Structural requirements for the extracellular interaction of plasminogen activator inhibitor 1 with endothelial cell matrix-associated vitronectin.
The interaction of plasminogen activator inhibitor-1 (PAI-1) with its binding protein vitronectin (VN) (Declerck, P. J., De Mol, M., Alessi, M.-C., Baudner, S., Paques, E.-P., Preissner, K. T., Müller-Berghaus, G., and Collen, D. (1988) J. Biol. Chem. 263, 15454-15461) in the extracellular matrix (ECM) of cultured human endothelial cells (HUVEC) was studied. Like PAI-1, VN was found associated with the ECM as evidenced by direct antibody binding, by Western blot analysis as well as by diffuse immunofluorescence staining in permeabilized HUVEC. The specific interaction of VN with confluent monolayers of HUVEC was found to be saturable within 2-4 h at 37 degrees C only with respect to binding to the cells, while no saturable binding to the underlying ECM was observed, indicating that the majority if not all ECM-associated VN was derived from the culture medium. In contrast to PAI-1, ECM-associated VN was resistant toward glycine (pH 2.3), guanidine or urokinase treatment, suggesting that VN was tightly associated with the ECM network. Binding of recombinant PAI-1 (rPAI-1) was largely blocked by anti-VN IgG and only partly by anti-collagen IgG but not by antibodies against other ECM components, indicating that VN constitutes the primary binding protein for ECM-associated PAI-1. This contention was supported by ligand blotting experiments in which rPAI-1 was reacted with nitrocellulose replicas of electrophoretically separated ECM components. Protein band(s) (Mr = 63,000-67,000), comigrating with bovine VN (i.e. medium-derived VN) rather than with human VN were identified as major binding component(s). Moreover, binding studies with purified components revealed that PAI-1 did not show any affinity for collagen (type I/III) alone, whereas VN collagen coating was a much better template for PAI-1 binding than VN alone and that conformationally extended VN provides maximal PAI-1 binding capacity. Binding of rPAI-1 to surface-coated VN was saturable and revealed that (unlike urokinase) heparin or the synthetic peptide Gly-Arg-Gly-Asp-Ser did not inhibit PAI-1 binding. Ligand binding of rPAI-1 to nitrocellulose replicas from sodium dodecyl sulfate-polyacrylamide gels containing electrophoretically separated peptides from VN digests documented the association of PAI-1 with Mr = 10,000-20,000 fragments originating from the heparin-binding domain of VN. These results indicate that the exposure of the glycosaminoglycan-binding domain in VN may allow the concomitant binding of PAI-1 and heparin-like molecules to this region of the VN molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
Preissner
K T
KT
Clinical Research Unit for Blood Coagulation and Thrombosis, Max-Planck-Gesellschaft, Giessen, West Germany.
Grulich-Henn
J
J
Ehrlich
H J
HJ
Declerck
P
P
Justus
C
C
Collen
D
D
Pannekoek
H
H
Müller-Berghaus
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000378:metabolism; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D019096:Vitronectin; D014841:von Willebrand Factor / Q000378:metabolism
D005353:Fibronectins; D006023:Glycoproteins; D006025:Glycosaminoglycans; D009842:Oligopeptides; D015849:Plasminogen Inactivators; D011971:Receptors, Immunologic; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; D019096:Vitronectin; D014841:von Willebrand Factor; D005340:Fibrinogen; D006493:Heparin; D003094:Collagen; D014568:Urokinase-Type Plasminogen Activator