In vitro characterization of a novel factor Xa inhibitor, RPR 130737.
RPR 130737 inhibited factor Xa (FXa) with a Ki of 2.4 nM and also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C, plasmin, tissue-plasminogen activator and trypsin. RPR 130737 prolonged plasma activated partial thromboplastin time and prothrombin time in a dose-dependent fashion. In the activated partial thromboplastin time assay, the concentrations required for doubling coagulation time were 0.32 microM (human), 0.61 microM (monkey), 0.44 microM (dog), 0.15 microM (rabbit), and 0.82 microM (rat). The concentrations required to double prothrombin time were 0.86 microM (human), and 1.26 microM (monkey), 1.15 microM (dog), 0.39 microM (rabbit) and 7.31 microM (rat). Kinetic studies revealed that RPR 130737 was a fast binding, reversible and competitive inhibitor for FXa when Spectrozyme FXa, a chromogenic substrate, was used. A coupled-enzyme assay measuring thrombin activity following prothrombinase conversion of prothrombin to thrombin indicated that RPR 130737 was a potent inhibitor for prothrombinase-bound FXa. In this assay, RPR 130737 showed IC50s of 17 nM and 35.9 nM, respectively when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or gel-filtered platelets were used as the phospholipid source. An FX-deficient plasma clotting-time correction assay further demonstrated that RPR 130737 was a specific inhibitor of FXa. RPR 130737 showed no effect on platelet aggregation in vitro. These results indicate that RPR 130737 has the potential to be developed as an antithrombotic agent based on its potent and selective inhibitory effect against FXa.
Chu
V
V
Cardiovascular Drug Discovery, Rhône-Poulenc Rorer Central Research, Collegeville, PA, USA. Valeria.Chu@aventis.com
Brown
K
K
Colussi
D
D
Choi
Y M
YM
Green
D
D
Pauls
H W
HW
Spada
A P
AP
Perrone
M H
MH
Leadley
R J
RJ
Dunwiddie
C T
CT
D000578:Amidines / Q000494:pharmacology*; D000925:Anticoagulants / Q000494:pharmacology; D001780:Blood Coagulation Tests; D004305:Dose-Response Relationship, Drug; D005171:Factor X Deficiency / Q000097:blood; D065427:Factor Xa Inhibitors; D006801:Humans; D007700:Kinetics; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors; D013925:Thromboplastin / Q000378:metabolism
D000578:Amidines; D000925:Anticoagulants; D065427:Factor Xa Inhibitors; C121695:RPR 130737; D015842:Serine Proteinase Inhibitors; D013450:Sulfones; D013925:Thromboplastin; D012697:Serine Endopeptidases; D013917:Thrombin