Recombinant RGD-Hirudin, A Novel Anticoagulant

Hirudin is an anticoagulative product from salivary glands of medicinal leech. It is a powerful and specific thrombin inhibitor. Unlike heparin, which has to bind to anti-thrombin III to exert its actions, hurudin binds directly to thrombin, inhibits fibrin-bound as well as fluid-phase thrombin. It also inhibits fibrinogen clotting and thrombin-catalysed haemostatic reactions.The naturally occurring hirudin (wt-Hirudin) is a single-chain, carbohydrate-free polypeptide containing 65-66 amino acids with a molecular weight of 7,000Da. Wt-Hirudin has a trisulfide-linked core structure, forming a compact NH₂-terminal domain and a long COOH-terminal extended domain. The crystallographic structure of hirudin-thrombin complex showed abundant interactions of these two molecules, both polar and apolar, which may account for the tight affinity and specificity of hirudin.Adhesion of platelets to vessel walls, their activation, and aggregation is a central primary event in blood coagulation. On activated platelets, the glycoprotein IIb/IIIa (GP IIb/IIIa) functions as a receptor for fibrinogen to mediate platelet aggregation and crosslinking. The capability of GP IIb/IIIa to bind to fibrinogen and other adhesive proteins is due to its ability to recognize Arg-Gly-Asp (RGD) motif within their sequences. Peptides containing the RGD sequence or RGD alone compete and inhibit the binding of fibrinogen to GP IIb/IIIa on platelets, thus inhibiting platelet aggregation. Adelman B. proved that peptide with RGD remarkably prevented platelet aggregation induced by ADP.In the present study, we engineered a novel bi-functional hirudin molecule, r-RGD-Hirudin, by inserting a RGD tripeptide to a given domain of the wt-Hirudin molecular. In theory, r-RGD-Hirudin has been able to inhibit both thrombin and platelet aggregation. It is anticipated that r-RGD-Hirudin replaces heparin or wt-Hirudin with higher efficacy and safety because of its bi-functional action and its low effective dosage.Section 1: Design of the novel Bi-Functional-Hirudin molecularBased on the research of structure and function of wt-Hirudin, a novel Bi-Functional-Hirudin was designed.We fused RGD tripeptide in a proper location of loop domain within wt-Hirudin molecular, and made the fusion protein by recombinant DNA technique and proteinengineering. The coding sequence of RGD was inserted into Hirudin cDNA. The expression plasmid r-RGD-Hirudin-pPIC9K was constructed. By means of electroporation, the recombinant DNA was transferred into the Pichia pastoris strain GS115 (his-4), His⁺ transformants were recovered on MD plate, then plated on YPD plate containing increasing concentrations of the aminoglycoside antibiotic G418 for screening multiple plasmid integration. The resulting His⁺ G₄₁₈-resistant transformants were ensured the integration of r-RGD-Hirudin cDNA into yeast chromosome DNA by PCR.Methanol utilization plus (Mut⁺) transformants were grown in shake flasks and screened for those that secreted r-RGD-Hirudin to the growth medium. After 2 days methanol induction, the expression product was up to 0.1 g/L supernatant. Fibrinogen solidification assay showed the specific activity of anti-thrombin was more than 10,000ATU/mg, and it was similar with wt-Hirudin. Nephelometery was used to determine its anti-platelet aggregation activity. Inhibitory rate was 10-20% with final concentration 1.5μg/mL of r-RGD-Hirudin.The experiment of competitive inhibition showed r-RGD-Hirudin was capable of inhibiting fibrinogen bind to GP IIb/IIIa of platelet, and LC50 was 0.4μmol/L. Flow Cytometry was used to study the K_m of r-RGD-Hirudin bind to GP IIb/IIIa. Compared with Clopidogrel Hydrogen Sulfate, a specific GP IIb/IIIa inhibitor, the LC50 which r-RGD-Hirudin inhibited GP IIb/IIIa was 0.3μmol/L.The conformation of r-RGD-Hirudin was studied by NMR which showed the structure of r-r-RGD-Hirudin was consistent with wt-Hirudin.Section 2: Pilot Research of r-RGD-HirudinThe clone with high expression level was picked up and fermented for 3 days in 30-liter fermenter. The optimal parameters of fermentation, DO, pH, the concentration of methanol et al. were studied. The culture was centrifuged and the supernatant was treated by ultra-filtration, gel filtration and anion exchange chromatography. Finally, we got purified r-RGD-Hirudin with 97% of purity and 12,000 ATU/mg of specific activity. The output was 1.0 g purified r-RGD-Hirudin from a liter culture. The purity of r-RGD-Hirudin was identified by reductive SDS-PAGE, HPLC, LC/MS, isoelectrofocusing assay, Western-Blot and UV absorptivity assay etc. And anti-thrombin activity assay and anti-platelet aggregation assay showed its double function.The r-RGD-Hirudin was added Mannitol, then freeze-dried and formed a preparation for injection.HPLC showed r-RGD-Hirudin included two peaks, and it was very hard to separated them by gel filtration or ion exchange chromatography. LC/MS was used to separate them. The molecular weight (MW) of the first peak was 7,030Da, the other is 7,180Da. These profiles mean that the material with MW 7,030Da should be product of r-RGD-Hirudin hydrolyzed by yeast carboxypeptidase and lost the COOH-terminal glutamic acid, since the MW of glutamic acid is 150Da.We separated two peaks by HPLC, and determined the anti-thrombin and anti-platelet aggregation specific activity. Their anti-thrombin specific activities were 12,030ATU/mg and 11,960ATU/mg. The inhibitive ratios of platelet aggregation with final concentration 2.5×10^-3mg/mL were 21.79% and 20.51%. The result proved both of them were r-RGD-Hirudin.Section 3: Pharmacodynamics and Safety Evaluation of r-RGD-HirudinThe r-RGD-Hirudin was used as anticoagulant for the prevention of thrombosis after femoral anastomosis. Rats and rabbits were used as experiment animal. Blood samples were collected before and after surgery in 1, 24 and 72 hours. Thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (aPTT), plasm concentration of fibrinogen (Fg) and the maximum platelet aggregation (PAGm) induced by ADP were determined. After three days of surgery, the vessels at the anastomosis area were separated and prepared slices. Haematological assay showed that TT, PT and aPTT were prolonged after infusion of r-RGD-Hirudin, wt-Hirudin or Heparin; only r-RGD-Hirudin was capable of inhibiting PAGm. Histopathological results showed that the dosage of r-RGD-Hirudin for prevention of thrombosis was half to one third of the doses of wt-Hirudin. Furthermore, no bleeding complication was observed in r-RGD-Hirudin treatment rabbits, in contrast to those treated with wt-hirudin.Classical Immunological research showed that guinea pigs were injected 3 times of clinical dosage of r-RGD-Hirudin and attacked after 2 or 3 weeks by 6 times of clinical dosage, anaphylaxis were not found.Section 4: Special Formulation of r-RGD-HirudinRapid Expansion of Supercritical Solution was used to prepared oral delivery of r-RGD-Hirudin. This special preparation was absorbed in colon, since the absorbed peak appeared at the 6^th-8^thhr after oral administration. At the peak stage, the plasma concentration of r-RGD-Hirudin was increased and aPTT was prolonged double.The conclusion: r-RGD-Hirudin has been potentially used as antithrombosis and anticoagulant agent for the prevention of thrombosis after artery anastomosis or other thrombotic events.