A novel hirudin derivative characterized with anti-platelet aggregations and thrombin inhibition

Abstract

Background Hirudin is an anti-coagulative product of the salivary glands of the medicinal leech Hirudo medicinalis. It is a powerful and specific thrombin inhibitor. Peptides containing the RGD motif competitively inhibit the binding of fibrinogen to GP IIb/IIIa on the platelets, thus inhibiting platelet aggregation. Results We have constructed a recombinant RGD-hirudin (r-RGDhirudin) by fusing the tripeptide RGD sequence to the native hirudin (wt-hirudin). The r-RGD-hirudin was expressed at high levels in Pichia pastoris, and was purified to *97% homogeneity. The specific anti-thrombin activity of purified r-RGD-hirudin is 12,000 ATU/mg, which is equivalent to wt-hirudin, but only r-RGD-hirudin can inhibit platelet aggregation. The biological effects of r-RGD-hirudin on Thrombin Time (TT), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), Bleeding Time (BT), maximum platelet aggregation (PAGm) induced by ADP were studied in rabbit model and compared with that of wt-hirudin. The rabbits were infused r-RGD-hirudin had prolonged TT, PT, and aPTT which were similar to that of wt-hirudin; but only r-RGD-hirudin was capable of inhibiting PAGm. Histopathological analyses showed that r-RGD-hirudin was two to three times more effective than wt-hirudin in preventing thrombosis. Conclusions r-RGD-hirudin can potentially be used as a novel anti-coagulant for the prevention of thrombosis after carotid artery anastomosis or in other thrombotic events.

Expression, Purification, and Mass Spectrometric Analysis of 15N, 13C-Labeled RGD-Hirudin, Expressed in Pichia pastoris, for NMR Studies

“…The abilities of this protein to inhibit thrombin and the aggregation of platelets were confirmed in our previous study [8]. More recent studies have focused on the structure-function relationship of RGD-hirudin.…”

Biotechnological production of recombinant analogues of hirudin-1 from Hirudo medicinalis

“…Structural analy sis of the hirudin 1 complex with thrombin revealed amino acid residues responsible for inhibitor affinity to the enzyme [10][11][12]. Numerous modified ana logues with increased antithrombotic activity were created on the basis of these works [13][14][15][16][17][18]. Medical preparations that are already registered include recombinant hirudin 1 (desirudin, Iprivask, Canyon Pharmaceuticals) [19][20][21] and lepirudin (Refludan, Bayer Schering Pharma, Sanofi Aventis, etc.)…”

Structural basis of RGD-hirudin binding to thrombin: Tyr3 and five C-terminal residues are crucial for inhibiting thrombin activity

“…It may be a more effective agent than wild-type hirudin for anti-coagulation and anti-thrombosis in post-anastomosis surgery. We propose that RGD-hirudin will be able to replace heparin or wild-type hirudin and reduce bleeding rates, due to its bi-functional action at a much lower dosage than wild-type hirudin [ 15 ]. However, RGD-hirudin needed to be modified to reduce the molecular weight to be suitable for advanced oral administration, rather than intravenous injection.…”

“…Thus, consumption of 1 NIH unit of thrombin is equivalent to 1 unit of anti-thrombin activity. A classic turbidity assay was used to measure the anti-platelet aggregation activity of RGD-hirudin and the mutant variants [ 15 ]. Aggregation of rabit platelets in response to antagonists was analyzed using lumi-aggregometer (Model 400VS, Chrono-Log, Haverston, PA, USA).…”

“…Asp 53 was mutated to Gln 53 , Glu 58 was mutated to Pro 58 , and Glu 66 was added. These changes improve the hydrophobicity of the protein and allow the recombinant RGD-hirudin to interact more effectively with the fibrinogen recognition exosite of thrombin, resulting in a specific activity of 12,000 ATU/mg [ 15 ]. Given these changes, we hypothesized that the interaction between RGD-hirudin and thrombin would be similar to that between wild-type hirudin and thrombin.…”