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IN FOCUS: A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant. I. In vitro study
Author(s) -
SZEMRAJ J.,
WALKOWIAK B.,
KAWECKA I.,
JANISZEWSKA G.,
BUCZKO W.,
BARTKOWIAK J.,
CHABIELSKA E.
Publication year - 2005
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2005.01480.x
Subject(s) - staphylokinase , recombinant dna , hirudin , fibrin , chemistry , fibrinolytic agent , antithrombin , biochemistry , in vitro , microbiology and biotechnology , thrombin , pharmacology , tissue plasminogen activator , biology , heparin , platelet , immunology , gene
Summary. 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 125 I‐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.