Chimeric antithrombin peptide. Characterization of an Arg-Gly-Asp (RGD)- and hirudin carboxyl terminus-containing synthetic peptides

We investigated the properties of an artificial chimeric peptide that contains an Arg-Gly-Asp (RGD)-tripeptide, the versatile cell recognition signal of extracellular matrix protein components, coupled to a carboxyl-terminal fragment of the highly specific alpha-thrombin inhibitor, hirudin (residues 53-64): WGRGDSANGDFEEIPEEYL (RGD-hirudin53-64). Hirudin53-64 and RGD-hirudin53-64 inhibited the fibrinogen clotting activity of alpha-thrombin and prolonged the activated partial thromboplastin time of human plasma. In addition, both peptides afforded total protection to thrombin from trypsionolysis. Neither hirudin53-64 nor RGD-hirudin53-64 dramatically interfered with the thrombin-antithrombin inhibition reaction either in the absence or presence of added heparin. alpha-Thrombin-induced platelet aggregation was effectively inhibited by hirudin53-64 and RGD-hirudin53-64. Unlike hirudin53-64, RGD-hirudin53-64 in solution inhibited integrin-mediated endothelial cell and fibroblast cell attachment to polystyrene wells in the presence of fetal bovine serum. Collectively, our results demonstrate that RGD-hirudin53-64 has anticoagulant/antiplatelet aggregation activity attributable to its hirudin sequence and integrin-directed cell attachment activity due to its RGD site. Our results suggest that this chimeric motif may serve as a prototype for a new class of anticoagulants where an integrin-specific sequence "targets" the peptide to a cell (ultimately through the platelet integrin alpha IIb beta 3) trapped amid a thrombus with ensuing proteinase inhibition.