Specific interaction of vitronectin with the cell-secreted protease inhibitor glia-derived nexin and its thrombin complex.
Interaction of vitronectin with glia-derived nexin (GDN), thrombin, and the complex GDN-thrombin was demonstrated in direct binding assays that indicated the formation of binary and ternary complexes. The concentration of vitronectin necessary to obtain 50% saturation of the immobilized GDN-thrombin complex binding sites (EC50) was about 1 nM. Under similar experimental conditions, the EC50 of vitronectin for the immobilized antithrombin-III-thrombin complex was about fivefold higher. A tight complex was also formed between vitronectin and immobilized GDN (EC50 approximately 1.5 nM) but when vitronectin was immobilized, GDN displayed a reduced affinity for vitronectin (EC50 approximately 10 nM). These results suggest differences between the immobilized and free conformations of GDN and/or vitronectin. In contrast, vitronectin displayed negligible affinity for antithrombin III. Biotinylated GDN was used to characterize further the binding of GDN or the GDN-thrombin complex to vitronectin. The interaction of the biotinylated GDN-thrombin complex with immobilized vitronectin (EC50 approximately 2 nM) was completely blocked by nonbiotinylated complexes of thrombin with either GDN or antithrombin III, whereas free GDN, free thrombin and the GDN-trypsin complex were only weak competitors. Active-site-blocked urokinase and the complex GDN-urokinase also strongly competed for binding of the biotinylated GDN-thrombin complex to vitronectin. Binding of biotinylated GDN to immobilized vitronectin was specific, saturable and was competed with decreasing efficiency by the GDN-thrombin complex, free GDN and free antithrombin III. These interactions between the adhesive component vitronectin and the serine protease inhibitor GDN may relate to localized control of thrombin and/or urokinase action at certain extravascular sites. These results are discussed in terms of binding sites for vitronectin on GDN, thrombin, and the GDN-thrombin complex.
Rovelli
G
G
Friedrich Miescher-Institut, Basel, Switzerland.
Stone
S R
SR
Preissner
K T
KT
Monard
D
D
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor; D000990:Antithrombin III / Q000737:chemistry; D001665:Binding Sites; D001710:Biotin / Q000032:analysis; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry*; D002478:Cells, Cultured; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D013917:Thrombin / Q000032:analysis / Q000737:chemistry*; D019096:Vitronectin
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D006023:Glycoproteins; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D019096:Vitronectin; D001710:Biotin; D000990:Antithrombin III; D013917:Thrombin