Methodological aspects in the studies on the mechanism of action of synthetic direct thrombin antagonists.

Requirements reported for an ideal anticoagulant [12] and for an ideal antithrombotic [18] show the necessity of many-sided methodological approach in order to detect the molecular mechanism of action of a novel synthetic antagonist of thrombin. The lack of a protocol internationally accepted, on the one hand, and with regard to a general proposal accepted [16], on the other hand, authors applied a complex methodological system involving also the study on the possible interactions at molecular level of some novel thrombin antagonists with the main components of their site of action. The surprising contradiction found between in vitro and in vivo efficacy of several antagonists could be attributed and explained by the significant differences in Ki and IC50 values determined in complex clotting assays containing plasma proteins and/or blood cells versus those measured in reaction mixtures consisting of a synthetic chromogenic substrate, the target enzyme, thrombin, and the antagonist compound in buffer solution.

Inhibition by D-MePhe-Pro-Arg-H (GYKI-14766) of thrombus growth in experimental models of thrombosis.

The antithrombotic action of the highly effective synthetic thrombin inhibitor D-MePhe-Pro-Arg-H (GYKI-14766) was studied in various models of experimental thrombosis. The compound administered to rats and rabbits by i.v. bolus injections, continuous i.v. infusions, subcutaneously and orally, respectively, induced significant decrease in thrombus weight (i) in a quantitative venous thrombosis model with stasis based on vascular lesion in rats, (ii) in an extracorporeal arterio-venous shunt model in rabbits, and (iii) prevented the occlusion of the vessel in arterial thrombosis induced by mechanical damage in rats. By using the arterio-venous shunt model in rabbits the inhibitory effect on thrombus growth could be demonstrated as a function of dose and time in self-controlled experiments. Blood level of the inhibitor determined by a bioassay varied between 0.09-0.67 microgram/ml whole blood when doses of 15 and 20 mg/kg were administered orally. A correlation was found between thrombin time, platelet aggregation induced by thrombin ex vivo and the weight of thrombi formed.

Comparative studies in vitro and ex vivo on the anticoagulant effect of a reversible and an irreversible tripeptide inhibitor of thrombin.

Comparative studies on the anticoagulant effect of D-Phe-Pro-Arg-H (ALD) and D-Phe-Pro-Arg-CH2Cl (CMK) were carried out in order to estimate whether the reversible or the irreversible tripeptide inhibitor of thrombin would be more suitable to develop as a novel anticoagulant. Conventional screening assay methods in vitro were focused on the functional stability of the compounds in whole blood and blood components while ex vivo the changes in whole blood clotting time under parenteral application of the inhibitors were investigated. The efficacy of ALD relative to that of CMK was found to depend on the complexity of the test systems. Thus CMK was the more inhibitory in citrated plasma, but ALD showed the higher potency in whole blood. When incubated in various systems such as human whole blood, serum, solutions of isolated plasma proteins, digestive juices and tissue homogenates, respectively, the inhibitory activity of ALD showed only slight decreases for several hours while marked or substantial loss of activity was observed with CMK under identical conditions. ALD administered parenterally to rabbits proved to be powerful anticoagulant; CMK exhibited only a weak and transient anticoagulant effect presumably due to its ability to bind irreversibly to various plasma and tissue proteins. Accordingly, the reversible inhibitor ALD should be more suitable to develop as an anti-coagulant than CMK, its irreversibly acting analogue.

In vitro inhibition of blood coagulation by tripeptide aldehydes--a retrospective screening study focused on the stable D-MePhe-Pro-Arg-H.H2SO4.

A series of peptide aldehydes synthetized in our institute during the last 15 years were screened to detect their inhibitory effect on blood coagulation. Simple conventional clotting assays, platelet function tests and fibrinolytic methods were used to evaluate the inhibitory potency of the compounds in complex clotting systems as well as their supposed antifibrinolytic effect in vitro. Special attention was paid to the possible interactions with blood cells and plasma proteins, and to the functional stability of the inhibitors in several tissue homogenates. D-Phe-Pro-Arg-H (GYKI-14166, RGH-2958), Boc-D-Phe-Pro-Arg-H (GYKI-14451) and D-MePhe-Pro-Arg-H (GYKI-14766) were found to be the most potent inhibitors. The peptide aldehydes via formation of reversible complexes with thrombin impede the enzyme to react with the coagulation factors, platelet membrane and vessel wall. The compounds inhibit platelet aggregation induced by thrombin specifically without changing the sensitivity of platelets to other inducers. D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H showed no antifibrinolytic effect. D-MePhe-Pro-Arg-H and Boc-D-Phe-Pro-Arg-H proved to be stable in dry state for years and in solution at room temperature for several days. The anticoagulant activity of the compounds was declared in NIH antithrombin units.

In vivo anticoagulant and antiplatelet effect of D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H.

D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H synthetized in our institute were administered to mice, rats, rabbits and beagle dogs. The kinetics of the anticoagulant and antiplatelet effect was recorded by measuring various clotting parameters, platelet count and aggregation, and evaluated as proposed by Verstraete and Verwilghen. The minimum effective doses were found to be 0.25-0.5 mg kg-1h-1 by intravenous continuous infusions and 0.5-1.0 mg/kg by single injections. The dose-dependent prolongation of clotting times appeared after application within minutes and returned to baseline values as a function of dose. Blood level of the inhibitors was determined by a bioassay. Unlike heparin, no higher starting dose was required to reach the anticoagulant threshold level, i.e. 0.03-0.1 microgram/ml whole blood. The peptides did not cause significant changes in platelet count and function or in hemodynamic parameters (blood pressure, heart rate and ECG) and in respiration. They blocked platelet aggregation induced by thrombin ex vivo specifically. No rebound effect or bleeding could be demonstrated even after subtoxic doses of the compounds. The onset of the anticoagulant and antithrombotic effect appeared within 60 min after single oral doses and lasted for 3-6 h. In close correlation with the anticoagulant effect a complete or significant inhibition of platelet aggregation induced by thrombin ex vivo could also be recorded by using 5-10 mg/kg doses.

Highly active and selective anticoagulants: D-Phe-Pro-Arg-H, a free tripeptide aldehyde prone to spontaneous inactivation, and its stable N-methyl derivative, D-MePhe-Pro-Arg-H.

D-Phe-Pro-Arg-H sulfate (GYKI-14166) is a highly active and selective inhibitor of thrombin both in vitro and in vivo. Recent studies on the stability of D-Phe-Pro-Arg-H in neutral aqueous solution at higher temperature have revealed that it is transformed into inactive 5,6,8,9,10,10a-hexahydro-2-(3'- guanidinopropyl)-5-benzyl-6-oxo- imidazo[1,2-a]pyrrolo[2,1-c]pyrazine. No such inactivation could be observed with Boc-D-Phe-Pro-Arg-H (GYKI-14451), but this compound was far less specific than the free peptide as it inhibited thrombin and, for instance, plasmin equally well. Assuming that the transformation of free tripeptide aldehyde, mentioned above, can only be initiated by a primary amino terminus, the N-alkyl derivatives of D-Phe-Pro-Arg-H were prepared. Of the new analogues, D-MePhe-Pro-Arg-H (GYKI-14766) proved to be as highly active and selective anticoagulant as its parent compound and was not inactivated by transformation into a heterocyclic compound.

Experimental oral anticoagulation by a directly acting thrombin inhibitor (RGH-2958).

D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate (RGH-2958), a directly acting synthetic thrombin inhibitor proved to be effective by experimental oral application. The rapid onset of its action has a special importance both from theoretical and practical point of view. Its antiplatelet effect relates to thrombin induced PA and runs parallel with the anticoagulant effect. RGH-2958 significantly reduced the thrombus weight in an experimental thrombosis model in rabbits. In the light of the therapeutic indices of the compound there is a real possibility to open a third way in the prevention and therapy of thrombosis.

Beta-2-microglobulin-specific autoantibodies cause platelet aggregation and interfere with ADP-induced aggregation.

The anti-platelet activity of beta-2-microglobulin (beta 2m) specific autoantibodies isolated from sera of patients with autoimmune diseases was tested in direct and ADP-induced aggregation assays. It was established that human anti-beta 2m autoantibodies and heterologous rabbit anti-beta 2m antibodies evoke a dose-dependent aggregation of human platelets. Anti-beta 2m autoantibodies also impaired ADP-induced platelet aggregation. Antibodies with anti-beta 2m activity could be desorbed from the platelets and lymphocytes of a patients with systemic lupus erythematosus who was not thrombocytopenic. The possibility that such autoantibodies may alter platelet function is considered.

Design and synthesis of peptide inhibitors of blood coagulations.

Inhibition of blood coagulation by peptide aldehydes has been studied. Amino acid sequences were assembled from the P1-P2 portion of the cleavage sites(s) of clotting factors and residues selected experimentally. The thrombin-fibrinogen reaction could effectively be inhibited by D-Phe-Pro-Arg-H (GYKI-14,166) and Boc-D-Phe-Pro-Arg-H (GYKI-14,451). Plasmin digestion of fibrin could be retarded by Boc-Gln-Phe-Lys-H (GYKI-14,605) derived from a susceptible fragment, i.e. Asn-Phe-Lys decreases to Ser. However, such peptides could not retard the zymogen activations proceeding in Ca++ complexes (which seemed to be uneffected by heparin-antithrombin III, too). Inhibition of enzymes by peptide aldehydes showed marked substrate dependence.

Inhibition of thrombin and trypsin by tripeptide aldehydes.

Inhibitory effects of certain tripeptide aldehydes on both thrombin and trypsin have been found to be strongly substrate-dependent. These compounds should therefore be considered as inhibitors of the particular proteolytic reaction for which they had been designed rather than real enzyme inhibitors, i.e. protein or polypeptide proteinase inhibitors of natural origin.