USE OF ANNEXIN-V TO DEMONSTRATE THE ROLE OF PHOSPHATIDYLSERINE EXPOSURE IN THE MAINTENANCE OF HEMOSTATIC BALANCE BY ENDOTHELIAL-CELLS

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 K(d) in the 10(-10) M range. The purpose of the present study was to use annexin-V as a probe to assess the catalytic...

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Published in:Biochemical journal 1992-02, Vol.282, p.7-13
Main Authors: RAVANAT, C, ARCHIPOFF, G, BERETZ, A, FREUND, G, CAZENAVE, JP, FREYSSINET, JM
Format: Article
Language:English
Subjects:
Biochemistry & Molecular Biology
Life Sciences & Biomedicine
Science & Technology
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Summary: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 K(d) 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-mu-M-phospholipid concentration, the apparent K(i) being 0.5-mu-M at 75-mu-M-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-mu-M, annexin-V totally inhibited the generation of activated protein C by Factor Xa in the presence of 75-mu-M-lipid, the saturating inhibitory concentration being dependent on phospholipid concentration. At 0.1-mu-M, 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-mu-M-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-mu-M, 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
ISSN:0264-6021
DOI:10.1042/bj2820007