BACKGROUND Factor VIII (FVIII) procoagulant activity is commonly assessed by measuring the activated partial thromboplastin time (APTT) to clot formation using one of the many available but differently composed reagents. The majority of APTT reagents also accurately recover the activity of the extended half-life molecule N-glycoPEGylated FVIII (N8-GP; turoctocog alfa pegol), while a few silica-based reagents give a low recovery. OBJECTIVE To identify the cause of N8-GP activity underestimation in the presence of certain silica-based APTT reagents. METHODS Development of FVIIIa-dependent tenase activity and appearance of FVIIIa from N8-GP and its non-PEGylated counterpart (N8) were compared using clotting assays, a factor Xa (FXa) activity assay mimic thereof, and thrombin activation time courses. RESULTS A strong correlation was demonstrated between clotting and FXa activity assays based on similar recoveries of N8-GP activity and equal responses to an altered duration of the contact activation phase, validating the latter as a useful clotting assay mimic. Contact activation phase duration influenced, and could even eliminate, N8-GP activity underestimation. Thrombin-catalyzed conversion of N8-GP to FVIIIa was considerably slower than that of N8 despite similar extents of adsorption to silica particles in APTT-SP, suggesting different modes and/or orientations of adsorption. CONCLUSIONS Some contact activators reduce thrombin's ability to cleave N8-GP more than native FVIII. Decelerated thrombin activation of N8-GP is reflected in delayed FVIIIa-dependent appearance of FXa activity in plasma, in turn leading to prolonged clotting time. This forms the basis for underestimation of N8-GP activity as measured by one-stage clotting assay against a FVIII standard.