Abstract
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.
Highlights
The formation of pathological thrombi in the circulatory system can produce significant unwanted consequences like embolism, ischemia, heart attack, stroke, etc
The docking models obtained from GRAMM-X Protein-Protein Docking Web Server v.1.2.0 [56] using available three dimensional structural information of murine antiplasmin as well as human plasminogen catalytic domain (PDB ID. 1DDJ) [55] were used to interpret interacting residues preferably lying on loop structures of micro-plasminogen
The present study illustrates the effect of targeted covalent grafting of polyethylene glycol (PEG) chains on human microplasminogen so as to slow the antiplasmin mediated inhibition of its activated form microplasmin
Summary
The micro-plasminogen (truncated plasminogen derivative) previously cloned in T7 RNA polymerase inducible promoter based expression vector pET11a was obtained from lab [54]. Methoxy-PEG maleimide reagent was purchased from JenKem Technology, USA. All the materials required for the SDS-PAGE were purchased from Bio-RAD, USA. Chromozym PL was purchased from Roche Diagnostics, USA. Zeba Spin Desalting Columns were purchased from Thermo Fisher Scientific, USA. Micro-plasminogen previously cloned in E.coli [54] was obtained after IPTG (isopropyl-thiogalactopyranoside) induction in the form of inclusion bodies, which were solubilised in 8M urea and 10mM DTT. Refolded micro-plasminogen was purified by cationexchange chromatography on SP-Sepharose (GE-Amersham Biosciences). The protein eluted with 1M NaCl in 20mM Sodium acetate buffer (pH 5.5) was further desalted in 50mM PB (pH7.4) using Zeba desalting columns
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