Covalent binding of human thrombin to a human endothelial cell-associated protein.
Binding of 125I-thrombin to endothelial cells derived from human umbilical vein was studied in tissue culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed covalent binding of thrombin in a 72-kDa complex. This binding is specific and requires the catalytically active site of the enzyme. Formation of the complex could be detected as early as 3 min after addition of thrombin or with a thrombin concentration as low as 0.5 nM. This irreversible binding exhibits thrombin dose-dependence and reaches maximum levels at a concentration of 50 nM (10 fmol/10(5) cells). Some characteristics of the 72-kDa complex were compared to those of the complexes formed between thrombin and protease nexin originating from fibroblasts or platelets: (i) its electrophoretic mobility on SDS-PAGE is identical to that of the thrombin-platelet protease nexin complex, (ii) heparin prevents the appearance of the complex on the cell surface, (iii) plasmin in a 100-fold molar excess prevents the covalent linkage of thrombin, suggesting that the protease specificity of the endothelial component involved in the complex might not be restricted to thrombin. Yet no release, nor any secretion of the endothelial protein, could be detected. These results indicate that active thrombin binds covalently to a specific endothelial protein that is in several respects similar to fibroblast or platelet protease nexin and provides a thrombin binding site distinct from thrombomodulin and glycosaminoglycans.
Leroy-Viard
K
K
Hémostase et Thrombose, Faculté Xavier Bichat, Paris, France.
Jandrot-Perrus
M
M
Tobelem
G
G
Guillin
M C
MC
D016564:Amyloid beta-Protein Precursor; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D005341:Fibrinolysin / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D058255:Protease Nexins; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D011956:Receptors, Cell Surface; D013917:Thrombin / Q000378:metabolism*
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D058255:Protease Nexins; D011506:Proteins; D011956:Receptors, Cell Surface; D006493:Heparin; D013917:Thrombin; D005341:Fibrinolysin