Functional role of SLFN14 in thrombosis. (A) Tail bleeding time assay. Two to 3 millimeters of tail was removed, and bleeding time until first stop was measured. Each data point represents 1 animal; n = 18 to 20 mice per genotype. (B) Clot retraction of SLFN14^K208N/+ mouse platelets in PRP. Clots were formed by stimulating 2 × 10⁸ platelets per milliliter with 0.1 U/mL thrombin; monitoring took place for 2 hours. Representative images (Bi) and final clot weight (Bii). Data are mean ± SEM; n = 8 or 9 mice per genotype. (C) Laser-induced thrombus formation in vivo. (Ci) Representative composite brightfield and fluorescence images of thrombus formation. Mice were injected with anti-GPIbβ DyLight488 (0.1µg/g body weight). Arterioles of the cremaster muscle were subsequently injured by laser (arrowheads) and thrombi fluorescence was measured. Scale bars, 10 µm. (Cii) Graph showing median integrated thrombus formation fluorescence intensity in arbitrary units (a.u.) for 31 or 32 injuries in 4 mice per genotype. (D) FeCl₃-induced thrombus formation. Mice were injected with DyLight488-conjugated anti-GPIbβ antibody (0.1 µg/g body weight), and the carotid artery was subsequently injured with 10% FeCl₃ solution for 3 minutes. (Di) Representative fluorescence images of platelets (GPIbβ). Scale bars, 200 µm. (Dii) Graph showing median integrated thrombus fluorescence. (Diii) Area under the curve (AUC) of the integrated fluorescence density (in a.u). Data are mean; n = 11 or 12 mice per genotype. See supplemental Videos 3 and 4 for wild-type and mutants, respectively.