Ruggedized Self-Propelling Hemostatic Gauze Delivers Low Dose of Thrombin and Systemic Tranexamic Acid and Achieves High Survival in Swine With Junctional Hemorrhage.

INTRODUCTION: Hemorrhage is responsible for 91% of preventable prehospital deaths in combat. Bleeding from anatomic junctions such as the groin, neck, and axillae make up 19% of these deaths, and reports estimate that effective control of junctional hemorrhage could have prevented 5% of fatalities in Afghanistan. Hemostatic dressings are effective but are time-consuming to apply and are limited when proper packing and manual pressure are not feasible, such as during care under fire. CounterFlow-Gauze is a hemostatic dressing that is effective without compression and delivers thrombin and tranexamic acid into wounds. Here, an advanced prototype of CounterFlow-Gauze, containing a range of low thrombin doses, was tested in a lethal swine model of junctional hemorrhage. Outcomes were compared with those of Combat Gauze, the current dressing recommended by Tactical Combat Casualty Care. MATERIALS AND METHODS: CounterFlow-Gauze containing thrombin doses of 0, 20, 200, and 500 IU was prepared. Swine received femoral arteriotomies, and CounterFlow-Gauze was packed into wounds without additional manual compression. In a separate study using a similar model of junctional hemorrhage without additional compression, CounterFlow-Gauze containing 500 IU thrombin was tested and compared with Combat Gauze. In both studies, the primary outcomes were survival to 3 h and volume of blood loss. RESULTS: CounterFlow-Gauze with 200 and 500 IU had the highest 3-h survival, achieving 70 and 75% survival, respectively. CounterFlow-Gauze resulted in mean peak plasma tranexamic acid concentrations of 9.6 ± 1.0 µg/mL (mean ± SEM) within 3 h. In a separate study with smaller injury, CounterFlow-Gauze with 500 IU achieved 100% survival to 3 h compared with 92% in Combat Gauze animals. CONCLUSIONS: An advanced preclinical prototype of CounterFlow-Gauze formulated with a minimized thrombin dose is highly effective at managing junctional hemorrhage without compression. These results demonstrate that CounterFlow-Gauze could be developed into a feasible alternative to Combat Gauze for hemorrhage control on the battlefield.

Self-propelling thrombin powder enables hemostasis with no observable recurrent bleeding or thrombosis over 3 days in a porcine model of upper GI bleeding.

BACKGROUND AND AIMS: Hemostatic powders used to manage upper GI bleeding continue to exhibit high recurrent bleeding rates. Previously, self-propelling thrombin powder (SPTP) sprayed endoscopically managed severe Forrest class 1A bleeding. Here, we evaluate SPTP in a 3-day recovery model of diffuse ulcerated bleeding. METHODS: Five anesthetized pigs underwent an endoscopic mucosal snare resection to trigger diffuse ulcer bleeding and were treated with SPTP. The time to hemostasis and the amount of powder delivered were measured. Pigs were recovered and monitored. RESULTS: Five pigs achieved hemostasis in 4.5 ± 1.2 minutes At 3 days after the procedure, the pigs were rescoped and showed no recurrent bleeding. Measured blood parameters were not significantly different from baseline. There were no signs of foreign bodies or thromboembolism during gross necropsy and histopathology of key organs. CONCLUSIONS: SPTP is a promising novel material that stopped diffuse ulcer bleeding in 5 pigs without recurrent bleeding or adverse local or systemic events.

Percutaneous delivery of self-propelling thrombin-containing powder increases survival from noncompressible truncal hemorrhage in a swine model of coagulopathy and hypothermia.

BACKGROUND: Noncompressible truncal hemorrhage (NCTH) remains a leading cause of preventable death on the battlefield. Definitively managing severe NCTH requires surgery within the first hour after injury, which is difficult when evacuating casualties from remote and austere environments. During delays to surgery, hemostatic interventions that are performed prehospital can prevent coagulopathy and hemorrhagic shock and increase the likelihood that casualties survive to receive definitive care. We previously reported that a self-propelling thrombin-containing powder (SPTP) can be delivered percutaneously into the abdomen as a minimally invasive intervention and can self-disperse through pooled blood to deliver the hemostatic agents thrombin and tranexamic acid locally to noncompressible intracavitary wounds. We hypothesized that, in swine with massive NCTH, dilutional coagulopathy, and hypothermia, delivering SPTP could extend survival times. METHODS: Ten swine (n = 5 per group) underwent NCTH from a Grade V liver injury following a midline laparotomy. The laparotomy was closed with sutures afterwards, creating a hemoperitoneum, and animals were managed with crystalloid fluid resuscitation, or crystalloid resuscitation and SPTP. Self-propelling thrombin-containing powder was delivered into the closed abdomen using a CO 2 -powered spray device and a catheter placed into the hemoperitoneum, entering through the upper right quadrant using the Seldinger technique. Survival to 1 and 3 hours was recorded. In an additional animal, hemorrhage was created laparoscopically, and SPTP was imaged in situ within the abdomen to visually track dispersion of the particles. RESULTS: Self-propelling thrombin-containing powder dispersed as far as 35 ± 5.0 cm within the abdomen. It increased survival to 1 and 3 hours (Kaplan-Meier p = 0.007 for both). The median survival time was 61 minutes with SPTP and 31 minutes without ( p = 0.016). CONCLUSION: Self-propelling thrombin-containing powder effectively disperses medications throughout a hemoperitoneum and increases survival in a model of NCTH. It is a promising strategy for nonsurgical management of NCTH, warranting further testing of its safety and efficacy.

Videoconferencing for Large Animal Trauma Experiments During COVID-19: A Cross-Continent Experience.

INTRODUCTION: COVID-19 shutdowns in many research facilities across North America impacted preclinical trauma-related research and development. Shutdown limited the speed and resources available for large animal experiments necessary for advancing medical devices and technologies. However, the pandemic led to the rapid adoption and expansion of videoconferencing in social circles, workplaces, and primary care health settings. Here, we describe the use of simple videoconferencing equipment to plan and carry out 3 total weeks of large animal experiments with a large, cross-continent, interdisciplinary team testing a novel technology in swine models of noncompressible intraabdominal hemorrhage and junctional hemorrhage. MATERIALS AND METHODS: Animal experiments using swine were scheduled over 3 weeks in February and March 2021 to take place in Toronto, Canada. All relevant animal protocols and COVID-19 site-specific risk assessments were completed and approved by the responsible institutional committees. Experiments were conducted by connecting 12 total research personnel from 3 sites by a simple video conferencing setup which included low-cost, high-definition webcams and standard smartphones streaming to Zoom. RESULTS: Video conferencing allowed for 3 weeks of trauma experiments to take place at the height of Toronto's third peak of COVID-19 cases. Up to 3 experiments were completed for models requiring 6 hours of monitoring, and up to 5 experiments were completed for models requiring 3 hours of monitoring. The large amount of digital data collected during these experiments was rapidly shared with our network of collaborators, who analyzed results and interpreted findings in real time. CONCLUSIONS: The system described in this paper has the potential to reduce costs of trauma animal model development and allow for rapid testing and implementation of life-saving devices in settings with limited onsite personnel as experienced during the COVID-19 pandemic.

Percutaneous delivery of self-propelling hemostatic powder for managing non-compressible abdominal hemorrhage: a proof-of-concept study in swine.

INTRODUCTION: Non-compressible intra-abdominal hemorrhage (NCIAH) is a major cause of preventable death on the battlefield and in civilian trauma. Currently, it can only be definitively managed with surgery, as there are limited strategies for controlling ongoing NCIAH in the prehospital environment. We hypothesized that a self-propelling thrombin-containing powder (SPTP) could increase survival in a swine model of NCIAH when delivered percutaneously into the closed abdomen using an engineered spray system. MATERIALS AND METHODS: Nineteen swine underwent surgical laparotomy followed by a Grade V liver injury that created massive hemorrhage, before closing the abdomen with sutures. Animals either received treatment with standard of care fluid resuscitation (n=9) or the SPTP spray system (n=10), which consisted of a spray device and a 14 Fr catheter. Using the spray system, SPTP was delivered into a hemoperitoneum identified using a focused assessment with sonography in trauma (FAST) exam. Lactated Ringer's solution was administered to all animals to maintain a mean arterial pressure (MAP) of >50 mmHg. The primary outcome was percentage of animals surviving at three hours following injury. RESULTS: In the swine model of NCIAH, a greater percentage of animals receiving SPTP survived to three hours, although differences were not significant. The SPTP spray system increased the median survival of animals from 1.6 hr in the fluid resuscitation group to 4.3 hr. The SPTP spray system delivered a total mass of 18.5 ± 1.0 g of SPTP. The mean change in intra-abdominal pressure following SPTP delivery was 5.2 ± 1.8 mmHg (mean ± SEM). The intervention time was 6.7 ± 1.7 min. No adverse effects related to the SPTP formulation or the spray system were observed. SPTP was especially beneficial in animals that had either severely elevated lactate concentrations or low mean arterial pressure of <35 mmHg shortly after injury. CONCLUSIONS: This demonstrates proof-of-concept for use of a new minimally invasive procedure for managing NCIAH, which could extend survival time to enable patients to reach definitive surgical care.

The adhesion of clots in wounds contributes to hemostasis and can be enhanced by coagulation factor XIII.

The adhesion of blood clots to wounds is necessary to seal injured vasculature and achieve hemostasis. However, it has not been specifically tested if adhesive failure of clots is a major contributor to rebleeding and what mechanisms prevent clot delamination. Here, we quantified the contribution of adhesive and cohesive failure to rebleeding in a rat model of femoral artery injury, and identified mechanisms that contribute to the adhesive strength of bulk clots in a lap-shear test in vitro. In the rat bleeding model, the frequency of clot failures correlated positively with blood loss (R = 0.81, p = 0.014) and negatively with survival time (R = - 0.89, p = 0.0030), with adhesive failures accounting for 51 ± 14% of rebleeds. In vitro, adhesion depended on fibrinogen and coagulation factor XIII (FXIII), and supraphysiological FXIII improved adhesive strength. Furthermore, when exogenous FXIII was topically applied into the wound pocket of rats, eleven adhesive failures occurred between eight rats, compared to seventeen adhesive failures between eight untreated rats, whereas the number of cohesive failures remained the same at sixteen in both groups. In conclusion, rebleeding from both adhesive and cohesive failure of clots decreases survival from hemorrhage in vivo. Both endogenous and exogenous FXIII improves the adhesive strength of clots.

Bleeding is increased in amyloid precursor protein knockout mouse.

BACKGROUND: Amyloid precursor protein (APP) is highly expressed in platelets. APP is the precursor to amyloid beta (Aß) peptides that accumulate in cerebral amyloid angiopathy and plaques in Alzheimer disease. APP and its metabolites interact with many components of the coagulation system, and have both anticoagulant and procoagulant properties, but it is unclear if APP contributes to hemostasis in vivo. OBJECTIVES: To determine whether APP contributes to hemostasis in mice, including when inhibitors of coagulation are administered. METHODS: Blood loss in APP knockout (KO) mice was measured in liver laceration and tail transection models of hemorrhage. Blood loss was also measured following tail transection in mice given an inhibitor of coagulation factor Xa (apixaban), platelet inhibitors (aspirin + clopidogrel), tissue-type plasminogen activator (t-PA), or the antifibrinolytic tranexamic acid (TXA). RESULTS AND DISCUSSION: Blood loss from liver lacerations was similar between APP KO mice and wild-type (WT) mice, but APP KO mice bled more from tail transections. When mice were challenged with aspirin + clopidogrel, the difference in bleeding between APP KO and WT mice was abrogated. In contrast, a difference in bleeding between the strains persisted when mice were treated with apixaban, t-PA, or TXA. Blood collected from APP KO mice and analyzed with thromboelastography had longer clotting times, and the clots were less stiff and more susceptible to fibrinolysis compared to blood from WT mice. CONCLUSIONS: The absence of APP measurably increases bleeding in mice, which is consistent with a role for platelet-derived APP and Aß peptides in hemostasis.

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

BACKGROUND: Endogenous 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. OBJECTIVES: To determine whether self-propelling particles could increase the efficacy of TXA. METHODS: Using 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. RESULTS: Self-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. CONCLUSIONS: A 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.

Localization of Short-Chain Polyphosphate Enhances its Ability to Clot Flowing Blood Plasma.

Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10-200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown.

Self-Propelled Dressings Containing Thrombin and Tranexamic Acid Improve Short-Term Survival in a Swine Model of Lethal Junctional Hemorrhage.

Hemorrhage is the leading cause of preventable death in trauma, and hemorrhage from noncompressible junctional anatomic sites is particularly difficult to control. The current standard is QuikClot Combat Gauze packing, which requires 3 min of compression. We have created a novel dressing with calcium carbonate microparticles that can disperse and self-propel upstream against flowing blood. We loaded these microparticles with thrombin and tranexamic acid and tested their efficacy in a swine arterial bleeding model without wound compression. Anesthetized immature female swine received 5 mm femoral arteriotomies to induce severe junctional hemorrhage. Wounds were packed with kaolin-based QuikClot Combat Gauze (KG), propelled thrombin-microparticles with protonated tranexamic acid (PTG), or a non-propelling formulation of the same thrombin-microparticles with non-protonated tranexamic acid (NPTG). Wounds were not compressed after packing. Each animal then received one 15 mL/kg bolus of hydroxyethyl starch solution followed by Lactated Ringer as needed for hypotension (maximum: 100 mL/kg) for up to 3 h. Survival was improved with PTG (3-h survival: 8/8, 100%) compared with KG (3/8, 37.5%) and NPTG (2/8, 25%) (P <0.01). PTG animals maintained lower serum lactate and higher hemoglobin concentrations than NPTG (P <0.05) suggesting PTG decreased severity of subsequent hemorrhagic shock. However, total blood loss, Lactated Ringer infusion volumes, and mean arterial pressures of surviving animals were not different between groups (P >0.05). Thus, in this swine model of junctional arterial hemorrhage, gauze with self-propelled, prothrombotic microparticles improved survival and 2 indicators of hemorrhagic shock when applied without compression, suggesting this capability may enable better treatment of non-compressible junctional wounds.

Halting hemorrhage with self-propelling particles and local drug delivery.

Approaches to locally deliver drugs to specific regions of the body are being developed for many clinical applications, including treating hemorrhage. Increasing the concentration of therapeutic coagulants in areas where clots are forming and growing can be achieved by directing them to the injury, such as with catheters or external delivery devices, or by systemically administering therapeutics that target molecular signals of vascular damage. Treating severe hemorrhage by external measures is challenging because blood flow pushes hemostatic agents outward, reducing their efficacy. This review explains that self-propelling particles may be used for delivering therapeutics, such as coagulation factors, small molecules, or other chemical or biological agents, deep into wounds during hemorrhage. A recent example of self-propelling particles is highlighted, where propulsion enhanced the efficacy of a formulation of thrombin and tranexamic acid in treating bleeding in two murine models of hemorrhage and a porcine model of fatal, non-compressible hemorrhage. Many agents exist which modulate clotting, and novel approaches that facilitate their safe delivery to sites of vascular injury could reduce the enormous number of deaths from hemorrhage that occur globally.

Self-propelled particles that transport cargo through flowing blood and halt hemorrhage.

Delivering therapeutics deep into damaged tissue during bleeding is challenging because of the outward flow of blood. When coagulants cannot reach and clot blood at its source, uncontrolled bleeding can occur and increase surgical complications and fatalities. Self-propelling particles have been proposed as a strategy for transporting agents upstream through blood. Many nanoparticle and microparticle systems exhibiting autonomous or collective movement have been developed, but propulsion has not been used successfully in blood or used in vivo to transport therapeutics. We show that simple gas-generating microparticles consisting of carbonate and tranexamic acid traveled through aqueous solutions at velocities of up to 1.5 cm/s and delivered therapeutics millimeters into the vasculature of wounds. The particles transported themselves through a combination of lateral propulsion, buoyant rise, and convection. When loaded with active thrombin, these particles worked effectively as a hemostatic agent and halted severe hemorrhage in multiple animal models of intraoperative and traumatic bleeding. Many medical applications have been suggested for self-propelling particles, and the findings of this study show that the active self-fueled transport of particles can function in vivo to enhance drug delivery.