bivalirudin and arginyl-glycyl-aspartic-acid

The RGD sequence was used to design potent hirudin isoform 3 mimetic peptides with both antithrombin activity and antiplatelet aggregation activity. The RGD and proline were inserted between the catalytic active binding domain (D-Phe-Pro-Arg-Pro) on the N-terminus and the anion-binding exosite binding domain (QGDFEPIPEDAYDE) on the Cterminus. Thrombin titration assay and ATP-induced platelet aggregation test revealed that the peptide with the linker RGDWP or RGDGP possessed potent antithrombin and antiplatelet activities, while other peptides without the Pro residue in the linker only showed antithrombin activity. Similar results were obtained in the RGD-containing hirulog-1 variants. Our study indicates that the inserted Pro residue facilitates the exposure of RGD and the binding of the peptide to glycoprotein IIb/IIIa (GPIIb/IIIa). The strategy of combining the RGD sequence and the Pro residue may be used for future designs of bifunctional antithrombotic agents.

Topics: Animals; Antithrombin Proteins; Antithrombins; Catalytic Domain; Hirudins; Integrin beta3; Male; Oligopeptides; Peptide Fragments; Platelet Aggregation Inhibitors; Platelet Membrane Glycoprotein IIb; Protein Isoforms; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Whole Blood Coagulation Time

Reocclusion is one of the major root causes for secondary complications that arise during thrombolytic therapy. A multifunctional staphylokinase variant SRH (staphylokinase (SAK) linked with tripeptide RGD and didecapeptide Hirulog) with antiplatelet and antithrombin activities in addition to clot specific thrombolytic function, was developed to address the reocclusion problem. We preferred to use Escherichia coli GJ1158 as the host in this study for economic production of SRH by osmotic (0.3 mol/L sodium chloride) induction, to overcome the problems associated with the yeast expression system. The therapeutic potential of SRH was evaluated in the murine model of vascular thrombosis. The SAK protein (1 mg/kg body mass) and SRH protein (1 mg/kg and 2 mg/kg) were administered intravenously to the different treatment groups. The results have shown a dose-dependent antithrombotic effect in carrageenan-induced mouse tail thrombosis. The thrombin time, activated partial thromboplastin time, and prothrombin time were significantly prolonged (p < 0.05) in the SRH-infused groups. Moreover, SRH inhibited platelet aggregation in a dose-dependent manner (p < 0.05), while the bleeding time was significantly (p < 0.05) prolonged. All of these results inferred that the osmotically produced multifunctional fusion protein SRH (SAK-RGD-Hirulog) is a promising thrombolytic agent, and one which sustained its multifunctionality in the animal models.

Topics: Animals; Antithrombins; Blood Coagulation; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Escherichia coli; Hirudins; Male; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Oligopeptides; Partial Thromboplastin Time; Peptide Fragments; Platelet Aggregation; Platelet Aggregation Inhibitors; Prothrombin Time; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Recombinant Proteins; Sodium Chloride; Thrombin Time; Thrombosis; Time Factors