Predictors and outcomes of acute pulmonary embolism in COVID-19; insights from US National COVID cohort collaborative.

OBJECTIVES: To investigate whether COVID-19 patients with pulmonary embolism had higher mortality and assess the utility of D-dimer in predicting acute pulmonary embolism. PATIENTS AND METHODS: Using the National Collaborative COVID-19 retrospective cohort, a cohort of hospitalized COVID-19 patients was studied to compare 90-day mortality and intubation outcomes in patients with and without pulmonary embolism in a multivariable cox regression analysis. The secondary measured outcomes in 1:4 propensity score-matched analysis included length of stay, chest pain incidence, heart rate, history of pulmonary embolism or DVT, and admission laboratory parameters. RESULTS: Among 31,500 hospitalized COVID-19 patients, 1117 (3.5%) patients were diagnosed with acute pulmonary embolism. Patients with acute pulmonary embolism were noted to have higher mortality (23.6% vs.12.8%; adjusted Hazard Ratio (aHR) = 1.36, 95% CI [1.20-1.55]), and intubation rates (17.6% vs. 9.3%, aHR = 1.38[1.18-1.61]). Pulmonary embolism patients had higher admission D-dimer FEU (Odds Ratio(OR) = 1.13; 95%CI [1.1-1.15]). As the D-dimer value increased, the specificity, positive predictive value, and accuracy of the test increased; however, sensitivity decreased (AUC 0.70). At cut-off D-dimer FEU 1.8 mcg/ml, the test had clinical utility (accuracy 70%) in predicting pulmonary embolism. Patients with acute pulmonary embolism had a higher incidence of chest pain and history of pulmonary embolism or deep vein thrombosis. CONCLUSIONS: Acute pulmonary embolism is associated with worse mortality and morbidity outcomes in COVID-19. We present D-dimer as a predictive risk tool in the form of a clinical calculator for the diagnosis of acute pulmonary embolism in COVID-19.

Deep learning, 3D ultrastructural analysis reveals quantitative differences in platelet and organelle packing in COVID-19/SARSCoV2 patient-derived platelets.

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients. Statistical analysis reveals that the α-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller -by 35% in volume - with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets.

COVID-19 patients exhibit a range of symptoms including microclotting. Clotting is a complex process involving both circulating proteins and platelets, a cell within the blood. Increased clotting is suggestive of an increased level of platelet activation. If this were true, we reasoned that parts of the platelet involved in the release of platelet contents during clotting would have lost their content and appear as expanded, empty "ghosts." To test this, we drew blood from severely ill COVID-19 patients and compared the platelets within the blood draws to those from healthy volunteers. All procedures were done under careful attention to biosafety and approved by health authorities. We looked within the platelets for empty ghosts by the high magnification technique of electron microscopy. To count the ghosts, we developed new computer software. In the end, we found little difference between the COVID patient platelets and the healthy donor platelets. The results suggest that circulating proteins outside of the platelet are more important to the strong clotting response. The software developed will be used to analyze other disease states.

Extracellular Histones Trigger Disseminated Intravascular Coagulation by Lytic Cell Death.

Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue-factor (TF)-dependent mechanism. We used a combination of various deficient mice to elucidate the molecular mechanism of histone-induced coagulation. We showed that histones trigger coagulation activation in vivo, as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on intracellular inflammasome pathways involving caspase 1/11 and gasdermin D (GSDMD), nor on cell surface receptor TLR2- and TLR4-mediated host immune response, as the deficiency of these genes in mice did not protect against histone-induced coagulopathy. The incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. The neutralization of TF diminished the histone-induced coagulation. Our findings revealed lytic cell death as a novel mechanism of histone-induced coagulation activation and thrombosis.

S100B as a biomarker of blood-brain barrier disruption after thoracoabdominal aortic aneurysm repair: a secondary analysis from a prospective cohort study.

PURPOSE: The objective of this study was to determine whether the magnitude of the peripheral inflammatory response to cardiovascular surgery is associated with increases in blood-brain barrier (BBB) permeability as reflected by changes in cerebrospinal fluid (CSF)/plasma S100B concentrations. METHODS: We conducted a secondary analysis from a prospective cohort study of 35 patients undergoing elective thoracoabdominal aortic aneurysm repair with (n = 17) or without (n = 18) cardiopulmonary bypass (CPB). Plasma and CSF S100B, interleukin-6 (IL-6), and albumin concentrations were measured at baseline (C0) and serially for up to five days. RESULTS: Following CPB, the median [interquartile range] plasma S100B concentration increased from 58 [32-88] pg·mL-1 at C0 to a maximum concentration (Cmax) of 1,131 [655-1,875] pg·mL-1 over a median time (tmax) of 6.3 [5.9-7.0] hr. In the non-CPB group, the median plasma S100B increased to a lesser extent. There was a delayed increase in CSF S100B to a median Cmax of 436 [406-922] pg·mL-1 in the CPB group at a tmax of 23.7 [18.5-40.2] hr. In the non-CPB group, the CSF concentrations were similar at all time points. In the CPB group, we did not detect significant correlations between plasma and CSF S100B with plasma IL-6 [r = 0.52 (95% confidence interval [CI], -0.061 to 0.84)] and CSF IL-6 [r = 0.53 (95% CI, -0.073 to 0.85)] concentrations, respectively. Correlations of plasma or CSF S100B levels with BBB permeability were not significant. CONCLUSION: The lack of parallel increases in plasma and CSF S100B following CPB indicates that S100B may not be a reliable biomarker for BBB disruption after thoracoabdominal aortic aneurysm repair employing CPB. TRIAL REGISTRATION: www.clinicaltrials.gov (NCT00878371); registered 7 April 2009.

RéSUMé: OBJECTIF: L'objectif de cette étude était de déterminer si l'intensité de la réponse inflammatoire périphérique à la chirurgie cardiovasculaire était associée à une augmentation de la perméabilité de la barrière hémato-encéphalique (BHE), telle que reflétée par des changements dans les concentrations de S100B dans le liquide céphalorachidien (LCR) et le plasma. MéTHODE: Nous avons mené une analyse secondaire à partir d'une étude de cohorte prospective portant sur 35 patients bénéficiant d'une réparation élective d'un anévrisme aortique thoraco-abdominal avec (n = 17) ou sans (n = 18) circulation extracorporelle (CEC). Les concentrations plasmatiques et dans le LCR de S100B, d'interleukine-6 (IL-6) et d'albumine ont été mesurées au départ (C0) et en série jusqu'à cinq jours. RéSULTATS: Après la CEC, la concentration médiane [écart interquartile] plasmatique de S100B est passée de 58 [32­88] pg·mL-1 au départ (C0) à une concentration maximale (Cmax) de 1131 [655­1875] pg·mL-1 sur une période médiane (tmax) de 6,3 [5,9­7,0] h. Dans le groupe sans CEC, la concentration plasmatique médiane de S100B a augmenté dans une moindre mesure. Il y a eu une augmentation retardée de S100B dans le LCR à une Cmax médiane de 436 [406­922] pg·mL-1 dans le groupe CEC à une tmax de 23,7 [18,5­40,2] h. Dans le groupe sans CEC, les concentrations dans le LCR étaient similaires à tous les moments. Dans le groupe CEC, nous n'avons pas détecté de corrélations significatives entre la concentration de S100B dans le plasma et le LCR avec les concentrations plasmatiques d'IL-6 [r = 0,52 (intervalle de confiance [IC] à 95 %, -0,061 à 0,84)] et d'IL-6 dans le LCR [r = 0,53 (IC 95 %, -0,073 à 0,85)], respectivement. Les corrélations entre les taux plasmatiques ou dans le LCR de S100B et la perméabilité de la BHE n'étaient pas significatives. CONCLUSION: L'absence d'augmentations parallèles de la concentration de S100B dans le plasma et le LCR après la CEC indique que la S100B pourrait ne pas être un biomarqueur fiable de la perturbation de la BHE après une réparation d'anévrisme aortique thoraco-abdominal sous CEC. ENREGISTREMENT DE L'éTUDE: www.clinicaltrials.gov (NCT00878371); enregistrée le 7 avril 2009.

Medevac Utilization and Patient Characteristics in Rural Alaska 2010 to 2018.

OBJECTIVE: Little is known about medevac utilization in remote, rural Alaska where there is no road access and communities are reliant on medevacs for emergency care. With high financial costs and risks to flight crews, there is an urgent need to understand medevac utilization in rural Alaska. This article aimed to describe medevac utilization and patient characteristics over 9 years in the remote, air transport dependent in Alaska. METHODS: Deidentified data (2010-2018) were obtained for all medevacs originating within the Yukon-Kuskokwim Delta. Descriptive statistics were calculated, and chi-square tests of independence were conducted to identify differences. RESULTS: Four thousand nine hundred ninety-one medevacs were performed, averaging 555 (standard deviation = 67.7) per year. Medevacs for respiratory complaints were predominant for children, whereas trauma predominated for adults 18 to 40 years old. Traumatic injury was more common in males than females aged < 65 years but was more common in females than males aged ≥ 65 years. Significant variability occurred in medevacs based on the community and the hour of the day. CONCLUSION: Medevacs are a critical part of health care in rural, remote Alaska but appear subject to clinical and nonclinical determinants. These baseline data provide a foundation for future studies aiming to increase medevac safety and provide decision-making support.

An observational study examining the effects of a surgically induced inflammatory response on the distribution of morphine and its metabolites into cerebrospinal fluid.

PURPOSE: Morphine is administered intravenously for pain management in the perioperative period. The effect of the inflammatory response to surgery on morphine distribution across the blood-brain barrier (BBB) in humans was investigated. We hypothesized that a graded surgically induced, systemic inflammatory response alters cerebrospinal fluid (CSF) levels of morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) through a temporary reduction in BBB drug efflux transporter function. METHODS: We conducted a prospective pharmacokinetic study of the plasma and CSF distribution of the P-glycoprotein (PGP) substrate morphine in 33 patients undergoing open thoracic (n = 18) or endovascular (n = 15) aortic aneurysm repair. Morphine was administered with induction of anesthesia and in the intensive care unit. Plasma and CSF concentrations of interleukin (IL)-6, morphine, M3G, M6G, and albumin were measured prior to surgery (baseline), during surgery, and postoperatively every six hours until removal of the CSF drain. The area under the curve (AUC) was determined for plasma and CSF IL-6, morphine, M3G, and M6G concentrations vs time. The primary endpoint measures were the correlations between the morphine, M6G, and M3G AUC CSF/plasma ratios and systemic inflammation as quantified by the time-normalized IL-6 exposure, which was calculated for each individual by dividing the total exposure (AUC) by time (t). A Bonferroni corrected P < 0.017 indicated a significant correlation. RESULTS: Plasma and CSF IL-6 concentrations increased postoperatively. The median [interquartile range] IL-6 exposures were significantly higher in the open vs endovascular surgical group for plasma (105 [40-256] pg·mL-1 vs 29 [16-70] pg·mL-1, respectively; P = 0.013) and CSF (79 [26-133] pg·mL-1 vs 16 [9-80] pg·mL-1, respectively; P = 0.013). For the primary endpoint, the plasma IL-6 AUC/t did not correlate with the CSF accumulation of morphine (r = -0.009; P = 0.96) or M3G (r = 0.37; P = 0.04) when corrected for surgical procedure, age, and sex. There were insufficient data on CSF concentration to complete the primary analysis for M6G. CONCLUSION: Morphine distribution into the CSF was not significantly altered in patients undergoing thoracic aortic aneurysm repair. This suggests that BBB PGP function may not be affected by the perioperative inflammatory response. TRIAL REGISTRATION: www.clinicaltrials.gov , NCT 00878371. Registered 7 April 2009.

Blocking inhibition of prothrombinase by tissue factor pathway inhibitor alpha: a procoagulant property of heparins.

Unfractionated heparin (UFH) has procoagulant activity in antithrombin/heparin cofactor II (HCII)-depleted plasma. UFH prevents tissue factor pathway inhibitor alpha (TFPIα) from inhibiting the procoagulant enzyme complex, prothrombinase, providing a possible mechanism for its procoagulant activity. The procoagulant potential of UFH and various low molecular weight heparins (LMWHs) were characterized for TFPIα dependence, using thrombin generation assays performed with antithrombin/HCII-depleted plasma. UFH, the LMWHs enoxaparin and dalteparin, and the low anticoagulant LMWH 2-O, 3-O desulphated heparin (ODSH) all promoted thrombin generation, but fondaparinux did not, and this activity was blocked by a TFPIα antibody. UFH, enoxaparin, and dalteparin were anticoagulant in reactions containing 1-2% normal plasma. In prothrombinase activity assays, UFH, enoxaparin, dalteparin and ODSH blocked prothrombinase inhibition by TFPIα, while again fondaparinux did not. In both the plasma and purified assays, LMWHs displayed greater procoagulant potential than UFH, even when normalized to saccharide concentration. These biochemical data reveal that UFH and LMWHs, but not fondaparinux, block prothrombinase inhibition by TFPIα, thereby producing their paradoxical procoagulant activity observed in the absence of antithrombin/HCII. The findings may help to understand the complex pathophysiology and treatment of patients that are simultaneously bleeding and clotting, such as those with disseminated intravascular coagulation.

Biology of tissue factor pathway inhibitor.

Recent studies of the anticoagulant activities of the tissue factor (TF) pathway inhibitor (TFPI) isoforms, TFPIα and TFPIß, have provided new insight into the biochemical and physiological mechanisms that underlie bleeding and clotting disorders. TFPIα and TFPIß have tissue-specific expression patterns and anticoagulant activities. An alternative splicing event in the 5' untranslated region allows for translational regulation of TFPIß expression. TFPIα has 3 Kunitz-type inhibitor domains (K1, K2, K3) and a basic C terminus, whereas TFPIß has the K1 and K2 domains attached to a glycosylphosphatidyl inositol-anchored C terminus. TFPIα is the only isoform present in platelets, whereas endothelial cells produce both isoforms, secreting TFPIα and expressing TFPIß on the cell surface. TFPIα and TFPIß inhibit both TF-factor VIIa-dependent factor Xa (FXa) generation and free FXa. Protein S enhances FXa inhibition by TFPIα. TFPIα produces isoform-specific inhibition of prothrombinase during the initiation of coagulation, an anticoagulant activity that requires an exosite interaction between its basic C terminus and an acidic region in the factor Va B domain. Platelet TFPIα may be optimally localized to dampen initial thrombin generation. Similarly, endothelial TFPIß may be optimally localized to inhibit processes that occur when endothelial TF is present, such as during the inflammatory response.

Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation.

Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIß. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIß and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIß in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIß acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.

Protein S is a cofactor for platelet and endothelial tissue factor pathway inhibitor-α but not for cell surface-associated tissue factor pathway inhibitor.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is produced in 2 isoforms: TFPIα, a soluble protein in plasma, platelets, and endothelial cells, and TFPIß, a glycosylphosphatidylinositol-anchored protein on endothelium. Protein S (PS) functions as a cofactor for TFPIα, enhancing the inhibition of factor Xa. However, PS does not alter the inhibition of prothrombinase by TFPIα, and PS interactions with TFPIß are undescribed. Thus, the physiological role and scope of the PS-TFPI system remain unclear. APPROACH AND RESULTS: Here, the cofactor activity of PS toward platelet and endothelial TFPIα and endothelial TFPIß was quantified. PS enhanced the inhibition of factor Xa by TFPIα from platelets and endothelial cells and stabilized the TFPIα/factor Xa inhibitory complex, delaying thrombin generation by prothrombinase. By contrast, PS did not enhance the inhibitory activity of TFPIß or a membrane-anchored form of TFPI containing the PS-binding third Kunitz domain (K1K2K3) although PS did function as a cofactor for K1K2K3 enzymatically released from the cell surface. CONCLUSIONS: The PS-TFPI anticoagulant system is limited to plasma TFPIα and TFPIα released from platelets and endothelial cells. PS likely functions to localize solution-phase TFPIα to the cell surface, where factor Xa is bound. PS does not alter the activity of membrane-associated TFPI. Because activated platelets release TFPIα and PS, the PS-TFPIα anticoagulant system may act physiologically to dampen thrombin generation at the platelet surface.

Inhibitors of the Thioesterase Activity of Mycobacterium tuberculosis Pks13 Discovered Using DNA-Encoded Chemical Library Screening.

DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.

Unfolded Von Willebrand Factor Binds Protein S and Reduces Anticoagulant Activity.

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca(2+)-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC(50) = 0.3µM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

Caveolae optimize tissue factor-Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor.

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIß and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.