STAPHYLOKINASE REQUIRES NH2-TERMINAL PROTEOLYSIS FOR PLASMINOGEN ACTIVATION

Staphylokinase (Sak), a single-chain protein comprising 136 amino acid s with NH2-terminal sequence, [GRAPHICS] forms a complex with plasmin, that is endowed with plasminogen activating properties, Plasmin is pr esumed to process mature (high molecular weight, HMW) Sak to low molec ular weight derivatives (LMW-Sak), primarily by hydrolyzing the Lys(10 )-Lys(11) peptide bond, but the kinetics of plasminogen activation by HMW-Sak and LMW-Sak are very similar. Here, the requirement of NH2-ter minal proteolysis of Sak for the induction of plasminogen activating p otential was studied by mutagenesis of Lys(10) and Lys(11) in combinat ion with NH2-terminal microsequence analysis of equimolar mixtures of Sak and plasminogen and determination of kinetic parameters of plasmin ogen activation by catalytic amounts of Sak, Substitution of Lys(10) w ith Arg did not affect processing of the Arg(10)-Lys(11) site nor plas minogen activation, whereas substitution with His resulted in cleavage of the Lys(11)-Gly(12) peptide bond and abolished plasminogen activat ion. Substitution of Lys(11) with Arg did not affect Lys(10)-Arg(11) p rocessing or plasminogen activation, whereas replacement with His did not prevent Lys(10)-His(11) hydrolysis but abolished plasminogen activ ation, Substitution of Lys(11) with Cys yielded ale inactive processed derivative which was fully activated by aminoethylation. Deletion of the 10 NH2-terminal amino acids did not affect plasminogen activation, but additional deletion of Lys(11) eliminated plasminogen activation. Thus generation of plasminogen activator potential in Sak proceeds vi a plasmin-mediated removal of the 10 NH2-terminal amino acids with exp osure of Lys(11) as the new NH2 terminus, This provides a structural b asis for the hypothesis, derived from kinetic measurements, that plasm inogen activation by Sak needs to be primed by plasmin and a mechanism for the high fibrin selectivity of Sak in a plasma milieu.