Contribution of RBC-exposed phosphatidylserine to TG. (A) TG was triggered by 0.1 pM tissue factor in the WB in the presence of annexin A5 (A5; 0.47-7.50 μg/mL) or mutant annexin A5 (MA5; 7.50 μg/mL), which lacks Ca²⁺- and phosphatidylserine-binding sites. Shown are representative calibrated curves of 3 independent experiments. (B) Schematic procedure of using isolated RBC, treatment with annexin A5 triple wash in the presence of 2 mM CaCl₂, and reconstitution with autologous PRP. (C,F) TG effect of RBC treatment with different annexin A5 concentrations (A5, 18.5-75.0 μg/mL) reconstituted at 35% hematocrit in the presence of platelets. Indicated are representative TG curves (C) and thrombin peak levels (F). Means ± SD (n = 3), 1-way ANOVA, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (D,G) TG effect of RBC treatment with vehicle or 37.5 μg/mL annexin A5, reconstituted at 20% or 40% hematocrit. Representative TG curves (D) and quantified thrombin peak levels (G). Means ± SD (n = 3); 2-way ANOVA, ∗P < .05; ∗∗P < .01. (E,H) TG effect of RBC treatment with different annexin A5 concentrations (A5, 18.5-75.0 μg/mL) reconstituted at 35% hematocrit in the absence of platelets. Representative TG curves (E) and quantified thrombin peak levels (H). Means ± SD (n = 3), 1-way ANOVA, ∗P < .05. Full data are in Datafile 1. Note: 0% RBC plus annexin A5 refers to a triple washing with Hepes buffer. Reconst, reconstituted.