Global coagulation function potentials on FVIII K1813A, K1818A, and K1813A/K1818A mutants. (A). FVIII WT (1 nM) or mutants (K1813A; 0.5 and 1 nM; K1818A, 1 nM; K1813A/K1818A, 1 nM) were added to commercial FVIII-deficient plasma. These samples were incubated with TF (1 pM) and PL vesicles (4 μM) before the addition of fluorogenic substrate and CaCl₂ at the start of the assay. Experiments were performed 3 separate times, and the mean values are shown thrombin generation curves are shown (black; WT 1 nM, red; K1813A 1 nM, tangerine; K1813A 0.5 nM, blue; K1818A 1 nM, green; K1813A/K1818A 1 nM, gray; buffer). (B) ROTEM. CaCl₂ was added to citrated whole blood sample (residual FVIII activity of 2.3 and <1 IU/dL) in 2 patients with severe HA, together with various concentrations of FVIII mutants (WT and K1813A), followed by a ROTEM assay. The raw blood data in 2 patients and the representative curves in 1 patient are shown. The average parameters (CT and CT+CFT) in 2 patients are shown below each figure. The CT and CT+CFT obtained from normal controls (n=20) were 938±128 seconds and 1273±217 seconds, respectively. CT, clot time; CFT, clot formation time.