In Vitro Inhibition of Blood Coagulation by Tripeptide Aldehydes - A Retrospective Screening Study Focused on the Stable D-MePhe-Pro-Arg-H · H2SO4

Abstract

SummaryA 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.

Comparative Studies on the Anticoagulant and Protease Generation Inhibitory Actions of Newly Developed Site-Directed Thrombin Inhibitory Drugs

“…Despite their differences in their site of action and kinetics, all of these anticoagulants are potent throm bin inhibitors. 6,11,14,15 The objective of these studies was to determine whether the differences in the mechanism of thrombin inhibition of each thrombin inhibitor correlate with their anticoagulant effects. It is clear from these studies that conventional tests such as the PT, PTT, and Heptest are of limited value in the potency evaluation of newly devel oped antithrombin agents, since the anticoagulant pro files of each of the thrombin inhibitors in the global tests do not correlate with their ability to inhibit coagulation feedback loops and generation of thrombin and factor Xa after extrinsic or intrinsic activation, as studied in defined systems.…”

Inhibition by D-MePhe-Pro-Arg-H (GYKI-14766) of Thrombus Growth in Experimental Models of Thrombosis

“…In contrast to the expectations only a very few of them proved to be effective also in vivo, opposed to the favourable results obtained in vitro biochemical experiments. The in vitro and in vivo anticoagulant and antiplatelet effects based on thrombin inhibition by D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H were thoroughly proved and reported (9,10). The present studies were carried out to estimate the antithrombotic potency of the most efficient tripeptide aldehyde in several models of experimental thrombosis in rats and rabbits.…”

“…Control thrombus weight used in the calculation of data included the wet weight of the thread which was deducted from the total thrombus weight. Coagula tion parameters (whole blood clotting time, activated partial thromboplas tin time, thrombin time as well as cell counts -RBC and platelets) were determined as described in Bagdy et al (9).…”

Direct Thrombin Inhibitors for the Prevention of Venous Thromboembolism after Major Orthopaedic Surgery

“…The most extensively investigated direct antithrombins are the polypeptides hirudin and hirulog [11,12] and the low molecular weight inhibitors of the active site of thrombin melagatran, with its oral prodrug ximelagatran [13], and dabigatran etexilate [14]. Other direct thrombin inhibitors, including argatroban [15], napsagatran [16], inogatran [17], and efegatran [18] have been either investigated in different clinical conditions or withdrawn from the clinical development.…”

“…Melagatran is a competitive and rapid inhibitor of the active site of thrombin with a molecular weight of 429 daltons [13]. Argatroban (argidipine, MD805), a derivative of arginine with a molecular weight of 526 daltons [15], napsagatran (RO 46-6240), a cyclopropyl derivative with a molecular weight of 559 daltons [15,22], inogatran, (H 314/27) is a synthetic dipeptide with a molecular weight of 439 daltons [17,23] and efegatran sulfate (GYKI 14766, LY294468), a tripeptide aldehyde containing arginine [18,23] are direct, selective and reversible inhibitors of the active site of thrombin. The antithrombotic effect of direct antithrombins in the experimental model is reviewed in details elsewhere [24].…”