Enhanced protein C activation and inhibition of fibrinogen cleavage by a thrombin modulator

Science

Volumn 273, Issue 5280, 1996, Pages 1389-1391

  Berg, David T ^a   Wiley, Michael R ^a   Grinnell, Brian W ^a  

^a ELI LILLY AND COMPANY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANTICOAGULANT AGENT; FIBRINOGEN; LY 254603; PROCOAGULANT; PROTEIN C; THROMBIN; UNCLASSIFIED DRUG;

ANTICOAGULATION; ARTICLE; BLOOD CLOTTING; DRUG RESEARCH; ENZYME ACTIVATION; FIBRINOGEN METABOLISM; PRIORITY JOURNAL; THROMBIN TIME;

EID: 0029817671     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5280.1389     Document Type: Article

References (28)

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17. A diverse screening set of mutually dissimilar compounds was chosen from Eli Lilly & Co. chemical archives by a clustering algorithm (R. Bruns, personal communication). Substructures were identified, and compounds with similar structural features were chosen from a nonselected set of compounds synthesized both at Lilly and from commercial sources using MACCS-II software (Molecular Design). LY254603 had been synthesized during the development of propanamine chemical synthetic methodologies.
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23. 2, 150 to 300 μg/ml of bovine serum albumin, and purified protein C at a concentration of 0.4 μM. After inhibition of thrombin with hirudin (100-fold molar excess), the amount of aPC generated was determined by means of a tripeptide chromogenic substrate (S-2366) at a concentration of 0.75 mM. Reactions were performed in sterile microtiter plates, and the changes in absorbance units per minute at 405 nm were measured in a ThermoMax kinetic microtiter plate reader (Molecular Devices). The amount of aPC generated was less than 10% of the initial zymogen in all experiments. LY254603 was dissolved in dimethyl sulfoxide (DMSO) or methanol (MeOH) and diluted into the reaction buffer with the thrombin 10 min before the addition of protein C. The final concentrations of DMSO or MeOH were maintained at levels at or below 0.5%, a level that had no effect on the thrombindependent activation of protein C or on protein C substrate cleavage in control reactions.
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26. Fibrinogen-clotting reactions were performed at 37°C with the use of 3 to 10 nM human α-thrombin in a reaction mix containing 20 mM tris, pH 7.4, 150 mM NaCl, and bovine fibrinogen (4 mg/ml). The cleavage and subsequent formation of fibrin were monitored by light scatter at 405 nm by means of a ThermoMax kinetic microtiter plate reader. All reactions were performed with compound in 0.5% or less MeOH, a level that did not alter thrombin-catalyzed fibrinogen clotting in control reactions.
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28. We gratefully thank B. Gerlitz and J. Jakubowski for helpful discussion. We also thank R. Bruns for supplying the selected cassette of synthetic compounds analyzed in this study.