Abstract
The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK alpha domain and converts SKDelta59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK alpha domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SKDelta59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SKDelta59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SKDelta59 does not protect plasmin from inhibition by alpha(2)-antiplasmin. At the same time, SKDelta59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SKDelta59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl(-)-deficient buffers. Taken together, these studies indicate that an intact alpha domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact alpha domain makes SKDelta59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by alpha(2)-antiplasmin.
Highlights
The enzyme plasmin plays a central role in degrading fibrin, the protein matrix of blood clots or thrombi
We have previously reported that the NH2 terminus of SK contains a “catalytic switch” that allows Pg activation in the presence or absence of fibrin [9]
1 The abbreviations used are: Pg, plasminogen; Glu-Pg, native plasminogen with amino-terminal Glu; Lys-Pg, plasmin-modified plasminogen with amino-terminal Lys; SK, streptokinase; (DD)E, complex of fibrin D-dimer noncovalently associated with fragment E; MBP, maltose-binding protein; EACA, ⑀-amino-n-caproic acid; Ab, antibody; S-2251, H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride; TPA, tissue Pg activator
Summary
Plasminogen; Glu-Pg, native plasminogen with amino-terminal Glu; Lys-Pg, plasmin-modified plasminogen with amino-terminal Lys; SK, streptokinase; (DD)E, complex of fibrin D-dimer noncovalently associated with fragment E; MBP, maltose-binding protein; EACA, ⑀-amino-n-caproic acid; Ab, antibody; S-2251, H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride; TPA, tissue Pg activator. In the presence of fibrin, TPA acquires greater substrate affinity by forming a ternary TPA1⁄7Pg1⁄7fibrin complex that more efficiently cleaves the Arg561Val562 bond of Pg [3] Another example of fibrin-dependent pro-enzyme activation occurs with pro-urokinase, which remains inactive until cleaved by plasmin on fibrin (or other binding sites) to a functional Pg activator [20, 21]. Unlike the parent SK molecule, SK⌬59 employs several fibrin-dependent mechanisms, similar to both TPA and staphylokinase
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