Succinate Activation of SUCNR1 Predisposes Severely Injured Patients to Neutrophil-mediated ARDS.

OBJECTIVES: Identify the metabolites that are increased in the plasma of severely injured patients that developed ARDS versus severely injured patients that did not, and assay if these increased metabolites prime pulmonary sequestration of neutrophils (PMNs) and induce pulmonary sequestration in an animal model of ARDS. We hypothesize that metabolic derangement due to advanced shock in critically injured patients leads to the PMNs, which serves as the first event in the ARDS. Summary of Background Data: Intracellular metabolites accumulate in the plasma of severely injured patients. METHODS: Untargeted metabolomics profiling of 67 critically injured patients was completed to establish a metabolic signature associated with ARDS development. Metabolites that significantly increased were assayed for PMN priming activity in vitro. The metabolites that primed PMNs were tested in a 2-event animal model of ARDS to identify a molecular link between circulating metabolites and clinical risk for ARDS. RESULTS: After controlling for confounders, 4 metabolites significantly increased: creatine, dehydroascorbate, fumarate, and succinate in trauma patients who developed ARDS ( P < 0.05). Succinate alone primed the PMN oxidase in vitro at physiologically relevant levels. Intravenous succinate-induced PMN sequestration in the lung, a first event, and followed by intravenous lipopolysaccharide, a second event, resulted in ARDS in vivo requiring PMNs. SUCNR1 inhibition abrogated PMN priming, PMN sequestration, and ARDS. Conclusion: Significant increases in plasma succinate post-injury may serve as the first event in ARDS. Targeted inhibition of the SUCNR1 may decrease ARDS development from other disease states to prevent ARDS globally.

Hormones, age, and sex affect platelet responsiveness in vitro.

BACKGROUND: Female sex confers a survival advantage following severe injury in the setting of trauma-induced coagulopathy, with female platelets having heightened responsiveness likely due to estrogen. The effects of testosterone on platelet biology are unknown, and platelets express both estradiol and androgen receptors on the plasma membrane. We hypothesize testosterone decreases platelet responses in vitro, and there are baseline differences in platelet function and metabolism stratified by sex/age. STUDY DESIGN AND METHODS: Apheresis platelets were collected from: older males (OM) ≥45 years, younger males (YM) <45 years, older females (OF) ≥54 years, and younger females (YF) <54 years, and testosterone and estradiol were measured. Platelets were incubated with testosterone (5.31 ng/ml), estradiol (105 pg/ml) or vehicle and stimulated with buffer, adenosine diphosphate (20 µM), platelet activating factor (2 µM), or thrombin (0.3 U/ml). Aggregation, CD62P surface expression, fibrinogen receptor surface expression, and platelet mitochondrial metabolism were measured. RESULTS: Testosterone significantly inhibited aggregation in OF and OM (p < .05), inhibited CD41a expression in YF, YM, and OM (p < .05), and affected a few of the baseline amounts of CD62P surface expression but not platelet activation to platelet-activating factor and adenosine diphosphate, and variably changed platelet metabolism. DISCUSSION: Platelets have sex- and age-specific aggregation, receptor expression, and metabolism. Testosterone decreases platelet function dependent on the stimulus, age, and sex. Similarly, platelet metabolism has varying responses to sex hormones with baseline metabolic differences dependent upon sex and age.

A pilot study of the metabolic profiles of apheresis platelets modified by donor age and sex and in vitro short-term incubation with sex hormones.

BACKGROUND: Platelets are part of innate immunity and comprise the cellular portion of hemostasis. Platelets express sex hormone receptors on their plasma membrane and sex hormones can alter their function in vitro. Little is known about how age and sex may affect platelet biology; thus, we hypothesized that platelets from males and females have different metabolomic profiles, which may be altered by age and in vitro treatment with sex hormones. METHODS: Day 1 apheresis platelets were drawn from five 18-53-year-old, premenopausal younger females (YF), five ≥54-year-old, postmenopausal, older females (OF), five 18-44-year-old younger males (YM), and four ≥45-year-old older males (OM). Platelets were normalized to a standard concentration and metabolomics analyses were completed. Unsupervised statistical analyses and hierarchical clustering with principal component analyses were completed. RESULTS: Platelets from OM had (1) elevated mono-, di- and tri-carboxylates, (2) increased levels of free fatty acids, acyl-carnitines, and free amino acids, and (3) increased purine breakdown and deamination products. In vitro incubation with sex hormones only affected platelets from OM donors with trends towards increased ATP and other high-energy purines and decreases in L-proline and other amino acids. CONCLUSION: Platelets from OM's versus YF, OF, and YM have a different metabolome implying increased energy metabolism, more free fatty acids, acylcarnitines, and amino acids, and increased breakdown of purines and deamination products. However, only platelets from OM were affected by sex hormones in vitro. Platelets from OM are metabolically distinct, which may impart functional differences when transfused.

Apolipoprotein A-I, elevated in trauma patients, inhibits platelet activation and decreases clot strength.

Apolipoprotein A-I (ApoA-I) is elevated in the plasma of a subgroup of trauma patients with systemic hyperfibrinolysis. We hypothesize that apoA-I inhibits platelet activation and clot formation. The effects of apoA-I on human platelet activation and clot formation were assessed by whole blood thrombelastography (TEG), platelet aggregometry, P-selectin surface expression, microfluidic adhesion, and Akt phosphorylation. Mouse models of carotid artery thrombosis and pulmonary embolism were used to assess the effects of apoA-I in vivo. The ApoA-1 receptor was investigated with transgenic mice knockouts (KO) for the scavenger receptor class B member 1 (SR-BI). Compared to controls, exogenous human apoA-I inhibited arachidonic acid and collagen-mediated human and mouse platelet aggregation, decreased P-selectin surface expression and Akt activation, resulting in diminished clot strength and increased clot lysis by TEG. ApoA-I also decreased platelet aggregate size formed on a collagen surface under flow. In vivo, apoA-I delayed vessel occlusion in an arterial thrombosis model and conferred a survival advantage in a pulmonary embolism model. SR-BI KO mice significantly reduced apoA-I inhibition of platelet aggregation versus wild-type platelets. Exogenous human apoA-I inhibits platelet activation, decreases clot strength and stability, and protects mice from arterial and venous thrombosis via the SR-BI receptor.

28-day thawed plasma maintains α2 -antiplasmin levels and inhibits tPA-induced fibrinolysis.

INTRODUCTION: Evidence supports the use of plasma-first resuscitation in the treatment of trauma-induced coagulopathy (TIC). While thawed plasma (TP) has logistical benefits, the ability of plasma proteins to attenuate fibrinolysis and correct TIC remain unknown. We hypothesize that TP retains the ability to inhibit tissue plasminogen activator(tPA)-induced fibrinolysis at 28-day storage. METHODS: Healthy volunteers underwent blood draws followed by 50% dilution of whole blood (WB) with TP at 28-, 21-, 14-, 7-, 5-, and, 0-day storage, normal saline (NS), and WB control. Samples underwent citrated tPA-challenge (75 ng/ml) thromboelastography (TEG). Plasminogen activator inhibitor-1 (PAI-1) and α2 -antiplasmin (α2 -AP) concentrations in thawed or stored plasma were determined. RESULTS: In the presence of tPA, 28-day TP inhibited tPA-induced coagulopathy as effectively as WB. 28-day TP had a similar R-time, MA, and fibrinolysis (P > 0·05 for all) compared to WB, while angle was enhanced (P = 0·02) compared to WB. Significant correlations were present between storage time and clot strength (P = 0·04) and storage time and fibrinolysis (P = 0·0029). Active PAI-1 levels in thawed plasma were 1·10 ± 0·54 ng/mL while total PAI-1 levels were 4·79 ± 1·41 ng/mL. There was no difference of α2 -AP levels in FFP (40·45 ± 3·5 µg/mL) compared to plasma thawed for 14 (36·78 ± 5·39 µg/mL, P = 0·65) or 28 days (45·16 ± 5·61 µg/mL, P = 0·51). DISCUSSION: Thawed plasma retained the ability to inhibit tPA-induced fibrinolysis over 28-day storage at 1-4°C. α2 -AP levels were maintained in plasma thawed for 28 days and FFP. These in vitro results suggest consideration should be made to increasing the storage life of TP.

Effects of Blood Components and Whole Blood in a Model of Severe Trauma-Induced Coagulopathy.

BACKGROUND: Plasma resuscitation ameliorates hyperfibrinolysis (HF) and trauma-induced coagulopathy (TIC). However, the use of other blood components to reduce HF has not been evaluated. Therefore, our aim was to determine the effect of individual blood components and whole blood (WB) on an in vitro model of severe HF/TIC. METHODS: A "TIC" solution was made with 1:1 dilution of WB with saline and exacerbated with tissue plasminogen activator (tPA). Components were added in proportions equivalent to the thromboelastography (TEG) based goal-directed resuscitation used at our institution. Whole blood was added at proportions equal to what has been transfused in injured patients. Samples (n = 9) underwent citrated native and tPA-challenge (75 ng/mL) TEG with analysis of R-time, angle, MA, and LY30. Statistical analyses were completed employing the nonparametric Kruskal-Wallis and Dunn's multiple comparisons tests. RESULTS: TIC solution, when compared to control, had a decrease in clot strength (MA 41 mm versus 51.5 mm, P < 0.01). The addition of tPA resulted in a severe coagulopathy (MA 24.5 mm versus 41 mm and LY30 52.8% versus 2.4%, P < 0.03 for all). The addition of 4U of WB improved clot strength compared to TIC + tPA (P = 0.03). No individual blood component resulted in improved fibrinolysis (P > 0.7). Cryoprecipitate improved R-time (7.5 versus 11.9 min, P < 0.01), angle (56.8 versus 30.2°) and MA (49 mm versus 36.25 mm), while platelets improved MA (44 mm versus 36.25 mm) compared to TIC + tPA (P < 0.03 for all). CONCLUSIONS: No single blood component or volume of whole blood led to attenuation of tPA-mediated fibrinolysis in an in vitro model of TIC. Cryoprecipitate was the most effective at improving coagulation function.

PAS-C platelets contain less plasma protein, lower anti-A and anti-B titers, and decreased HLA antibody specificities compared to plasma platelets.

BACKGROUND: Platelets (PLTs) collected and stored in PLT additive solution Intersol (PAS-C) are presumed to reduce recipient exposure to donor plasma components; however, the effects of PAS-C on PLT supernatant composition are poorly defined. Therefore, we compared the total protein concentration, isohemagglutinin titers, and HLA antibodies in supernatants of PAS-C PLTs to plasma PLTs. STUDY DESIGN AND METHODS: Apheresis PLTs from group O blood donors were collected into either 100% donor plasma (n = 50) or a mixture of 65% PAS-C/35% donor plasma (n = 50). Within 12 hours of collection, samples of the PLT supernatant were frozen and stored. PLT supernatants were assayed for total protein concentration and anti-A and anti-B titers and screened for HLA antibodies. Samples positive for HLA antibodies were further tested using single-antigen bead assays to determine antibody strength and specificity. RESULTS: Supernatants of PAS-C PLTs had significantly lower total protein concentration and anti-A and anti-B titers compared to plasma PLTs. There was no significant difference in the number of HLA antibody screen-positive PAS-C (3/50) compared to plasma PLT supernatants (2/50); however, the HLA antibody screen-positive supernatants of PAS-C PLTs had fewer HLA specificities (2) compared to those of the plasma PLTs (18). CONCLUSION: Decreased plasma proteins likely underlie lower rates of allergic and febrile, nonhemolytic transfusion reactions from the infusion of PAS-C PLTs. Decreased anti-A and anti-B titers may prevent hemolysis from minor ABO mismatch. Lower HLA antibody specificities may mitigate transfusion-related acute lung injury.

A comparison of different methods of red blood cell leukoreduction and additive solutions on the accumulation of neutrophil-priming activity during storage.

BACKGROUND: Three methods of leukoreduction (LR) are used worldwide: filtration, buffy coat removal (BCR), and a combination of the previous two methods. Additionally, there are a number of additive solutions (ASs) used to preserve red blood cell (RBC) function throughout storage. During RBC storage, proinflammatory activity accumulates; thus, we hypothesize that both the method of LR and the AS affect the accumulation of proinflammatory activity. STUDY DESIGN AND METHODS: Ten units of whole blood were drawn from healthy donors, the RBC units were isolated, divided in half by weight, and leukoreduced by: 1) BCR, 2) filtration, or 3) BCR and filtration (combination-LR); stored in bags containing AS-3 per AABB criteria; and sampled weekly. The supernatants were isolated and frozen (-80°C). RBC units drawn from healthy donors into AS-1-, AS-3-, or AS-5-containing bags were also stored and sampled weekly, and the supernatants were isolated and frozen. The supernatants were assayed for neutrophil (PMN)-priming activity and underwent proteomic analyses. RESULTS: Filtration and combination LR decreased priming activity accumulation versus buffy coat LR, although the accumulation of priming activity was not different during storage. Combination LR increased hemolysis versus filtration via proteomic analysis. Priming activity from AS-3 units was significant later in storage versus AS-1- or AS-5-stored units. CONCLUSIONS: Although both filtration and combination LR decrease the accumulation of proinflammatory activity versus buffy coat LR, combination LR is not more advantageous over filtration, has increased costs, and may cause increased hemolysis. In addition, AS-3 decreases the early accumulation of PMN-priming activity during storage versus AS-1 or AS-5.

Thrombin stimulates increased plasminogen activator inhibitor-1 release from liver compared to lung endothelium.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1) is a major regulator of the fibrinolytic system, covalently binding to tissue plasminogen activator and blocking its activity. Fibrinolysis shutdown is evident in the majority of severely injured patients in the first 24 h and is thought to be due to PAI-1. The source of this PAI-1 is thought to be predominantly endothelial cells, but there are known organ-specific differences, with higher levels thought to be in the liver. Thrombin generation is also elevated in injured patients and is a potent stimulus for PAI-1 release in human umbilical endothelial cells. We hypothesize that thrombin induces liver endothelial cells to release increased amounts of PAI-1, versus pulmonary endothelium, consisting of both stored PAI-1 and a larger contribution from de novo PAI-1 synthesis. METHODS: Human liver sinusoidal endothelial cells (LSECs) and human microvascular lung endothelial cells (HMVECs) were stimulated in vitro ± thrombin (1 and 5 IU/mL) for 15-240 min, the supernatants were collected, and PAI-1 was measured by enzyme-linked immunosorbent assays. To elucidate the PAI-1 contribution from storage versus de novo synthesis, cycloheximide (10 µg/mL) was added before thrombin in separate experiments. RESULTS: While both LSECs and HMVECs rapidly stimulated PAI-1 release, LSECs released more PAI-1 than HMVECs in response to high-dose thrombin, whereas low-dose thrombin did not provoke immediate release. LSECs continued to release PAI-1 over the ensuing 240 min, whereas HMVECs did not. Cycloheximide did not inhibit early PAI-1 release from LSECs but did at the later time points (30-240 min). CONCLUSIONS: Thrombin elicits increased amounts of PAI-1 release from liver endothelium compared with lung, with a small presynthesized stored contribution and a later, larger increase in PAI-1 release via de novo synthesis. This study suggests that the liver may be an important therapeutic target for inhibition of the hypercoagulable surgical patient and the associated complications that result.

Supernatants and lipids from stored red blood cells activate pulmonary microvascular endothelium through the BLT2 receptor and protein kinase C activation.

BACKGROUND: Although transfusion is a lifesaving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells (RBCs) induce proinflammatory activation of human pulmonary microvascular endothelial cells (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI). STUDY DESIGN AND METHODS: Ten units of RBCs were collected; 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). An additional 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10%-40%]FINAL of the supernatants on Day (D)1 to D42 of storage, lipid extracts, and purified lipids. Endothelial surface expression of intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. RESULTS: The supernatants [10%-40%]FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20%-40%]FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused coprecipitation of ß-arrestin-1 with BLT2, protein kinase C (PKC)ßI , and PKCδ and served as the first event in a two-event rodent model of ALI. CONCLUSION: Lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients.

Experimental prestorage filtration removes antibodies and decreases lipids in RBC supernatants mitigating TRALI in vivo.

Transfusion-related acute lung injury (TRALI) remains a significant cause of transfusion-related mortality with red cell transfusion. We hypothesize that prestorage filtration may reduce proinflammatory activity in the red blood cell (RBC) supernatant and prevent TRALI. Filters were manufactured for both small volumes and RBC units. Plasma containing antibodies to human lymphocyte antigen (HLA)-A2 or human neutrophil antigen (HNA)-3a was filtered, and immunoglobulins and specific HNA-3a and HLA-2a neutrophil (PMN) priming activity were measured. Antibodies to OX27 were added to plasma, and filtration was evaluated in a 2-event animal model of TRALI. RBC units from 31 donors known to have antibodies against HLA antigens and from 16 antibody-negative controls were filtered. Furthermore, 4 RBC units were drawn and underwent standard leukoreduction. Immunoglobulins, HLA antibodies, PMN priming activity, and the ability to induce TRALI in an animal model were measured. Small-volume filtration of plasma removed >96% of IgG, antibodies to HLA-A2 and HNA-3a, and their respective priming activity, as well as mitigating antibody-mediated in vivo TRALI. In RBC units, experimental filtration removed antibodies to HLA antigens and inhibited the accumulation of lipid priming activity and lipid-mediated TRALI. We conclude that filtration removes proinflammatory activity and the ability to induce TRALI from RBCs and may represent a TRALI mitigation step.

Antibodies to major histocompatibility complex class II antigens directly prime neutrophils and cause acute lung injury in a two-event in vivo rat model.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a significant cause of mortality, especially after transfusions containing antibodies to major histocompatibility complex (MHC) class II antigens. We hypothesize that a first event induces both 1) polymorphonuclear neutrophils (PMNs) to express MHC class II antigens, and 2) activation of the pulmonary endothelium, leading to PMN sequestration, so that the infusion of specific MHC class II antibodies to these antigens causes PMN-mediated acute lung injury (ALI). STUDY DESIGN AND METHODS: Rats were treated with saline (NS), endotoxin (lipopolysaccharide [LPS]), or cytokines (interferon-γ [IFNγ], macrophage colony-stimulating factor [MCSF], tumor necrosis factor-α [TNFα]); the PMNs were isolated; and the surface expression of the MHC class II antigen OX6 and priming by OX6 antibodies were measured by flow cytometry or priming assays. RESULTS: A two-event model of ALI was completed with NS, LPS, or IFNγ/MCSF/TNFα (first events) and the infusion of OX6 (second event). Compared with NS incubation, rats treated with either LPS or IFNγ/MCSF/TNFα exhibited OX6 PMN surface expression, OX6 antibodies primed the formyl-methionyl-leucyl phenylalanine (fMLF)-activated respiratory burst, and PMN sequestration was increased. OX6 antibody infusion into LPS-incubated or IFNγ/MCSF/TNFα-incubated rats elicited ALI, the OX6 antibody was present on the PMNs, and PMN depletion abrogated ALI. CONCLUSION: Proinflammatory first events induce PMN MHC class II surface expression, activation of the pulmonary endothelium, and PMN sequestration such that the infusion of cognate antibodies precipitates TRALI.

Neutrophil function in children following allogeneic hematopoietic stem cell transplant.

HSCT is a lifesaving procedure for children with malignant and non-malignant conditions. The conditioning regimen renders the patient severely immunocompromised and recovery starts with neutrophil (PMN) engraftment. We hypothesize that children demonstrate minimal PMN dysfunction at engraftment and beyond, which is influenced by the stem cell source and the conditioning regimen. Peripheral blood was serially collected from children at 1 to 12 months following allogeneic HSCT. PMN superoxide (O2-) production, degranulation (elastase), CD11b surface expression, and phagocytosis were assessed. Twenty-five patients, mean age of 10.5 yr with 65% males, comprised the study and transplant types included: 14 unrelated cord blood stem cells (cords), seven matched related bone marrow donors, three matched unrelated bone marrow donors, and one peripheral blood progenitor cells. Engraftment occurred at 24 days. There were no significant differences between controls and patients in PMN O2- production, phagocytosis, CD11b surface expression, and total PMN elastase. Elastase release was significantly decreased <6 months vs. controls (p < 0.05) and showed normalization by six months for cords only. The conditioning regimen did not affect PMN function. PMN function returns with engraftment, save elastase release, which occurs later related to the graft source utilized, and its clinical significance is unknown.

Routine storage of red blood cell (RBC) units in additive solution-3: a comprehensive investigation of the RBC metabolome.

BACKGROUND: In most countries, red blood cells (RBCs) can be stored up to 42 days before transfusion. However, observational studies have suggested that storage duration might be associated with increased morbidity and mortality. While clinical trials are under way, impaired metabolism has been documented in RBCs stored in several additive solutions (ASs). Here we hypothesize that, despite reported beneficial effects, storage in AS-3 results in metabolic impairment weeks before the end of the unit shelf life. STUDY DESIGN AND METHODS: Five leukofiltered AS-3 RBC units were sampled before, during, and after leukoreduction Day 0 and then assayed on a weekly basis from storage Day 1 through Day 42. RBC extracts and supernatants were assayed using a ultra-high-performance liquid chromatography separations coupled online with mass spectrometry detection metabolomics workflow. RESULTS: Blood bank storage significantly affects metabolic profiles of RBC extracts and supernatants by Day 14. In addition to energy and redox metabolism impairment, intra- and extracellular accumulation of amino acids was observed proportionally to storage duration, suggesting a role for glutamine and serine metabolism in aging RBCs. CONCLUSION: Metabolomics of stored RBCs could drive the introduction of alternative ASs to address some of the storage-dependent metabolic lesions herein reported, thereby increasing the quality of transfused RBCs and minimizing potential links to patient morbidity.

Cytoprotective 3K3A-activated protein C and plasma: A comparison of therapeutics for the endotheliopathy of trauma.

BACKGROUND: Both healthy plasma and cytoprotective aPC (3K3A-aPC) have been shown to mitigate the endotheliopathy of trauma (EoT), but optimal therapeutics remain unknown. Our aim was therefore to determine optimal therapies to mitigate EoT by investigating the effectiveness of 3K3A-aPC with and without plasma-based resuscitation strategies. METHODS: Electric cell-substrate impedance sensing (ECIS) was used to measure real-time permeability changes in endothelial cells. Cells were treated with a 2 µg/mL solution of aPC 30 minutes prior to stimulation with plasma taken from severely injured trauma patients (ISS > 15 and BD < -6) (TP). Healthy plasma, or plasma frozen within 24 hours (FP24), was added concomitantly with TP. Cells treated with thrombin and untreated cells were included in this study as control groups. RESULTS: A dose-dependent difference was found between the 5% and 10% plasma-treated groups when HUVECs were simultaneously stimulated with TP (µd 7.346 95%CI 4.574 to 10.12). There was no difference when compared to TP alone in the 5% (µd 5.713 95%CI -1.751 to 13.18) or 10% group (µd -1.633 95%CI -9.097 to 5.832). When 3K3A-aPC was added to plasma and TP, the 5% group showed improvement in permeability compared to TP alone (µd 10.11 95%CI 2.642 to 17.57), but there was no difference in the 10% group (µd -1.394 95%CI -8.859 to 6.070). The combination of 3K3A-aPC, plasma, and TP at both the 5% plasma (µd -28.52 95%CI-34.72 to -22.32) and 10% plasma concentrations (µd -40.02 95%CI -46.22 to -33.82) had higher inter-cellular permeability than the 3K3A-aPC pre-incubation group. CONCLUSION: Our data shows that FP24, in a post-trauma environment, pre-treatment with 3K3A-aPC can potentially mitigate the EoT to a greater degree than FP24 with or without 3K3A-aPC. Although further exploration is needed, this represents a potentially ideal and perhaps superior therapeutic treatment for the dysregulated thromboinflammation of injured patients. LEVEL OF EVIDENCE: Prognostic/Epidemiological, Therapeutic/Care Management, Level III.

RESUSCITATIVE ENDOVASCULAR BALLOON OCCLUSION OF THE AORTA: ZONE 1 REPERFUSION-INDUCED COAGULOPATHY.

ABSTRACT: Objective: We sought to identify potential drivers behind resuscitative endovascular balloon occlusion of the aorta (REBOA) induced reperfusion coagulopathy using novel proteomic methods. Background: Coagulopathy associated with REBOA is poorly defined. The REBOA Zone 1 provokes hepatic and intestinal ischemia that may alter coagulation factor production and lead to molecular pathway alterations that compromises hemostasis. We hypothesized that REBOA Zone 1 would lead to reperfusion coagulopathy driven by mediators of fibrinolysis, loss of coagulation factors, and potential endothelial dysfunction. Methods: Yorkshire swine were subjected to a polytrauma injury (blast traumatic brain injury, tissue injury, and hemorrhagic shock). Pigs were randomized to observation only (controls, n = 6) or to 30 min of REBOA Zone 1 (n = 6) or REBOA Zone 3 (n = 4) as part of their resuscitation. Thromboelastography was used to detect coagulopathy. ELISA assays and mass spectrometry proteomics were used to measure plasma protein levels related to coagulation and systemic inflammation. Results: After the polytrauma phase, balloon deflation of REBOA Zone 1 was associated with significant hyperfibrinolysis (TEG results: REBOA Zone 1 35.50% versus control 9.5% vs. Zone 3 2.4%, P < 0.05). In the proteomics and ELISA results, REBOA Zone 1 was associated with significant decreases in coagulation factor XI and coagulation factor II, and significant elevations of active tissue plasminogen activator, plasmin-antiplasmin complex complexes, and syndecan-1 (P < 0.05). Conclusion: REBOA Zone 1 alters circulating mediators of clot formation, clot lysis, and increases plasma levels of known markers of endotheliopathy, leading to a reperfusion-induced coagulopathy compared with REBOA Zone 3 and no REBOA.

Biological response modifiers in photochemically pathogen-reduced versus untreated apheresis platelet concentrates.

BACKGROUND: Lipids and other biologically active substances accumulate in platelet concentrates (PCs) during storage. Some of these substances have been suggested to modulate immune responses and to play a pathogenic role in the development of transfusion-related acute lung injury. This study compared the content and impact of some biological response modifiers in PCs treated with pathogen reduction (PR) technology and nontreated PCs. STUDY DESIGN AND METHODS: Apheresis PCs (n = 12) were split in two: one split was subjected to PR treatment (INTERCEPT, Cerus Corp.) and the other split was left untreated. Basic characterization and content of vascular endothelial growth factor (VEGF) and sCD154 were measured. Lipopolysaccharide (LPS)-induced secretion of interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) was measured after incubation of heparinized whole blood with platelet (PLT) supernatants. The supernatants' neutrophil (PMN)-priming capacity, and thereby activation of the NADPH oxidase, was measured as the rate of superoxide anion production after formyl-Met-Leu-Phe activation. Lipids were extracted from the supernatants on Day 6 and tested for PMN-priming activity. RESULTS: Supernatants from PR-treated PCs demonstrated significantly higher mean PLT volume (MPV) and O(2) , lower pH, CO(2) , and HCO(3-) , and significantly less LPS-induced TNF-α secretion compared to untreated PCs. No differences in swirling, PLT count, potassium levels, glucose consumption, lactate production, IL-10, VEGF, sCD154, or PMN-priming activity were found between the groups over time. CONCLUSION: INTERCEPT PR treatment caused no substantial differences in PCs, except for minor changes in MPV and metabolic variables. Further studies are needed to explain the differences in the LPS-induced TNF-α secretion.

Elucidating the molecular mechanisms of fibrinolytic shutdown after severe injury: The role of thrombin-activatable fibrinolysis inhibitor.

BACKGROUND: The mechanisms underlying trauma-induced coagulopathy remain elusive. Hyperfibrinolysis has been linked to increased plasminogen activation and antiprotease consumption; however, the mechanistic players in its counterpart, fibrinolysis shutdown, remain unclear. We hypothesize that thrombin-activatable fibrinolysis inhibitor (TAFI) plays a major role in fibrinolytic shutdown after injury. METHODS: As part of this observational cohort study, whole blood was collected from trauma activation patients at a single, level 1 trauma center. Citrated rapid thrombelastography and the following enzyme-linked immunosorbent assays were conducted: thrombin, antithrombin, thrombin-antithrombin complex, TAFI, plasminogen, antiplasmin, plasmin-antiplasmin (PAP), tissue plasminogen activator, plasminogen activator inhibitor 1, and tissue plasminogen activator-plasminogen activator inhibitor 1 complex. Univariate and cluster analysis were performed. RESULTS: Overall, 56 patients (median age, 33.5 years; 70% male) were included. The majority (57%) presented after blunt mechanism and with severe injury (median New Injury Severity Score, 27). Two clusters of patients were identified: Group 1 (normal fibrinolysis, n = 21) and Group 2 (fibrinolysis shutdown, n = 35). Group 2 had significantly lower fibrinolysis with a median LY30 of 1.1% (interquartile range [IQR], 0.1-1.9%) versus 2.1% (IQR, 0.5-2.8%) in Group 1; while the median LY30 was within physiologic range, 45% of patients in Group 2 were in shutdown (vs. 24% in Group 1, p = 0.09). Compared with Group 1, Group 2 had significantly higher PAP (median, 4.7 [IQR, 1.7-9.3] vs. 1.4 [1.0-2.1] µg/mL in Group 1; p = 0.002) and higher TAFI (median, 152.5% [IQR, 110.3-190.7%] vs. 121.9% [IQR, 93.2-155.6%]; p = 0.04). There was a strong correlation between PAP and TAFI ( R2 = 0.5, p = 0.0002). CONCLUSION: The presented data characterize fibrinolytic shutdown, indicating an initial plasmin burst followed by diminished fibrinolysis, which is distinct from hypofibrinolysis (inadequate plasmin burst and fibrinolysis). After an initial thrombin and plasmin burst (increased PAP), fibrinolysis is inhibited, mediated in part by increased TAFI.

Bradykinin release following trauma and hemorrhagic shock causes pulmonary alveolar leak in a rodent model.

BACKGROUND: Hemorrhage accounts for 40% of the preventable death following severe injury. Activation of systemic coagulation produces bradykinin (BK), which may cause leak from the plasma to the extravascular space and to the tissues, which is part of the complex pathophysiology of trauma-induced end-organ injury. We hypothesize that BK, released during activation of coagulation in severe injury, induces pulmonary alveolar leak. METHODS: Isolated neutrophils (PMNs) were pretreated with a specific BK receptor B2 antagonist HOE-140/icatibant and BK priming of the PMN oxidase was completed. Rats underwent tissue injury/hemorrhagic shock (TI/HS), TI/icatibant/HS, and controls (no injury). Evans blue dye was instilled, and the percentage leak from the plasma to the lung was calculated from the bronchoalveolar lavage fluid (BALF). CINC-1 and total protein were measured in the BALF, and myeloperoxidase was quantified in lung tissue. RESULTS: The BK receptor B2 antagonist HOE140/icatibant inhibited (85.0 ± 5.3%) BK priming of the PMN oxidase ( p < 0.05). The TI/HS model caused activation of coagulation by increasing plasma thrombin-antithrombin complexes ( p < 0.05). Versus controls, the TI/HS rats had significant pulmonary alveolar leak: 1.46 ± 0.21% versus 0.36 ± 0.10% ( p = 0.001) and increased total protein and CINC-1 in the BALF ( p < 0.05). Icatibant given after the TI significantly inhibited lung leak and the increase in CINC-1 in the BALF from TI/icatibant/HS rats versus TI/HS ( p < 0.002 and p < 0.05) but not the total protein. There was no PMN sequestration in the lungs. Conclusions: This mixed injury model caused systemic activation of hemostasis and pulmonary alveolar leak likely due to BK release. CONCLUSION: This mixed injury model caused systemic activation of hemostasis and pulmonary alveolar leak likely due to BK release. LEVEL OF EVIDENCE: Original Article, Basic Science.

Platelet and cryoprecipitate transfusions from female donors improve coagulopathy in vitro.

BACKGROUND: Females are relatively hypercoagulable compared with males, with increased platelet aggregation and improved clot dynamics. However, sex differences in coagulation have not yet been considered in transfusion guidelines. Therefore, our objective was to evaluate hemostatic differences in sex concordant and sex discordant cryoprecipitate and platelet transfusions. We hypothesized that transfusion of blood products from female donors results in improved coagulopathy compared with male blood products. METHODS: This was a cohort study evaluating sex dimorphisms in coagulation assays and clotting factors in healthy volunteer plasma and cryoprecipitate. Sex dimorphisms in transfusions were evaluated using an in vitro coagulopathy model. Female or male platelets or single-donor cryoprecipitate was added to "recipient" whole blood after dilution of recipient blood with citrated saline to provoke a coagulopathic profile. Citrated native thromboelastography was then performed. Liquid chromatography/mass spectroscopy was performed on single-donor cryoprecipitate to evaluate sex dimorphisms in the proteome of cryoprecipitate. RESULTS: Females have an increased proportion of functional fibrinogen. Transfusion of female-donor platelets and cryoprecipitate induces a larger decrease in R time and greater increase in angle than male-donor platelets or cryoprecipitate. Female-donor cryoprecipitate has increased factor V and factor XIII compared with male cryoprecipitate, and comprehensive proteomics revealed sex differences in several proteins with potential immunological significance. CONCLUSION: Platelets and cryoprecipitate from female donors improve coagulopathy more than male blood products in vitro. Increased factor V and factor XIII activity as well as increased fibrinogen activity in female donors appears to drive this disparity. Sex differences in the proteome of cryoprecipitate may influence how transfusions modulate the thromboinflammation of trauma. The differing hemostatic profiles of female and male blood products suggest the potential role of sex-specific transfusions guidelines in hemostatic resuscitation.