A novel factor IXa-specific enzyme-linked immunosorbent assay detects factor IXa in human plasma.

Background: Factor (F)IXa activity has been detected in human plasma and may impact thrombotic risk. Current FIXa activity assays are complex and cumbersome. Objectives: To develop a reproducible enzyme-linked immunosorbent assay (ELISA) using a novel monoclonal antibody that detects total FIXa in human plasma. Methods: A monoclonal antibody was raised against the new N-terminus exposed upon activation of FIX to FIXa by cleavage after R226. This antibody is specific for FIXa protease and does not recognize FIX zymogen or FIXα. The antibody was used to develop a FIXa-specific ELISA capable of quantifying total FIXa (free FIXa and FIXa-antithrombin complex) in human plasma. Total FIXa quantified using the ELISA was compared to that of FIXa-antithrombin quantified using modifications of a previously described ELISA. Results: The FIXa-specific ELISA was reproducible and quantified total FIXa in human plasma. Total FIXa levels correlated with FIXa-antithrombin levels. Conclusion: A monoclonal antibody was developed that specifically detects human FIXa protease. A FIXa-specific ELISA using the new antibody is capable of reproducibly measuring total FIXa in human plasma (both free FIXa and FIXa-antithrombin). This assay should facilitate the evaluation of total FIXa levels in a variety of clinical circumstances.

Decreased plasma cartilage acidic protein 1 in COVID-19.

Cartilage acidic protein-1 (CRTAC1) is produced by several cell types, including Type 2 alveolar epithelial (T2AE) cells that are targeted by SARS-CoV2. Plasma CRTAC1 is known based on proteomic surveys to be low in patients with severe COVID-19. Using an ELISA, we found that patients treated for COVID-19 in an ICU almost uniformly had plasma concentrations of CRTAC1 below those of healthy controls. Magnitude of decrease in CRTAC1 distinguished COVID-19 from other causes of acute respiratory decompensation and correlated with established metrics of COVID-19 severity. CRTAC1 concentrations below those of controls were found in some patients a year after hospitalization with COVID-19, long COVID after less severe COVID-19, or chronic obstructive pulmonary disease. Decreases in CRTAC1 in severe COVID-19 correlated (r = 0.37, p = 0.0001) with decreases in CFP (properdin), which interacts with CRTAC1. Thus, decreases of CRTAC1 associated with severe COVID-19 may result from loss of production by T2AE cells or co-depletion with CFP. Determination of significance of and reasons behind decreased CRTAC1 concentration in a subset of patients with long COVID will require analysis of roles of preexisting lung disease, impact of prior acute COVID-19, age, and other confounding variables in a larger number of patients.

Reengaging patients with forgotten filters through an institutional multidisciplinary approach.

OBJECTIVE: The aim of the present study was to evaluate the outcomes of a hospital-wide multidisciplinary initiative to reengage and manage patients with unretrieved chronic indwelling inferior vena cava (IVC) filters placed at a large tertiary care center, who had been lost to follow-up. METHODS: We performed a retrospective review of outcomes from a completed multidisciplinary quality improvement project. The quality improvement project identified and contacted (via letter) patients with chronic indwelling IVC filters placed at a single tertiary care center from 2008 to 2016 who were alive and without evidence of filter retrieval in the medical records. A total of 316 eligible patients were mailed a letter regarding their chronic indwelling IVC filter and the updated recommendations regarding IVC filter removal. The letter included institutional contact information, and all the patients who responded were offered a clinic visit to discuss potential filter retrieval. In the retrospective review, we assessed the outcomes of the quality improvement project, including the patient response rate, follow-up clinic visits, new imaging studies generated, retrieval rate, procedural success, and complications. The patient demographics and filter characteristics were collected and evaluated for correlations with the response and retrieval rates. RESULTS: The patient response rate to the letter was 32% (101 of 316). Of the 101 patients who responded, 72 (71%) were seen in clinic and 59 (82%) underwent new imaging studies. Using standard and advanced techniques, 34 of 36 filters after a median dwell time of 9.4 years (range, 3.3-13.3 years) were successfully retrieved (94% success rate). The patients with a documented IVC filter complication were more likely to respond to the letter (odds ratio, 4.34) and undergo IVC filter retrieval (odds ratio, 6.04). No moderate or severe procedural complications occurred during filter retrieval. CONCLUSIONS: An institutional, multidisciplinary quality initiative successfully identified and reengaged patients with chronic indwelling IVC filters who had been lost to follow-up. The filter retrieval success rate was high and procedural morbidity low. Institution-wide efforts to identify and retrieve chronic indwelling filters are feasible.

Predictors of intubation in COVID-19 patients undergoing awake proning in the emergency department.

BACKGROUND: Awake prone positioning (PP) has been used to avoid intubations in hypoxic COVID-19 patients, but there is limited evidence regarding its efficacy. Moreover, clinicians have little information to identify patients at high risk of intubation despite awake PP. We sought to assess the intubation rate among patients treated with awake PP in our Emergency Department (ED) and identify predictors of need for intubation. METHODS: We conducted a multicenter retrospective cohort study of adult patients admitted for known or suspected COVID-19 who were treated with awake PP in the ED. We excluded patients intubated in the ED. Our primary outcome was prevalence of intubation during initial hospitalization. Other outcomes were intubation within 48 h of admission and mortality. We performed classification and regression tree analysis to identify the variables most likely to predict the need for intubation. RESULTS: We included 97 patients; 44% required intubation and 21% were intubated within 48 h of admission. Respiratory oxygenation (ROX) index and P/F (partial pressure of oxygen / fraction of inspired oxygen) ratio measured 24 h after admission were the variables most likely to predict need for intubation (area under the receiver operating characteristic curve = 0.82). CONCLUSIONS: Among COVID-19 patients treated with awake PP in the ED prior to admission, ROX index and P/F ratio, particularly 24 h after admission, may be useful tools in identifying patients at high risk of intubation.

Intubation rate of patients with hypoxia due to COVID-19 treated with awake proning: A meta-analysis.

BACKGROUND: Awake prone positioning (PP), or proning, is used to avoid intubations in hypoxic patients with COVID-19, but because of the disease's novelty and constant evolution of treatment strategies, the efficacy of awake PP is unclear. We conducted a meta-analysis of the literature to assess the intubation rate among patients with COVID-19 requiring oxygen or noninvasive ventilatory support who underwent awake PP. METHODS: We searched PubMed, Embase, and Scopus databases through August 15, 2020 to identify relevant randomized control trials, observational studies, and case series. We performed random-effects meta-analyses for the primary outcome of intubation rate. We used moderator analysis and meta-regressions to assess sources of heterogeneity. We used the standard and modified Newcastle-Ottawa Scales (NOS) to assess studies' quality. RESULTS: Our search identified 1043 articles. We included 16 studies from the original search and 2 in-press as of October 2020 in our analysis. All were observational studies. Our analysis included 364 patients; mean age was 56.8 (SD 7.12) years, and 68% were men. The intubation rate was 28% (95% CI 20%-38%, I2 = 63%). The mortality rate among patients who underwent awake PP was 14% (95% CI 7.4%-24.4%). Potential sources of heterogeneity were study design and setting (practice and geographic). CONCLUSIONS: Our study demonstrated an intubation rate of 28% among hypoxic patients with COVID-19 who underwent awake PP. Awake PP in COVID-19 is feasible and practical, and more rigorous research is needed to confirm this promising intervention.

Early Predictors of Near-Shore Spinal Injuries Among Emergency Department Patients.

BACKGROUND: Spinal injuries (SIs) can pose a significant burden to patients and family; delayed surgical intervention, associated with interhospital transfer, results in worse outcomes. OBJECTIVE: This study aimed to identify early patient-centered factors associated with risk for near-shore SIs to assist clinicians with expeditious medical decision-making. METHODS: We performed a multicenter retrospective study of all adults transported from Ocean City, Maryland to two emergency departments (EDs) and one regional trauma center for evaluation of suspected SIs from 2006 to 2017. Outcomes were any SI and any spinal cord injury (SCI). Multivariable logistic regression was performed for association of environmental and clinical factors with outcomes. RESULTS: We analyzed 278 records, 102 patients (37%) were diagnosed with any SI and 41 (15%) were diagnosed with SCIs. Compared with patients without SI, patients with SI were more likely to be older (48 vs. 39 years), male (90% vs. 70%), with pre-existing spinal condition (62% vs. 33%), and injury caused by diving (11% vs. 2%). Multivariable logistic regression showed age (odd ratio [OR] 1.07; 95% confidence interval [CI] 1.04-1.11), diving (OR 3.5; 95% CI 3-100+), and wave height (OR 4.5; 95% CI 1.35-15.2) were associated with any SI, and a chief complaint of extremity numbness or tingling (OR 5.73; 95% CI 1.2-27.9) was associated with SCI. CONCLUSIONS: We identified older age, diving, and higher wave height as risk factors for any SI and symptoms of numbness and tingling were associated with SCIs. Clinicians should consider expediting these patients' transfers to a trauma center with neurosurgical capability.

Large-Scale Multi-omic Analysis of COVID-19 Severity.

We performed RNA-seq and high-resolution mass spectrometry on 128 blood samples from COVID-19-positive and COVID-19-negative patients with diverse disease severities and outcomes. Quantified transcripts, proteins, metabolites, and lipids were associated with clinical outcomes in a curated relational database, uniquely enabling systems analysis and cross-ome correlations to molecules and patient prognoses. We mapped 219 molecular features with high significance to COVID-19 status and severity, many of which were involved in complement activation, dysregulated lipid transport, and neutrophil activation. We identified sets of covarying molecules, e.g., protein gelsolin and metabolite citrate or plasmalogens and apolipoproteins, offering pathophysiological insights and therapeutic suggestions. The observed dysregulation of platelet function, blood coagulation, acute phase response, and endotheliopathy further illuminated the unique COVID-19 phenotype. We present a web-based tool (covid-omics.app) enabling interactive exploration of our compendium and illustrate its utility through a machine learning approach for prediction of COVID-19 severity.

Correlation of history and physical examination with imaging in traumatic near-shore aquatic head and spinal injury.

OBJECTIVE: It remains unclear whether clinicians can rely on specific symptoms and signs to detect or exclude serious head and spinal injury sustained during near-shore aquatic activities. Our study investigated patients' history of present illness (HPI) and physical examination (PE) for their utility in detecting serious head and spinal injury. METHODS: We conducted a multicenter retrospective comparative analysis of adult patients who were transported from the beach in Ocean City, Maryland, to three nearby emergency departments for possible spinal injury from 2006 through 2017. Patients suspected to have any spinal injury from beach activities were eligible. We excluded patients who could not verbalize their symptoms or with insufficient emergency department records. We compared components of each patient's HPI and PE with radiologic evidence of spinal injury. We calculated sensitivity, specificity, and negative and positive likelihood ratios (LRs). RESULTS: We analyzed 278 patients with suspected spinal injury. Midline spinal tenderness was associated with increased likelihood of thoracic (LR+ 2.6) and lumbar spinal fractures (LR+ 3.5). HPI complaints of paralysis (LR+ 13.9) and sensory loss (LR+ 5.8) had strong associations with spinal cord injuries. Weakness found through PE was also associated with spinal cord injury (LR+ 5.3). CONCLUSIONS: We identified several components of the clinical evaluation that had clinically significant association with spinal injuries from beach-related trauma. While prospective studies are needed to confirm our observations, clinicians may consider these high-risk features in patients with beach-related trauma and adjust testing and level of care appropriately.

Large-scale Multi-omic Analysis of COVID-19 Severity.

We performed RNA-Seq and high-resolution mass spectrometry on 128 blood samples from COVID-19 positive and negative patients with diverse disease severities. Over 17,000 transcripts, proteins, metabolites, and lipids were quantified and associated with clinical outcomes in a curated relational database, uniquely enabling systems analysis and cross-ome correlations to molecules and patient prognoses. We mapped 219 molecular features with high significance to COVID-19 status and severity, many involved in complement activation, dysregulated lipid transport, and neutrophil activation. We identified sets of covarying molecules, e.g., protein gelsolin and metabolite citrate or plasmalogens and apolipoproteins, offering pathophysiological insights and therapeutic suggestions. The observed dysregulation of platelet function, blood coagulation, acute phase response, and endotheliopathy further illuminated the unique COVID-19 phenotype. We present a web-based tool (covid-omics.app) enabling interactive exploration of our compendium and illustrate its utility through a comparative analysis with published data and a machine learning approach for prediction of COVID-19 severity.

A candidate activation pathway for coagulation factor VII.

The mechanism of generation of factor VIIa, considered the initiating protease in the tissue factor-initiated extrinsic limb of blood coagulation, is obscure. Decreased levels of plasma VIIa in individuals with congenital factor IX deficiency suggest that generation of VIIa is dependent on an activation product of factor IX. Factor VIIa activates IX to IXa by a two-step removal of the activation peptide with cleavages occurring after R191 and R226. Factor IXaα, however, is IX cleaved only after R226, and not after R191. We tested the hypothesis that IXaα activates VII with mutant IX that could be cleaved only at R226 and thus generate only IXaα upon activation. Factor IXaα demonstrated 1.6% the coagulant activity of IXa in a contact activation-based assay of the intrinsic activation limb and was less efficient than IXa at activating factor X in the presence of factor VIIIa. However, IXaα and IXa had indistinguishable amidolytic activity, and, strikingly, both catalyzed the cleavage required to convert VII to VIIa with indistinguishable kinetic parameters that were augmented by phospholipids, but not by factor VIIIa or tissue factor. We propose that IXa and IXaα participate in a pathway of reciprocal activation of VII and IX that does not require a protein cofactor. Since both VIIa and activated IX are equally plausible as the initiating protease for the extrinsic limb of blood coagulation, it might be appropriate to illustrate this key step of hemostasis as currently being unknown.

Neuroserpin Differentiates Between Forms of Tissue Type Plasminogen Activator via pH Dependent Deacylation.

Tissue-type plasminogen activator (t-PA), initially characterized for its critical role in fibrinolysis, also has key functions in both physiologic and pathologic processes in the CNS. Neuroserpin (NSP) is a t-PA specific serine protease inhibitor (serpin) found almost exclusively in the CNS that regulates t-PA's proteolytic activity and protects against t-PA mediated seizure propagation and blood-brain barrier disruption. This report demonstrates that NSP inhibition of t-PA varies profoundly as a function of pH within the biologically relevant pH range for the CNS, and reflects the stability, rather than the formation of NSP: t-PA acyl-enzyme complexes. Moreover, NSP differentiates between the zymogen-like single chain form (single chain t-PA, sct-PA) and the mature protease form (two chain t-PA, tct-PA) of t-PA, demonstrating different pH profiles for protease inhibition, different pH ranges over which catalytic deacylation occurs, and different pH dependent profiles of deacylation rates for each form of t-PA. NSP's pH dependent inhibition of t-PA is not accounted for by differential acylation, and is specific for the NSP-t-PA serpin-protease pair. These results demonstrate a novel mechanism for the differential regulation of the two forms of t-PA in the CNS, and suggest a potential specific regulatory role for CNS pH in controlling t-PA proteolytic activity.

Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy.

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

Priorities, strategies, and accountability measures in interprofessional education.

PURPOSE: The purpose of this pilot study was to identify the priorities, strategies, and accountability measures for interprofessional education (IPE) being used by health professions programs, allied health colleges, and/or universities. METHOD: An electronic survey was sent to 114 deans, associate deans, and directors (program, clinical education, graduate studies) at six institutions with allied health programs, including three academic medical centers and three comprehensive public institutions. The survey consisted of basic demographic questions and questions assessing knowledge of the Interprofessional Education Collaborative (IPEC) concepts of IPE, program-specific accreditation requirements for IPE, and institutional priorities, strategies, and accountability measures for IPE activities. RESULTS: An overall response rate of 50% (57/114) was achieved with representation from a total of 34 different allied health programs. Chi-squared statistics showed statistically significant differences (p<0.05) between the frequencies of survey responses and institutional types in the inclusion of IPE in the college/school's vision, the physical space available to accommodate IPE needs, and the commitment to set aside time for IPE. CONCLUSION: This study found that there is not a clear mandate or direction from most allied health disciplinary accrediting bodies for IPE. While there appears to be distinct movement by institutions to hold programs accountable for IPE and to integrate IPE into the curricula, barriers remain that have slowed the desired degree of implementation of an interprofessional curricula. While institutions, college, and/or programs may be slow to formally include IPE in its vision, this study found that, in general, support is being provided for IPE activities.

Structural differences between active forms of plasminogen activator inhibitor type 1 revealed by conformationally sensitive ligands.

Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) in which the reactive center loop (RCL) spontaneously inserts into a central beta-sheet, beta-sheet A, resulting in inactive inhibitor. Available x-ray crystallographic studies of PAI-1 in an active conformation relied on the use of stabilizing mutations. Recently it has become evident that these structural models do not adequately explain the behavior of wild-type PAI-1 (wtPAI-1) in solution. To probe the structure of native wtPAI-1, we used three conformationally sensitive ligands: the physiologic cofactor, vitronectin; a monoclonal antibody, 33B8, that binds preferentially to RCL-inserted forms of PAI-1; and RCL-mimicking peptides that insert into beta-sheet A. From patterns of interaction with wtPAI-1 and the stable mutant, 14-1B, we propose a model of the native conformation of wtPAI-1 in which the bottom of the central sheet is closed, whereas the top of the beta-sheet A is open to allow partial insertion of the RCL. Because the incorporation of RCL-mimicking peptides into wtPAI-1 is accelerated by vitronectin, we further propose that vitronectin alters the conformation of the RCL to allow increased accessibility to beta-sheet A, yielding a structural hypothesis that is contradictory to the current structural model of PAI-1 in solution and its interaction with vitronectin.

Maspin binds to urokinase-type and tissue-type plasminogen activator through exosite-exosite interactions.

Maspin is a member of the serpin family with a reactive center loop that is incompatible with proteinase inhibition by the serpin conformational change mechanism. Despite this there are reports that maspin might regulate uPA-dependent processes in vivo. Using exogenous and endogenous fluorescence, we demonstrate here that maspin can bind uPA and tPA in both single-chain and double-chain forms, with K(d) values between 300 and 600 nM. Binding is at an exosite on maspin close to, but outside of, the reactive center loop and is therefore insensitive to mutation of Arg(340) within the reactive center loop. The binding site on tPA does not involve the proteinase active site, with the result that maspin can bind to S195A tPA that is already complexed to plasminogen activator inhibitor-1. The ability of maspin to bind these proteinases without involvement of the reactive center loop leaves the latter free to engage in additional, as yet unidentified, maspin-protein interactions that may serve to regulate the properties of the exosite-bound proteinase. This may help to reconcile apparently conflicting studies that demonstrate the importance of the reactive center loop in certain maspin functions, despite the inability of maspin to directly inhibit tPA or uPA catalytic activity in in vitro assays through engagement between its reactive center loop and the active site of the proteinase.

Profound imbalance of pro-fibrinolytic and anti-fibrinolytic factors (tissue plasminogen activator and plasminogen activator inhibitor type 1) and severe bleeding diathesis in a patient with cirrhosis: correction by liver transplantation.

A 49-year-old male with alcoholic cirrhosis suffered several spontaneous, life-threatening, deep muscle bleeding episodes. Laboratory evaluation indicated excessive fibrinolysis with low plasminogen, low alpha2-antiplasmin, undetectable plasminogen activator inhibitor type 1 (PAI-1) activity, high tissue plasminogen activator (t-PA) activity and high t-PA antigen. Treatment with oral anti-fibrinolytic agents prevented further bleeding episodes. Decompensated cirrhosis eventually necessitated orthotopic liver transplantation. Post-operatively, the patient did not require oral anti-fibrinolytic agents, and there were no significant bleeding events. Circulating PAI-1 activity, t-PA activity and antigen normalized by 3 months post transplant. In short, the profound bleeding diathesis, as well as the imbalance in t-PA and PAI-1 levels, corrected after liver transplantation. Recognition of such patients is important, because the bleeding diathesis is an indication rather than a contraindication for orthotopic liver transplantation.

Acyl-enzyme complexes between tissue-type plasminogen activator and neuroserpin are short-lived in vitro.

The serine protease tissue-type plasminogen activator (t-PA) initiates the fibrinolytic protease cascade and plays a significant role in motor learning, memory, and neuronal cell death induced by excitotoxin and ischemia. In the fibrinolytic system, the serpin PAI-1 negatively regulates the enzymatic activity of both single-chain and two-chain t-PA (sct-PA and tct-PA). In the central nervous system, neuroserpin (NSP) is a serpin thought to regulate t-PA enzymatic activity. We report that although both sct-PA and tct-PA rapidly form acyl-enzyme complexes with NSP in vitro, the interactions are short-lived, rapidly progressing to complete cleavage of NSP and regeneration of fully active enzyme. All NSP molecules appear to transit through the detectable acyl-enzyme intermediate and progress to completion of cleavage; no subpopulation that functions as a pure substrate was detected. Likewise, all molecules were reactive, with no evidence of a latent subpopulation. The interactions between NSP and t-PA were distinct from those between plasmin and NSP, wherein the same peptide bond was cleaved but there was no evidence of a detectable plasmin-NSP acyl-enzyme complex. The interactions between t-PA and NSP contrast with the formation of long-lived, physiologically irreversible acyl-enzyme complexes between t-PA and PAI-1, suggesting that the physiologic effect of t-PA-NSP interactions may be more complex than previously thought.