Engineering Low Volume Resuscitants for the Prehospital Care of Severe Hemorrhagic Shock.

Globally, traumatic injury is a leading cause of suffering and death. The ability to curtail damage and ensure survival after major injury requires a time-sensitive response balancing organ perfusion, blood loss, and portability, underscoring the need for novel therapies for the prehospital environment. Currently, there are few options available for damage control resuscitation (DCR) of trauma victims. We hypothesize that synthetic polymers, which are tunable, portable, and stable under austere conditions, can be developed as effective injectable therapies for trauma medicine. In this work, we design injectable polymers for use as low volume resuscitants (LVRs). Using RAFT polymerization, we evaluate the effect of polymer size, architecture, and chemical composition upon both blood coagulation and resuscitation in a rat hemorrhagic shock model. Our therapy is evaluated against a clinically used colloid resuscitant, Hextend. We demonstrate that a radiant star poly(glycerol monomethacrylate) polymer did not interfere with coagulation while successfully correcting metabolic deficit and resuscitating animals from hemorrhagic shock to the desired mean arterial pressure range for DCR - correcting a 60% total blood volume (TBV) loss when given at only 10% TBV. This highly portable and non-coagulopathic resuscitant has profound potential for application in trauma medicine.

A novel melanocortin fusion protein inhibits fibrinogen oxidation and degradation during trauma-induced coagulopathy.

Immune cell inflammation is implicated in the pathophysiology of acute trauma-induced coagulopathy (TIC). We hypothesized that leukocyte inflammation contributes to TIC through the oxidation and proteolysis of fibrinogen. To test this hypothesis, antioxidants and a novel anti-inflammatory melanocortin fusion protein (AQB-565) were used to study the effects of interleukin-6 (IL-6)-stimulated human leukocytes on fibrinogen using single-cell imaging flow cytometry and multiplex fluorescent western blotting. We also studied the effects of AQB-565 on fibrinogen using an in vivo rat trauma model of native TIC. IL-6 induced cellular inflammation and mitochondrial superoxide production in human monocytes, causing fibrinogen oxidation and degradation in vitro. Antioxidants suppressing mitochondrial superoxide reduced oxidative stress and inflammation and protected fibrinogen. AQB-565 decreased inflammation, inhibited mitochondrial superoxide, and protected fibrinogen in vitro. Trauma with hemorrhagic shock increased IL-6 and other proinflammatory cytokines and chemokines, selectively oxidized and degraded fibrinogen, and induced TIC in rats in vivo. AQB-565, given at the onset of hemorrhage, blocked inflammation, protected fibrinogen from oxidation and degradation, and prevented TIC. Leukocyte activation contributes to TIC through the oxidation and degradation of fibrinogen, which involves mitochondrial superoxide and cellular inflammation. Suppression of inflammation by activation of melanocortin pathways may be a novel approach for the prevention and treatment of TIC.

Leukocyte activation primes fibrinogen for proteolysis by mitochondrial oxidative stress.

Critical illness leads to rapid fibrinogen consumption, hyperfibrinolysis, and coagulopathy that exacerbates bleeding and increases mortality. Immune cell activation and inflammation are associated with coagulopathy after injury but play an undetermined role. We performed high dimensional immunophenotyping and single-cell imaging flow cytometry to investigate for a pathophysiological mechanism governing the effects of leukocyte-associated inflammation on fibrinogen function. Fibrinogen was oxidized early, followed by its degradation after 3 hours of lipopolysaccharides (LPS)-induced sterile inflammation in a rat model in vivo. Fibrinogen incubated with human leukocytes activated by TNFα was similarly oxidized, and later proteolyzed after 3 hours in vitro. TNFα induced mitochondrial superoxide generation from neutrophils and monocytes, myeloperoxidase (MPO)-derived reactive oxygen species (ROS) from neutrophils, and nitric oxide from lymphocytes and monocytes. Inhibition of mitochondrial superoxide prevented oxidative modification and proteolysis of fibrinogen, whereas inhibition of MPO attenuated only fibrinogen proteolysis. Quenching of both mitochondrial superoxide and MPO-derived ROS prevented coagulopathy better than tranexamic acid. Collectively, these findings indicate that neutrophil and monocyte mitochondrial superoxide generation can rapidly oxidize fibrinogen as a priming step for fibrinogen proteolysis and coagulopathy during inflammation.

Phenotypic analysis of erythrocytes in sickle cell disease using imaging flow cytometry.

The morphology and other phenotypic characteristics of erythrocytes in sickle cell disease (SCD) have been analyzed for decades in patient evaluation. This involves a variety of techniques, including microscopic analysis of stained blood films, flow cytometry, and cell counting. Here, we analyzed SCD blood using imaging flow cytometry (IFC), a technology that combines flow cytometry and microscopy to enable simultaneous rapid-throughput analysis of cellular morphology and cell-surface markers. With IFC, we were able to automate quantification of poikilocytes from SCD blood. An important subpopulation of poikilocytes represented dense cells, although these could not be distinguished from other poikilocytes without first centrifuging the blood through density gradients. In addition, CD71-positive RBCs from SCD patients had two subpopulations: one with high CD71 expression and a puckered morphology and another with lower CD71 expression and biconcave morphology and presumably representing a later stage of differentiation. Some RBCs with puckered morphologies that were strongly positive for DAPI and CD49d were in fact nucleated RBCs. IFC identified more phosphatidylserine-expressing red cells in SCD than did conventional flow cytometry and these could also be divided into two subpopulations. One population had diffuse PS expression and appeared to be composed primarily of RBC ghosts; the other had lower overall PS expression present in intense, punctate dots overlying Howell-Jolly bodies. This study demonstrates that IFC can rapidly reveal and quantify RBC features in SCD that require numerous tedious methods to identify conventionally. Thus, IFC is likely to be a useful technique for evaluating and monitoring SCD.

A Multifunctional, Low-Volume Resuscitation Cocktail Improves Vital Organ Blood Flow and Hemostasis in a Pig Model of Polytrauma with Traumatic Brain Injury.

The resuscitation of polytrauma with hemorrhagic shock and traumatic brain injury (TBI) is a balance between permissive hypotension and maintaining vital organ perfusion. There is no current optimal solution. This study tested whether a multifunctional resuscitation cocktail supporting hemostasis and perfusion could mitigate blood loss while improving vital organ blood flow during prolonged limited resuscitation. Anesthetized Yorkshire swine were subjected to fluid percussion TBI, femur fracture, catheter hemorrhage, and aortic tear. Fluid resuscitation was started when lactate concentration reached 3-4 mmol/L. Animals were randomized to one of five groups. All groups received hydroxyethyl starch solution and vasopressin. Low- and high-dose fibrinogen (FBG) groups additionally received 100 and 200 mg/kg FBG, respectively. A third group received TXA and low-dose FBG. Two control groups received albumin, with one also including TXA. Animals were monitored for up to 6 h. Blood loss was decreased and vital organ blood flow was improved with low- and high-dose fibrinogen compared to albumin controls, but survival was not improved. There was no additional benefit of high- vs. low-dose FBG on blood loss or survival. TXA alone decreased blood loss but had no effect on survival, and combining TXA with FBG provided no additional benefit. Pooled analysis of all groups containing fibrinogen vs. albumin controls found improved survival, decreased blood loss, and improved vital organ blood flow with fibrinogen delivery. In conclusion, a low-volume resuscitation cocktail consisting of hydroxyethyl starch, vasopressin, and fibrinogen concentrate improved outcomes compare to controls during limited resuscitation of polytrauma.

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.

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.

Platelets retain inducible alpha granule secretion by P-selectin expression but exhibit mechanical dysfunction during trauma-induced coagulopathy.

Essentials Platelets in trauma-induced coagulopathy (TIC) are impaired, but the mechanism is not known. We performed comprehensive longitudinal platelet function testing in trauma patient samples. Platelets in TIC are widely impaired early after injury, but platelet activatability is intact. This suggests a mechanism of transient platelet cytoskeletal/integrin dysfunction during TIC. SUMMARY: Background Trauma-induced coagulopathy (TIC) is a common and deadly bleeding disorder. Platelet dysfunction is present during TIC, but its mechanisms remain unclear. Platelets are currently thought to become "exhausted," a state in which they have released their granule contents and can no longer aggregate or contract. Methods This prospective observational cohort study tested the hypothesis that platelet exhaustion is present during TIC and characterized the early time course of platelet dysfunction. Blood was collected from 95 adult trauma patients at a Level I trauma center at time of Emergency Department arrival and several time points over 72 h. Platelet activation state and function were characterized using CD62P (P-selectin) and PAC-1 surface membrane staining, platelet function analyzer (PFA-100), aggregometry, viscoelastic platelet mapping, and, to test for exhaustion, their ability to express CD62P after ex vivo adenosine diphosphate (ADP) agonism. Platelet function was compared between patients with and without TIC, defined by prothrombin time ≥18 s. Results Platelets in TIC showed no initial increase in their level of surface activation markers or impairment of their capacity to express CD62P in response to ADP stimulation. However, TIC platelets were impaired in nearly all functional assays, spanning adhesion, aggregation, and contraction. These effects largely remained after controlling for platelet count and fibrinogen concentration and resolved after 8 h. Conclusion The TIC platelets exhibit early impairment of adhesion, aggregation, and contraction with retained alpha granule secretion ability, suggesting a specific mechanism of cytoskeletal or integrin dysfunction that is not a result of more general platelet exhaustion.

Prehospital Lactate Predicts Need for Resuscitative Care in Non-hypotensive Trauma Patients.

INTRODUCTION: The prehospital decision of whether to triage a patient to a trauma center can be difficult. Traditional decision rules are based heavily on vital sign abnormalities, which are insensitive in predicting severe injury. Prehospital lactate (PLac) measurement could better inform the triage decision. PLac's predictive value has previously been demonstrated in hypotensive trauma patients but not in a broader population of normotensive trauma patients transported by an advanced life support (ALS) unit. METHODS: This was a secondary analysis from a prospective cohort study of all trauma patients transported by ALS units over a 14-month period. We included patients who received intravenous access and were transported to a Level I trauma center. Patients with a prehospital systolic blood pressure ≤ 100 mmHg were excluded. We measured PLac's ability to predict the need for resuscitative care (RC) and compared it to that of the shock index (SI). The need for RC was defined as either death in the emergency department (ED), disposition to surgical intervention within six hours of ED arrival, or receipt of five units of blood within six hours. We calculated the risk associated with categories of PLac. RESULTS: Among 314 normotensive trauma patients, the area under the receiver operator characteristic curve for PLac predicting need for RC was 0.716, which did not differ from that for SI (0.631) (p=0.125). PLac ≥ 2.5 mmol/L had a sensitivity of 74.6% and a specificity of 53.4%. The odds ratio for need for RC associated with a 1-mmol/L increase in PLac was 1.29 (95% confidence interval [CI] [0.40 - 4.12]) for PLac < 2.5 mmol/L; 2.27 (1.10 - 4.68) for PLac from 2.5 to 4.0 mmol/L; and 1.26 (1.05 - 1.50) for PLac ≥ 4 mmol/L. CONCLUSION: PLac was predictive of need for RC among normotensive trauma patients. It was no more predictive than SI, but it has certain advantages and disadvantages compared to SI and could still be useful. Prospective validation of existing triage decision rules augmented by PLac should be investigated.

Damage Control Resuscitation Supplemented with Vasopressin in a Severe Polytrauma Model with Traumatic Brain Injury and Uncontrolled Internal Hemorrhage.

INTRODUCTION: Traumatic brain injury (TBI) and hemorrhagic shock (HS) are the leading causes of traumatic death worldwide and particularly on the battlefield. They are especially challenging when present simultaneously (polytrauma), and clear blood pressure end points during fluid resuscitation are not well described for this situation. The goal of this study is to evaluate for any benefit of increasing blood pressure using a vasopressor on brain blood flow during initial fluid resuscitation in a swine polytrauma model. MATERIALS AND METHODS: We used a swine polytrauma model with simultaneous TBI, femur fracture, and HS with uncontrolled noncompressible internal bleeding from an aortic tear injury. Five animals were assigned to each of three experimental groups (hydroxyethyl starch only [HES], HES + 0.4 U/kg vasopressin, and no fluid resuscitation [No Fluids]). Fluids were given as two 10 mL/kg boluses according to tactical field care guidelines. Primary outcomes were mean arterial blood pressure (MAP) and brain blood flow at 60 min. Secondary outcomes were blood flows in the heart, intestine, and kidney; arterial blood lactate level; and survival at 6 hr. Organ blood flow was measured using injection of colored microspheres. RESULTS: Five animals were tested in each of the three groups. There was a statistically significant increase in MAP with vasopressin compared with other experimental groups, but no significant increase in brain blood flow during the first 60 min of resuscitation. The vasopressin group also exhibited greater total internal hemorrhage volume and rate. There was no difference in survival at 6 hours. CONCLUSION: In this experimental swine polytrauma model, increasing blood pressure with vasopressin did not improve brain perfusion, likely due to increased internal hemorrhage. Effective hemostasis should remain the top priority for field treatment of the polytrauma casualty with TBI.

Role of trauma team activation in poor outcomes of elderly patients.

INTRODUCTION: Elderly trauma patients suffer worse outcomes than younger patients. Trauma team activation (TTA) improves outcomes in younger patients. It is unclear whether decreased TTA effectiveness or under-activation in elderly patients could contribute to their poor outcomes. MATERIAL AND METHODS: This retrospective registry study examined all adult trauma patients admitted to a level 1 trauma center over 2 y. Analyses tested (1) whether age modifies the effect of TTA on poor outcomes, (2) whether elderly patients with severe injury were less likely to receive TTA than younger patients, and (3) which early variables were associated with poor outcomes among elderly patients who did not receive TTA. RESULTS: The study included 10,033 patients. The adjusted relative risk from TTA for all ages was 0.48 (95% confidence interval (CI) = 0.34-0.68, P < 0.001), and there was no effect modification by age (interaction term P value, 0.171). The adjusted odds ratio for the young was 0.49 (95% CI = 0.26-0.91, P = 0.024) and for the elderly was 0.80 (95% CI = 0.53-1.20, P = 0.282). The adjusted odds ratio for lack of TTA associated with old age was 1.37 (95% CI = 1.12-1.69, P = 0.003). The strongest associations with poor outcomes were seen with low heart rate, low minimum blood pressure, high injury severity score, and high Glasgow coma score. CONCLUSIONS: Lack of TTA could contribute to elderly patients' poor outcomes. Clinicians should not be reassured by normal heart rates and should be wary of even transiently lower blood pressures in the elderly. A large cohort study is needed to identify which additional elderly patients could benefit from TTA.

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.

Post-translational oxidative modification of fibrinogen is associated with coagulopathy after traumatic injury.

Victims of trauma often develop impaired blood clot formation (coagulopathy) that contributes to bleeding and mortality. Fibrin polymerization is one critical component of clot formation that can be impacted by post-translational oxidative modifications of fibrinogen after exposure to oxidants. In vitro evidence suggests that Aα-C domain methionine sulfoxide formation, in particular, can induce conformational changes that prevent lateral aggregation of fibrin protofibrils during polymerization. We used mass spectrometry of plasma from trauma patients to find that fibrinogen Aα-C domain methionine sulfoxide content was selectively-increased in patients with coagulopathy vs. those without coagulopathy. This evidence supports a novel linkage between oxidative stress, coagulopathy, and bleeding after injury.

Effects of rapid wound sealing on survival and blood loss in a swine model of lethal junctional arterial hemorrhage.

BACKGROUND: Hemostatic gauzes, which must be packed into wounds and compressed for several minutes, may be of limited use for noncompressible wounds in junctional anatomic locations. Rapid mechanical wound sealing is an alternative approach that seals the wound at the skin, allowing internal clot formation. We evaluate wound sealing for junctional hemorrhage control using a hemostatic clamp (iTClamp). METHODS: Severe junctional hemorrhage was induced in anesthetized immature female swine using a 5-mm femoral arteriotomy. After 30 seconds of free bleeding, animals were randomized to one of seven hemostatic interventions: no intervention (control), direct compression for 3 minutes (compression), plain gauze packing (packing), mechanical wound seal (seal), plain gauze packing + wound seal (packing + seal), plain gauze packing + compression (packing + compression), or hemostatic gauze packing (Combat Gauze) + compression (HS-packing + compression). All animals then received one 15-mL/kg bolus of Hextend, followed by lactated Ringer's solution for hypotension up to 100 mL/kg. Animals were monitored for 3 hours. RESULTS: Survival was similar between control (3-hour survival, 0%) and compression (0%, Kaplan-Meier survival analysis and log-rank test [KM-LR], p = 1.0) but marginally improved with packing (12.5%, KM-LR, p < 0.001). Survival improved with seal (62.5%) versus control (KM-LR, p < 0.001) and with packing + seal (100%) versus packing alone (KM-LR, p < 0.001). Survival was similar between packing + compression (87.5%), HS-packing + compression (62.5%), and packing + seal (100%) (KM-LR, p ≥ 0.05). Total hemorrhage volume was decreased for seal versus control (p < 0.001) and for packing + seal versus packing (p < 0.001). Hemorrhage was similar among packing + compression, HS-packing + compression, seal, and packing + seal (analysis of variance p ≥ 0.05). Application times (mean [SD]) were significantly faster with packing + seal (125.8 [56.2] seconds) than packing + compression (236.6 [7.2] seconds) and HS-packing + compression (223.0 [6.8] seconds) (analysis of variance, all p < 0.001). CONCLUSION: In this preclinical junctional hemorrhage model, rapid wound sealing improved survival and decreased hemorrhage in both packed and unpacked wounds and performed comparably with standard-of-care hemostatic bandages. Rapidly sealing junctional wounds may be a viable alternative to wound compression.

Comparison of rocuronium and succinylcholine on postintubation sedative and analgesic dosing in the emergency department.

OBJECTIVES: Rocuronium and succinylcholine are both commonly used neuromuscular blockers for rapid sequence intubation in the emergency department (ED). The objective of this study was to determine if patients who receive rocuronium are more likely to receive lower doses of postintubation sedatives and analgesics compared with patients who receive succinylcholine. METHODS: This was a retrospective cohort study carried out in a tertiary, academic ED. Consecutive adult patients, who were intubated using etomidate for induction of sedation, were included. Patients were categorized on the basis of whether they received (a) rocuronium or (b) succinylcholine for paralysis. The dosing of postintubation sedative and analgesic infusions were compared 30 min after initiation between the two groups. RESULTS: A total of 254 patients were included in the final analysis (rocuronium=127 and succinylcholine=127). In the overall cohort, 90.2% (n=229) of patients were administered a sedative postintubation in the ED. Most of these patients were initiated on propofol infusions. The mean propofol infusion rate at 30 min was 30±23 mcg/kg/min in the rocuronium group and 42±24 mcg/kg/min in the succinylcholine group (P=0.002). A total of 42.5% of patients (n=108) received an analgesic infusion (all patients received fentanyl). The mean fentanyl infusion rate at 30 min was 0.65±0.55 and 0.86±0.49 mcg/kg/h in the rocuronium and succinylcholine groups, respectively (P=0.041). CONCLUSION: Patients who receive rocuronium are more likely to receive lower doses of sedative and analgesic infusions after intubation. This may place them at risk of being awake under paralysis.