Soluble Sortilin is elevated in the acute phase of Kawasaki Disease

Authors: testMasayuki Nagasawa

E-mail:mnagasawa.ped@tmd.ac.jp

Citation: Soluble Sortilin is elevated in the acute phase of Kawasaki Disease,Global Scientific Research Journal of Pediatrics, 1(1), 2019, pp. 1-8.

Copyright: This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract

Soluble Sortilin is released from activated platelets, but its clinical significance remains to be elucidated. We measured serum soluble Sortilin of the acute and convalescent phase in 45 patients with Kawasaki disease (KD) and examined trend of soluble Sortilin/platelet-count ratio (sSort/Plt). Among 62 patients with KD admitted to our department between April, 2017 and March, 2018, serum samples were obtained prospectively from 45 patients.

sSort/Plt was increased in the acute phase and decreased in the convalescent phase. In the refractory cases, it was increased even after initial treatment, and decreased after additional treatment along with the improvement of clinical manifestations. In 28 patients whose samples were taken in both of the acute (within less than three days after treatment) and convalescent (7-12 days after treatment) phase, sSort/Plt was significantly decreased after treatment (1.08+/-0.32 vs. 0.84+/-0.14, p<0.003). sSort/Plt before treatment was significantly higher in patients with D-dimer level> 1 μg/mL (n=13) than those with D-dimer level <0.5 μg/mL (n=10) and D-dimer level between 0.6 and 1.0μg/mL (n=20) (1.16+/-0.33 vs 0.93+/-0.11 vs 0.93+/-0.24).

Conclusion: Soluble Sortilin is elevated in the acute phase of KD and may be useful as a new biomarker.

Key words: Soluble Sortilin, Kawasaki Disease, D-dimer, platelet,

What is Known: TNF-α is increased in the acute phase of Kawasaki disease. Sortilin is cleaved by TACE (TNF-α converting enzyme) and released as soluble Sortilin by the activation of platelet in vitro. Clinical significance of soluble Sortilin remains to be elucidated.

What is New: Soluble Sortilin is increased in the acute phase of Kawasaki disease and may be a valuable biomarker.

Description

INTRODUCTION

Sortilin is a type 1 membrane bound protein belonging to the VPS10P (vacuolar protein sorting 10 protein) receptor family and presents in cellular endoplasmic reticulum and cell membrane. It has VPS10P domain outside that can bind to a variety of protein ligands, and sorting motif inside that is associated with control of intracellular endoplasmic reticulum transport[1]. It is expressed in various cells and tissues, and involved in induction of nerve cell death[2], formation of glucose transporter (Glut-4) in the fatty cells, and glucose intake response by insulin stimulation[3]. Also, in the hepatocytes, some functions to regulate blood low-density lipoprotein (LDL) cholesterol level are reported [4; 5] and it is shown the LDL is taken up through Sortilin in the macrophage[6]. Furthermore, Sortilin is reported to control secretion of IL-6 and IFN-γ from T cells[7].

Sortilin on the cell membrane is cleaved by TACE (TNF-α converting enzyme) and released as soluble Sortilin (sSortilin)[8; 9]. Although, sSortilin is shown to bind various proteins through VPS10P domain[9], its physiological function remains to be determined. Whereas Sortilin is also present in a platelet, and it is secreted by collagen stimulation to aggregate.

We have recently established ELISA system for measurement of sSortilin, and reported that sSortilin is increased by the activation of platelet, and sSortilin level in blood may be correlated with a risk for atherosclerosis[10].

Kawasaki disease (KD) is an acute febrile illness of unknown origin which commonly develops in younger children less than four years old, and is classified as medium-sized vasculitis[11]. In severe cases, coronary artery aneurysm will develop and eventually progress to sudden death due to myocardial infarction[12].  It is reported that TNF-α is increased in an acute phase of KD[13] and anti-TNF-α antibody therapy is effective for refractory KD[14]. Because it has been recently reported that microparticle derived from a platelet is increased in the acute phase of KD [15; 16], we measured sSortilin to investigate clinical significance of sSortilin in KD.

Patients and methods

Among 62 patients with KD admitted to our department between April, 2017 and March, 2018, we obtained serum of the acute and convalescent phase from 45 patients from whom a written informed consent of the guardians was obtained and measured serum sSortilin. KD patients were treated according to the Kobayashi score (K-s) as described before[17]. Briefly, KD patients of K-s <5 were treated with intravenous high dose immunoglobulin (2g/kg; IVIG) and acetylsalicylic acid (30mg/kg/day; ASA), and KD patients of K-s>=5 were treated with IVIG, prednisolone (2mg/kg/day; PSL), and ASA. Unresponsiveness to IVIG was defined as follows; decline of fever (37.5℃) was not obtained or fever was re-increased again more than 37.5℃ within 24 to 36 hours after IVIG. Serum sSortilin was measured according to our method previously reported [10].

Statistical analysis    

The Mann-Whitney U-test was used for statistical comparisons between two data sets and the Dunn’s test was used for multiple comparisons between three data sets and correlation was analyzed by Pearson’s correlation coefficient (r).   A value of p < 0.05 was considered statistically significant. All statistical analyses were performed using Excel Statics ver6.0 (Microsoft, Redmomd, WA)

Results

We measured 149 specimens from 45 patients with KD in total. Summarized profiles of 45 patients with KD are presented in the Table 1.  


Unresponsive to initial IVIG treatment cases were 3/20 in K-s <5 group, and 6/25 in K-s >=5 group. IVIG: intravenous high dose immunoglobulin, ASA: Acetylsalicylic acid, PSL: prednisolone, K-s: Kobayashi score, SD: standard deviation

It has been reported that sSortilin is released during the aggregation process of the platelet and the level of sSortilin in the serum is affected artificially by a platelet count[10]. In this study, we examined a correlation of sSortilin with platelet count in the serum samples in KD patients obtained more than 10 days after the initial treatment. As shown in Figure 1,

Figure 1: Strong positive relation between serum soluble Sortilin and platelet count in the convalescent phase of Kawasaki disease.


sSortilin showed an extremely strong correlation with platelet count as presented before [10]. Therefore, sSortilin/platelet-count ratio (sSort/Plt) was used in the following analysis in order to avoid the effect of platelet count, because platelet count is known to increase in the convalescent phase of KD..

Figure 2 shows the four representative cases in which sequential sSort/Plt and C-reactive protein (CRP) after IVIG treatment are plotted. 

In the IVIG unresponsive case, sSort/Plt did not decrease after the first IVIG or even increased and then decreased only after the second IVIG treatment along with the improvement of clinical manifestation. In the IVIG responsive case,the sSort/Plt decreased after the first IVIG. On the contrary, CRP decreased after IVIG treatment even in the unresponsive cases as well as in the responsive cases.

Figure 2: Trends of soluble Sortilin/platelet count ratio (sSort/Plt) after IVIG treatment in the Kawasaki disease.


Kobayashi score was <5 in U-2, and >=5 in U-4, U-8, and R-32. In U-2, U-4, and U-8, patients were unresponsive to initial IVIG, but responsive to second IVIG. In R-32, patient was responsive to initial IVIG. Number of x-axis shows day after admission. IVIG: intravenous high dose immunoglobulin, PSL: prednisolone U-2 means unresponsive case-2. R-32 means responsive case-32.

We compared sSort/Plt before and after the treatment in 28 patients from whom samples were taken in both of the acute (within less than three days after treatment) and convalescent (7-12 days after treatment) phase. The sSort/Plt was significantly higher in the acute phase as shown in Figure 3.

Figure 3: Comparison of soluble Sortilin/platelet count ratio (sSort/Plt) between acute (within less than three days after treatment) and convalescent (7-12 days after treatment) phase of Kawasaki disease.

(a) sSort/Plt in the acute phase was significantly higher than that in the convalescent phase. (b) Trends of sSort/Plt in each patient were plotted. In most of the cases, sSort/Plt was decreased in the convalescent phase. *: statistically significant
It has been reported that D-dimer is elevated during the acute phase of KD, and related with the severity and the prediction of therapy-responsiveness and coronary aneurysm[18-20].   About 43 cases in whom D-dimer was measured before treatment, we examined a relation between D-dimer level and sSort/Plt. As presented in Figure 4, sSort/Plt was significant ly higher in KD patients with D-dimer > 1 μg/mL than those with D-dimer level <0.5 μg/mL and D-dimer level between 0.6 and 1.0μg/mL (1.16+/-0.33 vs 0.93+/-0.11 vs 0.93+/-0.24). Whereas, there was no significant difference about a platelet count between three groups (33.9+/-9.7 vs 31.9+/-6.4 vs 32.6+/-5.5).

Figure 4: sSort/Plt was significantly higher in the patients with D-dimer >1.0μg/mL than others.

sSort/Plt in the acute phase of KD patients with D-dimer >1.0mg/μL was compared to that with D-dimer =<1mg/μL. Normal value of D-dimer is less than 0.5μg/mL. *:statistically signifi - cant sSort/Plt in KD patients with K-s >=5 before the treatment was higher than those with K-s <5, but

Discussion

In this study, sSortilin/platelet-count ratio (sSort/Plt) was used as an indicator to correct a factor affected by artificially released Sortilin during the process of serum formation. Therefore, it is necessary to be careful about interpretation of the result. However, sSortilin is considered to increase in the acute phase of KD and decrease to normal level after treatment, when we put the analysis data from the various viewpoints together, as described above.

Acetylsalicylic acid (ASA) has an anti-platelet function and it inhibits platelet aggregation stimulated by collagen and thereby release of sSortilin in vitro [10]. ASA is given as a standard therapy for KD. The pharmacologic effect of ASA on a platelet is direct and rapid.  While, the change of sSort/Plt in KD was complicated and it was even increased under the ASA therapy in the IVIG unresponsive case, which indicates that a trend of sSort/Plt could not be explained only by pharmacological effect of ASA.

D-dimer is regarded as a useful and sensitive indicator of vasculitis and widely used clinically [21]. As shown in Figure 4, sSort/Plt was significantly higher in KD patients with D-dimer > 1 μ g/mL than those with D-dimer =< 1μg/mL. This observation seems interesting in the light of our previous report[18] and others which have reported that D-dimer level in KD before the treatment is associated with severity and refracnot statistically significant (1.04+/-0.27 vs 0.95+/-0.27, p=0.290).
toriness to IVIG[19; 20]. Furthermore, there is a report that expression of platelet activated marker of CD40L [22] is significantly increased in KD with coronary aneurysm [23].   Recent reports [15; 16] that platelet-derived microparticle is increased in the acute phase of KD indicate that platelet is activated in KD during the acute phase. It is well known that TNF-α is increased in the acute phase of KD[13], and anti-TNF-α antibody is used and effective for intractable KD[14]. Considering that both of TNF-α and sSortilin are produced by activated TACE, sSortilin may be a useful biomarker in the acute phase of KD. Furthermore, soluble Sortilin may be an interesting biomarker which links inflammation and coagulation.   In this context, it is noticeable that sSort/Plt increased even after initial IVIG treatment in the unresponsive cases, while CRP decreased both in the unresponsive and responsive cases, which indicates that sSort/Plt may reflect disease activity of KD more efficiently than CRP during the acute phase.

As mentioned first, this study is based on the tentative indicator of sSort/Plt and therefore preliminary and qualitative in nature.  Examination of sSortilin in plasma sample to avoid an artificial factor will provide more precise and quantitative analysis. 

AcknowledgementsWe appreciate Asami Shinbo, Hisae Nakatani, and Yumie Tamura for their help to collect serum samples.


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