Thioester Chromogenic Substrates for Human Factor VIIa: Substituted Isocoumarins Are Inhibitors of Factor VIIa and In Vitro Anticoagulants

Summary

Arginine thiobenzyl esters are convenient chromogenic substrates of factor Vila (Z-Arg-SBzl, k_cat/K_M = 1,600 M⁻¹ s⁻¹) and were used to study the kinetics of inhibition of factor Vila by several mechanism-based isocoumarin inhibitors of trypsin-like enzymes. Isocoumarin derivatives substituted with a 7-guanidino or 3-isothiureidopropoxy group were good inhibitors of factor Vila and acted as anticoagulants in human and rabbit plasma. With normal citrated human plasma, 4-chloro-3-ethoxy-7-guanidinoisocoumarin (3) and 7-amino-4-chloro-3-(3-isothiurei-dopropoxy) isocoumarin (ACITIC, 6) prolonged the prothrombin time (PT) ca. two-fold and prolonged the activated partial thromboplastin time (APTT) more than 4.5-fold at 20-30 pM. Both compounds had smaller effects in rabbit plasma. The short half-life of ACITIC and related isocoumarins in plasma should make these compounds uniquely useful as anticoagulants in therapeutic situations where it is desirable to have anticoagulant effects for a short defined time period.

• References

• 1 Cross PE. Antithrombotic agents. Annu Rep Med Chem 1982; 17: 79-88
• 2 Kikumoto R, Tamao Y, Tezuka T, Tonomura S, Hara H, Ninomiya K, Hijikata A, Okamoto S. Selective inhibition of thrombin by (2R,4R)-4-methyl-l-[N²-[(3-methyl-l,2,3,4-tetrahydro-8-quinolinyl)-sulfonyl]-L-arginyl)]-2-piperidinecarboxylic acid. Biochemistry 1984; 23: 85-90
• 3 Green D, Tsao CH, Reynolds N, Kohl H, Cohen I. In vitro studies of a new synthetic thrombin inhibitor. Thromb Res 1985; 37: 145-153
• 4 Hara H, Tamao Y, Kikumuto R, Okamoto S. Effect of a synthetic thrombin inhibitor MCI-9038 on experimental models of disseminated intravascular coagulation in rabbits. Thromb Haemostas 1987; 57: 165-170
• 5 Hauptmann J, Kaiser B, Markwardt F. Anticoagulant action of synthetic tight binding inhibitors of thrombin in vitro and in vivo. Thromb Res. 1985; 39: 771-775
• 6 Kaiser B, Markwardt F. Antithrombotic and haemorrhagic effects of synthetic and naturally occuring thrombin inhibitors. Thromb Res 1986; 43: 613-620
• 7 Bagdy D, Barabas E, Szeil E, Bajusz S. Biochemical pharmacology of tripeptide aldehydes. Folia Haematol (Leipzig) 1982; 109: 22-32
• 8 Collen D, Matsuo O, Stassen JM, Kettner C, Shaw E. In vivo studies of a synthetic inhibitor of thrombin. J Lab Clin Med 1982; 99: 76-83
• 9 Tidwell RR, Webster WP, Shaver SR, Geratz JD. Strategies for anticoagulation with synthetic protease inhibitors, Xa inhibitors versus thrombin inhibitors. Thromb Res 1980; 19: 339-349
• 10 Turner AD, Monroe DM, Roberts HR, Porter NA, Pizzo SV. p-Amidino esters as irreversible inhibitors of factor IXa and Xa and thrombin. Biochemistry 1986; 25: 4929-4935
• 11 Pizzo SV, Turner AD, Porter NA, Gonias SL. Evaluation of p-amidinophenyl esters as potential antithrombic agents. Thromb Haemostas 1986; 56: 387-390
• 12 Nemerson Y. Tissue factor and haemostasis. Blood 1988; 71: 1-8
• 13 Zur M, Nemerson Y. The esterase activity of coagulation factor VII. J Biol Chem 1978; 253: 2203-2209
• 14 Zur M, Radcliff RD, Oberdick J, Nemerson Y. The dual role of factor VII in blood coagulation. J Biol Chem 1982 257: 5623-5631
• 15 Kam C-M, Fujikawa K, Powers JC. Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants. Biochemistry 1988; 27: 2547-2557
• 16 McRae BJ, Kurach K, Heimark RL, Fujikawa K, Davie EW, Powers JC. Mapping the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein, and trypsin with amino acid and peptide thioesters: development of new sensitive substrates. Biochemistry 1981; 20: 7196-7206
• 17 Kam C-M, McRae BJ, Harper JW, Niemann MA, Volanakis JE, Powers JC. Human complement proteins D, C2, and B: active site mapping with peptide thioester substrates. J Biol Chem 1987; 262: 3444-3451
• 18 Harper JW, Hemmi K, Powers JC. Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor. Biochemistry 1985; 24: 1831-1841
• 19 Broze Jr GJ, Leykam JE, Schwartz BD, Miletich JP. Purification of human brain tissue factor. J Biol Chem 1985; 260: 10917-10920
• 20 Bach R, Oberdick J, Nemerson Y. Immunoaffinity purification of bovine factor VII. Blood 1984; 63: 393-398
• 21 Usharani P, Wam-Cramer BJ, Kasper CK, Bajaj SP. Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site. J Clin Invest 1985; 75: 76-83
• 22 Grassetti DR, Murray Jr JF. Determination of sulfhydryl groups with 2,2’ or 4,4’-dithiodipyridine. Arch Biochem Biophys 1967; 119: 41-49
• 23 Zur M, Nemerson Y. Kinetics of factor IXa activation via the extrinsic pathway. J Biol Chem 1980; 255: 5703-5707
• 24 Silverberg SA, Nemerson Y, Zur M. Kinetics of the activation of bovine coagulation factor X by components of the extrinsic pathway. J Biol Chem 1977; 252: 8481-8488
• 25 Rodbard D, Hutt DM. Statistical analysis of radioimmunoassays and immunoradiometric (labeled antibody) assays. A generalized weighted, iterative, least-squares method for logistic curve fitting. Radioassays: clinical concepts (Hunter, Q B, ed., Searle, Skokie, 111.) 1974; 209-233
• 26 McRae BJ, Lin T-Y, Powers JC. Mapping the substrate binding site of human Clr and Cls with peptide thioester. J Biol Chem 1981; 256: 12362-12366
• 27 Oesterud B, Rapaport SI. Activation of factor IX by the reaction product of tissue factor and factor VII: Additional pathway for initiating blood coagulation. Proc Natl Acad Sci USA 1977; 74: 5260-5264
• 28 Oweida S, Ku DN, Lumsden AB, Kam C-M, Powers JC. In vivo determination of the anticoagulant effect of a substituted isocoumarin (ACITIC). Thromb Res 1990; 58: 191-197
• 29 Mottaghy K, Oedekoven B, Bey R, Schmid-Schonbein H. Extracor-poreal anticoagulation using a serine protease inhibitor in hemodialysis in sheep. Trans Am Soc Artif Intern Organs 1985; 31: 534-536