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The objective of this review is to present the two-event model of transfusion-related acute lung injury (TRALI), a life-threatening complication of transfusions that has been the most common cause of transfusion-related death over the past 2 yrs in the United States. The two-event model of TRALI, which is identical to the pathogenesis of the acute respiratory distress syndrome (ARDS), is reviewed and contrasted to antibody-mediated TRALI. Laboratory studies, both in vitro and in vivo, are discussed as well as human studies of TRALI. Methods to avoid patient exposure to blood components that may cause TRALI are also discussed. [ABSTRACT FROM AUTHOR]

Introduction: Smoking is a public health threat because of its well-described link to increased oxidative stress-related diseases including peripheral vascular disease and coronary artery disease. Tobacco use has been linked to risk of inpatient trauma morbidity including acute respiratory distress syndrome; however, its mechanistic effect on comprehensive metabolic heterogeneity has yet to be examined., Methods: Plasma was obtained on arrival from injured patients at a Level 1 trauma center and analyzed with modern mass spectrometry-based metabolomics. Patients were stratified by nonsmoker, passive smoker, and active smoker by lower, interquartile, and upper quartile ranges of cotinine intensity peaks. Patients were substratified by high injury/high shock (Injury Severity Score, ≥15; base excess, <-6) and compared with healthy controls. p Value of <0.05 following false discovery rate correction of t test was considered significant., Results: Forty-eight patients with high injury/high shock (7 nonsmokers [15%], 25 passive smokers [52%], and 16 active smokers [33%]) and 95 healthy patients who served as controls (30 nonsmokers [32%], 43 passive smokers [45%], and 22 active smokers [23%]) were included. Elevated metabolites in our controls who were active smokers include enrichment in chronic inflammatory and oxidative processes. Elevated metabolites in active smokers in high injury/high shock include enrichment in the malate-aspartate shuttle, tyrosine metabolism, carnitine synthesis, and oxidation of very long-chain fatty acids., Conclusion: Smoking promotes a state of oxidative stress leading to mitochondrial dysfunction, which is additive to the inflammatory milieu of trauma. Smoking is associated with impaired mitochondrial substrate utilization of long-chain fatty acids, aspartate, and tyrosine, all of which accentuate oxidative stress following injury. This altered expression represents an ideal target for therapies to reduce oxidative damage toward the goal of personalized treatment of trauma patients., Level of Evidence: Prognostic and Epidemiological; Level IV., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

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., Competing Interests: Conflict of Interest: JTACS COI Disclosure forms for all authors have been supplied and are provided as supplemental digital content (http://links.lww.com/TA/D866)., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Patients with type O blood may have an increased risk of hemorrhagic complications due to lower baseline levels of von Willebrand Factor (vWF) and factor VIII, but the transition to a mortality difference in trauma is less clear. We hypothesized that type O trauma patients will have differential proteomic and metabolomic signatures in response to trauma beyond vWF and FVIII alone., Methods: Patients meeting the highest level of trauma activation criteria were prospectively enrolled. Blood samples were collected upon arrival to the emergency department. Proteomic and metabolomic (multi-omics) analyses of these samples were performed using liquid chromatography-mass spectrometry. Demographic, clinical, and multi-omics data were compared between patients with type O blood versus all other patients., Results: There were 288 patients with multi-omics data; 146 (51%) had type O blood. Demographics, injury patterns, and initial vital signs and laboratory measurements were not different between groups. Type O patients had increased lengths of stay (7 vs. 6 days, p = 0.041) and a trend towards decreased mortality secondary to traumatic brain injury compared to other causes (TBI, 44.4 % vs. 87.5%, p = 0.055). Type O patients had decreased levels of mannose-binding lectin (MBL) and MBL associated serine proteases 1 and 2 which are required for the initiation of the lectin pathway of complement activation. Type O patients also had metabolite differences signifying energy metabolism and mitochondrial dysfunction., Conclusion: Blood type O patients have a unique multi-omics signature, including decreased levels of proteins required to activate the lectin complement pathway. This may lead to overall decreased levels of complement activation and decreased systemic inflammation in the acute phase possibly leading to a survival advantage, especially in TBI. However, this may later impair healing. Future work will need to confirm these associations, and animal studies are needed to test therapeutic targets., Level of Evidence: Retrospective Comparative Study, Level IV., Competing Interests: Conflict of Interest Statement: All JTACS disclosure forms have been supplied and are provided as supplemental digital content. There are no direct conflicts of interest with this manuscript to report (http://links.lww.com/TA/D811)., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: We aimed to investigate if ex vivo plasma from injured patients causes endothelial calcium (Ca2+) influx as a mechanism of trauma-induced endothelial permeability., Summary Background Data: Endothelial permeability after trauma contributes to post-injury organ dysfunction. While the mechanisms remain unclear, emerging evidence suggests intracellular Ca2+ signaling may play a role., Methods: Ex vivo plasma from injured patients with "Low Injury/Low Shock" (injury severity score [ISS]<15, base excess [BE])≥-6mEq/L) and "High Injury/High Shock" (ISS≥15, BE<-6mEq/L) were used to treat endothelial cells. Experimental conditions included Ca2+ removal from the extracellular buffer, cyclopiazonic acid pre-treatment to deplete intracellular Ca2+ stores, and GSK2193874 pre-treatment to block the TRPV4 Ca2+ channel. Live cell fluorescence microscopy and ECIS were used to assess cytosolic Ca2+ increases and permeability, respectively. Western blot and live cell actin staining were used to assess myosin light chain (MLC) phosphorylation and actomyosin contraction., Results: Compared to Low Injury/Low Shock plasma, High Injury/High Shock induced greater cytosolic Ca2+ increase. Cytosolic Ca2+ increase, MLC phosphorylation, and actin cytoskeletal contraction were lower without extracellular Ca2+ present. High Injury/High Shock plasma did not induce endothelial permeability without extracellular Ca2+ present. TRPV4 inhibition lowered trauma plasma-induced endothelial Ca2+ influx and permeability., Conclusions: This study illuminates a novel mechanism of post-injury endotheliopathy involving Ca2+ influx via the TRPV4 channel. TRPV4 inhibition mitigates trauma-induced endothelial permeability. Moreover, widespread endothelial Ca2+ influx may contribute to trauma-induced hypocalcemia. This study provides the mechanistic basis for the development of Ca2+-targeted therapies and interventions in the care of severely injured patients., Competing Interests: Conflicts of Interest and Source of Funding: The authors report no conflicts of interest. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (T32 GM008315 and P50 GM049222 to M.C., R01HL136636 and RF1/R01NS129022 to F.D.). Other important funding sources include a series of grants through the Trans-agency Research Consortium for Trauma Induced Coagulopathy (TACTIC UM1-HL120877 to M.C.) from the National Heart, Lung and Blood Institute of the NIH, a research grant from the University of Pennsylvania Orphan Disease Center in partnership with the cureCADASIL to F.D., and a research grant from the Ludeman Family Center for Women’s Health Research CU AMC to F.D. The major funding source is an RM-1 grant (1RM1GM131968-01 to M.C.) that runs through May of 2024. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other sponsors of the project., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The coagulopathy of traumatic brain injury (TBI) remains poorly understood. Contradictory descriptions highlight the distinction between systemic and local coagulation, with descriptions of systemic hypercoagulability despite intracranial hypocoagulopathy. This perplexing coagulation profile has been hypothesized to be due to tissue factor release. The objective of this study was to assess the coagulation profile of TBI patients undergoing neurosurgical procedures. We hypothesize that dura violation is associated with higher tissue factor and conversion to a hypercoagulable profile and unique metabolomic and proteomic phenotype., Methods: This is a prospective, observational cohort study of all adult TBI patients at an urban, Level I trauma center who underwent a neurosurgical procedure from 2019 to 2021. Whole blood samples were collected before and then 1 hour following dura violation. Citrated rapid and tissue plasminogen activator (tPA) thrombelastography (TEG) were performed, in addition to measurement of tissue factory activity, metabolomics, and proteomics., Results: Overall, 57 patients were included. The majority (61%) were male, the median age was 52 years, 70% presented after blunt trauma, and the median Glasgow Coma Score was 7. Compared with pre-dura violation, post-dura violation blood demonstrated systemic hypercoagulability, with a significant increase in clot strength (maximum amplitude of 74.4 mm vs. 63.5 mm; p < 0.0001) and a significant decrease in fibrinolysis (LY30 on tPAchallenged TEG of 1.4% vs. 2.6%; p = 0.04). There were no statistically significant differences in tissue factor. Metabolomics revealed notable increases in metabolites involved in late glycolysis, cysteine, and one-carbon metabolites, and metabolites involved in endothelial dysfunction/arginine metabolism/responses to hypoxia. Proteomics revealed notable increase in proteins related to platelet activation and fibrinolysis inhibition., Conclusion: A systemic hypercoagulability is observed in TBI patients, characterized by increased clot strength and decreased fibrinolysis and a unique metabolomic and proteomics phenotype independent of tissue factor levels., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Abstract: Background: Trauma-induced hypocalcemia is common and associated with adverse outcomes, but the mechanisms remain unclear. Thus, we aimed to characterize the metabolomic and proteomic differences between normocalcemic and hypocalcemic trauma patients to illuminate biochemical pathways that may underlie a distinct pathology linked with this clinical phenomenon. Methods: Plasma was obtained on arrival from injured patients at a Level 1 Trauma Center. Samples obtained after transfusion were excluded. Multiple regression was used to adjust the omics data for injury severity and arrival base excess before metabolome- and proteome-wide comparisons between normocalcemic (ionized Ca 2+ > 1.0 mmol/L) and hypocalcemic (ionized Ca 2+ ≤ 1.0 mmol/L) patients using partial least squares-discriminant analysis. OmicsNet and Gene Ontology were used for network and pathway analyses, respectively. Results: Excluding isolated traumatic brain injury and penetrating injury, the main analysis included 36 patients (n = 14 hypocalcemic, n = 22 normocalcemic). Adjusted analyses demonstrated distinct metabolomic and proteomic signatures for normocalcemic and hypocalcemic patients. Hypocalcemic patients had evidence of mitochondrial dysfunction (tricarboxylic acid cycle disruption, dysfunctional fatty acid oxidation), inflammatory dysregulation (elevated damage-associated molecular patterns, activated endothelial cells), aberrant coagulation pathways, and proteolytic imbalance with increased tissue destruction. Conclusions: Independent of injury severity, hemorrhagic shock, and transfusion, trauma-induced hypocalcemia is associated with early metabolomic and proteomic changes that may reflect unique pathology in hypocalcemic trauma patients. This study paves the way for future experiments to investigate mechanisms, identify intervenable pathways, and refine our management of hypocalcemia in severely injured patients., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 by the Shock Society.)

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., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Even in the era of the COVID-19 pandemic, trauma remains the global leading cause of mortality under the age of 49. Trauma-induced coagulopathy is a leading driver of early mortality in critically ill patients, and transfusion of platelet products is a life-saving intervention to restore hemostasis in the bleeding patient. However, despite extensive functional studies based on viscoelastic assays, limited information is available about the impact of platelet transfusion on the circulating molecular signatures in trauma patients receiving platelet transfusion., Materials and Methods: To bridge this gap, we leveraged metabolomics and proteomics approaches to characterize longitudinal plasma samples (n = 118; up to 11 time points; total samples: 759) from trauma patients enrolled in the Control Of Major Bleeding After Trauma (COMBAT) study. Samples were collected in the field, in the emergency department (ED), and at intervals up to 168 h (7 days) post-hospitalization. Transfusion of platelet (PLT) products was performed (n = 30; total samples: 250) in the ED through 24 h post-hospitalization. Longitudinal plasma samples were subjected to mass spectrometry-based metabolomics and proteomics workflows. Multivariate analyses were performed to determine omics markers of transfusion of one, two, three, or more PLT transfusions., Results: Higher levels of tranexamic acid (TXA), inflammatory proteins, carnitines, and polyamines were detected in patients requiring PLT transfusion. Correlation of PLT units with omics data suggested sicker patients required more units and partially overlap with the population requiring transfusion of packed red blood cell products. Furthermore, platelet activation was likely increased in the most severely injured patients. Fatty acid levels were significantly lower in PLT transfusion recipients (at time of maximal transfusion: Hour 4) compared with non-recipients, while carnitine levels were significantly higher. Fatty acid levels restore later in the time course (e.g., post-PLT transfusion)., Discussion: The present study provides the first multi-omics characterization of platelet transfusion efficacy in a clinically relevant cohort of trauma patients. Physiological alterations following transfusion were detected, highlighting the efficacy of mass spectrometry-based omics techniques to improve personalized transfusion medicine. More specialized clinical research studies focused on PLT transfusion, including organized pre and post transfusion sample collection and limitation to PLT products only, are required to fully understand subsequent metabolomic and proteomic alterations., (© 2023 AABB.)

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., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Trauma-induced coagulopathy (TIC) has been the subject of intense study for greater than a century, and it is associated with high morbidity and mortality. The Trans-Agency Consortium for Trauma-Induced Coagulopathy, funded by the National Health Heart, Lung and Blood Institute, was tasked with developing a clinical TIC score, distinguishing between injury-induced bleeding from persistent bleeding due to TIC. We hypothesized that the Trans-Agency Consortium for Trauma-Induced Coagulopathy clinical TIC score would correlate with laboratory measures of coagulation, transfusion requirements, and mortality., Methods: Trauma activation patients requiring a surgical procedure for hemostasis were scored in the operating room (OR) and in the first ICU day by the attending trauma surgeon. Conventional and viscoelastic (thrombelastography) coagulation assays, transfusion requirements, and mortality were correlated to the coagulation scores using the Cochran-Armitage trend test or linear regression for numerical variables., Results: Increased OR TIC scores were significantly associated with abnormal conventional and viscoelastic measurements, including hyperfibrinolysis incidence, as well as with higher mortality and more frequent requirement for massive transfusion ( p < 0.0001 for all trends). Patients with OR TIC score greater than 3 were more than 31 times more likely to have an ICU TIC score greater than 3 (relative risk, 31.6; 95% confidence interval, 12.7-78.3; p < 0.0001)., Conclusion: A clinically defined TIC score obtained in the OR reflected the requirement for massive transfusion and mortality in severely injured trauma patients and also correlated with abnormal coagulation assays. The OR TIC score should be validated in multicenter studies., Level of Evidence: Prognostic and Epidemiological; Level IV., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a lifesaving therapy for hemorrhagic shock following pelvic/lower extremity injuries in military settings. However, Zone 1 aortic occlusion (AO; above the celiac artery), while providing brain/cardiac perfusion, may induce/worsen visceral ischemia and organ dysfunction. In contrast, AO Zone 3 (below the renal arteries) provides abdominal perfusion potentially minimizing ischemia/reperfusion injury. We hypothesized that, compared with AO Zone 1, AO Zone 3 provides neuro/cardioprotection while minimizing visceral ischemia and reperfusion coagulopathy after severe traumatic hemorrhage due to pelvic/lower extremity injuries., Methods: Fifty-kilogram male Yorkshire swine underwent a blast polytrauma injury followed by a resuscitation protocol with randomization to no AO (No AO, n = 6) or AO with REBOA at Zone 1 (AO Zone 1; n = 6) or Zone 3 (AO Zone 3; n = 4). Vital signs and intracranial pressure (ICP) were monitored for 240 minutes. Citrate native and tissue plasminogen activator challenge thrombelastography, prothrombin time, creatinine, lipase, total bilirubin, troponin, and enzyme-linked immunosorbent assays protein levels were measured at set intervals., Results: Both AO groups had significant increases in mean arterial pressure during aortic occlusion. All three groups had significant increases in ICP, but final ICP in the No AO group (26 ± 5.8 mm Hg) was significantly elevated compared with AO Zone 1 (17 ± 5.2 mm Hg) and AO Zone 3 (16 ± 4.2 mm Hg) ( p < 0.01). The final mean troponin in the No AO group (4.10 ± 5.67 ng/mL) was significantly higher than baseline (0.03 ± 0.02 ng/mL, p < 0.05), while the two AO groups had no significant changes ( p > 0.05). AO Zone 1 was the only group associated with hyperfibrinolysis ( p < 0.05) and significantly increased prothrombin time ( p < 0.05). Only AO Zone 1 group had significantly higher markers of organ damage., Conclusion: Compared with AO Zone 1, AO Zone 3 provided similar neuro/cardioprotection but with less organ dysfunction and coagulopathy. This study suggests that Zone 3 REBOA may be preferable over Zone 1 for treating military relevant blast polytrauma with minimal intra-abdominal and chest trauma, but further clinical investigation is warranted., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Tissue injury (TI) and hemorrhagic shock (HS) are the major contributors to trauma-induced coagulopathy (TIC). However, the individual contributions of these insults are difficult to discern clinically because they typically coexist. TI has been reported to release procoagulants, while HS has been associated with bleeding. We developed a large animal model to isolate TI and HS and characterize their individual mechanistic pathways. We hypothesized that while TI and HS are both drivers of TIC, they provoke different pathways; specifically, TI reduces time to clotting, whereas, HS decreases clot strength stimulates hyperfibrinolysis., Methods: After induction of general anesthesia, 50 kg male, Yorkshire swine underwent isolated TI (bilateral muscle cutdown of quadriceps, bilateral femur fractures) or isolated HS (controlled bleeding to a base excess target of - 5 mmol/l) and observed for 240 min. Thrombelastography (TEG), calcium levels, thrombin activatable fibrinolysis inhibitor (TAFI), protein C, plasminogen activator inhibitor 1 (PAI-1), and plasminogen activator inhibitor 1/tissue-type plasminogen activator complex (PAI-1-tPA) were analyzed at pre-selected timepoints. Linear mixed models for repeated measures were used to compare results throughout the model., Results: TI resulted in elevated histone release which peaked at 120 min (p = 0.02), and this was associated with reduced time to clot formation (R time) by 240 min (p = 0.006). HS decreased clot strength at time 30 min (p = 0.003), with a significant decline in calcium (p = 0.001). At study completion, HS animals had elevated PAI-1 (p = 0.01) and PAI-1-tPA (p = 0.04), showing a trend toward hyperfibrinolysis, while TI animals had suppressed fibrinolysis. Protein C, TAFI and skeletal myosin were not different among the groups., Conclusion: Isolated injury in animal models can help elucidate the mechanistic pathways leading to TIC. Our results suggest that isolated TI leads to early histone release and a hypercoagulable state, with suppressed fibrinolysis. In contrast, HS promotes poor clot strength and hyperfibrinolysis resulting in hypocoagulability., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

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., (Copyright © 2023 American Association for the Surgery of Trauma.)

Background: The collection of the first blood flow into a diversion pouch (DP) has become widely adopted in blood donation systems to reduce whole-blood unit contamination from skin bacteria. The strict control of pre-analytical variables, such as blood collection and proper anticoagulant selection, is critical to diminish experimental variability when studying different aspects of platelet biology. We hypothesize that the functional, mitochondrial, and metabolomic profiles of platelets isolated from the DP are not different from the ones isolated from standard venipuncture (VP), thus representing a suitable collection method of platelets for experimental purposes., Materials and Methods: Whole blood from the blood DP or VP was collected. Platelets were subsequently isolated and washed following standard protocols. Platelet function was assessed by flow cytometry, light transmission aggregometry, clot retraction, and under flow conditions using the total thrombus formation analyzer (T-TAS). Mitochondrial function and the platelet metabolome profiles were determined by the Seahorse extracellular flux analyzer (Agilent, Santa Clara, CA, USA) and ultra-high-pressure liquid chromatography-mass spectrometry metabolomics, respectively., Results: Platelets isolated from VP and the DP have similar functional, mitochondrial, and metabolic profiles with no significant differences between both groups at baseline and upon activation by any of the assays mentioned above., Discussion: The findings of our study support the use of platelets from the DP for performing functional and metabolic studies on platelets from a wide range of blood donors. The DP may serve as an alternative blood collection method to standard VP, allowing the study of diverse aspects of platelet biology, such as age, sex, race, and ethnicity, in many eligible individuals for blood donation.

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., (Copyright © 2022 American Association for the Surgery of Trauma.)

Background: Postpartum hemorrhage is a leading cause of morbidity and mortality worldwide, yet the associated early coagulopathy is not well defined., Objective: We hypothesized that women who develop postpartum hemorrhage have a distinct derangement of thrombin generation and coagulation factors compared with postpartum women without postpartum hemorrhage., Study Design: This prospective study of pregnant patients with postpartum hemorrhage was completed at a single urban hospital. Blood was drawn on postpartum hemorrhage diagnosis and 2 and 4 hours later. Assays of patients with postpartum hemorrhage included thrombelastography, whole blood thrombin generation, coagulation factor activity, tissue factor levels and activity, and tissue factor pathway inhibitor levels, which were compared with that of patients without postpartum hemorrhage., Results: A total of 81 patients were included in this study. Of those patients, 66 had postpartum hemorrhage, and 15 served as controls. Compared with patients without PPH, patients with postpartum hemorrhage had lower fibrinogen levels (469.0 mg/dL vs 411.0 mg/dL; P=.02), increased tissue plasminogen activator resistance (fibrinolysis 30 minutes after maximal clot strength: 8.7% vs 4.2%; P=.02), decreased peak thrombin concentration (150.2 nM vs 40.7 nM; P=.01), and decreased maximal rate of thrombin generation (60.1 nM/minute vs 2.8 nM/minute; P=.02). Furthermore, compared with patients without postpartum hemorrhage, patients with postpartum hemorrhage had decreased tissue factor levels (444.3 pg/mL vs 267.1 pg/mL; P=.02) and increased tissue factor pathway inhibitor levels (0.6 U/mL vs 0.8 U/mL; P=.04), with decreased tissue factor pathway inhibitor ratios (624 vs 299; P=.01)., Conclusion: PPH is not only an issue of uterine tone and mechanical bleeding but also a distinct coagulopathy that is characterized by decreased fibrinogen level, clot breakdown resistance, and markedly low thrombin generation. This pathology seemed to be driven by low tissue factor and high tissue factor pathway inhibitor levels., (Copyright © 2022. Published by Elsevier Inc.)

Background: Sex dimorphisms in coagulation are well established, with female-specific hypercoagulability conferring a survival benefit in the setting of trauma-induced coagulopathy (TIC). The mechanism behind these phenomena remains to be elucidated. We hypothesize that estradiol provokes a hypercoagulable profile and alters clot proteomics and fibrin crosslinking., Methods: Whole blood was collected from healthy adult volunteers (n = 30). A battery of thrombelastography (TEG) assays (native, kaolin, platelet-mapping, functional fibrinogen), whole blood thrombin generation, proteomics, and clot structure architecture (via analysis of fibrin crosslinks and fluorescent fibrinogen-visualized clots) were performed after pre-treatment of the blood with physiologic concentrations of beta-estradiol. In addition, a prospective study of coagulation through the menstrual cycle was conducted by collecting blood from women on peak and nadir estrogen days in the standard 28-day menstrual cycle., Results: On TEG, in females, estradiol provoked a hypercoagulable phenotype, specifically a shorter time to clot formation and greater thrombin generation, greater rate of clot propagation and functional fibrinogen, higher clot strength, and diminished clot fibrinolysis. In both males and females, estradiol increased platelet hyperactivity. Similar changes were seen in time to clot formation and clot strength in vivo during peak estrus of the menstrual cycle. On proteomic analysis, in both males and females, estradiol was associated with increases in abundance of several procoagulant and antifibrinolytic proteins. Crosslinking mass spectrometry analysis showed addition of estradiol increased the abundance of several FXIII crosslinks within the FIBA alpha chain in both sexes. Fluorescent fibrinogen analysis revealed a trend toward increased fiber resolvability index after addition of estradiol., Conclusion: Estradiol provokes a hypercoagulable phenotype, affecting time to clot formation, clot propagation, clot strength, clot fibrinolysis, and clot structure. In sum, these data highlight the role of estradiol is driving female-specific hypercoagulability and highlights its potential role as a therapeutic adjunct in resuscitation of TIC., (Copyright © 2022 American Association for the Surgery of Trauma.)

Abstract: Background: Severe injury can provoke systemic processes that lead to organ dysfunction, and hemolysis of both native and transfused red blood cells (RBCs) may contribute. Hemolysis can release erythrocyte proteins, such as hemoglobin and arginase-1, the latter with the potential to disrupt arginine metabolism and limit physiologic NO production. We aimed to quantify hemolysis and arginine metabolism in trauma patients and measure association with injury severity, transfusions, and outcomes. Methods: Blood was collected from injured patients at a level I trauma center enrolled in the COMBAT (Control of Major Bleeding After Trauma) trial. Proteomics and metabolomics were performed on plasma fractions through liquid chromatography coupled with mass spectrometry. Abundances of erythrocyte proteins comprising a hemolytic profile as well as haptoglobin, l -arginine, ornithine, and l -citrulline (NO surrogate marker) were analyzed at different timepoints and correlated with transfusions and adverse outcomes. Results: More critically injured patients, nonsurvivors, and those with longer ventilator requirement had higher levels of hemolysis markers with reduced l -arginine and l -citrulline. In logistic regression, elevated hemolysis markers, reduced l -arginine, and reduced l -citrulline were significantly associated with these adverse outcomes. An increased number of blood transfusions were significantly associated with elevated hemolysis markers and reduced l -arginine and l -citrulline independently of New Injury Severity Score and arterial base excess. Conclusions: Severe injury induces intravascular hemolysis, which may mediate postinjury organ dysfunction. In addition to native RBCs, transfused RBCs can lyse and may exacerbate trauma-induced hemolysis. Arginase-1 released from RBCs may contribute to the depletion of l -arginine and the subsequent reduction in the NO necessary to maintain organ perfusion., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 by the Shock Society.)

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., (© 2022 AABB.)

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Abstract: Background: Blood type O is the most common blood type and has lower von Willebrand factor (vWF) levels (25%-35% lower than non-O blood types). von Willebrand factor is important for initiating platelet attachment and binding factor VIII. We hypothesized that patients with type O blood are at an increased risk of trauma-induced coagulopathy and bleeding post injury. Study Design: Adult trauma activations with known blood type at a level I trauma center with field systolic blood pressure < 90 mm Hg were studied retrospectively. The relationships of blood group O versus non-O to coagulation assays, massive transfusion (MT), ventilator-free days, and mortality were adjusted for confounders. Hyperfibrinolysis (HF) was defined as thromboelastogram of percent lysis in 30 min > 3%, and fibrinolysis shutdown was defined as percent lysis in 30 min < 0.9%. von Willebrand factor activity was quantified on 212 injured patients using a STAGO apparatus. Results: Overall, 268 patients met criteria. Type O patients were more likely to develop HF than non-type O blood patients (43% vs. 29%, P = 0.06) and had significantly lower vWF activity (222% vs. 249%, P = 0.01). After adjustment for New Injury Severity Score and blunt mechanism, type O had higher odds of HF (odds ratio, 1.94, 95% confidence interval, 1.09-3.47) and increased odds of MT (odds ratio, 3.02; 95% confidence interval, 1.22-7.49). Other outcomes were not significantly affected. Conclusion: Type O patients with hypotension had increased HF and MT post injury, and these were associated with lower vWF activity. These findings have implications for the monitoring of HF in patients receiving type O whole-blood transfusions post injury., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 by the Shock Society.)

Abstract: Introduction: Severely injured patients develop a dysregulated inflammatory state characterized by vascular endothelial permeability, which contributes to multiple organ failure. To date, however, the mediators of and mechanisms for this permeability are not well established. Endothelial permeability in other inflammatory states such as sepsis is driven primarily by overactivation of the RhoA GTPase. We hypothesized that tissue injury and shock drive endothelial permeability after trauma by increased RhoA activation leading to break down of endothelial tight and adherens junctions. Methods: Human umbilical vein endothelial cells (HUVECs) were grown to confluence, whereas continuous resistance was measured using electrical cell-substrate impedance sensing (ECIS) Z-Theta technology, 10% ex vivo plasma from severely injured trauma patients was added, and resistance measurements continued for 2 hours. Areas under the curve (AUCs) were calculated from resistance curves. For GTPase activity analysis, HUVECs were grown to confluence and incubated with 10% trauma plasma for 5 minutes before harvesting of cell lysates. Rho and Rac activity were determined using a G-LISA assay. Significance was determined using Mann-Whitney tests or Kruskal-Wallis test, and Spearman ρ was calculated for correlations. Results: Plasma from severely injured patients induces endothelial permeability with plasma from patients with both severe injury and shock contributing most to this increased permeability. Surprisingly, Injury Severity Score (ISS) does not correlate with in vitro trauma-induced permeability (-0.05, P > 0.05), whereas base excess (BE) does correlate with permeability (-0.47, P = 0.0001). The combined impact of shock and injury resulted in a significantly smaller AUC in the injury + shock group (ISS > 15, BE < -9) compared with the injury only (ISS > 15, BE > -9; P = 0.04) or minimally injured (ISS < 15, BE > -9; P = 0.005) groups. In addition, incubation with injury + shock plasma resulted in higher RhoA activation ( P = 0.002) and a trend toward decreased Rac1 activation ( P = 0.07) compared with minimally injured control. Conclusions: Over the past decade, improved early survival in patients with severe trauma and hemorrhagic shock has led to a renewed focus on the endotheliopathy of trauma. This study presents the largest study to date measuring endothelial permeability in vitro using plasma collected from patients after traumatic injury. Here, we demonstrate that plasma from patients who develop shock after severe traumatic injury induces endothelial permeability and increased RhoA activation in vitro . Our ECIS model of trauma-induced permeability using ex vivo plasma has potential as a high throughput screening tool to phenotype endothelial dysfunction, study mediators of trauma-induced permeability, and screen potential interventions., Competing Interests: Funding disclosure and conflict of interest statement: The Trauma Research Center has received extramural funding since its inception, mainly from the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, MD, in the form of program project grants. Other important funded research programs include the Control of Major Bleeding after Trauma (COMBAT; W81XWH-12-2-2008), which was funded by the Department of Defense; a research grant from the Foundation for Women and Girls with Bleeding Disorders (FWGBD); and a series of grants through the Trans-agency Research Consortium for Trauma Induced Coagulopathy (TACTIC; UM1-HL120877) from the National Heart, Lung, and Blood Institute, National Institutes of Health. The current major funding source is an RM-1 grant 1RM1GM131968-01 that runs through May of 2024. We otherwise report no financial or other conflicts of interest., (Copyright © 2022 by the Shock Society.)

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.

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.

Background: Complement activation after trauma promotes hemostasis but is associated with increased morbidity and mortality. However, the specific pathways and downstream mediators remain unclear. Recently, the anaphylatoxin C4a has been shown to bind to thrombin receptors. While plasma-based resuscitation has been shown to modify the endotheliopathy of trauma, it may provide complement zymogens that fuel ongoing inflammatory cascades. We sought to characterize the activation of complement after injury and the effect of fresh frozen plasma (FFP) on this inflammatory response. We hypothesized that trauma induces C4 activation, which is associated with worse outcomes and influenced by FFP resuscitation., Methods: Blood was collected from injured patients at a single level I trauma center enrolled in the Control of Major Bleeding after Trauma (COMBAT) randomized clinical trial. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry. For the present observational study, concentrations of complement proteins were analyzed at multiple time points, compared between treatment groups, and correlated with outcomes., Results: C4 activation occurred over the first 6 hours postinjury with peak activation 6 to 24 hours. Tissue hypoperfusion, defined as base deficit >10 mEq/L, and requirement for massive transfusion were associated with greater C4 activation. C4 activation was associated with mortality, multiple organ failure, and longer ventilator requirement. In addition, temporal trends of C1q, factor B, and C3 by outcome groups support the prevailing theory of primary classical pathway activation with alternative pathway amplification. Resuscitation with FFP over the first 6 hours was associated with increased C4 activation at 12 and 24 hours., Conclusion: C4 activation has an important inflammatory role postinjury, and FFP has the potential to augment this complement activation during resuscitation., Level of Evidence: Prognostic/epidemiological, level III., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

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., (© 2022 AABB.)