Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia‐reperfusion physiology is a key component of the inflammatory and vaso‐occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro‐inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso‐occlusion. We hypothesize that hypoxia‐reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso‐occlusion. At baseline and after H/R, sickle Townes‐SS mice had increased C3 activation fragments and C5b‐9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA‐mice. Activated complement promoted vaso‐occlusion (microvascular stasis) in SS‐mice; infusion of zymosan‐activated, but not heat‐inactivated serum, induced substantial vaso‐occlusion in the skin venules of SS‐mice. Infusion of recombinant C5a induced stasis in SS, but not AA‐mice that was blocked by anti‐C5a receptor (C5aR) IgG. C5a‐mediated stasis was accompanied by inflammatory responses in SS‐mice including NF‐κB activation and increased expression of TLR4 and adhesion molecules VCAM‐1, ICAM‐1, and E‐selectin in the liver. Anti‐C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up‐regulated Weibel‐Palade body P‐selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS‐mice, a blocking antibody to P‐selectin inhibited C5a‐induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R‐induced stasis in SS‐mice. These results suggest that inhibition of C5a may be beneficial in SCD.

Background Trauma‐induced hypocalcemia is an underappreciated complication of severe injury but is well known to result in the derangement of an array of physiological regulatory mechanisms. Existing literature provides a compelling link between hypocalcemia and worse trauma‐induced coagulopathy and increased mortality after injury. Study Design and Methods This narrative review evaluates available data related to the risk factors, mechanisms, and treatment of hypocalcemia after severe injury. The authors did not perform a systemic review or meta‐analysis. Results and Discussion The interplay of acidosis, hypothermia, and coagulopathy with hypocalcemia potentiates the bloody vicious cycle of hemorrhagic shock which has been the paradigm of trauma resuscitation for over half a century. However, current screening and treatment of postinjury hypocalcemia are relegated to a secondary consideration in trauma resuscitation. We conclude calcium supplementation should be a primary tier intervention for life‐threatening injury.

Red blood cell (RBC) transfusion is a life-saving intervention for millions of trauma patients every year worldwide. While hemoglobin thresholds are clinically driving the need for RBC transfusion, limited information is available with respect to transfusion efficacy at the molecular level in clinically relevant cohorts. Here, we combined plasma metabolomic and proteomic measurements in longitudinal samples (n = 118; up to 13 time points; total samples: 690) from trauma patients enrolled in the control of major bleeding after trauma (COMBAT) study. Samples were collected in the emergency department and at continuous intervals up to 168 h (seven days) post-hospitalization. Statistical analyses were performed to determine omics correlate to transfusions of one, two, three, five, or more packed RBC units. While confounded by the concomitant transfusion of other blood components and other iatrogenic interventions (e.g., surgery), here we report that transfusion of one or more packed RBCs—mostly occurring within the first 4 h from hospitalization in this cohort—results in the increase in circulating levels of additive solution components (e.g., mannitol, phosphate) and decreases in the levels of circulating markers of hypoxia, such as lactate, carboxylic acids (e.g., succinate), sphingosine 1-phosphate, polyamines (especially spermidine), and hypoxanthine metabolites with potential roles in thromboinflammatory modulation after trauma. These correlations were the strongest in patients with the highest new injury severity scores (NISS > 25) and lowest base excess (BE < −10), and the effect observed was proportional to the number of units transfused. We thus show that transfusion of packed RBCs transiently increases the circulating levels of plasticizers—likely leaching from the blood units during refrigerated storage in the blood bank. Changes in the levels of arginine metabolites (especially citrulline to ornithine ratios) are indicative of an effect of transfusion on nitric oxide metabolism, which could potentially contribute to endothelial regulation. RBC transfusion was associated with changes in the circulating levels of coagulation factors, fibrinogen chains, and RBC-proteins. Changes in lysophospholipids and acyl-carnitines were observed upon transfusion, suggestive of an effect on the circulating lipidome—though cell-extrinsic/intrinsic effects and/or the contribution of other blood components cannot be disentangled. By showing a significant decrease in circulating markers of hypoxia, this study provides the first multi-omics characterization of RBC transfusion efficacy in a clinically relevant cohort of trauma patients.

BackgroundEndothelium-derived microparticles (EMPs) are submicron vesicles released from the plasma membrane of endothelial cells in response to injury, apoptosis or activation. We have previously demonstrated EMP-induced acute lung injury (ALI) in animal models and endothelial barrier dysfunction in vitro. Current treatment options for ALI are limited and consist of supportive therapies. We hypothesize that standard clinical continuous venovenous hemofiltration (CVVH) reduces serum EMP levels and may be adapted as a potential therapeutic intervention.Materials and methodsEMPs were generated from plasminogen activation inhibitor-1 (PAI-1)-stimulated human umbilical vein endothelial cells (HUVECs). Flow cytometric analysis was used to characterize EMPs as CD31- and annexin V-positive events in a submicron size gate. Enumeration was completed against a known concentration of latex beads. Ultimately, a concentration of ~650,000 EMP/mL perfusate fluid (total 470 mL) was circulated through a standard CVVH filter (pore size 200 μm, flow rate 250 mL/hr) for a period of 70 minutes. 0.5 mL aliquots were removed at 5- to 10-minute intervals for flow cytometric analysis. EMP concentration in the dialysate was measured at the end of 4 hours to better understand the fate of EMPs.ResultsA progressive decrease in circulating EMP concentration was noted using standard CVVH at 250 mL/hr (a clinical standard rate) from a 470 mL volume modelling a patient's circulation. A 50% reduction was noted within the first 30 minutes. EMPs entering the dialysate after 4 hours were 5.7% of the EMP original concentration.ConclusionThese data demonstrate that standard CVVH can remove EMPs from circulation in a circuit modelling a patient. An animal model of hemofiltration with induction of EMP release is required to test the therapeutic potential of this finding and potential of application in early treatment of ALI.

BACKGROUND: Release of neutrophil extracellular traps (NETosis) may mediate postinjury organ dysfunction, but mechanisms remain unclear. The intracellular serine protease inhibitor (serpin) B1 is vital to neutrophil function and has been shown to restrict NETosis in inflammatory settings.In this study, we used discovery proteomics to identify the proteomic signature of trauma-induced NETosis. We hypothesized that serpinB1 would be a major component of this NET protein profile and associated with adverse outcomes. METHODS:This was a post hoc analysis of data collected as part of the COMBAT randomized clinical trial. Blood was collected from injured patients at a single Level I Trauma Center. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry. Abundances of serpinB1 and known NETosis markers were analyzed with patient and injury characteristics, clinical data, and outcomes. RESULTS:SerpinB1 levels on emergency department (ED) arrival were significantly correlated with proteomic markers of NETosis, including core histones, transketolase, and S100A8/A9 proteins. More severely injured patients had elevated serpinB1 and NETosis markers on ED arrival. Levels of serpinB1 and top NETosis markers were significantly elevated on ED arrival in nonsurvivors and patients with fewer ventilator-and ICU-free days. In proteome-wide receiver operating characteristic analysis, serpinB1 was consistently among the top proteins associated with adverse outcomes. Among NETosis markers, levels of serpinB1 early in the patient's course exhibited the greatest separation between patients with fewer and greater ventilator-and ICU-free days. Gene Ontology analysis of top predictors of adverse outcomes further supports NETosis as a potential mediator of postinjury organ dysfunction. CONCLUSION: We have identified a proteomic signature of trauma-induced NETosis, and NETosis is an early process following severe injury that may mediate organ dysfunction. In addition, serpinB1 is a major component of this NET protein profile that may serve as an early marker of excessive NETosis after injury.