Journal of Thrombosis and Haemostasis 2008-05-01

Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo.

M T Nieman, F Burke, M Warnock, Y Zhou, J Sweigart, A Chen, D Ricketts, B R Lucchesi, Z Chen, E Di Cera, J Hilfinger, J S Kim, H I Mosberg, A H Schmaier

Index: J. Thromb. Haemost. 6(5) , 837-45, (2008)

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Abstract

Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin - RPPGF.These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at >or=0.78, 1.6, and 1.6 microm, respectively. They competitively inhibit alpha-thrombin-induced cleavage of a chromogenic substrate at 4.4-8.2 microm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p-Me)], blocks alpha-thrombin-induced calcium flux in fibroblasts with an IC(50) of 6.9 +/- 1.2 microm. FM19 achieved 100% inhibition of threshold alpha- or gamma-thrombin-induced platelet aggregation at 8.4 +/- 4.7 microm and 16 +/- 4 microm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p-methyl Phe engages thrombin's aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis.FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.