title: Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106-7284, USA.
authors.forename: M T
authors.initials: MT
authors.lastname: Nieman
authors.forename: F
authors.initials: F
authors.lastname: Burke
authors.forename: M
authors.initials: M
authors.lastname: Warnock
authors.forename: Y
authors.initials: Y
authors.lastname: Zhou
authors.forename: J
authors.initials: J
authors.lastname: Sweigart
authors.forename: A
authors.initials: A
authors.lastname: Chen
authors.forename: D
authors.initials: D
authors.lastname: Ricketts
authors.forename: B R
authors.initials: BR
authors.lastname: Lucchesi
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.forename: E
authors.initials: E
authors.lastname: Di Cera
authors.forename: J
authors.initials: J
authors.lastname: Hilfinger
authors.forename: J S
authors.initials: JS
authors.lastname: Kim
authors.forename: H I
authors.initials: HI
authors.lastname: Mosberg
authors.forename: A H
authors.initials: AH
authors.lastname: Schmaier
mesh_terms: D000818:Animals; D001665:Binding Sites; D001920:Bradykinin / Q000008:administration & dosage / Q000737:chemistry* / Q000494:pharmacology*; D002341:Carotid Artery Thrombosis / Q000188:drug therapy; D018360:Crystallography, X-Ray; D051379:Mice; D015394:Molecular Structure; D010446:Peptide Fragments / Q000008:administration & dosage / Q000737:chemistry* / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013918:Thrombin Time
chemical_list: D010446:Peptide Fragments; C079000:bradykinin (1-5); D013917:Thrombin; D001920:Bradykinin