Factor VIII activation in the presence or absence of fibrinogen. One NIH unit/mL α-thrombin in 100 μL buffer containing 1 mM CaCl₂, and 1 mM GPRP peptide to prevent fibrin polymerization,⁵  was incubated at room temperature with 43 nM factor XIII (×), 86 nM γ_A/γ_A fibrinogen (▪), 86 nM γ_A/γ′ fibrinogen (□), 86 nM γ_A/γ_A fibrinogen + 43 nM factor XIII (•), or 86 nM γ_A/γ′ fibrinogen + 43 nM factor XIII (○). Thrombin was inactivated at the indicated times with 0.5 mM PPACK. Factor XIIIa activity was measured by the incorporation of 0.5 mM 5-(biotinamido)pentylamine into immobilized N, N′-dimethylcasein, and detected at 405 nm by P-nitrophenyl phosphate hydrolysis following incubation with streptavidin-conjugated alkaline phosphatase.⁶