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ABSTRACT: SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul are recombinant proteins that are derivatives of r-SAK (recombinant staphylokinase). They are characterized by their fibrin-specific plasminogen activation properties and their antithrombin and antiplatelet activities. The difference between these proteins is the presence of the antithrombotic fragment (hirudin or hirulog) in the C-terminal portion of the r-SAK. The aim of the present study was to examine the thrombolytic potentials of SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul in an electrically induced carotid artery thrombosis model in rats and to compare the potentials to that of r-SAK. We determined that a bolus injection of SAK-RGD-K2-Hirul was more effective than one of r-SAK in the improvement and maintenance of carotid patency and in arterial thrombus weight reduction; however, it had the same potency as SAK-RGD-K2-Hir. The bleeding time, prothrombin time and activated partial thromboplastin time were significantly prolonged in the animals that were treated with either dose (1.5 or 3.0 mg/kg) of SAK-RGD-K2-Hir or SAK-RGD-K2-Hirul, whereas no changes were observed in the plasma fibrinogen concentration or the α2 plasmin inhibitor level. r-SAK alone did not change the bleeding time or coagulation parameters. In conclusion, our findings demonstrate the thrombolytic activity of intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hirul in rats. Although this protein compares favorably with r-SAK, we were unable to show the presence of any beneficial effects of SAK-RGD-K2-Hirul over those of SAK-RGD-K2-Hir. Furthermore, our results suggest that high doses of SAK-RGD-K2-Hirul bear the risk of bleeding.
Pharmacological reports: PR 09/2011; 63(5):1169-79. · 2.44 Impact Factor
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ABSTRACT: To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of (125)I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir.
Acta biochimica Polonica 12/2008; 56(1):41-53. · 1.49 Impact Factor
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ABSTRACT: In this study we examined the effects of exogenous nitric oxide (sodium nitroprusside, SNP) and hydrogen peroxide (H2O2) on the expression level of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVEC). The expression of selected genes involved in fibrynolysis under the influence of oxidative stress was analyzed at the levels of mRNA, protein, and promoter activity. The results of the conducted studies revealed that oxidative stress in endothelial cells causes a significant increase in PAI-1 and u-PAR expression and a moderate increase in t-PA and u-PA expression at all of the investigated levels. We attempted to elucidate the molecular signaling mechanisms by which SNP and H2O2 regulate expression of the respective fibrinolytic factors. Therefore, we tested the protein levels of AP-1, NF-kappaB, and HIF-1 and their DNA-binding activity in endothelial cells subjected to oxidative stress. We found strong correlation between AP-1, NF-kappaB, and HIF-1 in the contribution of regulation of selected genes. In addition, we also found that the inhibition of PAI-1 synthesis by antisense oligonucleotide to PAI-1 mRNA results in markedly increased u-PAR expression and that NF-kappaB and AP-1 are involved in this regulation.
Biochemistry and Cell Biology 12/2008; 86(6):477-86. · 2.67 Impact Factor
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ABSTRACT: In this study we examined the effects of exogenous nitric oxide (sodium nitroprusside, SNP) and hydrogen peroxide (H2O2) on the expression level of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVEC). The expression of selected genes involved in fibrynolysis under the influence of oxidative stress was analyzed at the levels of mRNA, protein, and promoter activity. The results of the conducted studies revealed that oxidative stress in endothelial cells causes a significant increase in PAI-1 and u-PAR expression and a moderate increase in t-PA and u-PA expression at all of the investigated levels. We attempted to elucidate the molecular signaling mechanisms by which SNP and H2O2 regulate expression of the respective fibrinolytic factors. Therefore, we tested the protein levels of AP-1, NF-κB, and HIF-1 and their DNA-binding activity in endothelial cells subjected to oxidative stress. We found strong correlation between AP-1, NF-κB, and HIF-1 in the contribution of regulation of selected genes. In addition, we also found that the inhibition of PAI-1 synthesis by antisense oligonucleotide to PAI-1 mRNA results in markedly increased u-PAR expression and that NF-κB and AP-1 are involved in this regulation.Nous avons examiné lors de cette étude les effets de l'oxyde nitrique (nitroprusside de sodium, NPS) et du peroxyde d'hydrogène exogènes sur l'expression de l'activateur de plasminogène de type tissulaire (t-PA), de l'activateur du plasminogène de type urokinase (u-PA), du récepteur de l'activateur de plasminogène de type urokinase (u-PAR) et de l'inhibiteur de l'activateur de plasminogène de type 1 (PAI-1) dans des cellules endothéliales de cordon ombilical humain (« HUVEC »). L'expression des gènes sélectionnés, impliqués dans la fibrinolyse, soumis à l'influence d'un stress oxydatif a été analysée au niveau de l'ARNm, de la protéine et de l'activité de leur promoteur. Les résultats de ces études ont révélé que le stress oxydatif produisait chez les cellules endothéliales une augmentation significative de l'expression de PAI-1 et du u-PAR et une augmentation modérée de l'expression du t-PA et du u-PA à tous les niveaux examinés. Nous avons tenté d'élucider les signaux moléculaires par lesquels le NPS et le H2O2 règlent l'expression de ces facteurs fibrinolytiques respectifs. C'est ainsi que nous avons mesuré les niveaux de AP-1, de NF-κB et de HIF-1 ainsi que leur activité de liaison à l'ADN dans les cellules endothéliales soumises à un stress oxydatif. Nous avons trouvé une forte corrélation entre AP-1, NF-κB et HIF-1 dans la contribution à la régulation des gènes sélectionnés. De plus, nous avons aussi trouvé que l'inhibition de la synthèse de PAI-1 par un oligonucléotide antisens à l'ARNm de PAI-1 résulte en une forte augmentation de l'expression du u-PAR, et que NF-κB et AP-1 sont impliqués dans cette régulation.
Biochemistry and Cell Biology 11/2008; 86(6):477-486. · 2.67 Impact Factor
