Staphylokinase

Staphylokinase is a protein produced by a number of strains of S. aureus that also displays therapeutic potential as a thrombolytic agent. The protein has been purified from its natural source by a combination of ammonium sulfate precipitation and cation-exchange chromatography on CM cellulose. Affinity chromatography using plasmin or plasminogen immobilized to sepharose beads has also been used. The pure product is a 136 amino acid polypeptide displaying a molecular mass in the region of 16.5 kDa. Lower molecular mass derivatives lacking the first 6 or 10 NH2-terminal amino acids have also been characterized. All three appear to display similar thrombolytic activity in vitro at least.

The staphylokinase gene has been cloned in E. coli, as well as various other recombinant systems. The protein is expressed intracellularly in E. coli at high levels, representing 10-15 per cent of total cellular protein. It can be purified directly from the clarified cellular homogenate by a combination of ion-exchange and hydrophobic interaction chromatography.

Although staphylokinase shows no significant homology with streptokinase, it induces a throm-bolytic effect by a somewhat similar mechanism: it also forms a 1:1 stoichiometric complex with plasminogen. The proposed mechanism by which staphylokinase induces plasminogen activation is outlined in Figure 12.13. Binding of the staphylokinase to plasminogen appears initially to yield an inactive staphylokinase-plasminogen complex. However, complex formation somehow induces subsequent proteolytic cleavage of the bound plasminogen to form plasmin, which remains com-plexed to the staphylokinase. This complex (via the plasmin) then appears to catalyse the conversion of free plasminogen to plasmin, and it may even accelerate the process of conversion of other staphylokinase-plasminogen complexes into staphylokinase-plasmin complexes. The net effect is generation of active plasmin, which displays a direct thrombolytic effect by degrading clot-based fibrin, as described previously (Figure 12.11).

The serum protein a2-antiplasmin can inhibit the activated plasmin-staphylokinase complex. It appears that the a2-antiplasmin can interact with the active plasmin moiety of the complex, resulting in dissociation of staphylokinase and consequent formation of an inactive plasmin-a2-antiplasmin complex.

Staphylokinase

Staphylokinase

Staphylokinase-plasminogen complex (inactive)

Free plasminogen

Staphylokinase-plasminogen complex (inactive)

Free plasminogen

Staphylokinase-plasmin complex (active)

Free plasmin

Staphylokinase-plasmin complex (active)

Free plasminogen

Figure 12.13 Schematic representation of the mechanism by which staphylokinase appears to activate the thrombolytic process via the generation of plasmin. See text for details

Free plasmin

Free plasminogen

Figure 12.13 Schematic representation of the mechanism by which staphylokinase appears to activate the thrombolytic process via the generation of plasmin. See text for details

The thrombolytic ability of (recombinant) staphylokinase has been evaluated in initial clinical trials with encouraging results. Some 80 per cent of patients suffering from acute myocardial infarction who received staphylokinase responded positively (10 mg staphylokinase was administered by infusion over 30 min). The native molecule displays a relatively short serum half-life (of 6.3 min), although covalent attachment of PEG reduces the rate of serum clearance, hence effectively increasing the molecule's half-life significantly. As with streptokinase, patients administered staphylokinase develop neutralizing antibodies. A number of engineered (domain-deleted) variants have been generated that display significantly reduced immunogenicity.

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