Clinical Efficacy of Soluble Thrombomodulin, Tissue Plasminogen
Activator Inhibitor complex, Thrombin-Antithrombin
complex,α2-Plasmininhibitor-Plasmin complex in Pediatric Sepsis
Sepsis is defined as a life-threatening organ dysfunction that is caused by a host
immune disorder due to infection, and it is a common and serious disease in pediatric intensive care units
(PICUs), with a high morbidity and mortality. Epidemiological surveys over the past
10 years have shown that the incidence rate of sepsis in children is increasing annually. Twenty-six international multicenter studies revealed that the prevalence of
pediatric severe sepsis increased to 8.2%, and the mortality rate was as high as 25%
in between 2013 and 2014, with sepsis-induced coagulation disorder accounting for 50%–70% of these deaths. Sepsis-induced coagulopathy (SIC) constitutes a series of diseases, from the
early activation of the coagulation system to the formation of microthromboses to
the massive consumption of coagulation factors and the occurrence of overt
disseminated intravascular coagulation (DIC). As up to 70% of patients die of overt DIC in sepsis, SIC remains one of the
main causes of death in sepsis. SIC is a hypercoagulable condition due to inhibition of the anticoagulant system
after early endothelial system injury. Since conventional or classical coagulation
laboratory tests (CCTs) cannot detect activation of the early rising coagulation
system or the inhibition of the fibrinolytic system, coagulation markers that
accurately reflect the coagulation status of children with sepsis are sorely needed.
Soluble thrombomodulin (sTM), thrombin-antithrombin complex (TAT),
α2-plasmininhibitor-plasmin complex (PIC), and tissue plasminogen activator
inhibitor complex (t-PAI·C) have been useful in a variety of adult
diseases; – however, there is little research on sTM, TAT, PIC, or t-PAI·C
in children. In the present study, we assessed the coagulation status of children
with sepsis by determining sTM, TAT, PIC, and t-PAI·C combined with CCTs, and
determined their values in the diagnosis of pSIC and the outcome of sepsis in
children. 

Study Design This was a single-center prospective study in which patients were diagnosed as
having sepsis or severe sepsis (including septic shock) at the Department of
Critical Care Medicine of Shanghai Children's Medical Center, Affiliated with
Shanghai Jiaotong University, between June 2019 and June 2021. Sepsis and severe
sepsis were diagnosed in accordance with the 2005 International Guidelines for
the Diagnosis of Sepsis in Children. This study was approved by the Ethics Committee of Shanghai Children's
Medical Center (Shanghai, China, approval number: SCMCIRB-K2021065-1). Patients Children who fulfilled the following criteria were included: 1) aged over 28 days
and under 18 years; 2) afflicted with acute diseases that required PICU
admission; and 3) who received parental consent. The exclusion criteria included
1) patients with congenital coagulation dysfunction; 2) long-term use of
anticoagulant drugs (heparin, antithrombin, etc); 3) undergoing blood
purification and establishment of in vitro life support, and requiring systemic
heparin anticoagulant therapy; and 4) the guardian refusing to sign an informed
consent form. Laboratory Test Methods The novel coagulation markers sTM, TAT, PIC, and t-PAI·C were determined using
matching reagents by a chemiluminescence method on a Sysmex HISCL-5000
chemiluminescence immunoassay analyzer (Sysmex Corporation, Kobe, Hyogo, Japan).
CCTs were assessed using a CA-1500 whole-blood coagulation analyzer from the
Sysmex Company in Japan (Sysmex Corporation, Kobe, Hyogo, Japan). The timing of
coagulation markers measurement was when the child was admitted to PICU to
diagnose sepsis or septic shock. Study Groups The children were divided into a pSIC group and non-pSIC group according to their
sepsis coagulation scores (pSIC score, ). Also, consonant with the outcomes of sepsis patients at discharge, they
were divided into survival and non-survival groups. Observation Indicators The primary outcome indicators were sTM, TAT, PIC, and t-PAI·C. The secondary outcome indicators were CCTs that included Plt count (Plt),
activated partial thromboplastin time(aPTT), Prothrombin time(PT), Thrombin
time(TT), International Normalized Ratio(INR), Fibrinogen(Fib), D-dimers (DD),
and Fibrin degradation products(FDP). Other secondary outcome indicators were inflammation markers, organ-function
markers, clinical characteristics, anticoagulant therapy and blood product
infusion index, critical-illness scores (PRISM III, pSOFA,P-MODS), – DIC scores, mechanical ventilation time, vasoactive drug score (VIS), duration in the PICU, total hospital stay, and PICU outcome. Safety indicators included the presence of active bleeding and thromboses. Statistical Methods Measurement data that conformed to a normal or approximately normal distribution
are expressed as (x ± s), and we compared two independent samples using the
independent-sample t test. Measurement data following a
non-normal distribution are expressed as median (interquartile interval; P25,
P75), and we performed the Mann-Whitney U test to compare two independent
samples. Logistic regression analysis was used to analyze the independent risk
factors of pSIC and mortality outcome due to sepsis following discharge. We
implemented the area under the ROC curve (AU-ROC) method to evaluate the
sensitivity and specificity of sTM, TAT, and PIC, and t-PAI·C without or
combined with CCTs, to predict the mortality outcome of children with pSIC and
sepsis following discharge. Data were analyzed using SPSS 21.0 for Windows
(SPSS, Inc.). 

This was a single-center prospective study in which patients were diagnosed as
having sepsis or severe sepsis (including septic shock) at the Department of
Critical Care Medicine of Shanghai Children's Medical Center, Affiliated with
Shanghai Jiaotong University, between June 2019 and June 2021. Sepsis and severe
sepsis were diagnosed in accordance with the 2005 International Guidelines for
the Diagnosis of Sepsis in Children. This study was approved by the Ethics Committee of Shanghai Children's
Medical Center (Shanghai, China, approval number: SCMCIRB-K2021065-1). 

Children who fulfilled the following criteria were included: 1) aged over 28 days
and under 18 years; 2) afflicted with acute diseases that required PICU
admission; and 3) who received parental consent. The exclusion criteria included
1) patients with congenital coagulation dysfunction; 2) long-term use of
anticoagulant drugs (heparin, antithrombin, etc); 3) undergoing blood
purification and establishment of in vitro life support, and requiring systemic
heparin anticoagulant therapy; and 4) the guardian refusing to sign an informed
consent form. 

The novel coagulation markers sTM, TAT, PIC, and t-PAI·C were determined using
matching reagents by a chemiluminescence method on a Sysmex HISCL-5000
chemiluminescence immunoassay analyzer (Sysmex Corporation, Kobe, Hyogo, Japan).
CCTs were assessed using a CA-1500 whole-blood coagulation analyzer from the
Sysmex Company in Japan (Sysmex Corporation, Kobe, Hyogo, Japan). The timing of
coagulation markers measurement was when the child was admitted to PICU to
diagnose sepsis or septic shock. 

The children were divided into a pSIC group and non-pSIC group according to their
sepsis coagulation scores (pSIC score, ). Also, consonant with the outcomes of sepsis patients at discharge, they
were divided into survival and non-survival groups. 

The primary outcome indicators were sTM, TAT, PIC, and t-PAI·C. The secondary outcome indicators were CCTs that included Plt count (Plt),
activated partial thromboplastin time(aPTT), Prothrombin time(PT), Thrombin
time(TT), International Normalized Ratio(INR), Fibrinogen(Fib), D-dimers (DD),
and Fibrin degradation products(FDP). Other secondary outcome indicators were inflammation markers, organ-function
markers, clinical characteristics, anticoagulant therapy and blood product
infusion index, critical-illness scores (PRISM III, pSOFA,P-MODS), – DIC scores, mechanical ventilation time, vasoactive drug score (VIS), duration in the PICU, total hospital stay, and PICU outcome. Safety indicators included the presence of active bleeding and thromboses. 

Measurement data that conformed to a normal or approximately normal distribution
are expressed as (x ± s), and we compared two independent samples using the
independent-sample t test. Measurement data following a
non-normal distribution are expressed as median (interquartile interval; P25,
P75), and we performed the Mann-Whitney U test to compare two independent
samples. Logistic regression analysis was used to analyze the independent risk
factors of pSIC and mortality outcome due to sepsis following discharge. We
implemented the area under the ROC curve (AU-ROC) method to evaluate the
sensitivity and specificity of sTM, TAT, and PIC, and t-PAI·C without or
combined with CCTs, to predict the mortality outcome of children with pSIC and
sepsis following discharge. Data were analyzed using SPSS 21.0 for Windows
(SPSS, Inc.). 

Clinical Characteristics  Primary outcomes in the pSIC and non-pSIC groups There was a significant difference in t-PAI·C between the two groups (P <
0.05) . There were significant differences in Plt, PT, INR, aPTT, TT, and DIC scores
between the two groups (P < 0.05) . Logistic regression analysis of sTM, TAT, and PIC; and t-PAI·C combined with CCTs
in predicting pSIC. Multivariate logistic regression analysis showed that Plt
(P = 0.032) was an independent risk factor for pSIC . Comparison of outcomes due to sepsis at discharge; 44 children survived and 15
died at discharge. There were significant differences in sTM, TAT, Plt, PT, INR,
DIC score, pSIC score, PRISM III, and pSOFA between the two groups (P < 0.05)
. Logistic regression analysis of sTM, t-PAI·C,TAT and PIC combined with CCTs in
predicting the mortality outcome of children with sepsis at discharge. Multivariate logistic regression analysis showed that sTM (P = 0.007) and Plt
(P = 0.016) were independent risk factors for mortality outcome due to sepsis at
discharge . AU-ROC was used to evaluate the efficacy of sTM combined with Plt in predicting
the mortality outcome with sepsis at discharge. The area under the ROC curves for sTM combined with Plt in predicting the
mortality outcome of children with sepsis at discharge was 0.889 (95%CI:
0.781,0.956), which was higher than that for PRISM III (AUC, 0.723), pSOFA (AUC,
0.764), and blood Lac (AUC, 0.717) when sepsis was diagnosed in the PICU  Primary outcomes in the pSIC and non-pSIC groups There was a significant difference in t-PAI·C between the two groups (P <
0.05) . There were significant differences in Plt, PT, INR, aPTT, TT, and DIC scores
between the two groups (P < 0.05) . Logistic regression analysis of sTM, TAT, and PIC; and t-PAI·C combined with CCTs
in predicting pSIC. Multivariate logistic regression analysis showed that Plt
(P = 0.032) was an independent risk factor for pSIC . Comparison of outcomes due to sepsis at discharge; 44 children survived and 15
died at discharge. There were significant differences in sTM, TAT, Plt, PT, INR,
DIC score, pSIC score, PRISM III, and pSOFA between the two groups (P < 0.05)
. Logistic regression analysis of sTM, t-PAI·C,TAT and PIC combined with CCTs in
predicting the mortality outcome of children with sepsis at discharge. Multivariate logistic regression analysis showed that sTM (P = 0.007) and Plt
(P = 0.016) were independent risk factors for mortality outcome due to sepsis at
discharge . AU-ROC was used to evaluate the efficacy of sTM combined with Plt in predicting
the mortality outcome with sepsis at discharge. The area under the ROC curves for sTM combined with Plt in predicting the
mortality outcome of children with sepsis at discharge was 0.889 (95%CI:
0.781,0.956), which was higher than that for PRISM III (AUC, 0.723), pSOFA (AUC,
0.764), and blood Lac (AUC, 0.717) when sepsis was diagnosed in the PICU . 

Sepsis leads to endothelial cell damage, activation of the procoagulant system, and
inhibition of anticoagulant substances such as antithrombin (AT), protein C (PC),
and tissue factor pathway inhibitor (TFPI). Coagulopathy is a disorder of coagulation that includes both bleeding and
thrombosis, and the prevalence rates of adult SIC and DIC have been reported to be
as high as 50%–70% and 35%, respectively. Early recognition of coagulopathy is therefore critical for clinical
diagnosis and treatment. Since the diagnostic criteria for sepsis in children are
different from sepsis-3.0 in adults, , we proposed a pSIC scoring
scheme based on pSOFA scores in children and SIC scores in adults. pSIC was then used to evaluate the occurrence of pSIC in the group of
children in our study. As classical coagulation indicators such as PT or aPTT do not reflect the state of
overall coagulatory function, , the ability to diagnose pSIC
early remains limited. Among the new coagulation markers, TAT directly reflects the
activation of the coagulation system and can be used as a marker of coagulation
initiation. When the SIC anticoagulation system is inhibited, the fibrinolytic
system also changes. Plasmin is an important component of the fibrinolytic system.
The half-life of activated plasmin is very short; and plasmin reacts rapidly with
fibrinolytic system α 2 plasmin inhibitor to combine 1:1 and thereby form PIC, which
then reflects the early rise in plasmin and measurement of which can be used to
evaluate the degree of fibrinolytic activation in vivo. In addition, the
fibrinolytic system is principally mediated by plasminogen activator inhibitor-1
(PAI-1) and PIC, and thrombin activator fibrinolysis inhibitor (TAFI) leads to
fibrinolytic inhibition. Activated platelets and endothelial cells then promote the
rapid increase of PAI-1 in sepsis patients; and PAI-1 forms t-PAI·C by combining
with tissue plasminogen activator (t-PA) to inactivate it, thereby inhibiting the
dissolution of fibrin. The emergence of t-PAI·C can provide an early indication of the occurrence of
fibrinolytic inhibition, and is closely related to endothelial cell injury. Therefore, sTM, TAT, PIC, and t-PAI·C can be utilized to evaluate the changes
in coagulation status from the critical environments of many pathophysiological
mechanisms that underly SIC—including endothelial cell injury, early-coagulation
activation, and fibrinolytic-system inhibition. Asakura recommended that TAR, PIC, and t-PAI·C be detected in sepsis-related DIC to
evaluate the status of coagulation disorders. In this study, the new coagulation marker t-PAI·C was significantly increased in the
pSIC and non-pSIC groups (P = 0.001). Semeraro et al reported in 280 adult patients with sepsis that the changes in fibrinolytic
state in sepsis was mainly due to the increase in PAI-1 and the activation of TAFI.
In an animal model of endotoxemia, PAI-1 expression was primarily localized to
vascular endothelial cells, suggesting that plasma PAI-1 originated from endothelial cells; and this coincided with the theory of endothelial-cell injury in sepsis.
Madoiwa et al reported that increased PAI-1 in sepsis was also associated with organ
dysfunction and a poor prognosis in sepsis. The results of our study are thus very
similar to those reported in the literature. Combined with the results from CCTs, Plt was shown to be a risk factor for pSIC in
children with sepsis. Mei et al demonstrated that TAT, t-PAI·C, and sTM increased in patients with DIC in
adult sepsis, revealing endothelial injury, coagulation activation, and fibrinolytic
inhibition in patients with sepsis. The reasons for not obtaining the same research
conclusions in the present study may include the following: 1) The diagnostic
criteria for sepsis in adults and children were not completely congruent. The
diagnosis of sepsis in adults depends upon SOFA score, while sepsis-2.0 is still
used for children, and pSOFA is not involved in the diagnosis of sepsis in children. 2) There
were many children with hematological malignancies included in our study, and the
underlying status of their coagulatory function was different from that of the study
by Mei et al and other patients. There may also be damage to hematopoietic function,
especially with respect to the impact of platelet count on coagulatory function.
Therefore, we herein concluded that platelet count was a risk factor for pSIC. 3)
Coagulatory function in children is greatly affected by age-related
factors, , and use of the pSIC score has not yet been proposed by the
International Society for Thrombosis and Hemostasis (ISTH). Additional clinical
studies are therefore needed to confirm the methods for the early diagnosis of pSIC
and to provide timely treatment of sepsis in children. In the current study, we demonstrated that Plt and sTM were independent risk factors
for mortality outcome following discharge after sepsis. We showed that sTM in the
non-survival group was significantly higher than in the survival group, and studies
have revealed that decreased platelet count and prolonged PT were associated with
increased mortality due to sepsis. sTM can also be used as an early predictor of pediatric sepsis death, and the
level of sTM in non-survival groups has been significantly increased. Khattab et al depicted a higher level of sTM in pediatric sepsis relative to their control
group, and the mortality of children with increased sTM was higher than that for
surviving patients ( P = 0.005). Our AU-ROC for sTM in predicting
the outcome of sepsis death in children was 0.711, which was higher than the AU-ROCs
for PRISM III and PIM scores (0.918 and 0.960, respectively). The results of this
study were also consistent with the conclusions drawn by Khattab et al
Thrombomodulin is a transmembrane glycoprotein mainly produced by vascular
endothelial cells, and indicates injury to vascular endothelial cells. Vascular
endothelium is the principal target of sepsis, and one of the characteristics of SIC
is endothelial dysfunction. Therefore, the augmented increase in sTM that we observed was consistent with
the pathophysiological mechanism underlying endothelial cell injury caused by
sepsis. The results of our study thus show that sTM is a reliable biomarker of
endothelial cell injury. pSOFA,PRISM III and P-MODS are commonly used scoring methods for organ-function
evaluation and early-warning prognosis in children with sepsis. For example, studies
have shown that pSOFA score, PRISM III, and hyperlactatemia are reliable indicators
for predicting sepsis death in children. , In the present study, sTM,
TAT, PIC, and t-PAI·C combined with CCTs possessed the strongest predictive
capability for mortality outcomes of children with sepsis following discharge
(AU-ROC 0.892), followed by pSOFA (AU-ROC 0.764); CCTs (AU-ROC 0.740); sTM, TAT,
PIC, and t-PAI·C (AU-ROC 0.738); PRISM III (AU-ROC 0.723); and blood Lac (AU-ROC
0.717). This shows that the prediction of sTM, TAT, PIC, and t-PAI·C combined with
CCTs on the prognosis of sepsis patients at discharge is superior to pSOFA, PRISM
III, and Lac. The results of our study are thus consistent with the conclusions of
Khattab et al. The clinical significance of this study is that after endothelial cell injury
caused by sepsis, starting from the initiating links of endothelial cell injury,
coagulation factors activation and fibrinolysis function inhibition, through new
coagulation markers and combined with CCTs, it can play an early warning role in the
prognosis of sepsis and pSIC in children, We recommend further multicenter clinical
research to explore the early warning efficacy of the cut-off values of
sTM,t-PAI·C,TAT and PIC combined with CCTs on the diagnosis of sepsis and pSIC in
children. It should be noted that the pathophysiology of coagulation disorder in sepsis is the
formation of microthrombosis, in which fibrinolytic inhibition constitutes the
primary cause of sepsis-related organ dysfunction. After the activation of the coagulation system in sepsis, a large number of
immunothromboses are formed in microcirculatory vessels, blocking oxygen delivery
and resulting in organ dysfunction and increasing the mortality from sepsis. The
physiological coagulation status in children is more likely to encompass a mild
hypercoagulable state than in adults, and children's thrombotic diseases are more often ignored than those of adults. Studies have shown that FDPs and D-dimers are significantly increased during immunothrombosis, but the increase observed in CCTs lacks specificity as these indices are also
increased in trauma, tumor, infection, and other diseases; thus, they cannot fully reflect the changes caused by abnormal fibrinolysis
in sepsis. The accuracy of FDPs and D-dimers in evaluating the damage to the
fibrinolytic system in sepsis has therefore been questioned. sTM, TAT, PIC, and
t-PAI·C reflect the initial changes in endothelial cell injury, coagulation-system
activation, and fibrinolytic system changes from different aspects. Combined with
FDPs and D-dimers, these markers are more conducive to the evaluation of the
prethrombotic state. The evaluation of the fibrinolytic state of pSIC should
therefore be further investigated in the future to guide more accurate
treatment. We had been paying attention to the influence of developmental hemostasis theory on
bleeding and coagulation function in children of different ages. , This mainly
affects the coagulation function in infants. In this study, 12 infants aged less
than 1 year were included. because of its relationship to sample size, age
stratification was also not delineated. Therefore, we hope to expand the included
sample size, reduce the influence of confounding factors, including age, on
pediatric sepsis and pSIC as much as possible, and obtain clinical research results
that can be used to guide clinical decision-making. There were several limitations to the present study: 1) this study was a
single-center study, and the number of child cases was small; 2) there were many
children with hematologic malignancies that were not discussed with respect to
disease stratification; 3) changes in sTM, TAT, and t-PAI·C were not continuously
monitored so as to reflect their dynamic changes in pediatric sepsis;4) there were
many confounding factors in clinical research on sepsis, such as underlying
diseases, clinical interventions, enrollment time, age, and gender. Therefore, In
the process of logistic regression, we assumed linearity but may not hold true in
real world data. We should increase the included sample size in order to control
confounding factors; 5) In this study, the ROC curve was based on a training sample.
Due to the small sample size, there will be the problem of overfitting. However, the
clinical research data of sTM, PIC, t-PAIC, and TAT tests in pediatric sepsis are
very limited, especially for the special patient population of pediatric sepsis. In
future research, we will further increase the included sample size and carry out
multi-center clinical research to obtain more detailed clinical research data. 

We herein demonstrated that the novel coagulation marker t-PAI·C increased in
children with pSIC. The prognostic prediction for sepsis patients using sTM, TAT,
PIC, and t-PAI·C—combined with CCTs—was superior to that for PRISM III, pSOFA, or
Lac, particularly with respect to biomarkers of endothelial cell injury. sTM was
also related to the mortality outcome with respect to sepsis at discharge. Further
research is, however, still needed to explore the clinical value of sTM, TAT, PIC,
and t-PAI·C in the diagnosis and prognosis of sepsis and pSIC in children. 

