Arixtra

5 μM human α1-antitrypsin (Sigma) or human antithrombin (Aclotin^®, LFB) were incubated with 50 μM annonacinone (or 0.5% DMSO in reaction buffer as control) for 20 min at 37 °C. The mixtures containing α1-antitrypsin were diluted in N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma) containing solution and substrate hydrolysis kinetic was triggered by addition of neutrophil elastase (Sigma) to a final volume of 100 μL containing 13 nM α1-antitrypsin, 130 nM annonacinone, 82 μM chromogenic substrate and 5 nM neutrophil elastase. The mixtures containing antithrombin were diluted in fondaparinux (Arixtra^®, GlaxoSmithKline) and CS-11(22)-FXa (Hyphen Biomed) containing solution and substrate hydrolysis kinetic was triggered by addition of factor Xa (Stago BNL) to a final volume of 100 μL containing 51 nM antithrombin, 510 nM annonacinone, 200 μM chromogenic substrate, 5 μM fondaparinux and 2 nM factor Xa.

The assay was carried out in a 96-well plate in which the formation of the disaccharide product upon cleavage of fondaparinux (Arixtra, GlaxoSmithKline, Brentford, UK) was estimated using WST-1 tetrazolium salt as described earlier [22 (link),23 (link)]. In brief, the reaction system (100 μL) comprised sodium acetate buffer (pH 5.0, 40 mM) and fondaparinux (100 mM) with or without different doses of chemicals under investigation. The assay was initiated by the addition of recombinant heparanase to make the final concentration of 140 pM. The reaction vessels were placed at 37 °C for 18 h, followed by the addition of stop solution (100 μL). WST-1 prepared in 0.1 M NaOH served as a stop solution. Thereafter, the plates were incubated at 60 °C for 60 min, and absorbance was measured at 584 nm. N-(4-([4-(1H-Benzoimidazol-2-yl)-arylamino]-methyl)-phenyl)-benzamide (compound #98) was used as a reference compound. The title compounds were examined for their inhibitory efficacy towards heparanase at 10 and 50 μg/mL. Compounds that yielded inhibition at these concentrations were further tested at lower concentrations to determine their IC₅₀.

LMWH preparations used in this study were enoxaparin, supplied by Sanofi-Aventis Pharma (Milan, Italy) as injectable Clexane; tinzaparin, supplied by LEO-Pharma (Ballerup, Denmark) as a powder; dalteparin, from Pharmacia AB (Uppsala, Sweden) as injectable Fragmin. The synthetic pentasaccharide Arixtra (fondaparinux) was from GlaxoSmithKline (Verona, Italy). Heparin low-molecular-mass for calibration CRS batch 1, (Mn = 3700 Da) was purchased from European Pharmacopoeia (EP) LMWH standard.

We performed MIS-TKA for all patients at an accessible level while they were under general anesthesia. A tourniquet was not used for any patient. A slightly medial straight skin incision was made from the superior pole of the patella to the tibial tuberosity (range 8–10 cm long for individual cases). The mini-subvastus approach without patella eversion was used in all patients, and bone cutting was performed according to the MIS Quad-Sparing TKA technique [10 (link)].
In all cases, the prosthesis was a NexGen Complete Knee Solution Legacy Knee Posterior Stabilized (LPS) LPS-Flex Fixed Bearing Knee (Zimmer, Warsaw, IN, USA). In each case, the patella was resurfaced, and all components were fixed with cement. A total of 800 g of autologous blood was prepared before the operation, and this was transfused to each patient after surgery (400 g transfused twice). An epidural catheter was inserted during the operation and remained in place for 48 h after. Intermittent pneumatic compression was applied for the same period. The anticoagulant fondaparinux (Arixtra; GlaxoSmithKline plc, London, UK) was administered for 14 days (2.5 mg daily) from 24 h after removal of the epidural catheter. All patients received rehabilitation therapy sessions each lasting 60–120 min per day on 5–6 days per week throughout their hospitalization, and clinical symptoms were observed every day until discharge.