The Mechanism of a Bacterial Plasminogen Activator Intermediate between Streptokinase and Staphylokinase

Abstract

The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (α, β, and γ) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by α2-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase γ-domain, we examined the mechanism of action of a novel two-domain (α,β) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by α2-antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase γ-domain bound to the b-plasminogen·SUPA complex and significantly reduced these lag phases. The SUPA-b·plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b·plasmin complex also activated h-plasminogen but with a lower kcat (25-fold) and kcat/Km (7.9-fold) than SK. We conclude that a γ-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase γ-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.