Redirection of the reaction between activated protein C and a serpin to the substrate pathway |
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Authors: | Komissarov Andrey A Andreasen Peter A Declerck Paul J Kamikubo Yuichi Zhou Aiwu Gruber András |
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Affiliation: | Department of Chemistry, Portland State University, P. O. Box 751, Portland, OR 97207-0751, USA. akomiss@pdx.edu |
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Abstract: | BACKGROUND: Activated protein C (APC) reduces mortality in severe sepsis. Protecting APC in the circulatory system from inactivation by serine protease inhibitors (serpins) could improve its therapeutic efficiency. Significantly elevated levels of a serpin plasminogen activator inhibitor 1 (PAI-1) correlate with a lethal outcome in severe sepsis and disseminated intravascular coagulation. Intermolecular mechanisms were employed to redirect the reaction between APC and PAI-1 from the inhibitory to the substrate pathway, which results in the catalytic neutralization of the serpin. METHODS: The effects of anti-PAI-1 monoclonal antibodies (mAbs) and vitronectin, as well as their fragments, on the kinetics and stoichiometry of the reaction between PAI-1 and APC were studied using SDS PAGE and fluorescence spectroscopy. RESULTS: MAbs with epitopes at alpha-helix F redirected 70-80% of the reaction between PAI-1 and APC, to the substrate pathway. Vitronectin and its SMB domain did not affect the stoichiometry of acyl-enzyme formation, but enhanced the effect of mAbs. While vitronectin induced a more than two-fold increase in the rate of the reaction between PAI-1 and APC, neither mAbs (mAb fragments), nor SMB domain of vitronectin affected it. CONCLUSIONS: Ligands interacting with alpha-helix F of PAI-1 demonstrated a potential for the protection of APC from inactivation by PAI-1. Since the mechanism of proteinase/serpin interaction is universal, a similar design and approach could be employed for enhancing the inactivation of other serpins in order to preserve APC activity in the circulation. Rational pharmacological targeting of the inhibitors of APC could have therapeutic utility. |
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Keywords: | α1AT, α1-antitrypsin APC, activated protein C DIC, disseminated intravascular coagulation mAb, monoclonal antibody NBD, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole NBD P9 PAI-1, Ser338Cys (position P9 of RCL) mutant variant of wt-rPAI-1 with NBD group attached to the cysteine residue PAI-1, plasminogen activator inhibitor-1 PCI, protein C inhibitor RCL, reactive center loop scFv, single chain Fv fragment SI, stoichiometry of inhibition SMB domain, somatomedin B domain of vitronectin tPA, two chain tissue type plasminogen activator uPA, urokinase type plasminogen activator Vn, vitronectin wt-rPAI-1, human recombinant wild-type PAI-1. |
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