A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity--a staphylokinase variant. I. In vitro study.
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.
Szemraj
J
J
Department of Medical Biochemistry, Medical University of Lódz, Lódz, Poland. jszemraj@csk.am.lodz.pl
Walkowiak
B
B
Kawecka
I
I
Janiszewska
G
G
Buczko
W
W
Bartkowiak
J
J
Chabielska
E
E
D003001:Cloning, Molecular; D015195:Drug Design; D005337:Fibrin / Q000378:metabolism*; D005342:Fibrinolysis; D005343:Fibrinolytic Agents / Q000378:metabolism / Q000494:pharmacology*; D006629:Hirudins / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D009842:Oligopeptides / Q000235:genetics / Q000627:therapeutic use; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use*; D013917:Thrombin / Q000037:antagonists & inhibitors; D015912:Thrombolytic Therapy / Q000379:methods*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000627:therapeutic use
D005343:Fibrinolytic Agents; D006629:Hirudins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C047981:arginyl-glycyl-aspartic acid; D005337:Fibrin; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus