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.

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.

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.

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.

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.

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.

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.

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).

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.