The effect of Affimer F5 on clot lysis in FVIII-deficient plasma, and on lysis and platelet activation in whole blood. (A) Turbidimetric experiments in which clot formation was initiated with tissue factor (5 pM) were performed using normal plasma and FVIII-deficient plasma. The effect of increasing concentrations of Affimer F5 were tested in FVIII-deficient plasma, with scaffold-only protein included as a control. Numbers on the x-axis represent Affimer:fibrinogen molar ratios. Data are presented as the mean ± SD of 3 independent experiments. Unpaired Student t test was used to compare buffer-only controls in normal and FVIII-deficient plasma; **P < .01. 1-way ANOVA was used to determine the significance of Affimer F5 effect in FVIII-deficient plasma at different concentrations. Asterisks represents difference from buffer-only control; *P < .05; **P < .01. (B) The effect of F5 on platelet activation was tested using whole blood from 3 healthy volunteers. Blood was incubated with buffer only (green peak), scaffold (blue peak), or F5 (red peak) and then stimulated with thrombin receptor activating peptide. CD62P expression was compared with basal platelets (purple peak). Affimer proteins were used at 10:1 Affimer:fibrinogen molar ratio. (C) The effect of increasing concentrations of Affimer F5 on whole-blood lysis was tested using human whole blood from 3 healthy individuals using ROTEM. Tissue factor and tPA were used for initiation of clotting and lysis, respectively. (D) Maximum clot firmness from ROTEM experiments. (E) Lysis time of whole-blood clots formed in the presence of Affimer F5. Numbers on the x-axis represent Affimer protein:fibrinogen molar ratios. Data are presented as the mean ± SD of 3 independent experiments. Statistical analysis was performed using 1-way ANOVA. Asterisks represents difference from buffer-only control; *P < .05; **P < .01.