Thrombin generation assay

FIX-deficient plasma was obtained from George King Bio-Medical, Inc (Overland Park, KS). Plasma-derived factor IXa and human thrombin were purchased from Haematologic Technologies (Essex Junction, Vermont). Thrombin activity was determined by the calibrated automated thrombogram (CAT) method described by Hemker et al. using the standard assay protocol and reagents from Thrombinoscope^® (Stago, Parsippany, NJ) [18]. Final concentrations of reagents were 1 pM tissue factor and 4 μM phospholipids for assay wells, or 630 nM thrombin calibrator for calibration wells. When thrombin generation was triggered with factor IXa, increasing concentrations (0–100 pM) of plasma-derived factor IXa, followed by 5 nM human thrombin, were added to FIX-deficient plasma in the presence or absence of factor IX variants 3 min prior to recalcification.