Three-dimensional structure of Staphylokinase (Sakstar variant). (From Berman, H., et al., The protein data bank [PDB], Nucleic Acids Res., 28, 235-242, 2000. PDB ID: 2SAK. With permission.)
other plasminogen molecules. Staphylokinase and streptokinase display very little homology despite their similar plasminogen activation mechanism . When staphylokinase is added to human plasma containing fibrin clot, it binds to plasminogen, creating the plas-minogen-staphylokinase complex. This complex reacts poorly with free plasminogen. However, the staphylokinase-plasminogen complex reacts with traces of plasmin at the clot surface with high affinity, converting it into the plasmin-staphylokinase complex. This complex activates free plasminogen at the clot surface, forming plasmin (process outlined in Figure 13.3). Free plasmin is inhibited from cleaving the plasminogen-staphy-lokinase complex in plasma by the inhibitor a2-antiplasmin. The plasmin-staphylokinase complex and plasmin are protected from inhibition by a2-antiplasmin while they are bound to fibrin. However, once they are liberated from the clot surface or generated in plasma, they are rapidly inhibited. Inhibition by a2-antiplasmin is mediated by interaction between the plasmin moiety of the plasmin-staphylokinase complex. This interaction results in the plasmin dissociating from staphylokinase and forming an inactive plas-min-a2-antiplasmin complex [1,64]. Owing to this mechanism, the process of plasminogen activation is confined to the thrombus, preventing excessive plasmin generation and fibrinogen degradation in plasma .
R719 of plasminogen plays a pivotal role in the formation of the plasminogen-staphylo-kinase complex. The amino acid in position 26 appears to be of crucial importance for the activation of plasminogen by staphylokinase. The formation of the highly active plasmin-staphylokinase complex requires the conversion of plasminogen to plasmin and hydrolysis of the K10-K11 peptide bond of staphylokinase by plasmin. The N-terminal amino acids of staphylokinase play a role in conferring plasminogen activation ability on plasmin when bound to staphylokinase. Activation of plasminogen by the preformed plasmin-staphylo-kinase complex obeys Michaelis-Menten kinetics with a Km of 7.0 pmol/L [64-67].
Natural strains of Staphylococcus aureus produce very low levels of staphylokinase. As a result, many efforts have been made to clone and overexpress the gene . The staphylokinase (sak) gene has been cloned from the serotype B bacteriophage sak0C, from the serotype F bacteriophage sak42D, and from the genomic DNA (sakSTAR) of a staphyloki-nase-secreting Staphylococcus aureus strain . These three natural staphylokinase gene variants differ at amino acid positions 34, 36, and 43 but have very similar fibrin-dissolving potency . The gene has been cloned into E. coli, Bacillus sp., and various other recombinant systems. The protein has been produced at high levels (10 to 15% of total cellular protein) intracellularly in E. coli TGI cells and has been purified from cytosol fractions by a combination of ion-exchange and hydrophobic-interaction chromatography, with yields of 50 to 70% [1,68]. Natural staphylokinase has been purified by precipitation with ammonium sulfate followed by cation-exchange chromatography on CM-cellulose. Affinity chromatography using a plasmin-Sepharose and a plasminogen-Sepharose ligand has also been successful in obtaining pure staphylokinase .
Mechanism of staphylokinase clot specific generation of plasmin. Plg, plasminogen; Sak, staphylokinase; Pli, plasmin; Pli.Sak, plasmin-staphylokinase complex; Oj-AP, Oj-antiplasmin; PAP, inactive plasmin-Oj-antiplasmin complex. (From Collen, D. and Lijnen, H., Blood, 84, 680-686, 1994. With permission)
Plg + Sak * » Plg.Sak-► Pli.Sak " ► PAP + Sak fibrin fibrin fibrin a2-AP a2-AP Plg-► Pli
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