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Stability of the thrombin-thrombomodulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926.

ABSTRACT: Protein C activation by alpha-thrombin on the surface of endothelial cells depends on an essential membrane-glycoprotein cofactor, thrombomodulin. In the present study we have monitored the activity of thrombin-thrombomodulin complexes on human saphenous-vein endothelial cells (HSVEC) or on the endothelial cell line EA.hy 926. Cell monolayers were exposed for 5 min to 8.5 nM human alpha-thrombin and then washed to remove unbound thrombin. The cells were then incubated at 37 degrees C for 5-180 min. At the end of the respective incubation periods, purified human protein C (120 nM) was added in order to assay the activity of the thrombin-thrombomodulin complexes present on the cell surface. HSVEC pre-exposed to thrombin retained their full capacity to promote protein C activation up to 90 min after free thrombin was removed. This capacity then decreased slowly to reach 56% of control value after 180 min of incubation. Original activity was 3.8 +/- 0.9 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 5). The capacity of protein C activation of EA.hy 926 cells remained constant for 120 min after free thrombin was removed, then decreased to 76% of control after 180 min. Original activity was 2.0 +/- 0.4 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 3). Similar results were obtained with cells fixed with 3% paraformaldehyde. However, during the 5-180 min incubation period, non-fixed cells of both types were capable of significantly internalizing fluorescent acetylated low-density lipoprotein. In the experimental protocol used here, an eventual inhibition of thrombin internalization by protein C can be excluded, as protein C is only added at the end of the incubation period. We conclude that there is no evidence of rapid internalization of thrombin-thrombomodulin complexes on HSVEC or the EA.hy 926 cell line, as assessed by the ability of membrane-bound thrombin to activate protein C.

SUBMITTER: Beretz A

PROVIDER: S-EPMC1138469 | biostudies-other | 1989 Apr

REPOSITORIES: biostudies-other

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Project description: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.

Project description:Cysteinyl leukotrienes (cysLT), i.e. LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway. The cysLT receptors cysLT1-R and cysLT2-R are expressed on different target cells and mediate inflammatory reactions in tissue- and LT-R-specific ways. Though endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be defined. To delineate cysLT2-R´s action, we stimulated human umbilical vein EC with 100 nM LTD4 for 60 min, determined gene signatures by microarrays, and characterized the resulting EC phenotypes. As controls, we compared LTD4-induced genes with those induced by 10 nM thrombin, a prototype vasoactive activator of EC that binds to protease-activated receptor 1 (PAR-1). Following application of stringent filters 37 LTD4-upregulated genes were identified (> 2.5fold stimulation). Surprisingly, most of the LTD4-regulated genes were also induced by thrombin and expression of cysLT2-R- and PAR-1-regulated genes strongly correlated (Pearson correlation coefficient: r = 0.90). Moreover, LTD4 + thrombin, when added together, augmented expression of LTD4- or thrombin-stimulated genes (Wilcoxon signed rank test: p < 0.01). Prominently induced genes that may play roles in vascular injury were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A; E-selectin; CXC ligand 2; interleukin 8 (IL-8); a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS-1); and tissue factor (TF). Transcripts of these genes peaked at approximately 60 min, were unaffected by the cysLT1-R antagonist montelukast, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus in LTD4-stimulated cells; LTD4 upregulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC pro-coagulant activity. These data show that cysLT2-R activation results in a pro-inflammatory EC phenotype through activation of immediate-early genes that resemble those induced by PAR-1. As LTD4 and thrombin are formed concomitantly during vascular injury and pro-thrombotic states, cysLT2-R and PAR-1 may collaborate in vivo to mediate vascular injury and repair. Keywords: Leukotriene Transcriptome, Thrombin Transcriptome, HUVEC, Immediate-Early Gene Expression, Cysteinyl Leukotriene 2 Receptor Gene Signature in HUVEC

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Stability of the thrombin-thrombomodulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926.

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