Document Detail


Characterization of wild-type and mutant alpha2-antiplasmins: fibrinolysis enhancement by reactive site mutant.
MedLine Citation:
PMID:  10381509     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
During human blood clotting, alpha2-antiplasmin (alpha2AP) becomes covalently linked to fibrin when activated blood clotting factor XIII (FXIIIa) catalyzes the formation of an isopeptide bond between glutamine at position two in alpha2AP and a specific epsilon-lysyl group in each of the alpha-chains of fibrin. This causes fibrin to become resistant to plasmin-mediated lysis. We found that chemically Arg-modified alpha2AP, which lacked plasmin-inhibitory activity, competed effectively with native alpha2AP for becoming cross-linked to fibrin and as a consequence, enhanced fibrinolysis. Recombinant alpha2AP reported to date by other groups either lacked or possessed a low level of FXIIIa substrate activity. As a first step in the development of an engineered protein that might have potential as a localized fibrin-specific fibrinolytic enhancer, we expressed recombinant alpha2AP in Pichia pastoris yeast. Two forms of nonglycosylated recombinant alpha2AP were expressed, isolated and characterized: (1) wild-type, which was analogous to native alpha2AP, and (2) a mutant form, which had Ala substituted for the reactive-site Arg364. Both the wild-type and mutant forms of alpha2AP functioned as FXIIIa substrates with affinities and kinetic efficiencies comparable to those of native alpha2AP, despite each having an additional acetylated Met blocking group at their respective amino-termini. Wild-type recombinant alpha2AP displayed full plasmin inhibitory activity, while mutant alpha2AP had none. Neither the absence of glycosylation nor blockage of the amino-terminus affected plasmin-inhibitory or FXIIIa substrate activities of wild-type alpha2AP. When our mutant alpha2AP, which lacked plasmin-inhibitory function, was added to human plasma or whole blood clots, urokinase (UK)-induced clot lysis was enhanced in a dose-dependent manner, indicating that mutant alpha2AP augmented lysis by competing with native alpha2AP for FXIIIa-catalyzed incorporation into fibrin.
Authors:
K N Lee; W C Tae; K W Jackson; S H Kwon; P A McKee
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Blood     Volume:  94     ISSN:  0006-4971     ISO Abbreviation:  Blood     Publication Date:  1999 Jul 
Date Detail:
Created Date:  1999-07-23     Completed Date:  1999-07-23     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  7603509     Medline TA:  Blood     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  164-71     Citation Subset:  AIM; IM    
Affiliation:
William K. Warren Medical Research Institute and the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA. kyung-lee@ouhsc.edu
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MeSH Terms
Descriptor/Qualifier:
Cross-Linking Reagents
Fibrin / metabolism
Fibrinolysis* / genetics
Humans
Mutation*
Recombinant Proteins / genetics,  metabolism
Substrate Specificity / genetics
Transglutaminases / metabolism
alpha-2-Antiplasmin / genetics*,  metabolism
Chemical
Reg. No./Substance:
0/Cross-Linking Reagents; 0/Recombinant Proteins; 0/alpha-2-Antiplasmin; 9001-31-4/Fibrin; EC 2.3.2.13/Transglutaminases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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