B Ünlü1, V Y Bogdanov2, H H Versteeg3. 1. Einthoven Laboratory for Experimental Vascular Medicine, Department of Internal Medicine, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands. 2. Division of Hematology/Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States. 3. Einthoven Laboratory for Experimental Vascular Medicine, Department of Internal Medicine, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands. Electronic address: h.h.versteeg@lumc.nl.
Abstract
BACKGROUND: Full length Tissue factor (flTF) is a key player in hemostasis and also likely contributes to venous thromboembolism (VTE), the third most common cardiovascular disease. flTF and its minimally coagulant isoform, alternatively spliced TF (asTF), have been detected in thrombi, suggesting participation of both isoforms in thrombogenesis, but data on participation of asTF in hemostasis is lacking. Therefore, we assessed the role of asTF in flTF cofactor activity modulation, using a co-expression system. OBJECTIVE: To investigate the interplay between flTF and asTF in hemostasis on endothelial cell surface. METHODS: Immortalized endothelial (ECRF) cells were adenovirally transduced to express asTF and flTF, after which flTF cofactor activity was measured on cells and microvesicles (MVs). To study co-localization of flTF/asTF proteins, confocal microscopy was performed. Finally, intracellular distribution of flTF was studied in the presence or absence of heightened asTF levels. RESULTS: Levels of flTF antigen and cofactor activity were not affected by asTF co-expression. asTF and flTF were found to localize in distinct subcellular compartments. Only upon heightened overexpression of asTF, lower flTF protein levels and cofactor activity were observed. Heightened asTF levels also induced a shift of flTF from non-raft to lipid raft plasma membrane fractions, and triggered the expression of ER stress marker BiP. Proteasome inhibition resulted in increased asTF - but not flTF - protein expression. CONCLUSION: At moderate levels, asTF appears to have negligible impact on flTF cofactor activity on endothelial cells and MVs; however, at supra-physiological levels, asTF is able to reduce the levels of flTF protein and cofactor activity.
BACKGROUND: Full length Tissue factor (flTF) is a key player in hemostasis and also likely contributes to venous thromboembolism (VTE), the third most common cardiovascular disease. flTF and its minimally coagulant isoform, alternatively spliced TF (asTF), have been detected in thrombi, suggesting participation of both isoforms in thrombogenesis, but data on participation of asTF in hemostasis is lacking. Therefore, we assessed the role of asTF in flTF cofactor activity modulation, using a co-expression system. OBJECTIVE: To investigate the interplay between flTF and asTF in hemostasis on endothelial cell surface. METHODS: Immortalized endothelial (ECRF) cells were adenovirally transduced to express asTF and flTF, after which flTF cofactor activity was measured on cells and microvesicles (MVs). To study co-localization of flTF/asTF proteins, confocal microscopy was performed. Finally, intracellular distribution of flTF was studied in the presence or absence of heightened asTF levels. RESULTS: Levels of flTF antigen and cofactor activity were not affected by asTF co-expression. asTF and flTF were found to localize in distinct subcellular compartments. Only upon heightened overexpression of asTF, lower flTF protein levels and cofactor activity were observed. Heightened asTF levels also induced a shift of flTF from non-raft to lipid raft plasma membrane fractions, and triggered the expression of ER stress marker BiP. Proteasome inhibition resulted in increased asTF - but not flTF - protein expression. CONCLUSION: At moderate levels, asTF appears to have negligible impact on flTF cofactor activity on endothelial cells and MVs; however, at supra-physiological levels, asTF is able to reduce the levels of flTF protein and cofactor activity.
Authors: T H Bugge; Q Xiao; K W Kombrinck; M J Flick; K Holmbäck; M J Danton; M C Colbert; D P Witte; K Fujikawa; E W Davie; J L Degen Journal: Proc Natl Acad Sci U S A Date: 1996-06-25 Impact factor: 11.205
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Authors: Betül Ünlü; Begüm Kocatürk; Araci M R Rondon; Clayton S Lewis; Nathalie Swier; Rob F P van den Akker; Danielle Krijgsman; Iris Noordhoek; Erik J Blok; Vladimir Y Bogdanov; Wolfram Ruf; Peter J K Kuppen; Henri H Versteeg Journal: Oncogene Date: 2022-10-21 Impact factor: 8.756