(A) Schematic representation of the hemostatic efficacy studies in BALB/c mice. Mice were intravenously administered with saline, HA, or HA-peptide conjugates, and a tail vein laceration was induced following 1- or 20-min circulation time. Bleeding times (B, D, and F) and total blood loss (C, E, and G) were quantified. (B and C) Hemostatic efficacy of HA-CBP, HA-VBP, HA-VBP/HA-CBP, and HAPPI with untreated, saline, native HA, and free peptides (CBP and VBP) as controls. In HA-CBP, HA-VBP, and HAPPI groups, mice were administered with 4.6 mg/kg of equivalent HA dose. HA-VBP/HA-CBP was formulated by mixing the desired amounts of HA-VBP and HA-CBP conjugates to obtain final individual concentrations equal to the HA-VBP group and HA-CBP group, respectively (n = 5 mice). (D and E) HA-CBP*, HA-VBP*, and HAPPI* were dosed with the same amount of peptides as detailed in Materials and Methods (n ≥ 3 mice). (F and G) Efficacy of HAPPI with 20 min circulation time was compared with untreated mice and mice treated with saline of 20 min circulation and HAPPI of 1 min circulation time (n ≥ 4 mice). All data are means ± SEM; statistics by two-tailed, nonparametric Mann-Whitney test (*P < 0.05 and **P < 0.01) and Kruskal-Wallis test followed by Dunn’s multiple comparisons test (#P < 0.05, ##P < 0.01, and ###P < 0.01). ns, not significant; Hb, hemoglobin.