FXII inhibition attenuates stroke severity in HbSS mice after tMCAO. Quantification of (A) stroke score, (B) proportion of stroke severity, (C) and brain infarct in HbAA and HbSS mice subjected to brain ischemia/reperfusion injury after treatment with IgG or 15D10 antibodies (10 mg/kg, IV). Intravital microscopy analysis was performed to assess the number of (D) rolling and (E) adherent leukocytes. Data represent mean ± SEM; n = 6 mice per group. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 by one-way ANOVA and Tukey post-hoc test. (F) Microscale thermophoresis was performed to measure direct binding between uPAR and FXII, in the absence (blue line) or presence (red line) of 15D10. Recombinant His-tagged murine FXII was fluorescently labeled with RED-tris-nitrilotriacetic acid and subsequently incubated with 15 μM Zn²⁺ and serially diluted murine uPAR. Where indicated, 1 μM of 15D10 was added to the reaction mixture. Initial fluorescence intensity of RED-FXII was used to normalize fluorescence changes (ΔFnorm representing the bound fraction). Binding constants (Kd) over time were determined for FXII-uPAR based on triplicate measurements of n = 3 individual experiments. ANOVA, analysis of variance.