Research Article: Site-specific PEGylation of micro-plasmin for improved thrombolytic therapy through engineering enhanced resistance against serpin mediated inhibition

Date Published: May 29, 2019

Publisher: Public Library of Science

Author(s): Navneet Kaur, Prakash Kumar Sinha, Girish Sahni, Marco Rito-Palomares.


The relatively rapid inhibition of microplasmin by α2-AP leads to short functional half-life of the molecule in vivo, causing inefficient clot dissolution, even after site-specific, local catheter-based delivery. Here, we describe a PEGylation approach for improving the therapeutic potential via improving the survival of microplasmin in presence of its cognate inhibitor, α2-AP, wherein a series of strategically designed cysteine analogs of micro-plasminogen were prepared and expressed in E. coli, and further modified by covalent grafting in vitro with PEG groups of different molecular sizes so as to select single or double PEG chains that increase the molecular weight and hydrodynamic radii of the conjugates, but with a minimal discernible effect on intrinsic plasmin activity and structural framework, as explored by amidolytic activity and CD-spectroscopy, respectively. Interestingly, some of the purified PEG-coupled proteins after conversion to their corresponding proteolytically active forms were found to exhibit significantly reduced inhibition rates (up to 2-fold) by α2-AP relative to that observed with wild-type microplasmin. These results indicate an interesting, and not often observed, effect of PEG groups through reduced/altered dynamics between protease and inhibitor, likely through a steric hindrance mechanism. Thus, the present study successfully identifies single- and double-site PEGylated muteins of microplasmin with significantly enhanced functional half-life through enhanced resistance to inactivation by its in vivo plasma inhibitor. Such an increased survival of bioactivity in situ, holds unmistakable potential for therapeutic exploitation, especially in ischemic strokes where a direct, catheter-based deposition within the cranium has been shown to be promising, but is currently limited by the very short in vivo bioactive half-life of the fibrin dissolving agent/s.

Partial Text

The formation of pathological thrombi in the circulatory system can produce significant unwanted consequences like embolism, ischemia, heart attack, stroke, etc. Currently available thrombolytic treatments using plasminogen activators are associated with high cerebral bleeding risks and a 2–3 h, narrow therapeutic time-window especially in case of ischemic stroke [1–4].

The ability to modify protein structure away from the active site expands the realm of possibilities for preventing unwanted molecular interactions near the active site of an enzyme especially where relatively distant exosites are targeted. The rationale behind the present study was to investigate the effect of site selective PEGylation of microplasmin through a protein engineering approach.

The present study illustrates the effect of targeted covalent grafting of PEG chains on human microplasminogen so as to slow the antiplasmin mediated inhibition of its activated form microplasmin. We have identified some functional hot spots in microplasminogen that allow effective attachment of PEG moieties to the surface of the microplasmin without dramatically affecting its intrinsic enzymatic activity. The experiments suggest that physical steric hindrance caused by the relatively mobile but appropriately placed PEG group affect the association of PEGylated microplasmin with α2 –antiplasmin when attached at these site/s, without any marked alteration of the former’s fibrinolytic potency. Overall, the outcome of the present investigation emphasizes that microplasmin interactions with antiplasmin can be inhibited even by the non-rigid PEG polymer through sterically effective positional placements in the former.