Coatest sp4 factor 8 kit

APTT clotting assays and video microscopy of clot growth assays were performed as described [34 (link)]. CS assays were performed with Coatest SP4 Factor VIII kit (Chromogenix; Diapharma, West Chester, OH, USA). An in-house FVIII standard was used as a calibrator. All samples were diluted in FVIII-deficient plasma to ~1 IU/mL, and then serially diluted in a buffer included in the kit.
For the TG assay, citrated FVIII-deficient plasma (50% vol/vol) was mixed with corn trypsin inhibitor (50 μg/mL), fluorogenic substrate Z-Gly-Gly-Arg-AMC (800 μM), phospholipid vesicles (4 μM) and tissue factor (0.2 pM). Plasma was warmed to 37 °C, and TG in plasma was activated by the solution of CaCl₂ and activated coagulation factor XI (10 mM and 10 pM final concentrations, respectively). Immediately after the activation, plasma was mixed with the samples (35% vol/vol) containing FVIII and scFv iKM33 variant or thrombin calibrator. AMC fluorescence (460 nm) was recorded at 37 °C with a microplate reader. Thrombin peak height was used to characterize TG as described [62 (link)].

Forty-eight hours after transfection, conditioned media were collected for FVIII activity and antigen assays. The Coatest SP4 Factor VIII kit (Chromogenix, Bedford, MA) was used to measure FVIII activity in conditioned media. WT mouse plasma was used as standards for activity assays. The VisuLize FVIII antigen kit (Affinity Biologicals, Ancaster, Canada) was used to analyze the antigen levels of FVIII in cell lysates and conditioned media. Activities and antigen levels of WT and mutant FVIII were subtracted from mock-controls.