Blood Loss In Mice Research Articles

Traumatic Bleeding in Mice Is Amplified By Iron Deficiency Anemia and Can be Mitigated By Tranexamic Acid Prophylaxis

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.

Topical tranexamic acid inhibits fibrinolysis more effectively when formulated with self‐propelling particles

BackgroundEndogenous fibrinolytic activation contributes to coagulopathy and mortality after trauma. Administering tranexamic acid (TXA), an antifibrinolytic agent, is one strategy to reduce bleeding; however, it must be given soon after injury to be effective and minimize adverse effects. Administering TXA topically to a wound site would decrease the time to treatment and could enable both local and systemic delivery if a suitable formulation existed to deliver the drug deep into wounds adequately. ObjectivesTo determine whether self‐propelling particles could increase the efficacy of TXA. MethodsUsing previously developed self‐propelling particles, which consist of calcium carbonate and generate CO2 gas, TXA was formulated to disperse in blood and wounds. The antifibrinolytic properties were assessed in vitro and in a murine tail bleeding assay. Self‐propelled TXA was also tested in a swine model of junctional hemorrhage consisting of femoral arteriotomy without compression. ResultsSelf‐propelled TXA was more effective than non‐propelled formulations in stabilizing clots from lysis in vitro and reducing blood loss in mice. It was well tolerated when administered subcutaneously in mice up to 300 to 1000 mg/kg. When it was incorporated in gauze, four of six pigs treated after a femoral arteriotomy and without compression survived, and systemic concentrations of TXA reached approximately 6 mg/L within the first hour. ConclusionsA formulation of TXA that disperses the drug in blood and wounds was effective in several models. It may have several advantages, including supporting local clot stabilization, reducing blood loss from wounds, and providing systemic delivery of TXA. This approach could both improve and simplify prehospital trauma care for penetrating injury.

Reversal of Novel Oral Anticoagulant (NOAC)-Induced Bleeding in Mice By Engineered superfactor Va

Introduction and Objectives: Novel oral anticoagulants (NOACs), such as Factor (F) Xa inhibitors (Rivaroxaban and Apixaban) or the direct thrombin inhibitor (Dabigatran), are used for prevention of venous thromboembolism and ischemic strokes. However, conventional hemostatic reversal strategies in case of bleeding or surgery are ineffective, posing a major unmet clinical need. We recently demonstrated that superFVa, a novel FVa variant engineered with mutations of 3 activated protein C (APC) cleavage sites and a disulfide bond between the A2 and A3 domains, has enhanced biological activity and APC resistance. SuperFVa provided efficient normalization of hemostasis in hemophilia A mice and in animal models of APC-induced bleeding. Moreover, superFVa synergistically reduced bleeding in combination with recombinant human (rh) FVIIa in hemophilia mice. Thus, superFVa fits the criteria for a prohemostatic biologic. Therefore, the in vitro and in vivo effects of superFVa alone and in combination with other prohemostatic agents such as rhFVIIa or 4-Factor prothrombin complex concentrates (4F-PCC) as a novel hemostatic reversal strategy for NOAC-induced bleeding were determined.Materials and Methods: In vitro procoagulant properties of superFVa alone and in combination with rhFVIIa or 4F-PCC were studied using thrombin generation assays in normal human plasma (NHP) in the presence of FXa inhibitors (Rivaroxaban, Apixaban) or the direct thrombin inhibitor (Dabigatran, active form). In vivo prevention of blood loss by superFVa after intravenous injection of Rivaroxaban, Apixaban, or Dabigatran was studied using the tail clip model in BalbC mice.Results: Rivaroxaban and Apixaban each dose-dependently reduced thrombin generation in NHP and reduced the Endogenous Thrombin Potential (ETP) by ~60% at therapeutic concentrations (200 nM). Addition of either superFVa (50 nM) or rhFVIIa (40 nM, equivalent to a 90 µg/kg therapeutic dose in hemophilia patients), increased thrombin generation to some extent. However, ETP and thrombin peak height increased dose-dependently when increasing concentrations of superFVa (6.25 to 400 nM) were added with rhFVIIa (40 nM). superFVa effects appeared synergistic, with a plateau reached at 25-50 nM superFVa and ETP restored to ~93% of normal. Synergistic effects of superFVa were also present and even more pronounced in combination with 4F-PCC (1.35 U/ml; therapeutic plasma concentration).In the presence of Dabigatran (1 µM),superFVa (0.1 nM-100 nM) in combination with rhFVIIa (40 nM) or 4F-PCC (1.35 U/ml) showed a concentration dependent reduction of the lag time of thrombin generation. However, in contrast to the FXa inhibitors, in the presence of Dabigatran, no effects on ETP and thrombin peak height were observed for the addition of superFVa or combinations of superFVa and rhFVIIa or 4F-PCC.The in vivo efficacy of superFVa to reduce NOAC-induced bleeding was determined by blood loss after tail transection in BalbC mice injected i.v. with Rivaroxaban (40 mg/kg), Apixaban (20 mg/kg), or Dabigatran (0.4 mg/kg). Mean blood loss in mice injected with Rivaroxaban (16 µL/g), Apixaban (16.5 µL/g) or Dabigatran (14.5 µL/g) was significantly higher than baseline bleeding (4 µL/g, p<0.001). superFVa reduced blood loss after Rivaroxaban or Apixaban administration significantly in a dose dependent manner, e.g., superFVa at 40 U/kg significantly reduced bleeding to the baseline control level (5 µL/g). RhFVIIa (1 mg/kg) was able to reduce bleeding caused by Rivaroxaban but not by Apixaban. Neither superFVa nor rhFVIIa was able to reduce bleeding caused by Dabigatran.Conclusion:superFVa alone or in combination with rhFVIIa significantly improved in vitro thrombin generation in the presence of FXa inhibitors and to some extent in the presence of a direct thrombin inhibitor. SuperFVa alone consistently reduced bleeding in mice treated with FXa inhibitors, whereas a mixed response dependent on the type of FXa inhibitor was obtained with rhFVIIa. None of the agents was able to decrease bleeding in mice induced by Dabigatran. Because superFVa exerts a potent class effect as a hemostatic reversal strategy for FXa inhibitor-induced bleeding, it deserves further consideration for potential development as a hemostatic agent. DisclosuresGale:University of California, San Diego: University of California, San Diego Patents & Royalties. Griffin:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties. Mosnier:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties. von Drygalski:University of California, San Diego: University of California, San Diego Patents & Royalties.

Prothrombin complex concentrates reduce blood loss in murine coagulopathy induced by warfarin, but not in that induced by dabigatran etexilate

Both established oral anticoagulants such as warfarin and newer agents such as dabigatran etexilate (DE) effectively prevent thromboembolic disease, but may provoke bleeding. Limited clinical data exist linking oral anticoagulant reversal and bleeding tendency, as opposed to surrogate laboratory markers. To quantify bleeding in warfarin-anticoagulated and DE-anticoagulated mice by tail transection with or without pretreatment with potential reversal agents: prothrombin complex concentrate (PCC); activated PCC (APCC); recombinant factor VIIa (rFVIIa); or murine fresh-frozen plasma (FFP). CD1 mice were given warfarin or DE by gavage, and the effects on in vitro coagulation assays, volume of blood loss and the bleeding time following tail transection injury were evaluated with different reversal agents. PCC (14.3 IU kg(-1) ), but not rFVIIa (3 mg kg(-1) ) or FFP (12 mL kg(-1) ), normalized blood loss and bleeding time in mice with warfarin-induced elevations of mean prothrombin time at two intensities (prothrombin time ratios of either 4.3 or 24). Neither separate nor combined PCC and/or rFVIIa treatment nor APCC (100 U kg(-1) ) treatment significantly reduced blood loss in mice anticoagulated with 60 mg kg(-1) DE 75 min prior to tail transection. Both combined PCC plus rFVIIa treatment and APCC treatment significantly reduced bleeding time in the DE-treated mice. Our data suggest that PCC treatment prevents excess bleeding much more effectively in warfarin-induced coagulopathy than in DE-induced coagulopathy.

Granulocytes and Vascularization Regulate Uterine Bleeding and Tissue Remodeling in a Mouse Menstruation Model

Menstruation-associated disorders negatively interfere with the quality of life of many women. However, mechanisms underlying pathogenesis of menstrual disorders remain poorly investigated up to date. Among others, this is based on a lack of appropriate pre-clinical animal models. We here employ a mouse menstruation model induced by priming mice with gonadal hormones and application of a physical stimulus into the uterus followed by progesterone removal. As in women, these events are accompanied by menstrual-like bleeding and tissue remodeling processes, i.e. disintegration of decidualized endometrium, as well as subsequent repair. We demonstrate that the onset of bleeding coincides with strong upregulation of inflammatory mediators and massive granulocyte influx into the uterus. Uterine granulocytes play a central role in regulating local tissue remodeling since depletion of these cells results in dysregulated expression of matrix modifying enzymes. As described here for the first time, uterine blood loss can be quantified by help of tampon-like cotton pads. Using this novel technique, we reveal that blood loss is strongly reduced upon inhibition of endometrial vascularization and thus, is a key regulator of menstrual bleeding. Taken together, we here identify angiogenesis and infiltrating granulocytes as critical determinants of uterine bleeding and tissue remodeling in a mouse menstruation model. Importantly, our study provides a technical and scientific basis allowing quantification of uterine blood loss in mice and thus, assessment of therapeutic intervention, proving great potential for future use in basic research and drug discovery.

The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

AbstractTwo major pathways contribute to Ras-proximate-1–mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, RasGRP2) mediates the rapid but reversible activation of integrin αIIbβ3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI−/− blood, even in the presence of exogenous adenosine diphosphate and thromboxane A2. In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI−/− mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro- and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from athero-thrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel.

Ability of peritoneal exudate cells to trigger various mechanisms of erythroid differentiation at different terms after massive blood loss in mice.

Thirty minutes after massive blood loss, peritoneal cells gained the ability to trigger reparative erythropoiesis in the bone marrow of normal syngeneic recipients. This was manifested in proliferation of oxyphilic erythroblasts and activation of the reserve pathway of erythroid differentiation 4 days after cell transplantation. The maximum transfer capacity of peritoneal cells was observed 4 h after blood loss, but 4 days later they only initiated mitoses in oxyphilic erythroblasts.

The role of peritoneal T lymphocytes and macrophages in the initiation of stress erythropoiesisin vivo following a single massive blood loss in mice

Peritoneal cells of anemic donors enriched with macrophages at the expense of T-cell lysis using anti-Thy-1.2 monoclonal antibodies are capable of triggering various routes of terminal erythroid differentiation characteristic of stress erythropoiesis during their adoptive transfer to normal syngeneic recipients. Stimulation of the proliferation of polychromatophilic erythroblasts and initiation of mitoses in oxyphilic erythroblasts are regulated by interacting T cells and macrophages, and the “reserve erythropoiesis” mainly by macrophages.

Hemorrhage produces abnormalities in lymphocyte function and lymphokine generation.

Hemorrhage has been shown to produce abnormalities in lymphocyte function, particularly in the proliferative response to mitogens such as PHA and Con A. In order to better examine the hemorrhage-induced alterations in immune function, we determined the effects of blood loss in mice without any surgical manipulation on lymphocyte populations and subpopulations, cellular activation, and lymphokine production. Hemorrhage induced no changes in cell numbers in the spleen, thymus, lymph nodes, and bone marrow. No alterations in the relative percentages of B (B220+, mu+) and T (Lyt-1+, Lyt-2+, T3+, L3T4+) cell subpopulations were found in any organ after blood loss. Significant decreases in splenocyte proliferation in response to Con A, IL-2R expression and blast formation occurred after hemorrhage. IFN-gamma production increased 24 and 48 h post hemorrhage. Decreases in IL-2, IL-3, and IL-5 generation were present 2 h after blood loss. IL-2 production remained significantly decreased for 48 h posthemorrhage, then increased to more than twice normal levels 72 h posthemorrhage, and subsequently returned to prehemorrhage values. These results demonstrate that hemorrhage produces widespread alterations in immune function without affecting lymphocyte population and subpopulation numbers.