Traumatic injury results in prolonged circulation of ultralarge von Willebrand factor and a reduction in ADAMTS13 activity.

BACKGROUND: Increases in plasma von Willebrand Factor (VWF) levels, accompanied by decreases in the metalloprotease ADAMTS13, have been demonstrated soon after traumatic injury while downstream effects remain unclear. STUDY DESIGN AND METHODS: A cohort of 37 injured trauma patients from a randomized control trial investigating the use of prehospital plasma transfusion were analyzed for activity and antigen levels of ADAMTS13 and VWF at 0 and 24 hours after admission. Relevant clinical data were abstracted from the medical records. Trauma patient plasma was analyzed via agarose gel electrophoresis to evaluate the effects of injury on VWF multimer composition compared to healthy controls. RESULTS: von Willebrand factor levels were elevated at presentation (189% [110%-263%] vs. 95% [74%-120%]), persisting through 24 hours (213% [146%-257%] vs. 132% [57%-160%]), compared to healthy controls. Ultralarge VWF (UL-VWF) forms were elevated in trauma patients at both 0 and 24 hours, when compared to pooled normal plasma (10.0% [8.9%-14.3%] and 11.3% [9.1%-21.2%], respectively, vs. 0.6%). Circulating plasma ADAMTS13 activity was decreased at 0 hours (66% [47%-86%] vs. 100% [98%-100%]) and at 24 hours (72.5% [56%-87.3%] vs. 103% [103%-103%]) in trauma patients. ADAMTS13 activity independently predicted the development of coagulopathy and correlated with international normalized ratio, thromboelastography values, injury severity, and blood product transfusion. CONCLUSION: Traumatic injury is associated with acute coagulopathy that is characterized by increased UL-VWF multimers and reduction in ADAMTS13, which correlates with blood loss, transfusion requirement, and injury severity. These findings suggest the potential for future trials targeting ADAMTS13 repletion to enhance clearance of VWF multimers.

MACROPHAGE SWITCHING: POLARIZATION AND MOBILIZATION AFTER TRAUMA.

ABSTRACT: Introduction: Trauma alters the immune response in numerous ways, affecting both the innate and adaptive responses. Macrophages play an important role in inflammation and wound healing following injury. We hypothesize that macrophages mobilize from the circulation to the site of injury and secondary sites after trauma, with a transition from proinflammatory (M1) shortly after trauma to anti-inflammatory (M2) at later time points. Methods: C57Bl6 mice (n = 6/group) underwent a polytrauma model using cardiac puncture/hemorrhage, pseudofemoral fracture, and liver crush injury. The animals were killed at several time points: uninjured, 24 h, and 7 days. Peripheral blood mononuclear cells, spleen, liver nonparenchymal cells, and lung were harvested, processed, and stained for flow cytometry. Macrophages were identified as CD68 + ; M1 macrophages were identified as iNOS + ; M2 macrophages as arginase 1 + . Results: We saw a slight presence of M1 macrophages at baseline in peripheral blood mononuclear cells (6.6%), with no significant change at 24 h and 7 days after polytrauma. In contrast, the spleen has a larger population of M1 macrophages at baseline (27.7%), with levels decreasing at 24 h and 7 days after trauma (20.6% and 12.6%, respectively). A similar trend is seen in the lung where at baseline 14.9% of CD68 + macrophages are M1, with subsequent continual decrease reaching 8.7% at 24 h and 4.4% at 7 days after polytrauma. M1 macrophages in the liver represent 14.3% of CD68 + population in the liver nonparenchymal cells at baseline. This percentage increases to 20.8% after trauma and decreases at 7 days after polytrauma (13.4%). There are few M2 macrophages in circulating peripheral blood mononuclear cells and in spleen at baseline and after trauma. The percentage of M2 macrophages in the lungs remains constant after trauma (7.2% at 24 h and 9.2% at 7 days). In contrast, a large proportion of M2 macrophages are seen in the liver at baseline (36.0%). This percentage trends upward and reaches 45.6% acutely after trauma and drops to 21.4% at 7 days. The phenotypic changes in macrophages seen in the lungs did not correlate with a functional change in the ability of the macrophages to perform oxidative burst, with an increase from 2.0% at baseline to 22.1% at 7 days after polytrauma ( P = 0.0258). Conclusion: Macrophage phenotypic changes after polytrauma are noted, especially with a decrease in the lung M1 phenotype and a short-term increase in the M2 phenotype in the liver. However, macrophage function as measured by oxidative burst increased over the time course of trauma, which may signify a change in subset polarization after injury not captured by the typical macrophage phenotypes.

EFFECT OF IRRIGATION FLUID COMPOSITION ON HEMOSTASIS IN MOUSE BLEEDING MODELS.

ABSTRACT: Introduction: Intraoperative irrigation, usually with normal saline (NS), aids in bleeding identification and management. We investigated the effect of different irrigation fluids, with additives, on hemostasis using two bleeding models. Methods: C57BL/6 J mice were subjected to a tail bleed model or uncontrolled abdominal hemorrhage via liver laceration followed by abdominal cavity irrigation. We compared NS, lactated Ringer's (LR), and PlasmaLyte. We examined NS and LR at different temperatures. Normal saline or LR with calcium (Ca 2+ ) or tranexamic acid (TXA) was studied. Results: Compared with room temperature (RT), increasing the temperature of the irrigation fluid to 37°C and 42°C reduced tail vein bleeding times substantially in both NS and LR (all P < 0.001), with no significant differences between the two fluids. At RT, LR, but not PlasmaLyte, substantially reduced bleeding times in comparison to NS ( P < 0.0001). Liver injury blood loss was lower with LR ( P < 0.01). Normal saline supplemented with 2.7 mEq/L of Ca 2+ decreased bleeding time and blood loss volume ( P < 0.001 and P < 0.01, respectively) to similar levels as LR. Normal saline with 150 mg/mL of TXA markedly reduced bleeding time ( P < 0.0001), and NS with 62.5 mg/mL TXA decreased blood loss ( P < 0.01). Conclusion: Whereas Ca 2+ - and TXA-supplemented NS reduced bleeding, LR remained superior to all irrigation fluid compositions. As LR contains Ca 2+ , and Ca 2+ -supplemented NS mirrored LR in response, Ca 2+ presence in the irrigation fluid seems key to improving solution's hemostatic ability. Because warming the fluids normalized the choice of agents, the data also suggest that Ca 2+ -containing fluids such as LR may be more suitable for hemostasis when used at RT.

Reduced cleavage of von willebrand factor by ADAMTS13 is associated with microangiopathic acute kidney injury following trauma.

Acute kidney injury (AKI) is common after trauma, but contributory factors are incompletely understood. Increases in plasma von Willebrand Factor (vWF) with concurrent decreases in ADAMTS13 are associated with renal microvascular thrombosis in other disease states, but similar findings have not been shown in trauma. We hypothesized that molecular changes in circulating vWF and ADAMTS13 promote AKI following traumatic injury. VWF antigen, vWF multimer composition and ADAMTS13 levels were compared in plasma samples from 16 trauma patients with and without trauma-induced AKI, obtained from the Prehospital Air Medical Plasma (PAMPer) biorepository. Renal histopathology and function, vWF and ADAMTS13 levels were assessed in parallel in a murine model of polytrauma and haemorrhage. VWF antigen was higher in trauma patients when compared with healthy controls [314% (253-349) vs. 100% (87-117)] [median (IQR)], while ADAMTS13 activity was lower [36.0% (30.1-44.7) vs. 100.0% (83.1-121.0)]. Patients who developed AKI showed significantly higher levels of high molecular weight multimeric vWF at 72-h when compared with non-AKI counterparts [32.9% (30.4-35.3) vs. 27.8% (24.6-30.8)]. Murine plasma cystatin C and vWF were elevated postpolytrauma model in mice, with associated decreases in ADAMTS13, and immunohistologic analysis demonstrated renal injury with small vessel plugs positive for fibrinogen and vWF. Following traumatic injury, the vWF-ADAMTS13 axis shifted towards a prothrombotic state in both trauma patients and a murine model. We further demonstrated that vWF-containing, microangiopathic deposits were concurrently produced as the prothrombotic changes were sustained during the days following trauma, potentially contributing to AKI development.

Platelet-mimicking procoagulant nanoparticles augment hemostasis in animal models of bleeding.

Treatment of bleeding disorders using transfusion of donor-derived platelets faces logistical challenges due to their limited availability, high risk of contamination, and short (5 to 7 days) shelf life. These challenges could be potentially addressed by designing platelet mimetics that emulate the adhesion, aggregation, and procoagulant functions of platelets. To this end, we created liposome-based platelet-mimicking procoagulant nanoparticles (PPNs) that can expose the phospholipid phosphatidylserine on their surface in response to plasmin. First, we tested PPNs in vitro using human plasma and demonstrated plasmin-triggered exposure of phosphatidylserine and the resultant assembly of coagulation factors on the PPN surface. We also showed that this phosphatidylserine exposed on the PPN surface could restore and enhance thrombin generation and fibrin formation in human plasma depleted of platelets. In human plasma and whole blood in vitro, PPNs improved fibrin stability and clot robustness in a fibrinolytic environment. We then tested PPNs in vivo in a mouse model of thrombocytopenia where treatment with PPNs reduced blood loss in a manner comparable to treatment with syngeneic platelets. Furthermore, in rat and mouse models of traumatic hemorrhage, treatment with PPNs substantially reduced bleeding and improved survival. No sign of systemic or off-target thrombotic risks was observed in the animal studies. These findings demonstrate the potential of PPNs as a platelet surrogate that should be further investigated for the management of bleeding.

Nanomedicine platform for targeting activated neutrophils and neutrophil-platelet complexes using an α1-antitrypsin-derived peptide motif.

Targeted drug delivery to disease-associated activated neutrophils can provide novel therapeutic opportunities while avoiding systemic effects on immune functions. We created a nanomedicine platform that uniquely utilizes an α1-antitrypsin-derived peptide to confer binding specificity to neutrophil elastase on activated neutrophils. Surface decoration with this peptide enabled specific anchorage of nanoparticles to activated neutrophils and platelet-neutrophil aggregates, in vitro and in vivo. Nanoparticle delivery of a model drug, hydroxychloroquine, demonstrated significant reduction of neutrophil activities in vitro and a therapeutic effect on murine venous thrombosis in vivo. This innovative approach of cell-specific and activation-state-specific targeting can be applied to several neutrophil-driven pathologies.

Platelet-derived extracellular vesicles released after trauma promote hemostasis and contribute to DVT in mice.

BACKGROUND: Traumatic injury can lead to dysregulation of the normal clotting system, resulting in hemorrhagic and thrombotic complications. Platelet activation is robust following traumatic injury and one process of platelet activation is to release of extracellular vesicles (PEV) that carry heterogenous cargo loads and surface ligands. OBJECTIVES: We sought to investigate and characterize the release and function of PEVs generated following traumatic injury. METHODS: PEV content and quantity in circulation following trauma in humans and mice was measured using flow cytometry, size exclusion chromatography, and nanoparticle tracking analysis. PEVs were isolated from circulation and the effects on thrombin generation, bleeding time, hemorrhage control, and thrombus formation were determined. Finally, the effect of hydroxychloroquine (HCQ) on PEV release and thrombosis were examined. RESULTS: Human and murine trauma results in a significant release of PEVs into circulation compared with healthy controls. These PEVs result in abundant thrombin generation, increased platelet aggregation, decreased bleeding times, and decreased hemorrhage in uncontrolled bleeding. Conversely, PEVs contributed to enhanced venous thrombus formation and were recruited to the developing thrombus site. Interestingly, HCQ treatment resulted in decreased platelet aggregation, decreased PEV release, and reduced deep vein thrombosis burden in mice. CONCLUSIONS: These data demonstrate that trauma results in significant release of PEVs which are both pro-hemostatic and pro-thrombotic. The effects of PEVs can be mitigated by treatment with HCQ, suggesting the potential use as a form of deep vein thrombosis prophylaxis.

Intravenous administration of synthetic platelets (SynthoPlate) in a mouse liver injury model of uncontrolled hemorrhage improves hemostasis.

BACKGROUND: Clinical resuscitative treatment of traumatic hemorrhage involves transfusion of RBC, platelets and plasma in controlled ratios. However, use of such blood components, especially platelets, present many challenges including availability, portability, contamination risks, and short shelf-life, which limit the use of platelet transfusions outside of large trauma centers such as remote civilian hospitals and austere prehospital settings. This has prompted significant research in platelet substitutes that may resolve the above issues while providing platelet-mimetic hemostatic action. In this framework, we have developed a synthetic platelet surrogate, SynthoPlate, by integrative decoration of platelet function mimetic peptides on a biocompatible lipid nanovesicle platform. We have previously demonstrated hemostatic capability of SynthoPlate in correcting tail-bleeding time in thrombocytopenic mice. Building on this, we hypothesized that SynthoPlate transfusion would decrease bleeding in a murine model of acute hemorrhagic shock. METHODS: A validated model of uncontrolled intraperitoneal hemorrhage, via liver laceration was used to induce hemorrhagic shock in mice. SynthoPlate, control (unmodified) particles, and normal saline were administered as pretreatment and recue infusions to mice undergoing liver laceration and evaluated for hemostatic benefit by determining differences in blood loss and monitoring real-time hemodynamic data. RESULTS: Pretreatment SynthoPlate transfusion resulted in significant reduction of blood loss following hemorrhage, compared with control particles or normal saline treatment (0.86 ± 0.16 g control particles [CP] vs. 0.84 ± 0.13 g normal saline [NS] vs. 0.68 ± 0.09 g SynthoPlate, p < 0.005). SynthoPlate transfused mice demonstrated improved hemodynamics taking significantly longer to develop post-injury hypotension (168.3 ± 106.6 seconds CP vs. 137 ± 58 seconds NS vs. 546.7 ± 329.8 seconds SynthoPlate, p < 0.05). SynthoPlate infusion following liver laceration, that is, rescue transfusion, also resulted in a significant decrease in blood loss (0.89 ± 0.17 g CP vs. 0.92 ± 0.19 g NS vs. 0.69 ± 0.18 g SynthoPlate, p < 0.05). CONCLUSION: Transfusion of SynthoPlate particles reduces blood loss in a murine model of liver injury, and SynthoPlates may represent a viable transfusion product for the mitigation of blood loss in acute, severe hemorrhagic shock.

Deep vein thrombosis in mice is regulated by platelet HMGB1 through release of neutrophil-extracellular traps and DNA.

Venous thromboembolic (VTE) disease, consisting of deep venous thrombosis (DVT) and pulmonary embolism (PE) is a leading cause of morbidity and mortality. Current prophylactic measures are insufficient to prevent all occurrence in part due to an incomplete understanding of the underlying pathophysiology. Mounting evidence describes interplay between activation of the innate immune system and thrombus development. Recent work has demonstrated that platelet release of HMGB1 leads to increased microvascular complications following injury. Additionally, platelet HMGB1 was found to enhance DVT and increase the formation of neutrophil extracellular traps (NETs), although the role of HMGB1 induced NET release in thrombosis remains unexplored. Utilizing a transgenic mouse lacking HMGB1 specifically from platelets and megakaryocytes we now demonstrate the specific role of platelet-derived HMGB1 in acute and subacute/chronic venous thrombosis. Platelets account for the majority of circulating HMGB1 and HMGB1 deposition within the developing clot. The pro-thrombotic effect of platelet-derived HMGB1 is mediated through enhanced neutrophil recruitment, NET formation and specifically release of extracellular DNA during NET formation. Taken together, these data suggest that platelet HMGB1 mediated NET release is a primary regulator of DVT formation in mice.

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring.

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival. Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals. Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.