title: Maximal PAI-1 inhibition in vivo requires neutralizing antibodies that recognize and inhibit glycosylated PAI-1.
abstract: Plasminogen activator inhibitor-1 (PAI-1) regulates the activity of t-PA and u-PA and is an important inhibitor of the plasminogen activator system. Elevated PAI-1 levels have been implicated in the pathogenesis of several diseases. Prior to the evaluation of PAI-1 inhibitors in humans, there is a strong need to study the effect of PAI-1 inhibition in mouse models. In the current study, four monoclonal antibodies previously reported to inhibit recombinant PAI-1 in vitro, were evaluated in an LPS-induced endotoxemia model in mice. Both MA-33H1F7 and MA-MP2D2 exerted a strong PAI-1 inhibitory effect, whereas for MA-H4B3 and MA-124K1 no reduced PAI-1 activity was observed in vivo. Importantly, the lack of PAI-1 inhibition observed for MA-124K1 and MA-H4B3 in vivo corresponded with the absence of inhibition toward glycosylated mouse PAI-1 in vitro. Three potential N-glycosylation sites were predicted for mouse PAI-1 (i.e. N209, N265 and N329). Electrophoretic mobility analysis of glycosylation knock-out mutants before and after deglycosylation indicates the presence of glycan chains at position N265. These data demonstrate that an inhibitory effect toward glycosylated PAI-1 is a prerequisite for efficient PAI-1 inhibition in mice. Our data also suggest that PAI-1 inhibitors for use in humans must preferably be screened on glycosylated PAI-1 and not on recombinant non-glycosylated PAI-1.
authors.affiliation: Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.
authors.forename: Britt
authors.initials: B
authors.lastname: Van De Craen
authors.forename: Ilse
authors.initials: I
authors.lastname: Scroyen
authors.forename: Christine
authors.initials: C
authors.lastname: Vranckx
authors.forename: Griet
authors.initials: G
authors.lastname: Compernolle
authors.forename: H Roger
authors.initials: HR
authors.lastname: Lijnen
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Declerck
authors.forename: Ann
authors.initials: A
authors.lastname: Gils
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D057134:Antibodies, Neutralizing / Q000627:therapeutic use*; D019446:Endotoxemia / Q000188:drug therapy* / Q000276:immunology*; D006031:Glycosylation / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D017395:Plasminogen Activator Inhibitor 1; C051321:SERPINE1 protein, human