Use of annexin-V to demonstrate the role of phosphatidylserine exposure in the maintenance of haemostatic 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 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)
Ravanat
C
C
INSERM U.311, Centre Régional de Transfusion Sanguine, Strasbourg, France.
Archipoff
G
G
Beretz
A
A
Freund
G
G
Cazenave
J P
JP
Freyssinet
J M
JM
D017304:Annexin A5; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D005167:Factor VII / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D005260:Female; D006487:Hemostasis / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000494:pharmacology; D008970:Molecular Weight; D010718:Phosphatidylserines / Q000494:pharmacology*; D010743:Phospholipids / Q000494:pharmacology*; D010920:Placenta / Q000502:physiology; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D011486:Protein C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D012501:Saphenous Vein; D013917:Thrombin / Q000378:metabolism*
D017304:Annexin A5; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D010718:Phosphatidylserines; D010743:Phospholipids; D011257:Pregnancy Proteins; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D005167:Factor VII; D005170:Factor X; D013917:Thrombin