Redirection of the reaction between activated protein C and a serpin to the substrate pathway.
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.
Komissarov
Andrey A
AA
Department of Chemistry, Portland State University, P. O. Box 751, Portland, OR 97207-0751, USA. akomiss@pdx.edu
Andreasen
Peter A
PA
Declerck
Paul J
PJ
Kamikubo
Yuichi
Y
Zhou
Aiwu
A
Gruber
András
A
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D004211:Disseminated Intravascular Coagulation / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011486:Protein C / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D018805:Sepsis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D008024:Ligands; D017395:Plasminogen Activator Inhibitor 1; D011486:Protein C; D011994:Recombinant Proteins; C051321:SERPINE1 protein, human