The Core Domain of Hirudin from the Leech Hirudinaria manillensis

Abstract

Hirudin is a small (∼ 7 kDa) disulfide-crosslinked polypeptide known as the most potent and specific inhibitor of thrombin, a serine protease that plays a key role in the coagulation cascade and pathology of thrombotic diseases (Tapparelli et al., 1993). We have previously shown that the N-terminal proteolytic fragment 1–47 of hirudin HM2 from Hirudinaria manillensis (Fig. 1) maintains inhibitory action towards thrombin (Vindigni et al., 1994). An analog of fragment 1–47 with a Tyr³ →Trp exchange (Y3W) was prepared by solid-phase chemical synthesis and shown to be ~5-fold more active than the Tyr³ natural fragment 1–47 in inhibiting thrombin (De Filippis et al., 1995). In this study, we report the results of experiments of chemical modification and peptide bond cleavage at the level of Trp³ of the synthetic Y3W peptide 1–47 with the aim to prepare modified and truncated species of the Y3 W peptide analog. Modification reactions were carried out also on fragment 1–41, prepared by V8-protease digestion of the synthetic Y3W peptide 1–47 at Glu⁴¹. All peptide derivatives prepared in the course of this work were isolated to homogeneity and assayed for biological activity. The results of this study revealed the critical role of the N-terminal portion of peptide 1–47 in the inhibition of thrombin, providing experimental support for the proposed mechanism of interaction between hirudin and thrombin advanced in previous studies.