A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity--a staphylokinase variant. I. In vitro study

• Published in: Journal of thrombosis and haemostasis 2005-10, Vol.3 (10), p.2156-2165
• Main Authors: Szemraj, J, Walkowiak, B, Kawecka, I, Janiszewska, G, Buczko, W, Bartkowiak, J, Chabielska, E
• Format: Article
• Language: English
• Subjects: Cloning, Molecular; Drug Design; Fibrin - metabolism; Fibrinolysis; Fibrinolytic Agents - metabolism; Fibrinolytic Agents - pharmacology; Hirudins - genetics; Hirudins - pharmacology; Humans; Kinetics; Metalloendopeptidases - genetics; Metalloendopeptidases - metabolism; Metalloendopeptidases - therapeutic use; Oligopeptides - genetics; Oligopeptides - therapeutic use; Platelet Aggregation - drug effects; Protein Binding; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Recombinant Fusion Proteins - therapeutic use; Thrombin - antagonists & inhibitors; Thrombolytic Therapy - methods; Tissue Plasminogen Activator - genetics; Tissue Plasminogen Activator - therapeutic use
• ISSN: 1538-7933
• DOI: 10.1111/j.1538-7836.2005.01480.x

We attempted to construct a new recombinant protein characterized by fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. To the C-terminal part of recombinant staphylokinase (r-SAK), which is a promising profibrinolytic agent, we assembled: (i) the Kringle 2 domain (K2) of tissue-type plasminogen activator (t-PA), containing a fibrin-specific binding site, (ii) the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation and (iii) the antithrombotic agent - hirudin. The cDNA for hybrid protein SAK-RGD-K2-Hir was cloned into pESP-3 yeast protein expression vector. The introduction of K2 t-PA, RGD sequence and hirudin into r-SAK molecule did not alter the SAK activity. The plasminogen activation rate (determined by K(M) and K(cat)) of SAK-RGD-K2-Hir was not significantly different from that of r-SAK. Affinity and binding strength of the recombinant protein to fibrin immobilized on the biosensor were higher than to r-SAK. We observed a higher clot lysis potency of SAK-RGD-K2-Hir as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirudin part of the recombinant fusion protein SAK-RGD-K2-Hir was the same as that of r-Hir alone. In conclusion, the results of the in vitro study suggest that the SAK-RGD-K2-Hir construct can be a more potent and faster-acting thrombolytic agent with antithrombin and antiplatelet properties compared with standard r-SAK.