium. This suggests that the simultaneous and opposite modulation of these proteins does not represent an unified response of the endothelial cells to procoagulant stimuli. These results also confirm the absence of effect of thrombin on the expression of thrombomodulin on the cell surface.

scholarly journals Use of annexin-V to demonstrate the role of phosphatidylserine exposure in the maintenance of haemostatic balance by endothelial cells

1992 ◽  
Vol 282 (1) ◽  
pp. 7-13 ◽  
Author(s):  
C Ravanat ◽  
G Archipoff ◽  
A Beretz ◽  
G Freund ◽  
J P Cazenave ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Protein C ◽  
Activated Protein C ◽  
Factor Xa ◽  
Annexin V ◽  
Factor Activity ◽  
Cells In Culture ◽  
Tissue Factor Activity

Annexin-V (PAP-I, lipocortin-V) acts as a potent anticoagulant in vitro by binding to negatively charged phospholipids with higher affinity than vitamin K-dependent proteins, with a Kd in the 10(-10) M range. The purpose of the present study was to use annexin-V as a probe to assess the catalytic potential of phospholipids in pro- and anti-coagulant reactions in purified systems and at the surface of endothelial cells in culture after stimulation. Procoagulant tissue factor and anticoagulant thrombomodulin activities were compared by using specific two-stage amidolytic assays performed with purified proteins. Procoagulant activity was estimated by the generation of Factor Xa by the Factor VII(a)-tissue factor complex. Anticoagulant activity was estimated by the generation of activated protein C by either the thrombin-thrombomodulin complex or Factor Xa. Annexin-V induced a decrease of 70% of thrombomodulin activity when thrombomodulin (5.4-214 nM) was reconstituted into phosphatidylcholine/phosphatidylserine (1:1, mol/mol) vesicles at 37.5 or 75 microM-phospholipid concentration, the apparent Ki being 0.5 microM at 75 microM-lipid. The saturating concentration of annexin-V was dependent on phospholipid concentration, but was independent of the phospholipid/thrombomodulin ratio. By contrast, when thrombomodulin was not reconstituted in vesicles, annexin-V had no effect. At 2 microM, annexin-V totally inhibited the generation of activated protein C by Factor Xa in the presence of 75 microM-lipid, the saturating inhibitory concentration being dependent on phospholipid concentration. At 0.1 microM, annexin-V totally inhibited tissue-factor activity present in crude brain thromboplastin. In the absence of stimulation, human endothelial cells in culture expressed significant thrombomodulin activity and no detectable tissue-factor activity. Basal thrombomodulin activity was only slightly inhibited (less than 15%) by 0.5 microM-annexin-V. Phorbol myristate acetate (PMA) induced the expression of tissue-factor activity and decreased thrombomodulin activity at the endothelial-cell surface. Annexin-V, at a concentration of 16 microM, caused an 80% decrease of tissue-factor activity induced by PMA at 10 ng/ml, whereas it inhibited thrombomodulin activity by only 15% on the same stimulated cells. Our results confirm that annexin-V inhibits, in vitro, procoagulant tissue-factor activity and anticoagulant activities (activation of protein C by the thrombin-thrombomodulin complex and by Factor Xa), through phospholipid-dependent mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Plasmin enhances cell surface tissue factor activity in mesothelial and endothelial cells

2009 ◽  
Vol 7 (1) ◽  
pp. 121-131 ◽  
Author(s):  
H. KOTHARI ◽  
G. KAUR ◽  
S. SAHOO ◽  
S. IDELL ◽  
L. V. M. RAO ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Tissue Factor ◽  
Factor Activity ◽  
Tissue Factor Activity

Incidence of Residual Clot Strands in Saphenous Vein Grafts after Endoscopic Harvest

2006 ◽  
Vol 1 (6) ◽  
pp. 323-327 ◽  
Author(s):  
Nicholas Burris ◽  
Kimberly Schwartz ◽  
Jamie Brown ◽  
Michael Kwon ◽  
Richard Pierson ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Saphenous Vein ◽  
Average Length ◽  
Common Finding ◽  
Fisher Exact Test ◽  
Factor Activity ◽  
Vein Grafts ◽  
Tissue Factor Activity ◽  
Saphenous Vein Grafts ◽  
Endothelial Integrity

Objective Strands of clot are frequently flushed out of saphenous vein grafts (SVG) during preparation for grafting, particularly those that are endoscopically harvested. However, saline distention at uncontrolled pressures increases graft thrombogenicity and the risk of early failure after coronary artery bypass grafting. The purpose of this prospective investigation was to define the incidence of intraluminal clot within endoscopically harvested SVG and the effect of attempted removal by saline distention. Methods Endoscopically harvested SVG were intraoperatively prepared for grafting by using saline distention at uncontrolled pressure (n = 24) or without distension (n = 20). Optical coherence tomography, a catheter-based infrared imaging system, was used to identify and characterize intraluminal clot strands in surplus SVG segments (average length for analysis, 4.9 ± 2.6 cm). These segments were also assessed for luminal tissue factor activity and percent endothelial integrity by CD31-directed immunohistochemistry. Results Clot strands were observed in 45.4% (20 of 44) of imaged SVG segments (severity of observed clots: 54%, mild; 32%, moderate; 14%, severe). Compared with grafts distended with saline, vein segments that were not distended displayed significantly higher endothelial integrity (60.1% ± 27.2% versus 24.7% ± 24.1%, P < 0.05) and lower tissue factor activity (1.28 ± 0.95 versus12.3 ±5.5 U/cm2, P < 0.001) despite having a higher incidence of clot stands (65.0% versus 29.1%, P < 0.02, Fisher exact test). Static flow was observed in veins during endoscopic harvest. Conclusions Clot strands of varying severity are a common finding after endoscopic vein harvest. Saline distention is not completely effective in removing clot strands and increases overall graft thrombogenicity. Therefore, prevention of clot or less traumatic methods of removing clot are indicated.

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