Characterization of wild-type and mutant alpha(2)-antiplasmins: Fibrinolysis enhancement by reactive site mutant

During human blood clotting, alpha(2)-antiplasmin (alpha(2)AP) becomes cova lently linked to fibrin when activated blood clotting factor XIII (FXIIIa) catalyzes the formation of an isopeptide bond between glutamine at position two in alpha(2)AP and a specific E-lysyl group in each of the a-chains of fibrin. This causes fibrin to become resistant to plasmin-mediated lysis. W e found that chemically Arg-modified alpha(2)AP, which lacked plasmin-inhib itory activity, competed effectively with native alpha(2)AP for becoming cr oss-linked to fibrin and as a consequence, enhanced fibrinolysis. Recombina nt alpha(2)AP 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-s pecific fibrinolytic enhancer, we expressed recombinant alpha(2)AP in Pichi a pastoris yeast. Two forms of nonglycosylated recombinant alpha(2)AP were expressed, isolated and characterized: (1) wild-type, which was analogous t o native alpha(2)AP, and (2) a mutant form, which had Ala substituted for t he reactive-site Arg364. Both the wildtype and mutant forms of alpha(2)AP f unctioned as FXIIIa substrates with affinities and kinetic efficiencies com parable to those of native alpha(2)AP, despite each having an additional ac etylated Met blocking group at their respective amino-termini. Wild-type re combinant alpha(2)AP displayed full plasmin inhibitory activity, while muta nt alpha(2)AP had none. Neither the absence of glycosylation nor blockage o f the amino-terminus affected plasmin-inhibitory or FXIIIa substrate activi ties of wild-type alpha(2)AP. When our mutant alpha(2)AP, which lacked plas min-inhibitory function, was added to human plasma or whole blood clots, ur okinase (UK)-induced clot lysis was enhanced in a dose-dependent manner, in dicating that mutant a2AP augmented lysis by competing with native alpha(2) AP for FXIIIa-catalyzed incorporation into fibrin. (C) 1999 by The American Society of Hematology.