Abstract
The mechanism for the conversion of plasminogen activator inhibitor-1 (PAI-1) from the active to the latent conformation is not well understood. Recently, a monoclonal antibody, 33B8, was described that rapidly converts PAI-1 to the latent conformation (Verhamme, I., Kvassman, J. O., Day, D., Debrock, S., Vleugels, N., Declerck, P. J., and Shore, J. D. (1999) J. Biol. Chem. 274, 17511-17517). In an attempt to understand this interaction, and more broadly to understand the mechanism of the natural transition of PAI-1 to the latent conformation, we have used random mutagenesis to identify the 33B8 epitope in PAI-1. This site involves at least 8 amino acids scattered over more than two-thirds of the linear sequence that form a compact epitope on the PAI-1 three-dimensional structure. Surface plasmon resonance studies indicate a high affinity interaction between latent PAI-1 and 33B8 that is approximately 100-fold higher than comparable binding to active PAI-1. Structural modeling results together with surface plasmon resonance analysis of parental and site-directed PAI-1 mutants with disrupted 33B8 binding suggest the existence of a specific PAI-1 intermediate structure that is stabilized by 33B8 binding. These analyses strongly suggest that this intermediate form of PAI-1 has a partial insertion of the reactive center loop into beta-sheet A, and together, these data have significant implications for the general serpin mechanism of proteinase inhibition.
Highlights
The mechanism for the conversion of plasminogen activator inhibitor-1 (PAI-1) from the active to the latent conformation is not well understood
Materials—Murine monoclonal antibodies MA-33B8 (33B8) and MA31C9 (31C9) as well as rabbit polyclonal antibodies directed against human PAI-1 were purchased from Molecular Innovations (Southfield, MI), horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antiserum were from Bio-Rad. type plasminogen activator (tPA) was from Genentech (South San Francisco, CA), and urokinase-type plasminogen activator (uPA) was from Molecular Innovations
Our data demonstrate that the identified 8 residues must comprise at least a significant part of the 33B8 binding epitope on the surface of PAI-1
Summary
With respect to pathological fibrosis, PAI-1 null mice are protected from the development of vascular plaque [12] and pulmonary fibrosis [13, 14] These studies, together with the aforementioned clinical observations, suggest that PAI-1 inhibition may restore endogenous stimulation of fibrinolysis by plasminogen activation, re-establishing a critical defense mechanism for the prevention of intravascular thrombosis and tissue fibrosis. The reported rate of inactivation by this antibody is ϳ4000-fold faster than the spontaneous conversion of PAI-1 to the latent structure Based on these studies it was suggested that exposure of the antibody epitope depended on an unfavorable equilibrium in PAI-1 that might involve an intermediate PAI-1 structure with partial insertion of the RCL into -sheet A.
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