New insights into the size and stoichiometry of the plasminogen activator inhibitor type-1.vitronectin complex.
Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that the physiologically relevant form of PAI-1 is in a complex with the abundant plasma glycoprotein, vitronectin. The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation. Although the complex is clearly significant, information is vague regarding the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo. Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M(r) approximately 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces.
Podor
T J
TJ
Department of Pathology and Molecular Medicine, McMaster University and the Hamilton Civic Hospitals Research Centre, Ontario, Canada.
Shaughnessy
S G
SG
Blackburn
M N
MN
Peterson
C B
CB
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D008956:Models, Chemical; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015843:Serpins / Q000737:chemistry; D014461:Ultracentrifugation; D019096:Vitronectin / Q000097:blood / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D015843:Serpins; D019096:Vitronectin