Pathogenic soluble tau peptide disrupts endothelial calcium signaling and vasodilation in the brain microvasculature.

The accumulation of the microtubule-associated tau protein in and around blood vessels contributes to brain microvascular dysfunction through mechanisms that are incompletely understood. Delivery of nutrients to active neurons in the brain relies on capillary calcium (Ca2+) signals to direct blood flow. The initiation and amplification of endothelial cell Ca2+ signals require an intact microtubule cytoskeleton. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced microtubule destabilization would impair endothelial Ca2+ signaling. We tested the hypothesis that tau disrupts the regulation of local cerebral blood flow by reducing endothelial cell Ca2+ signals and endothelial-dependent vasodilation. We used a pathogenic soluble tau peptide (T-peptide) model of tau aggregation and mice with genetically encoded endothelial Ca2+ sensors to measure cerebrovascular endothelial responses to tau exposure. T-peptide significantly attenuated endothelial Ca2+ activity and cortical capillary blood flow in vivo. Further, T-peptide application constricted pressurized cerebral arteries and inhibited endothelium-dependent vasodilation. This study demonstrates that pathogenic tau alters cerebrovascular function through direct attenuation of endothelial Ca2+ signaling and endothelium-dependent vasodilation.

Pathogenic soluble tau peptide disrupts endothelial calcium signaling and vasodilation in the brain microvasculature.

The accumulation of the microtubule-associated tau protein in and around blood vessels contributes to brain microvascular dysfunction through mechanisms that are incompletely understood. Delivery of nutrients to active neurons in the brain relies on capillary inositol 1,4,5-triphosphate receptor (IP3R)-mediated calcium (Ca2+) signals to direct blood flow. The initiation and amplification of endothelial cell IP3R-mediated Ca2+ signals requires an intact microtubule cytoskeleton. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced microtubule destabilization would impair endothelial IP3-evoked Ca2+ signaling. We tested the hypothesis that tau disrupts the regulation of local cerebral blood flow by reducing endothelial cell Ca2+ signals and endothelial-dependent vasodilation. We used a pathogenic soluble tau peptide (T-peptide) model of tau aggregation and mice with genetically encoded endothelial Ca2+ sensors to measure cerebrovascular endothelial responses to tau exposure. T-peptide significantly attenuated endothelial Ca2+ activity and cortical capillary blood flow in vivo within 120 seconds. Further, T-peptide application constricted pressurized cerebral arteries and inhibited endothelium-dependent vasodilation. This study demonstrates that pathogenic tau alters cerebrovascular function through direct attenuation of endothelial Ca2+ signaling and endothelium-dependent vasodilation.

The Polyanionic Drug Suramin Neutralizes Histones and Prevents Endotheliopathy.

Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but challenges with dosing or side effects such as bleeding limit clinical application. In this study, we demonstrate that suramin, a widely available polyanionic drug, completely neutralizes the toxic effects of individual histones, but not citrullinated histones from neutrophil extracellular traps. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sublethal doses of histones in vivo. Suramin also prevented histone-induced lung endothelial cell cytotoxicity in vitro and lung edema, intra-alveolar hemorrhage, and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels.

Alpha-methyltyrosine reduces the acute cardiovascular and behavioral sequelae in a murine model of traumatic brain injury.

BACKGROUND: Increased catecholamines contribute to heightened cardiovascular reactivity and behavioral deficits after traumatic brain injury (TBI); adrenergic receptor blockade has limited success in reducing adverse sequelae of TBI. Injury-induced increases in the synthesis of catecholamines could contribute to adverse outcomes in TBI. Inhibition of catecholamine synthesis with alpha-methyltyrosine (αMT) could offer a benefit after TBI. METHODS: Original research trial in mice randomized to αMT (50 mg·kg -1 ·d -1 ) or vehicle for 1 week after TBI induced by controlled cortical impact. Primary outcomes of cardiovascular reactivity and behavioral deficits were assessed after 1 week. Secondary outcomes included blood brain barrier permeability and quantification of gene transcription whose products determine intraneuronal chloride concentrations, the release of catecholamines, and activation of the sympathetic nervous system. These genes were the alpha-2 adrenergic receptor ("Adra2c"), the sodium-potassium-chloride cotransporter ("Nkcc1"), and the potassium chloride cotransporter ("Kcc2"). We also assessed the effect of TBI and αMT on the neuronal chloride/bicarbonate exchanger ("Ae3"). RESULTS: Traumatic brain injury-induced increases in blood pressure and cardiac reactivity were blocked by αMT. Inhibition of catecholamine synthesis decreased blood brain barrier leakage and improved behavioral outcomes after TBI. Traumatic brain injury diminished the transcription of Adra2c and enhanced expression of Nkcc1 while reducing Kcc2 transcription; αMT prevented the induction of the Nkcc1 by TBI without reversing the effects of TBI on Kcc2 expression; αMT also diminished Ae3 transcription. CONCLUSION: Traumatic brain injury acutely increases cardiovascular reactivity and induces behavioral deficits in an αMT-sensitive manner, most likely by inducing Nkcc1 gene transcription. Alpha-methyltyrosine may prove salutary in the treatment of TBI by attenuating the enhanced expression of Nkcc1, minimizing blood brain barrier leakage, and diminishing central catecholamine and sympathetic output. We also found an unreported relationship between Kcc2 and the chloride/bicarbonate exchanger, which should be considered in the design of trials planned to manipulate central intraneuronal chloride concentrations following acute brain injury.

Estradiol provokes hypercoagulability and affects fibrin biology: A mechanistic exploration of sex dimorphisms in coagulation.

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.

Digestion behavior, in vitro and in vivo bioavailability of cannabidiol in emulsions stabilized by whey protein-maltodextrin conjugate: Impact of carrier oil.

An emulsion delivery system may be affected significantly by oil phase composition in terms of digestion behavior and bioavailability of the delivered substance. In this study, emulsions loaded with cannabidiol (CBD) were prepared with medium chain triglyceride (MCT), long chain triglyceride (LCT) or MCT/LCT(1:1) as carrier oil and whey protein-maltodextrin conjugate as emulsifier, and the digestion behavior of emulsion and bioavailability of CBD were assessed in vitro and in vivo. The particle size of emulsions throughout the in vitro digestion process was in the order of MCT < MCT/LCT < LCT, and three emulsions showed consistent particle size changes: stable in oral phase, sharply increased in gastric phase, and decreased in small intestine. After intestinal digestion, about 90% of free fatty acids (FFA) was released in MCT emulsion, followed by MCT/LCT (76%) and then LCT (45%). CBD was degraded during gastrointestinal digestion and the transformation stability of CBD in oil phase was in the order of LCT > MCT/LCT > MCT. Although CBD had higher bioaccessibility in MCT and MCT/LCT emulsions, the bioavailability of CBD in LCT was the highest (43%), followed by MCT/LCT (39%), MCT (33%). In vivo pharmacokinetic study showed that MCT/LCT and LCT were more favorable for CBD transport and absorption. The results may provide useful information for the construction of delivery systems, protecting CBD molecules, and improving their bioavailability.

Beyond uterine atony: characterizing postpartum hemorrhage coagulopathy.

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.

Targeting hydrogen sulfide and nitric oxide to repair cardiovascular injury after trauma.

The systemic cardiovascular effects of major trauma, especially neurotrauma, contribute to death and permanent disability in trauma patients and treatments are needed to improve outcomes. In some trauma patients, dysfunction of the autonomic nervous system produces a state of adrenergic overstimulation, causing either a sustained elevation in catecholamines (sympathetic storm) or oscillating bursts of paroxysmal sympathetic hyperactivity. Trauma can also activate innate immune responses that release cytokines and damage-associated molecular patterns into the circulation. This combination of altered autonomic nervous system function and widespread systemic inflammation produces secondary cardiovascular injury, including hypertension, damage to cardiac tissue, vascular endothelial dysfunction, coagulopathy and multiorgan failure. The gasotransmitters nitric oxide (NO) and hydrogen sulfide (H2S) are small gaseous molecules with potent effects on vascular tone regulation. Exogenous NO (inhaled) has potential therapeutic benefit in cardio-cerebrovascular diseases, but limited data suggests potential efficacy in traumatic brain injury (TBI). H2S is a modulator of NO signaling and autonomic nervous system function that has also been used as a drug for cardio-cerebrovascular diseases. The inhaled gases NO and H2S are potential treatments to restore cardio-cerebrovascular function in the post-trauma period.

Plasma-based assays distinguish hyperfibrinolysis and shutdown subgroups in trauma-induced coagulopathy.

BACKGROUND: Trauma patients with abnormal fibrinolysis have increased morbidity and mortality. Knowledge of mechanisms differentiating fibrinolytic phenotypes is important to optimize treatment. We hypothesized that subjects with abnormal fibrinolysis identified by whole blood viscoelastometry can also be distinguished by plasma thrombin generation, clot structure, fibrin formation, and plasmin generation measurements. METHODS: Platelet-poor plasma (PPP) from an observational cross-sectional trauma cohort with fibrinolysis shutdown (% lysis at 30 minutes [LY30] < 0.9, n = 11) or hyperfibrinolysis (LY30 > 3%, n = 9) defined by whole blood thromboelastography were studied. Noninjured control subjects provided comparative samples. Thrombin generation, fibrin structure and formation, and plasmin generation were measured by fluorescence, confocal microscopy, turbidity, and a fluorescence-calibrated plasmin assay, respectively, in the absence/presence of tissue factor or tissue plasminogen activator (tPA). RESULTS: Whereas spontaneous thrombin generation was not detected in PPP from control subjects, PPP from hyperfibrinolysis or shutdown patients demonstrated spontaneous thrombin generation, and the lag time was shorter in hyperfibrinolysis versus shutdown. Addition of tissue factor masked this difference but revealed increased thrombin generation in hyperfibrinolysis samples. Compared with shutdown, hyperfibrinolysis PPP formed denser fibrin networks. In the absence of tPA, the fibrin formation rate was faster in shutdown than hyperfibrinolysis, but hyperfibrinolysis clots lysed spontaneously; these differences were masked by addition of tPA. Tissue plasminogen activator-stimulated plasmin generation was similar in hyperfibrinolysis and shutdown samples. Differences in LY30, fibrin structure, and lysis correlated with pH. CONCLUSION: This exploratory study using PPP-based assays identified differences in thrombin generation, fibrin formation and structure, and lysis in hyperfibrinolysis and shutdown subgroups. These groups did not differ in their ability to promote tPA-triggered plasmin generation. The ability to characterize these activities in PPP facilitates studies to identify mechanisms that promote adverse outcomes in trauma. LEVEL OF EVIDENCE: Prognostic/Epidemiological; Level III.

Enhanced Stability and Oral Bioavailability of Cannabidiol in Zein and Whey Protein Composite Nanoparticles by a Modified Anti-Solvent Approach.

Wide applications of cannabidiol (CBD) in the food and pharmaceutical industries are limited due to its low bioavailability, sensitivity to environmental pressures and low water solubility. Zein nanoparticles were stabilized by whey protein (WP) for the delivery of cannabidiol (CBD) using a modified anti-solvent approach. Particle size, surface charge, encapsulation efficiency, and re-dispersibility of nanoparticles were influenced by the zein to WP ratio. Under optimized conditions at 1:4, zein-WP nanoparticles were fabricated with CBD (200 µg/mL) and further characterized. WP absorbed on zein surface via hydrogen bond, hydrophobic forces, and electrostatic attraction. The zein-WP nanoparticles showed excellent storage stability (4 °C, dark) and effectively protected CBD degradation against heat and UV light. In vivo pharmacokinetic study demonstrated that CBD in zein-WP nanoparticles displayed 2-times and 1.75-fold enhancement in maximum concentration (C max) and the area under curve (AUC) as compared to free-form CBD. The data indicated the feasibility of developing zein-WP based nanoparticles for the encapsulation, protection, and delivery of CBD.

Assessing Factor V Antigen and Degradation Products in Burn and Trauma Patients.

INTRODUCTION: Proposed mechanisms of acute traumatic coagulopathy (ATC) include decreased clotting potential due to factor consumption and proteolytic inactivation of factor V (FV) and activated factor V (FVa) by activated protein C (aPC). The role of FV/FVa depletion or inactivation in burn-induced coagulopathy is not well characterized. This study evaluates FV dynamics following burn and nonburn trauma. METHODS: Burn and trauma patients were prospectively enrolled. Western blotting was performed on admission plasma to quantitate levels of FV antigen and to assess for aPC or other proteolytically derived FV/FVa degradation products. Statistical analysis was performed with Spearman's, Chi-square, Mann-Whitney U test, and logistic regression. RESULTS: Burn (n = 60) and trauma (n = 136) cohorts showed similar degrees of FV consumption with median FV levels of 76% versus 73% (P = 0.65) of normal, respectively. Percent total body surface area (TBSA) was not correlated with FV, nor were significant differences in median FV levels observed between low and high TBSA groups. The injury severity score (ISS) in trauma patients was inversely correlated with FV (ρ = -0.26; P = 0.01) and ISS ≥ 25 was associated with a lower FV antigen level (64% versus. 93%; P = 0.009). The proportion of samples showing proteolysis-derived FV was greater in trauma than burn patients (42% versus. 16%; P = 0.0006). CONCLUSIONS: Increasing traumatic injury severity is associated with decreased FV antigen levels, and a greater proportion of trauma patient samples exhibit proteolytically degraded FV fragments. These associations are not present in burns, suggesting that mechanisms underlying FV depletion in burn and nonburn trauma are not identical.

Increased histone-DNA complexes and endothelial-dependent thrombin generation in severe COVID-19.

Coagulopathy in severe COVID-19 is common but poorly understood. The purpose of this study was to determine how SARS-CoV-2 infection impacts histone levels, fibrin structure, and endogenous thrombin potential in the presence and absence of endothelial cells. We studied individuals with SARS-CoV-2 infection and acute respiratory distress syndrome at the time of initiation of mechanical ventilation compared to healthy controls. Circulating histone-DNA complexes were elevated in the plasma of COVID-19 patients relative to healthy controls (n=6, each group). Using calibrated automated thrombography, thrombin generation was altered in COVID-19 patient plasma samples. Despite having increased endogenous thrombin potential, patient plasma samples exhibited prolonged lag times and times to peak thrombin in the presence of added tissue factor and PCPS. Strikingly different results were observed when endothelial cells were used in place of tissue factor and PCPS. While healthy control plasma samples did not generate measurable thrombin after 60 min, plasma samples from COVID-19+ patients formed thrombin (mean lag time ~20 min). Consistent with the observed alterations in thrombin generation, clots from COVID-19 subjects exhibited a denser fibrin network, thinner fibers and lower fibrin resolvability. Elevated histones, aberrant fibrin formation, and increased endothelial-dependent thrombin generation may contribute to coagulopathy in COVID-19.

Thrombo-inflammatory biomarkers and D-dimer in a biracial cohort study.

BACKGROUND: Higher D-dimer is a risk factor for cardiovascular diseases and venous thromboembolism. In the general population, D-dimer and other thrombo-inflammatory biomarkers are higher among Black individuals, who also have higher risk of these conditions compared to White people. OBJECTIVE: To assess whether Black individuals have an exaggerated correlation between D-dimer and thrombo-inflammatory biomarkers characteristic of cardiovascular diseases. METHODS: Linear regression was used to assess correlations of 11 thrombo-inflammatory biomarkers with D-dimer in a cross-sectional study of 1068 participants of the biracial Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort. RESULTS: Adverse levels of most biomarkers, especially fibrinogen, factor VIII, C-reactive protein, N-terminal pro-B-type natriuretic peptide, and interleukin (IL)-6, were associated with higher D-dimer. Several associations with D-dimer differed significantly by race. For example, the association of factor VIII with D-dimer was more than twice as large in Black compared to White participants. Specifically, D-dimer was 26% higher per standard deviation (SD) higher factor VIII in Black adults and was only 11% higher per SD higher factor VIII in White adults. In Black but not White adults, higher IL-10 and soluble CD14 were associated with higher D-dimer. CONCLUSIONS: Findings suggest that D-dimer might relate to Black/White differences in cardiovascular diseases and venous thromboembolism because it is a marker of amplified thrombo-inflammatory response in Black people. Better understanding of contributors to higher D-dimer in the general population is needed.

Traumatic Brain Injury Impairs Systemic Vascular Function Through Disruption of Inward-Rectifier Potassium Channels.

Trauma can lead to widespread vascular dysfunction, but the underlying mechanisms remain largely unknown. Inward-rectifier potassium channels (Kir2.1) play a critical role in the dynamic regulation of regional perfusion and blood flow. Kir2.1 channel activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid that is degraded by phospholipase A2 (PLA2) in conditions of oxidative stress or inflammation. We hypothesized that PLA2-induced depletion of PIP2 after trauma impairs Kir2.1 channel function. A fluid percussion injury model of traumatic brain injury (TBI) in rats was used to study mesenteric resistance arteries 24 hours after injury. The functional responses of intact arteries were assessed using pressure myography. We analyzed circulating PLA2, hydrogen peroxide (H2O2), and metabolites to identify alterations in signaling pathways associated with PIP2 in TBI. Electrophysiology analysis of freshly-isolated endothelial and smooth muscle cells revealed a significant reduction of Ba2+-sensitive Kir2.1 currents after TBI. Additionally, dilations to elevated extracellular potassium and BaCl2- or ML 133-induced constrictions in pressurized arteries were significantly decreased following TBI, consistent with an impairment of Kir2.1 channel function. The addition of a PIP2 analog to the patch pipette successfully rescued endothelial Kir2.1 currents after TBI. Both H2O2 and PLA2 activity were increased after injury. Metabolomics analysis demonstrated altered lipid metabolism signaling pathways, including increased arachidonic acid, and fatty acid mobilization after TBI. Our findings support a model in which increased H2O2-induced PLA2 activity after trauma hydrolyzes endothelial PIP2, resulting in impaired Kir2.1 channel function.

An oropharyngeal device for airway management of conscious and semiconscious patients: A randomized clinical trial.

OBJECTIVE: No oropharyngeal devices exist for use in conscious and semiconscious trauma patients during emergency evacuation, transport, or resuscitation. We aimed to test the hypotheses that the ManMaxAirway (MMA) is better tolerated than the standard Guedel-style device in awake volunteers and that it produces a jaw thrust and improves air flow. METHODS: This was a randomized cross-over study of healthy volunteers with either the MMA or standard device. The primary outcome of tolerability was defined as maintaining the device in place for 60 seconds. Secondary outcomes included respiratory system function and jaw thrust. Resistance to airflow through the device lumen was measured in situ and when placed in subjects in the pulmonary laboratory alone. Jaw thrust was quantified as displacement between the mandibular condyle and condylar fossa apex relative to baseline visualized with magnetic resonance imaging (MRI). RESULTS: We enrolled 19 subjects. Of these, a convenience sample of 5 individuals was selected for MRI; the remaining individuals (n = 14) were randomized for the cross-over study. All 14 subjects were able to maintain the MMA for 60 seconds compared with 2/14 (14%) with the standard device (odds ratio, 145; 95% confidence interval, 6.3-3314). Subjects reported that the experimental device was more comfortable and its placement did not trigger the gag reflex. Airway resistance produced by the MMA in an oscillatory flow model was nearly an order of magnitude lower than that of the standard device (experimental vs standard, 8 Hz-0.092 vs 0.786 cmH20·s/L; 15 Hz-0.193 vs 1.321 cmH20·s/L). Rapid induction of the gag reflex precluded further measurements with the standard device. Forced oscillation pulmonary testing in conscious volunteers with and without the MMA demonstrated that the device decreased respiratory system resistance to airflow and reduced respiratory elastance (31% ± 8% and 44% ± 13.4%, respectively; P < 0.05). MRIs of the subjects (n = 5) with the MMA in place showed a significant jaw thrust compared with baseline (7 ± 1 mm). CONCLUSIONS: The MMA proved well tolerated in conscious subjects, resulting in an opening of the anatomic airway and a decreased resistance to airflow.

Viscoelastic hemostasis assays in septic, critically ill coronavirus disease 2019 patients: a practical guide for clinicians.

Coronavirus disease 2019 (COVID-19)-associated coagulopathy is unusual, poorly defined and is linked with significant hypercoagulability and microthrombotic and macrothrombotic complications leading to worse outcomes and higher mortality. Conventional coagulation assays do not always actively reflect these derangements and might fail to detect this coagulopathy. Viscoelastic hemostatic assays (VHA) provide a possible tool that adds to conventional coagulation assays in identifying this hypercoagulable state. VHA has been mostly used in surgery and trauma but it's still not well defined in sepsis patients with lack of large randomized trials. Few studies described VHA findings in patients with COVID-19 showing significant hypercoagulability and fibrinolysis shutdown. Clinicians taking care of these patients might have little experience interpreting VHA results. By reviewing the available literature on the use of VHA in sepsis, and the current knowledge on COVID-19-associated coagulopathy we provide clinicians with a practical guide on VHA utilization in patients with COVID-19.

Impaired capillary-to-arteriolar electrical signaling after traumatic brain injury.

Traumatic brain injury (TBI) acutely impairs dynamic regulation of local cerebral blood flow, but long-term (>72 h) effects on functional hyperemia are unknown. Functional hyperemia depends on capillary endothelial cell inward rectifier potassium channels (Kir2.1) responding to potassium (K+) released during neuronal activity to produce a regenerative, hyperpolarizing electrical signal that propagates from capillaries to dilate upstream penetrating arterioles. We hypothesized that TBI causes widespread disruption of electrical signaling from capillaries-to-arterioles through impairment of Kir2.1 channel function. We randomized mice to TBI or control groups and allowed them to recover for 4 to 7 days post-injury. We measured in vivo cerebral hemodynamics and arteriolar responses to local stimulation of capillaries with 10 mM K+ using multiphoton laser scanning microscopy through a cranial window under urethane and α-chloralose anesthesia. Capillary angio-architecture was not significantly affected following injury. However, K+-induced hyperemia was significantly impaired. Electrophysiology recordings in freshly isolated capillary endothelial cells revealed diminished Ba2+-sensitive Kir2.1 currents, consistent with a reduction in channel function. In pressurized cerebral arteries isolated from TBI mice, K+ failed to elicit the vasodilation seen in controls. We conclude that disruption of endothelial Kir2.1 channel function impairs capillary-to-arteriole electrical signaling, contributing to altered cerebral hemodynamics after TBI.

Actin is associated with tissue injury in trauma patients and produces a hypercoagulable profile in vitro.

BACKGROUND: While tissue injury provokes fibrinolysis shutdown in trauma, the mechanism remains elusive. Cellular death causes release of structural proteins, including actin and myosin, which may interact with clot formation and structure. We hypothesized that tissue injury is associated with high circulating actin and that actin produces a hypercoagulable profile with decreased fibrinolysis in vitro. METHODS: Blood was collected from trauma activation patients at a single Level I trauma center for thrombelastography and proteomics. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry using isotope-labeled standards for quantification of actin and its endogenous inhibitor gelsolin. Based on the results, we added physiologic concentrations of cytoskeletal G-actin to whole blood from healthy volunteers and analyzed changes in thrombelastography, as well as to plasma and examined clot architecture using confocal microscopy of fluorescently labeled fibrinogen. RESULTS: Overall, 108 trauma patients were included: majority (71%) men, median age of 32.7 years, 66% blunt mechanism, median New Injury Severity Score (NISS) of 41. Compared with patients without severe tissue injury (NISS < 15, n = 10), patients with severe tissue injury (NISS > 15, n = 98) had higher levels of circulating actin (0.0428 vs. 0.0301, p = 0.02). Further, there was a trend toward lower gelsolin levels in patients with fibrinolysis shutdown (0.1844 vs. 0.2052, p = 0.17) and tissue plasminogen activator resistance (0.1676 vs. 0.2188, p = 0.06).Ten healthy volunteers were included in the in vitro experiments (50% male; median age, 31.3 years). Actin significantly increased angle (40.0° to 52.9°, p = 0.002) and decreased fibrinolysis (percent clot lysis 30 minutes after reaching maximum amplitude, 4.0% to 1.6%; p = 0.002), provoking fibrinolytic shutdown in three patients. The addition of actin to control plasma decreased fiber resolvability of fibrin clots, monitored by microscopy, and decreased plasmin-mediated fibrinolysis. CONCLUSION: Actin increases clot propagation and provokes fibrinolysis shutdown in vitro, through a mechanism of plasmin inhibition. High circulating levels of actin are present in trauma patients with severe tissue injury, suggesting actin contributes to fibrinolysis shutdown in the setting of tissue injury.

Dense and dangerous: The tissue plasminogen activator-resistant fibrinolysis shutdown phenotype is due to abnormal fibrin polymerization.

BACKGROUND: Both hyperfibrinolysis and fibrinolysis shutdown can occur after severe trauma. The subgroup of trauma patients with fibrinolysis shutdown resistant to tissue plasminogen activator (t-PA)-mediated fibrinolysis have increased mortality. Fibrin polymerization and structure may influence fibrinolysis subgroups in trauma, but fibrin architecture has not been characterized in acutely injured subjects. We hypothesized that fibrin polymerization measured in situ will correlate with fibrinolysis subgroups. METHODS: Blood samples were collected from trauma patients and noninjured controls. We selected samples across a range of fibrinolysis phenotypes (shutdown, physiologic, hyperfibrinolysis) and t-PA sensitivities (sensitive, physiologic, resistant) determined by thrombelastography. Plasma clots were created in situ with fluorescent fibrinogen and imaged using confocal microscopy for analysis of clot architecture in three dimensions. For each clot, we quantified the fiber resolvability, a metric of fiber distinctness or clarity, by mapping the variance of fluorescence intensity relative to background fluorescence. We also determined clot porosity by measuring the size and distribution of the gaps between fibrin fibers in three-dimensional space. We compared these measures across fibrinolysis subgroups. RESULTS: Fiber resolvability was significantly lower in all trauma subgroups compared with controls (n = 35 and 5, respectively; p < 0.05). We observed markedly different patterns of fibrin architecture among trauma patients stratified by fibrinolysis subgroup. Subjects with t-PA-resistant fibrinolysis shutdown exhibited abnormal, densely packed fibrin clots nearly devoid of pores. Individuals with t-PA-hypersensitive fibrinolysis shutdown had highly irregular clots with pores as large as 2500 µm to 20,000 µm, versus 78 µm to 1250 µm in noninjured controls. CONCLUSION: Fiber resolvability was significantly lower in trauma patients than controls, and subgroups of fibrinolysis differ in the porosity of the fibrin clot structure. The dense fibrin network in the t-PA-resistant group may prevent access to plasmin, suggesting a mechanism for thrombotic morbidity after injury.

Diagnostic Performance of Carbon Monoxide Testing by Pulse Oximetry in the Emergency Department.

BACKGROUND: Carbon monoxide (CO) exposure causes roughly 40,000 emergency department (ED) visits annually and is commonly misdiagnosed. Whereas the standard method of carboxyhemoglobin (HbCO) measurement utilizes blood gas analysis, a noninvasive, FDA-cleared alternative exists. We evaluated the performance of pulse oximetry (SpCO) for identification of CO exposure in ED patients. METHODS: We compared pulse oximetry to blood HbCO levels in a prospective observational study of adult and pediatric subjects recruited from the ED. Nurses screened a convenience sample of patients and referred those with SpCO ≥ 10% to research staff. Researchers also approached individuals who presented with signs and symptoms of CO toxicity. We determined diagnostic performance with a Bland-Altman analysis and calculated sensitivity and specificity for detection of elevated HbCO at thresholds of ≥ 10% and ≥ 15%. To optimize the potential sensitivity of SpCO for detection of CO toxicity, research technicians performed 3 SpCO readings within 5 min of the blood draw for laboratory measurement. A positive SpCO test was defined as any SpCO ≥ 10%. RESULTS: 42,000 patients were screened, 212 were evaluated, and 126 subjects were enrolled. Median HbCO level was 6% (range 1.6-21.9%). Limits of agreement were -10.3% and 8.1%. Of 23 individuals with elevated HbCO ≥ 10%, 13 were not suspected based on clinical assessment. Critically elevated HbCO was present in 6 individuals. Based on our a priori threshold of 10% for a positive test, pulse oximetry identified 14 of 23 subjects with HbCO ≥ 10%, with a sensitivity of 61% (95% CI 39-80%) and a specificity of 86% (95% CI 78-92%), and 5 of 6 subjects with HbCO ≥ 15%, with a sensitivity of 83% (95% CI 36-100%) and a specificity of 81% (95% CI 73-87%). CONCLUSIONS: Pulse oximetry underestimated HbCO and produced false negative results (ie, SpCO < 10% for all three measurements) in 17% of ED subjects with elevated HbCO ≥ 15%. Triage screening with pulse oximetry detected cases of elevated HbCO that were not suspected by the clinical provider.