FIGURE 5.

In vitro and ex vivo activity characterization of CT‐001. FXa generation was measured in both (A) TF independent, and (B) TF dependent reactions to determine the ability of the FVIIa variants to generate FXa. FXa generation was determined using chromogenic substrate S‐2765 and a purified FXa standard curve. Independent of TF, CT‐001 had two times higher catalytic rate constant (k_cat), maximum reaction rate (V_max), and specificity constant (k_cat/K_M) in comparison to WT FVIIa. dWT FVIIa and WT FVIIa have similar k_cat, Michaelis constant (K_M), and V_max under this experimental condition. The data are displayed as the average of three experiments (ANOVA; *P < 0.01; **P < 0.0005). Under TF‐dependent conditions, similar k_cat, K_M, and V_max were observed among CT‐001, CT‐001(Sial), dWT FVIIa, and WT FVIIa. The data are displayed as the average of three to six experiments. (C) CT‐001 has enhanced thrombin generation activity relative to other FVIIa variants. Thrombin generation was measured in normal human plasma with 0.024 – 1000 nM of FVIIa in the presence 4 μM phospholipid. Thrombin generation was initiated with CaCl₂, and in the presence of thrombin fluorogenic substrate. The endogenous thrombin potential (ETP) was measured. CT‐001 had two times higher potency than CT‐001(Sial), dWT FVIIa, and WT FVIIa. The data are represented by the average of three independent experiments using a normal human plasma pool. (D‐G) CT‐001 has superior clotting activity to WT FVIIa across species. aPTT (STA‐PTT A) assays were performed in normal plasma pool from C57Bl/6 mice (D), cynomolgus monkeys (E), and humans (F). For all species, CT‐001 and CT‐001(Sial) were similar and demonstrated superior clotting time relative to dWT FVIIa or WT FVIIa. (G) In PT assays (STA‐Neoplastine CI Plus 5, rabbit brain thromboplastin), human plasma has an average PT clotting time of 12.8 seconds. CT‐001, CT‐001(Sial), dWT FVIIa, and WT FVIIa had similar potency, requiring 0.5, 0.6, 0.8, and 0.7 nM respectively to shorten PT clotting time to 10 seconds. (H) CT‐001 and CT‐001(Sial) showed enhanced clotting activity in rotational thromboelastometry assay in comparison to dWT FVIIa and WT FVIIa. INTEM assays were performed with ellagic acid for activating the intrinsic pathway. While all FVIIa variants shortened the average clotting time, CT‐001 and CT‐001(Sial) were superior to either dWT FVIIa or WT FVIIa (n=4 independent normal donors). ANOVA, analysis of variance; aPTT, activated partial thromboplastin time; dWT, desialylated wild‐type; FVIIa, activated factor VII; FXa, activated factor X; INTEM, intrinsic pathway thromboelastometry; PT, prothrombin time; TF, tissue factor; WT, wild‐type