CHEMICAL SYNTHESIS AND STRUCTURAL CHARACTERIZATION OF THE RGD-PROTEINDECORSIN - A POTENT INHIBITOR OF PLATELET-AGGREGATION

Decorsin is a 39-residue RGD-protein crosslinked by three disulfide br idges isolated from the leech Macrobdella decora belonging to the fami ly of GPIIb-IIIa antagonists and acting as a potent inhibitor of plate let aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse -phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synth etic protein, NH2-terminal sequencing and mass determination (4,377 Da ) by electrospray mass spectrometry were in full agreement with this t heory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mappin g using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited h uman platelet aggregation with an IC50, of similar to 0.1 mu M, a figu re quite similar to that determined utilizing decorsin from natural so urce. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platele t aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (T- m similar to 74 degrees C). The features of the oxidative refolding pr ocess of reduced decorsin, as well as the thermal stability of the oxi dized species. were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This frag ment shows similarity in size, pairing of the three disulfides and thr ee-dimensional structure with those of decorsin, even if very low sequ ence similarity. It is suggested that the less efficient oxidative fol ding and the enhanced thermal stability of decorsin in respect to thos e of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in th e hirudin domain. The results of this study indicate that decorsin can be obtained by solid-phase methodology in purity and quantities suita ble for structural and functional studies and thus open the way to pre pare by chemical methods novel decorsin derivatives containing unusual amino acids or even non-peptidic moieties.