Pinpointing The Structural Dynamics of Plasminogen Activator Inhibitor -1 upon binding to Heparin using Hydrogen/Deuterium Exchange Mass Spectrometry

The native conformation of plasminogen activator inhibitor (PAI-1) is an active metastable structure that transforms spontaneously to an inactive latent form within 1-2 hrs under physiological conditions. PAI-1 is a key player to regulate the activation of fibrinolysis, with broad influence effects on inflammation, hemostasis, tissue remodeling, and wound healing ¹.

The binding of endogenous cofactor vitronectin to PAI-1 causes delaying in the transition to the latent state and significantly boosts the protein's thermal stability ². This binding helps to extend PAI-1 half-life and delay its latency transition thus controlling the stability of the active form. Previous studies have suggested that low molecular weight heparin alters the levels of circulating PAI-1 and enhances endogenous fibrinolysis ³. However, the intrinsic dynamics of this binding are not completely understood.

We have used hydrogen/deuterium exchange mass spectrometry to elucidate the structural dynamics of PAI-1 and heparin binding. Heparin binding to PAI-1 induces significant attenuation of deuterium uptake in PAI-1. The strongest protection against isotopic exchange is observed in the lower half of PAI-1. Peptides covering the heparin binding interface are shielded from the solvent and show a slow exchange rate. In addition, we observed a moderate effect on the peptides remote from the heparin binding site. These peptides are topographically connected in a circle-like shape around the vertical axis of the protein structure.

Our findings reveal that heparin may contribute to the localization of PAI-1 at specific sites, hence involved in the regulation of plasminogen activation and its functional stability.