Sensolyte 520 factor xa assay kit

The Factor Xa inhibition assay was carried out using a commercial SensoLyte® 520 Factor Xa Assay Kit (AnaSpec). The Factor Xa activity was assessed by its ability to cleave the fluorogenic FRET peptide and the subsequent unquenching by QXL 520 quencher of the 5-FAM fluorophore, which emits fluorescence. All extracts and controls were performed with three replicates in a 384-well plate format, each with a total reaction mixture of 12.5 μL. To begin, the test extracts and Thrombin/Factor Xa solution were incubated for five minutes then the substrate was added. After substrate addition and gentle mixing the reaction was incubated at room temperature for one hour. The fluorophore was detected with the use of a SpectraMax® Paradigm® Multi-mode Microplate Detection Platform (Molecular Devices) by scanning at 490 nm excitation wavelength and 520 nm emission wavelength.

To compare the magnitude of antithrombotic effect of statins to a conventional anticoagulant, additional mice (N = 32) with stasis IVC VT received subcutaneous low molecular treatment daily (enoxaparin 10 mg/kg SQ) for three days. Control mice received PBS. In addition, evaluation of combination daily statin (atorvastatin 1.14mg/kg or rosuvastatin 0.84 mg/kg) and enoxaparin 10 mg/kg LMWH therapy was examined. To verify the systemic anticoagulant effects of LMWH, factor Xa activity levels were measured in blood samples obtained four hours after the final LMWH injection, and (SensoLyte 520 Factor Xa Assay Kit, AnaSpec Inc.).