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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 164-171
Characterization of Wild-Type and Mutant
 2-Antiplasmins: Fibrinolysis Enhancement by Reactive
Site Mutant
Kyung N. Lee,
Weon-Chan Tae,
Kenneth W. Jackson,
Soon H. Kwon, and
Patrick A. McKee
From the William K. Warren Medical Research Institute and the
Department of Medicine, University of Oklahoma Health Sciences Center,
Oklahoma City, OK.
During human blood clotting,  2-antiplasmin
(2AP) 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 2AP
and a specific  -lysyl group in each of the -chains of fibrin.
This causes fibrin to become resistant to plasmin-mediated lysis. We
found that chemically Arg-modified 2AP, which lacked
plasmin-inhibitory activity, competed effectively with native
2AP for becoming cross-linked to fibrin and as a
consequence, enhanced fibrinolysis. Recombinant
2AP 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 2AP in Pichia pastoris yeast. Two
forms of nonglycosylated recombinant 2AP were expressed,
isolated and characterized: (1) wild-type, which was analogous to
native 2AP, and (2) a mutant form, which had Ala
substituted for the reactive-site Arg364. Both the wild-type and mutant
forms of 2AP functioned as FXIIIa substrates with
affinities and kinetic efficiencies comparable to those of native
2AP, despite each having an additional acetylated Met
blocking group at their respective amino-termini. Wild-type recombinant
2AP displayed full plasmin inhibitory activity, while mutant 2AP had none. Neither the absence of
glycosylation nor blockage of the amino-terminus affected
plasmin-inhibitory or FXIIIa substrate activities of wild-type
2AP. When our mutant 2AP, 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 2AP
augmented lysis by competing with native 2AP for
FXIIIa-catalyzed incorporation into fibrin.
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