Insertion of the Asp-Ser/Phe sequence in the P' position of hirutonin provides molecules having both antithrombin and...

We have developed novel synthetic peptides that display both antithrombin and disintegrin activity. These peptides were derived from hirutonins, a class of potent proteolytically resistant thrombin inhibitors, in which a dipeptidyl sequence, Asp-Phe or Asp-Ser, was introduced after the proteolytically resistant ketomethylene arginyl glycine isostere. These modified hirutonins inhibited the amidolytic activity of alpha-thrombin (Ki approximately 35 nM), prevented fibrinogen clotting (dTT approximately 100 nM) and inhibited human platelet aggregation and 5-hydroxytryptamine secretion induced by alpha-thrombin (IC50 approximately 600 nM). Unlike their parent hirutonins, they inhibited SFLLR-NH2-induced human platelet aggregation (IC50 approximately 45 microM) without inhibition of 5-HT secretion. These peptides also competed for fibrinogen binding to purified GpIIbIIIa integrin (IC50 approximately microM) and prevented attachment of B16-F10 mouse melanoma cells to vitronectin. We conclude that addition of the dipeptidyl sequence, Asp-Phe or Asp-Ser, in hirutonin molecules confers disintegrin activity. However, this activity was not superior to the activity observed with the linear RGDS peptide and was achieved at the expense of direct antithrombin activity. Additional modifications around the RGD-like adhesion sequence may permit identification of the appropriate conformation for optimal binding to thrombin and to specific integrin receptors.