Complex Formation Between Prothrombin And Staphylocoagulase

Staphylocoagulase is a protein secreted by certain strains of Staphylococcus aureus. Addition of the extracellular protein to a human prothrombin preparation results in the generation of a thrombin-like activity.

Our investigations on the prothrombin-staphylocoagulase interaction was undertaken in order to establish the kinetics of the complex formation, the mechanism of activation of prothrombin by staphylocoagulase and the enzymatic properties of the complex.

Evidence for the formation of a equimolar complex between prothrombin and staphylocoagulase was obtained fromgelelec- trophoresis and titration experiments. The procedure,in order to study the kinetics of the complex formation, made use of a coupled assay in which D-Phe-Pip-L-Arg-p-nitroanilide hydrolysis by the complex was monitored. The second rate constant of the complex formation, as determined from pseudofirst order kinetics, is 4×l0⁶sec^-1M^-1. The dissociationconstant of the complex was determined to be orders of magnitude less than 10^-11M. Using ³H-DFP, no exchange was observed between prothrombin in the complex with free prothrombin. Analysis of the complex by gelelectrophoresisin the presence of Na-dodecyl sulfate and mercaptoethanol shows that proteolysis cannot account for the generation of thrombin-like activity. Small amounts of degradationproducts (prethrombin 1) were found.

Thrombin-like activity was generated as a result of the interactions between staphylocoagulase and human prethrombin 1, human prethrombin 2 and bovine prethrombin 2. Indications for an interaction between staphylocoagulase and thrombin were obtained from experiments with anti-thrombin III and hirudin. Identical enzymatic properties of thrombin and the prothrombin-staphylocoagulase complex were observed towards small synthetic substrates and fibrinogen. No activity of the complex towards factor V, factor VIII and platelets was found.