Importance of N-Terminal Residues in Plasminogen Activator Inhibitor 1 on its Antibody Induced Latency Transition

 

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Summary

The serpin plasminogen activator inhibitor-1 (PAI-1) is a wellknown risk factor for thromboembolic and cardiovascular diseases. Many efforts have been made to reveal structure-function relationship in PAI-1, including the understanding of its unique latency transition. In this study, we describe the molecular characterization of PAI-1 neutralization by MA-159M12, a monoclonal antibody against rat PAI-1.

Time-dependent inactivation of PAI-1, exposure of a trypsin cleavage site typically for the latent conformation and disappearance of elastase susceptibility revealed that MA-159M12 accelerated the active to latent, conformational transition (t_1/2 120 ± 12 min and 18 ± 3.6 min in the absence and presence of MA-159M12, p <0.0001).

Cross-reactivity analysis of the antibody with various rat/human PAI-1 chimeras revealed that the epitope resides in αhA of rat PAI-1. Subsequent alanine-scanning mutagenesis and binding studies demonstrated that Pro²- Leu³- Pro⁴-Glu⁵ constitute the major residues of the epitope for MA-159M12. In conclusion, these findings demonstrate that, even though unexpected based on current knowledge on PAI-1 stability and function, interference with αhA results in a destabilisation of its active, inhibitory conformation. Therefore, αhA forms a putative target for the rational development of PAI-1 neutralizing components.

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