ABSTRACT
An inhibitor of alpha-thrombin was designed on the basis of the X-ray crystal structures of thrombin and trypsin. The design strategy employed the geometric and electrostatic differences between the specificity pockets of the two enzymes. These differences arise due to the replacement of Ser 190 in trypsin by Ala 190 in thrombin. The new inhibitor contained a tryptophan side chain instead of the arginine side chain that is present in the prototypical thrombin inhibitors. This inhibitor had a Ki value of 0.25 microM, displayed more than 400-fold specificity for thrombin over trypsin, and doubled the rat plasma APTT at a concentration of 44.9 microM. The X-ray crystal structure of the inhibitor/alpha-thrombin complex was determined. This represents the first reported three-dimensional structure of a thrombin/ inhibitor complex where the specificity pocket of the enzyme is occupied by a chemical moiety other than a guanidino or an amidino group. As was predicted by the molecular model, the tryptophan side chain docks into the specificity pocket of the enzyme. This finding is in contrast with the indole binding region of thrombin reported earlier [Berliner, L. J., & Shen, Y. Y. L. (1977) Biochemistry 16, 4622-4626]. The lower binding affinity of the new inhibitor for trypsin, compared to that for thrombin, appears to be due to (i) the extra energy required to deform the smaller specificity pocket of trypsin to accommodate the bulky indole group and (ii) the favorable electrostatic interactions of the indole group with the more hydrophobic specificity pocket of thrombin. The neutral indole group may be of pharmacological significance because the severe hypotension and respiratory distress observed following the administration of some thrombin inhibitors have been linked to the positively charged guanidino or amidino functionalities.
Subject(s)
Oligopeptides/chemistry , Serine Proteinase Inhibitors/chemistry , Thrombin/antagonists & inhibitors , Thrombin/chemistry , Alanine , Animals , Binding Sites , Crystallography, X-Ray , Drug Design , Hirudins/chemistry , Hirudins/pharmacology , Models, Molecular , Oligopeptides/chemical synthesis , Oligopeptides/pharmacology , Partial Thromboplastin Time , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protein Conformation , Rats , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacology , Software , Trypsin/chemistry , Trypsin Inhibitors/chemical synthesis , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/pharmacology , TryptophanABSTRACT
Fluorinated ketone thrombin inhibitors based on the peptide sequence methyl-(D)-Phe-Pro-Arg-CF2R were synthesized: MDL 73,446 (1, R = F); MDL 73,775 (2, R = CF3); and MDL 75,579 (3, R = CH2CH2CH3). These were shown to be highly effective, slow binding inhibitors of thrombin. Anticoagulant activity was dose-dependent with 3 > 2 > 1 at doubling thrombin time and APTT, respectively. Anticoagulant activity corresponded with efficacy in a platelet-dependent (FeCl3-induced) rat carotid artery thrombosis model. Arterial occlusion was dose-dependently prolonged with 3 > 2 > 1 at doubling the occlusion time.
Subject(s)
Anticoagulants/chemical synthesis , Antithrombins/chemical synthesis , Carotid Artery Thrombosis/physiopathology , Oligopeptides/chemical synthesis , Amino Acid Sequence , Animals , Anticoagulants/chemistry , Anticoagulants/pharmacology , Antithrombins/chemistry , Antithrombins/pharmacology , Blood Platelets/physiology , Carotid Artery Thrombosis/drug therapy , Chlorides , Disease Models, Animal , Ferric Compounds , Fluorocarbons , Indicators and Reagents , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Oligopeptides/chemistry , Oligopeptides/pharmacology , RatsABSTRACT
The effect of heparin and the synthetic irreversible antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (FPRCH2Cl) was studied on FeCl3-induced thrombotic occlusion of rat carotid arteries. Thrombocytopenia prevented occlusion in five of 7 rats for the 60 min observation period after FeCl3 injury demonstrating platelet dependence in this model of thrombosis. Intravenous injection of heparin (250 units/kg) followed by continuous infusion (250 units/kg/hr) failed to prevent occlusion in four of 6 rats whereas intravenous FPRCH2Cl infusion prevented occlusion at a dose of 200 nmol/kg/min during infusion in 6/6 rats. These findings indicate that thrombin plays a principle role in the platelet-dependent process of arterial thrombosis in FeCl3-damaged rat carotid arteries. Neutralization of the thrombogenic stimulus in this model by the thrombin inhibitor FPRCH2Cl suggests selective thrombin inhibition may be useful in the treatment of arterial thrombosis.
Subject(s)
Carotid Artery Thrombosis/prevention & control , Heparin/therapeutic use , Thrombin/antagonists & inhibitors , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Blood Platelets/physiology , Carotid Artery Thrombosis/chemically induced , Disease Models, Animal , Ferrous Compounds/toxicity , Fibrin/physiology , Heparin/pharmacology , Male , Rats , Rats, Inbred Strains , Thrombocytopenia/bloodABSTRACT
A synthetic hirudin55-65 C-terminal fragment analog was evaluated for anticoagulant activity and in models of experimental thrombosis in mice and rats. Intravenous injection caused dose-related inhibition of thrombin and anticoagulation in rat blood samples, protection from thromboembolism in mice and inhibition of stasis-induced venous thrombosis in rats. Antithrombotic effectiveness corresponded with anticoagulant activity. Anephric animals exhibited longer duration of activity than normal animals suggesting the kidney as a major route of elimination.