Acute traumatic coagulopathy (ATC) occurs in ≈30% of trauma patients and is associated with increased mortality. Excessive generation of activated protein C (APC) and hyperfibrinolysis are believed to be driving forces for ATC. Two mouse models were used to investigate whether an engineered activated FV variant (superFVa) that is resistant to inactivation by APC and contains a stabilizing A2-A3 domain disulfide bond, is able to reduce traumatic bleeding and normalize hemostasis parameters in ATC. First, ATC was induced by the combination of trauma and shock. ATC was characterized by APTT prolongation and reductions of FV, FVIII, and fibrinogen, but not FII and FX. Administration of superFVa normalized the APTT, returned FV and FVIII clotting activity levels to their normal range, and reduced APC and thrombin-antithrombin (TAT) levels, indicating improved hemostasis. Next, a liver laceration model was used where ATC develops as the consequence of severe bleeding. SuperFVa prophylaxis prior to liver laceration reduced bleeding, prevented APTT prolongation, depletion of FV and FVIII, and excessive generation of APC. Thus, prophylactic administration of superFVa prevented the development of ATC. SuperFVa intervention started after the development of ATC stabilized bleeding, reversed the prolonged APTT, returned FV and FVIII levels to their normal range, and reduced TAT levels that were increased by ATC. In summary, superFVa prevented ATC and traumatic bleeding when administered prophylactically, and superFVa stabilized bleeding and reversed abnormal hemostasis parameters when administered while ATC was in progress. Thus, superFVa may be an attractive strategy to intercept ATC and mitigate traumatic bleeding.

Iron deficiency is the most common nutritional disorder worldwide, often resulting in severe anemia. At highest risk are children and pregnant women in developing countries where maternal mortality is very high (~500-1000 per 100,000 births) compared to developed countries (~5-10 per 100,000 births). Postpartum hemorrhage (PPH), defined as 500 -1000 mL of blood loss, is responsible for 30 - 50% of the deaths. Clinical evidence suggests that anemia increases the risk of PPH, which is associated with poor outcomes. These observations have large implications in areas like Sub-Saharan Africa, where the prevalence of iron deficiency anemia (IDA) approaches 80%. To study the effects of IDA on traumatic bleeding we developed a mouse model of IDA and traumatic injury, and tested the utility of prophylactic tranexamic acid (TXA) to mitigate bleeding. TXA is an anti-fibrinolytic agent that can be administered orally or intravenously, and reduces mortality when given shortly after trauma or PPH. Materials and Methods First, we generated mice with severe IDA. Different groups of C57BL/6J mice, both male and female, were subjected to three different diets starting at 3 weeks of age: "iron deficient diet" (4ppm iron), "standard iron diet" (48ppm iron) and usual laboratory chow. After 6 weeks, a complete blood count (CBC) was performed to document the degree of anemia and red cell indices and mice were subjected to an established liver laceration model causing severe bleeding. In some groups of mice, TXA or saline were administrated as bolus (10mg/kg) before liver laceration. Blood loss was assessed by weighing blood-soaked sponges in the abdominal cavity 10 minutes after injury. All data were expressed as mean ± SD and were compared using non-parametric Kruskal-Wallis one-way ANOVA or Mann-Whitney test. Results Only mice fed with the "iron deficient diet" developed hypochromic and microcytic anemia with a mean hematocrit of 32±1.7%. The hematocrit of mice fed with the "standard iron diet" or usual laboratory chow (control groups) was within normal range and similar (49±1.2%). Mice with IDA subjected to liver laceration demonstrated significantly increased bleeding when compared to the control groups. Blood loss in mice with IDA was 0.50±0.01g compared to ~ 0.40±0.01g in the control groups (p<0.0001). Prophylactic treatment with TXA reduced bleeding significantly in all groups of mice to ~ 0.33±0.01g (p<0.0001), demonstrating that TXA controlled bleeding similarly in anemic and non-anemic mice. Conclusion IDA causes enhanced bleeding in a murine trauma model which was mitigated significantly by administration of prophylactic TXA. While the mechanisms by which IDA increases traumatic bleeding are not well defined, our observations point towards a pragmatic, prophylactic role of TXA in areas with a high prevalence of IDA (i.e. Sub-Saharan Africa) and high mortality associated with potentially preventable bleeding situations like PPH. In fact, a clinical trial to test this hypothesis is currently ongoing (Women-2; https://clinicaltrials.gov/ct2/show/NCT03475342). It is in this context that our mouse model could be utilized to study the etiology, treatments and outcomes of augmented traumatic bleeding in IDA, the most prevalent cause of anemia in the developing world. Disclosures von Drygalski: Biomarin, Bioverativ/Sanofi-Genzyme, Novo Nordisk, Pfizer, Uniqure, Takeda: Consultancy; Hematherix Inc: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder; superFVa; Joint Activity and Damage Examination (JADE) Ultrasound measurement tool: Patents & Royalties.

Clinical evidence suggests that anemia increases surgical bleeding and is associated with poor outcomes. Globally, ~ 50% of anemia is attributed to iron deficiency as the most common nutritional disorder. To study the effects of iron deficiency anemia (IDA) on traumatic bleeding we developed a mouse model of IDA and traumatic injury, and tested the effects of prophylactic tranexamic acid (TXA), on blood loss, coagulopathy and survival. Materials and Methods C57BL/6J mice, both male and female, were fed with usual laboratory ("control mice") or iron deficient (4ppm iron) chow ("IDA mice") starting at 3 weeks of age. After 6 weeks, IDA was documented by blood count, red cell indices and liver iron content. Mice then were subjected to an established liver laceration model causing severe bleeding. Blood loss (weighing blood-soaked sponges in the abdominal cavity), seven-day survival and coagulation parameters (APTT, activity levels of Factor (F) V, FVIII, Fibrinogen) were determined at 15 and 60 minutes after liver laceration for "IDA" and "control mice", treated or not treated five minutes prior to injury with TXA (10mg/kg). All data were expressed as median and were compared using Mann-Whitney test. Results Compared to "control" mice, "IDA mice" developed hypochromic and microcytic anemia with a significantly lower mean hematocrit (32±1.7% vs 49±1.2%, p≤0.0001) and hepatic iron content (75.9±19.8µg/g vs 22.4±9.4 µg/g, p≤0.0001). "IDA mice" demonstrated significantly more bleeding after liver laceration compared to "control mice." Most of the bleeding had occurred at 15 minutes after injury with little incremental bleeding at 60 minutes. The blood loss in "IDA mice" was greater at both time points compared to "control mice" (15 minutes: 21.5±2.3µl/g vs 15.9±2.6µl/g, p≤0.0001; 60 minutes: 24.5±1.6µl/g vs 20.7±1.9µl/g, p≤0.0001). TXA reduced bleeding significantly in "IDA mice" at 15 and 60 min to ~15µl/g. In "control mice", bleed improvement was only present at 60 min (also reduced to ~15µl/g), without improvement below the ~15µl/g baseline threshold at 15 min. Coagulopathy developed in both groups over 60 min, with similar prolongation of the APTT from baseline (23.9±1.5s to 36.0±4.9s in "IDA mice"; 22.8±1.3s 35.0±3.8s in "control mice"), substantial depletion of FV and FVIII activity levels and partial reduction of fibrinogen. TXA administration normalized the APTT only in "control mice" at 60 minutes, but not in "IDA mice", although TXA reconstituted FV and fibrinogen to ~100% activity, with FVIII activity of ~50% in both groups. Survival of "IDA mice" was lower compared to "control mice" (50% vs 75%), but increased significantly with TXA (80%, p=0.04). Conclusion Enhanced bleeding and poorer survival was observed in "IDA mice" compared to "control mice" after liver laceration at similar degrees of coagulopathy. Prophylactic TXA corrected bleeding and reduced mortality in "IDA mice" to values similar to those observed for "control mice" despite incomplete correction of the coagulopathy in "IDA mice". This suggests that TXA may be of particular importance in the face of anemia, by mechanisms that may go beyond hemostasis correction alone, warranting further investigation. Disclosures von Drygalski: CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hematherix, Inc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Super FVa; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biomarin: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; uniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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