Background
Acute respiratory failure (ARF) can progress to pediatric acute respiratory distress syndrome (PARDS), multiorgan failure (MOF) and death [
1‐
3]. The heterogeneity of ARF and PARDS are potential impediments to the discovery of effective therapeutic options [
4], and consequently, recent studies have aimed to endotype, subclassify and prognostically enrich ARDS based on clinical and serum biomarkers [
5,
6]. In adults, clinical markers such as dead space fraction [
7‐
9] and the ventilatory ratio [
10‐
12] have highlighted the contribution of inefficient ventilation in the prognosis of ARDS and are starting to be used in clinical investigation. Serum biomarkers, by enabling mechanism-specific subclassification of ARDS, may also elucidate pathway-targeted therapies and enable predictive enrichment [
13]. A role for inflammation in the pathogenesis of PARDS has been supported by studies that showed plasma levels of interleukins (IL)-6, IL-8, IL-10, IL-18, soluble Tumor Necrosis Factor Receptor-2 and interleukin-1 receptor antagonist [
14‐
16] are associated with higher mortality in these patients. In addition, plasminogen activator inhibitor-1, soluble thrombomodulin (sTM) and von Willebrand factor-antigen, involved in endothelial injury and dysregulated coagulation, are also implicated in the pathogenesis of adult [
17‐
19] and pediatric [
20,
21] ARDS, potentially through microvascular thrombosis contributing to dead space ventilation and organ dysfunction.
Soluble thrombomodulin is an attractive candidate biological marker for respiratory failure and ARDS because thrombomodulin, an anti-thrombotic agent found in the endothelial cell surface, is cleaved into its soluble form in response to local endothelial damage [
22]. Both full length and the soluble form of thrombomodulin are protective against thrombosis; however, once thrombomodulin is cleaved and released into the circulation, it is assumed that the local anti-thrombotic effect is lost due to reduced avidity of the marker after cleavage, the generation of fragments of varying lengths and affinities, and whole-body redistribution. While it is likely that sTM levels increase in response to endothelial damage in a variety of organs, thrombomodulin is most prominently expressed in the human lung [
22]. Thrombomodulin also plays an important role in lung development [
23], which may imply a higher concentration of sTM in the pediatric lung, but this is not known.
Elevated levels of plasma sTM reflect inflammation, endothelial damage and loss of protection against thrombosis. A post hoc analysis of the FACTT trial revealed that elevated levels of plasma sTM were associated with higher mortality in adult patients with ARDS [
19], and another study reported that specific gene polymorphisms of thrombomodulin have been associated with increased mortality in adult ARDS [
24]. In children, a study in septic meningitis demonstrated the loss of local endothelial thrombomodulin and an elevation of plasma sTM [
25]. In addition, we reported preliminary findings that sTM levels are associated with increased mortality in children with ARDS caused by indirect lung injury [
21], though these findings have not yet been validated in an independent, heterogeneous cohort. A recent systematic review has highlighted the insufficient number of studies evaluating the role of sTM as a predictor of mortality in ARDS [
26]. Therefore, as part of the
Genetic Variation and Biomarkers in Children with Acute Lung Injury (
BALI; R01HL095410) which enrolled over 500 patients who were part of the
Randomized Evaluation of Sedation Titration for Respiratory Failure (
RESTORE; U01 HL086622) prospective clinical trial, we tested the hypothesis that plasma sTM is a predictor of ARDS severity, mortality and worse outcomes in pediatric patients with acute respiratory failure requiring mechanical ventilation.
Methods
Patients
This study,
Genetic Variation and Biomarkers in Children with Acute Lung Injury (
BALI; R01HL095410), was an ancillary study to the multisite clinical trial,
Randomized Evaluation of Sedation Titration for Respiratory Failure (
RESTORE; U01 HL086622) that enrolled intubated mechanically ventilated children [
16]. Details of the study methodology have been published previously [
27], and relevant details are summarized in the appendix.
Measurements
Blood samples were taken within 24 h of consent and again 24 and 48 h later, with the first blood sample drawn within three days of intubation (days 0–3) in most patients (98%). Plasma thrombomodulin levels were measured using two-antibody sandwich enzyme linked immunosorbent assays (ELISA, Asserchrome, Diagnostica Stago). The measurements were carried out in duplicate and followed the manufacturer’s protocol. For this study, we analyzed up to three sTM measurements per patient, collected within the first 5 days after intubation.
Primary outcomes
We examined the association between plasma sTM and 90-day in-hospital mortality adjusted for confounding variables.
Secondary outcomes
We examined the association of sTM with OI, the presence of non-pulmonary organ failure, ventilation-free days and PICU length of stay in survivors. We used the PALICC definition of ARDS. The determination of other secondary outcomes is as described in the supplement.
Confounding variables
The main analysis was adjusted for age, race, sex and PRISM-III scores by multivariable analyses. Additional multivariable models incorporated OI from the first 24 h after intubation, use of vasopressors at day 1 and use of neuromuscular blockade at day 1. These confounders were chosen a priori for their clinical significance and face validity. We used PRISM-III to adjust for baseline severity of illness.
Statistics
Given the unique nature of our dataset, which included repeated measurements of sTM along several days, and both continuous and binary outcomes with time varying covariates, we tested the relationship between sTM and primary and secondary outcomes using multiple approaches. We calculated odds ratio (OR) of mortality (alive or deceased at 90 days) given daily sTM level for days 0–2 by use of logistic regression. Receiver operating characteristic (ROC) curves were then evaluated to assess whether sTM drawn on these days could predict mortality. We also analyzed the relationship of sTM with mortality utilizing a composite estimate of all sTM levels in an individual patient using sTM intercept and slope. sTM intercept and sTM slope were determined by establishing a least square (LS) estimate between daily sTM values measured in the first 5 days. The intercept was the projected value of sTM where the LS line crossed t = 0. The slope indicates the rate of change of sTM in the first 5 days.
Finally, the hazard ratio (HR) for 90 day in-hospital mortality was assessed from sTM of all patient plasma samples collected between the day of intubation (day 0) and day 5 using counting process Cox proportional hazard model [
28].
sTM values on individual days up to day 3 were compared by Mann–Whitney U test between patients with PARDS and those without (days 4 and 5 were excluded due to low numbers).
Mixed effect modelling (MEM) was used to test the relationship of sTM with MOF and PICU length of stay. MEM was also used to evaluate the relationship between the initial sTM (intercept) or the rate of increase in sTM (slope) and maximum OI. Daily OIs (or if unavailable, converted OSIs) up until maximum value were analyzed using mixed effect modelling (MEM), with sTM intercepts and slopes as predictor variables and age, gender, race/ethnicity and PRISM-III score as confounding variables. The relationship between initial sTM or rate of change of sTM and daily number of failed organs within the first 28 days was also evaluated using MEM.
The outcome of ventilation free days was analyzed using Fine and Gray model with Cox proportional hazard regression. Death was utilized as a competing risk.
Study approval
Written informed consent was obtained from patients or their guardians prior to inclusion in the study. The study was approved by the Institutional Review Boards at all participating sites.
Discussion
In this study, higher initial values and rates of increase in soluble thrombomodulin (sTM) were associated with mortality in children with ARF. Moreover, elevated levels of sTM, particularly on day 1, independently associate with increased risk of in-hospital mortality after adjusting for several factors such as age, different markers of disease severity, severity of respiratory failure and use of neuromuscular blockade. There was also a statistically significant association between sTM and worsening oxygenation index, a validated marker of pulmonary dysfunction and ARDS severity [
29,
30]. Finally, higher initial values of sTM, and/or a greater rate of increase in sTM, were associated with multi-organ failure.
Thrombomodulin is an attractive candidate for assessment of ARF and ARDS given that the majority of thrombomodulin is found in the lung [
22] and its cleaved, soluble form (sTM) can be detected in patient plasma [
31]. We observed that at day 1, the area under the ROC curve was 0.7, which suggests moderate usefulness in prognosticating mortality from respiratory failure in this population. In context, this is similar to the AUC for procalcitonin in differentiating between bacterial and viral pneumonia in adults [
32]. It will be useful to evaluate the utility of sTM as a prognostic marker in combination with other biologic and clinical markers of ARDS in future studies.
In this study, levels of sTM correlated not only with mortality but also with severity of hypoxic respiratory failure. It is likely that pulmonary vascular damage would be a principal contributor to serum sTM in this study of pediatric acute respiratory failure from primary pulmonary or airways disease. Given the known association of sTM with vascular damage, and the loss of the anti-thrombotic molecule at the site of injury, it is conceivable that elevated sTM may reflect an increase in pulmonary dead space ventilation in ARDS. Dead space is a strong predictor of mortality in ARDS, even surpassing markers that measure oxygenation such as OI and P/F ratio [
8,
9,
33]. Since the
RESTORE trial did not record parameters for dead space ventilation, future studies on sTM would benefit from a prospective evaluation of sTM and dead space ventilation in ARDS or ARF.
Finally, sTM was associated with higher rates of extrapulmonary multiorgan failure. We posit whether this is a consequence of the pro-thrombotic state caused by the cleavage of thrombomodulin. Indeed, recombinant sTM, by replacing the vasculitis-induced depletion of membrane-bound local thrombomodulin, has been implicated in protection or reversal of vascular injury, disseminated intravascular coagulation (DIC) and in animal models of ARDS. In animal studies, recombinant sTM was shown to have a protective effect on septic rats by suppressing leukocyte adhesion to the microvasculature, reducing thrombus formation and preventing endothelial damage [
34]; and murine studies have suggested a protective role of sTM in LPS-induced ARDS [
35]. In humans, a randomized clinical trial evaluating patients with DIC suggested that treatment with recombinant sTM showed a more significant reversal of DIC than did heparin therapy, but did not evaluate the outcome of mortality [
36]. However, a large, multicenter clinical trial testing the therapeutic effect of recombinant sTM on 800 patients with sepsis-associated coagulopathy revealed no effect of sTM therapy on patient mortality, or secondary outcomes such as shock free, dialysis free and ventilator free days [
37]. Since the latter study enrolled patients presenting with sepsis complicated by DIC, it is very possible that the population was too heterogeneous to observe an effect on patients that would otherwise benefit from therapy. There was no effort in that trial to enrich for patients with an elevated thrombomodulin plasma level. In contrast, the
BALI cohort, in which we did find an association of elevated levels of sTM with higher mortality, included only children with a primary respiratory diagnosis. We postulate that since sTM is primarily derived from lung endothelium, patients with respiratory failure may be more likely to show a benefit from recombinant thrombomodulin compared to a population with non-pulmonary sources of sepsis. In addition, given the promising therapeutic effect of recombinant sTM on murine ARDs, it would be important to evaluate the therapeutic role of recombinant thrombomodulin specifically in patients with ARDS demonstrating elevated dead space ventilation and increased sTM as a marker of thrombomodulin depletion from the pulmonary vascular endothelium. Dead space could be measured at the bedside with the ventilatory ratio, an index that is associated with higher mortality in ARDS [
10].
The strength of this study lies in its relatively large sample size that includes a diverse study population in children. In addition, the study benefits from the availability of plasma samples from multiple time points and a well curated collection of data elements. The chosen outcomes of mortality, severity of hypoxic respiratory failure and multi-organ failure are of high clinical applicability and are arguably the most useful in assessing patient health. One study limitation is that we did not have access to data on ventilator parameters such as tidal volume and PEEP, which precluded our ability to investigate how ventilator changes may correlate with sTM levels. Another limitation was that all outcomes studied were measured in a population with some subtype of respiratory failure as a primary diagnosis, with almost 70% of the cohort developing PARDS within 5 days of intubation. As such, these findings can only be interpreted in the context of respiratory failure commonly leading to PARDS. Another limitation is that since over 90% of patients who developed PARDS did so by day 1, there was limited opportunity to assess the association of sTM with PARDS development.
Acknowledgements
We would like to thank all the patients and guardians of those patients for their participation in the study. We would also like to acknowledge the contribution of the BALI study investigators at the sites that participated in the RESTORE study including: Scot T. Bateman (University of Massachusetts Memorial Children's Medical Center, Worcester, MA), M. D. Berg (University of Arizona Medical Center, Tucson, AZ), Santiago Borasino (Children’s Hospital of Alabama, Birmingham, AL), G. Kris Bysani (Medical City Children's Hospital, Dallas, TX), Allison S. Cowl (Connecticut Children's Medical Center, Hartford, CT), Cindy Darnell Bowens (Children’s Medical Center of Dallas, Dallas, TX), E. Vincent S. Faustino (Yale-New Haven Children’s Hospital, New Haven, CT), Lori D. Fineman (University of California San Francisco Benioff Children’s Hospital at San Francisco, San Francisco, CA), A. J. Godshall (Florida Hospital for Children, Orlando, FL), Ellie Hirshberg (Primary Children’s Medical Center, Salt Lake City, UT), Aileen L. Kirby (Oregon Health & Science University Doernbecher Children's Hospital, Portland, OR), Gwenn E. McLaughlin (Holtz Children’s Hospital, Jackson Health System, Miami,FL), Shivanand Medar (Cohen Children's Medical Center of New York, Hyde Park, NY), Phineas P. Oren (St. Louis Children’s Hospital, St. Louis, MO), James B. Schneider (Cohen Children's Medical Center of New York, Hyde Park, NY), Adam J. Schwarz (Children’s Hospital of Orange County, Orange, CA), Thomas P. Shanley (C. S. Mott Children’s Hospital at the University of Michigan, Ann Arbor, MI), Lauren R. Sorce (Ann & Robert H. Lurie, Children’s Hospital of Chicago, Chicago, IL), Edward J. Truemper (Children’s Hospital and Medical Center, Omaha, NE), Michele A. Vander Heyden (Children's Hospital at Dartmouth, Dartmouth, NH), Kim Wittmayer (Advocate Hope Children’s Hospital, IL), Athena Zuppa (Children’s Hospital of Philadelphia, Philadelphia, PA) and the RESTORE data coordination center led by David Wypij, PhD (Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts; Department of Pediatrics, Harvard Medical School, Boston, Massachusetts; Department of Cardiology, Boston Children’s Hospital, Boston, Massachusetts).
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