Potential for application of direct thrombin inhibitors isolated from Euphorbia resinifera O.Berg latex in fibrin clot formation

Abstract

Direct thrombin inhibitors (designated as EuRL-DTIs) were partially purified from ethanol extracts of Euphorbia resinifera O.Berg latex. The obtained EuRL-DTIs comprised four major compounds: two isomers of phenolic compounds (C₁₉H₂₆O₁₂) and two amide compounds (tentatively identified as C₂₄H₄₄N₄O₄ and C₃₆H₆₆N₆O₆), as identified by liquid chromatography and electrospray ionisation quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, and/or nuclear magnetic resonance (NMR) spectroscopy. The effects of EuRL-DTIs on human thrombin-induced fibrin clot production were analysed using thrombin time, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), synchrotron radiation X-ray tomographic microscopy (SRXTM), and scanning electron microscopy (SEM). Kinetic studies revealed that EuRL-DTIs inhibited human thrombin from cleaving the chromogenic substrate S2238, with a K_i of 3.7 μg/mL, in a non-competitive inhibition manner. All results supported the hypothesis that the EuRL-DTIs directly abolished thrombin activity in the production of fibrin clots without requiring a cofactor. The cytotoxicity test showed that EuRL-DTIs were nontoxic to normal human foetal lung fibroblasts (IMR-90). Thus, EuRL-DTIs have potential as antithrombotic agents for application as drugs for thrombosis treatments or in medical devices such as coating surgical sutures.