Interactions between staphylokinase, plasmin(ogen), and fibrin. Staphylokinase discriminates between free plasminogen and plasminogen bound to partially degraded fibrin

Interactions between staphylokinase, plasmin(ogen), and fibrin. Staphylokinase discriminates between free plasminogen and plasminogen bound to partially degraded fibrin

Gaubius Instituut TNO
Sakharov, D.V.
Lijnen, H.R.
Rijken, D.C.

1996

Staphylokinase (STA), a protein of bacterial origin, induces highly fibrin-specific thrombolysis both in human plasma in vitro and in pilot clinical trials. Using fluorescence microscopy, we investigated the spatial distribution of fluorescein isothiocyanate (FITC)-labeled STA during lysis of a plasma clot and its binding to purified fibrin clots in the presence or in the absence of plasmin(ogen). STA highly accumulated in a thin superficial layer of the lysing plasma clot following the distribution of plasminogen (Pg) during lysis. Experiments with purified fibrin clots revealed that STA binds to Pg bound to partially degraded fibrin but not to Pg bound to intact fibrin. Binding of FITC-labeled STA to various forms of plasmin(ogen) in a buffer solution was studied by measuring fluorescence anisotropy. The binding constant for Glu-Pg was estimated as 7.4 uM and for Lys-Pg as 0.28 uM; for active-site blocked plasmin the binding constant was less than 0.05 uM. The much lower affinity of STA for Glu-Pg compared with that for active site- blocked plasmin was mainly due to a lower association rate constant, as assessed by real time biospecific interaction analysis. Gel filtration of a mixture of STA with a molar excess of Glu-Pg demonstrated that STA migrated as an unbound 18-kDa protein when activation of Pg into plasmin was precluded by inhibitors of plasmin. When gel-filtered under the same conditions with plasmin, STA migrated in complex with plasmin with an apparent molecular mass of 100 kDa. Confocal fluorescence microscopy finally demonstrated that when FITC-labeled STA was added to plasma before clotting, it did not bind to fibrin fibers during the first minutes (lag phase), although Pg bound to the fibers moderately. Then, both Pg and STA started to accumulate on the fibers progressively, followed by complete lysis of the clot. In conclusion, our results imply that, when STA is added to plasma, only a small percentage associates with Pg. In contrast, STA binds strongly to plasmin and to Pg, which is bound to partially degraded fibrin. These findings add a new mechanism to the known explanations for the inefficient Pg activation by STA in plasma and specify the mechanism for fibrin-dependent activation of Pg. Chemicals/CAS: auR protein, Staphylococcus aureus, EC 3.4.24.29; Fibrin, 9001-31-4; Fibrinolytic Agents; Fluorescein-5-isothiocyanate, 3326-32-7; Metalloendopeptidases, EC 3.4.24.-; Plasmin, EC 3.4.21.7; Plasminogen, 9001-91-6; Recombinant Proteins

Fluorescein isothiocyanate
Staphylokinase
Blood clot lysis
Fluorescence microscopy
Molecular weight
Plasma
Protein protein interaction
Chromatography, Gel
Fibrin
Fibrinolysis
Fibrinolytic Agents
Fluorescein-5-isothiocyanate
Fluorescence Polarization
Humans
Kinetics
Metalloendopeptidases
Microscopy, Confocal
Plasmin
Plasminogen
Protein Binding
Recombinant Proteins
Substrate Specificity
Bacteria (microorganisms)

http://resolver.tudelft.nl/uuid:90827c7f-dcb2-4d0c-bc15-6ae4f6156f73

https://doi.org/10.1074/jbc.271.44.27912

233554

0021-9258

Journal of Biological Chemistry, 271 (271), 27912-27918

article