HIRUDIN

Refludan was launched in Germany for heparin-associated thrombocytopenia. Originally isolated from the saliva of leeches (Hirudo medicinalis), the gene for recombinant hirudin was synthesized and expressed in Saccharomyces cerevisiae. The isolated protein is 65 amino acids differing from the native protein by the first two amino acids (Leu1 and Thr2) and Tyr63 is not sulfated, while it does retain the three disulfide bonds. Refludan is a potent, specific and almost irreversible inhibitor of thrombin with which it forms a 1:1 complex. The N-terminal domain binds to the active catalytic site of thrombin, while the C-terminal end binds to the anion binding exosite (fibrinogen binding cleft). It has several advantages over heparin: it can inhibit thrombin bound to extracellular matrices, does not require antithrombin Ⅲ as a cofactor, is not inhibited by activated platelet nor increases platelet activity, and is a pure, single compound with a single mechanism of action. It is useful for myocardial infarcts, unstable angia and cardiovascular events. Heparin treatment can be inhibited by platelets activated by the Fc fragment of antibodies developed to the antigen of heparin-platelet factor 4. A corresponding inactivation of Refuldan is not known.

Recombinant hirudin from yeast has been used in mannan-binding lectin serine protease 2 (MASP2) activation assay. It has also been used in pre-treating the nanosheets in phosphate buffered saline (PBS) for platelet adhesion assay.

Hirudin is a 7 kDa acidic protein containing 65 amino acid residues that has an isoelectric point of 3.5-4.0. It is not glycosylated and lacks tryptophan, arginine and methionine residues. It is chemically stable between pH 2-12 and at temperatures up to 80°C.