Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo.
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.
Nieman
M T
MT
Division of Hematology and Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106-7284, USA.
Burke
F
F
Warnock
M
M
Zhou
Y
Y
Sweigart
J
J
Chen
A
A
Ricketts
D
D
Lucchesi
B R
BR
Chen
Z
Z
Di Cera
E
E
Hilfinger
J
J
Kim
J S
JS
Mosberg
H I
HI
Schmaier
A H
AH
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
D010446:Peptide Fragments; C079000:bradykinin (1-5); D013917:Thrombin; D001920:Bradykinin