Binding of recombinant annexin V to endothelial cells: effect of annexin V binding on endothelial-cell-mediated thrombin formation.
Annexin V binds with high affinity to procoagulant phospholipid vesicles and thereby inhibits the procoagulant reactions catalysed by these surfaces in vitro. In vivo, vascular endothelial cells are known to catalyse the formation of thrombin by the expression of binding sites at which procoagulant complexes can assemble. Here, we have studied the binding capacity of recombinant annexin V (rANV) to quiescent, phorbol 12-myristate 13-acetate (PMA)- and tumour necrosis factor alpha (TNF-alpha)-stimulated cultured human umbilical-vein endothelial cells (HUVEC). The dissociation constant (Kd) was 15.5 +/- 3.3 nM and the number of binding sites was 8.8 (+/- 3.9) x 10(6)/cell. These binding parameters did not change significantly during a 30 h incubation period with PMA or TNF-alpha. rANV inhibited HUVEC-mediated factor Xa formation via the extrinsic as well as the intrinsic route. Activation of factor X by the tissue factor-factor VII-factor X complex and tenase complex was inhibited with IC50 values of 43 +/- 30 nM and 33 +/- 24 nM respectively. Endothelial-cell-mediated generation of thrombin by the prothrombinase complex was inhibited by rANV with an IC50 of 16 +/- 12 nM. Preincubation of rANV with the endothelial cells did not significantly influence the IC50 values. These results show that rANV binds to the same extent to quiescent, PMA- and TNF-stimulated HUVEC, and, as a result of this binding, rANV efficiently inhibits endothelial-cell-mediated thrombin formation.
van Heerde
W L
WL
Department of Haematology, University Hospital Utrecht, The Netherlands.
Poort
S
S
van 't Veer
C
C
Reutelingsperger
C P
CP
de Groot
P G
PG
D017304:Annexin A5 / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017304:Annexin A5; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate