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

We have developed novel synthetic peptides that display both antithrombin a nd disintegrin activity. These peptides were derived from hirutonins, a cla ss of potent proteolytically resistant thrombin inhibitors, in which a dipe ptidyl sequence, Asp-Phe or Asp-Ser, was introduced after the proteolytical ly resistant ketomethylene arginyl glycine isostere. These modified hiruton ins inhibited the amidolytic activity of a-thrombin (K(i)approximate to 35 nM), prevented fibrinogen clotting (dTT approximate to 100 nM) and inhibite d human platelet aggregation and 5-hydroxytryptamine secretion induced by c u-thrombin (ICS(50)approximate to 600 nM). Unlike their parent hirutonins, they inhibited SFLLR-NH2-induced human platelet aggregation (IC(50)approxim ate to 45 mu M) without inhibition of 5-HT secretion. These peptides also c ompeted for fibrinogen binding to purified GpIIbIIIa integrin (IC(50)approx imate to 10 mu M) and prevented attachment of B16-F10 mouse melanoma cells to vitronectin. We conclude that addition of the dipeptidyl sequence, Asp-P he or Asp-Ser, in hirutonin molecules confers disintegrin activity. However , this activity was not superior to the activity observed with the linear R GDS 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 thro mbin and to specific integrin receptors, (C) 1999 Elsevier Science Ltd. All rights reserved.