|Ahead of print publication
Platelet microparticles level in juvenile idiopathic arthritis: A pediatric population-based cross-sectional study in a tertiary care center
Naresh Kumar1, K Anu Punnen1, Sukesh Chandran Nair2, Visalakshi Jayaseelan3, T Sathish Kumar1
1 Department of Pediatrics, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Transfusion Medicine and Immuno-Hematology, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Biostatistics, Christian Medical College, Vellore, Tamil Nadu, India
K Anu Punnen,
Department of Pediatrics, Christian Medical College, Vellore - 632 004, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Platelet-derived microparticles (PMPs) are small vesicles that are released from the plasma membrane upon platelet activation, which are then involved in haemostasis, vascular health, and have recently been shown to be intimately involved in immune responses.
Aims and Objectives: We aimed to evaluate the level of plasma Platelet-derived micro particles in children with JIA and to assess the relationship between PMP levels and disease activity in JIA.
Materials and Methods: Children with JIA who fulfilled the International League of Associations for Rheumatology (ILAR) classification criteria for juvenile idiopathic arthritis were included. They were categorised into active disease group and inactive disease as assessed by Wallace criteria. Samples were run in Navios flow cytometer (Beckman Coulter). Platelet microparticles were identified by MPs positive for both Annexin V and CD41 antibodies.
Results: Out of 26 children with JIA, 12 had active disease group and 14 had inactive disease as assessed by Wallace criteria. Mean PMP level was 83507 cells/μl and 34904 cells/μl in active and inactive disease respectively (P = 0.06). There was no significant correlation between PMP and CRP levels (P = 0.75 and r = 0 .102) or PMP and ESR levels (P = 0.56 and r = -0.186) in JIA children with active disease.
Conclusion: PMP levels were significantly elevated in disease activity of JIA and could represent a new biomarker reflecting the state of cell activation in JIA. PMP role in the inflammatory processes needs to be further elucidated.
Keywords: Biomarkers, Juvenile idiopathic arthritis, Platelet-derived microparticles
|How to cite this URL:|
Kumar N, Punnen K A, Nair SC, Jayaseelan V, Kumar T S. Platelet microparticles level in juvenile idiopathic arthritis: A pediatric population-based cross-sectional study in a tertiary care center. Indian J Rheumatol [Epub ahead of print] [cited 2019 Aug 19]. Available from: http://www.indianjrheumatol.com/preprintarticle.asp?id=263908
| Introduction|| |
Platelet-derived microparticles (PMPs) are small vesicles that are released from the plasma membrane upon platelet activation. These membrane vesicles, ranging in size from 0.1 to 1.0 μL, are shed by platelets after stimulation with physiological agonists such as thrombin or collagen, or in response to high shear stress. PMPs are involved in hemostasis and play a role in the regulation of immunity. In addition to procoagulant functions, several studies suggest a role of PMPs in inflammatory processes during vascular pathogenesis.
Several studies have reported the increased level of platelet-derived microparticles (PMPs) in inflammatory diseases such as rheumatoid arthritis (RA), spondyloarthritis, systemic lupus erythematosus (SLE), antineutrophil cytoplasmic antibody-associated vasculitis, and inflammatory bowel disease, in which the increase in MPs is associated with disease severity.
Secretory phospholipase A2 (sPLA2) activity, which is derived predominantly from activated platelets are significantly increased in both the synovium and serum of RA patients compared with healthy controls (HCs) and is also correlated with disease activity. SPLA2 catalyzes the synthesis of arachidonic acid from phospholipids. Subsequently, arachidonic acid is converted by cyclooxygenase-2 (COX-2) into prostaglandins, which are essential for the development of inflammation. The activation of both COX-2 and sPLA2 is linked to platelet-activating factor (PAF), which is synthesized by various cells, including platelets. PAF is present in the synovial fluid of RA patients, and administration of a PAF receptor antagonist significantly reduces inflammation.,
Plasma levels of soluble P-selectin are elevated in RA patients compared with controls. Thus, sPLA2, PAF, and P-selectin are all associated with RA as well as with platelet activation. Since in various clinical conditions, platelet activation results in the production of MPs, PMPs may also play a role in the inflammatory processes of RA a prototype inflammatory arthritis, and a similar picture is expected in juvenile idiopathic arthritis (JIA) which is the most common chronic rheumatologic disease in children. In summary, several observations support the theory that PMPs are involved in the inflammatory processes of rheumatic diseases. No such studies are done in children with JIA. Until now, major studies are done in adult population with RA, and there is a scarcity of studies done in children.
JIA encompasses a complex group of disorders comprising several clinical entities with the common feature of arthritis. Assessment of disease activity in JIA typically includes measurement of inflammatory markers in peripheral blood, including either the erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP). Both of these inflammatory indices have low sensitivity and specificity, because they can be raised for many reasons other than JIA activity and in some children do not closely mirror disease activity. Several autoantibodies are already in widespread use as biomarkers in routine care of JIA, such as rheumatoid factor (RF), and anti-nuclear antibody.,
The pro-inflammatory S100 proteins, S100A8/9 (also known as calprotectin or myeloid-related protein [MRP 8/14] and S100A12, have been described to be sensitive measures for disease activity in JIA, and both correlate well with physicians' assessment of disease or with actively inflamed joint.,, Studies on ankylosing spondylitis (AS) also demonstrated the significance of circulating endothelial MPs (EMPs) and platelet MPs levels with disease activity. However, the standardization of the measurements and the detailed procedures for assay and dilution of serum samples from a wide range of patients, with varying disease activity, are nontrivial, complicating implementation of such a test in routine clinical care. However, there is no indisputable gold standard or biomarker for determining the stage of disease activity in JIA till now.
In JIA, there are currently no validated pediatric biomarkers available to help with setting up a more tailored approach on which drug choice could be based, to achieve remission early in the course of disease. Despite the huge potential of pediatric biomarkers, for JIA, there are currently no validated pediatric biomarkers available to help in setting up a tailored or “personalized” approach on which drug choice can be based. Hence, our study is one of the initial ventures to know the role of PMP, in pediatric population with JIA. Hence, we evaluated the level of plasma PMPs in children with JIA and their relationship with disease activity in JIA.
| Methodology|| |
Children with JIA were recruited consecutively after obtaining informed consent from the parents and assent from children above the age of 7 years, from the Paediatric Rheumatology Clinic of Christian Medical College, Vellore, for a period of 12 months for inclusion in this descriptive, cross-sectional study. The inclusion criteria were children with JIA who fulfilled the International League of Associations for Rheumatology (2001) classification criteria for JIA and the exclusion criteria were children with JIA with use of anticoagulants and/or corticosteroids. Children with JIA with active disease who had active joint involvement, elevated ESR and CRP were the cases (Group I) and children with inactive disease who had no active joint involvement and ESR and CRP within normal limits for age served as controls (Group 2) as assessed by Wallace criteria. Automatized complete blood count and serum analysis were carried out for all the participants. All patients gave informed consent and the study was approved by the Institutional Review Board (IRB) of Christian Medical College, Vellore (IRB No. 8730).
Platelet microparticle estimation
Platelett-free plasma (PEP) was Prepared by centrifugation of citrated whole blood at 2500 G, twice for 15 min. PFP was separated and stored at −80°C until used. When needed, PFP was thawed in a 37°C water bath before use. Procedure: 30 μl of PFP was incubated with 5 ul of fluorescein isothiocyanate-conjugated annexin V (Beckman Coulter) and 5 ul of phycoerythrin-conjugated CD41 (Biocytex, Marseille, France) monoclonal antibodies for 15 min at room temperature. Thirty microliters of counting beads (Biocytex, Marseille, France) were added to estimate the MP counts. About 500 ul of Annexin V binding buffer (with calcium) was added to the tube. Samples were run in Navios flow cytometer (Beckman Coulter). The procedure of differential lysis was not followed in this study.
Forward scatter and side scatter were set as a logarithmic gain, and Megamix-Plus FSC (Biocytex, Marseille, France), containing a mix of fluorescent microbeads of various diameters (0.1, 0.3, 0.5, and 0.9 μm), was used for initial settings and before each experiment to measure MPs, as an internal control. Gates were then set to include events between 0.3 and 1.0 μm with exclusion of background corresponding to debris usually present in buffers. Platelet MPs were identified by MPs positive for both Annexin V and CD41.
All categorical variables were presented using the frequencies and percentages. For continuous variables, results of descriptive statistics were expressed as mean ± standard deviation and median (range). Comparisons of continuous variables were carried out using Student's t-test or Mann–Whitney test; for categorical variables, the Chi-square statistics was used. Continuous variables were described as median and interquartile range, and categorical variables as absolute and relative (%) frequencies. Nonparametric tests (Mann–Whitney) were used to compare the values of PMPs in the various groups. Since data were not normally distributed, Spearman's test was applied to correlate the number of PMPs with disease activity. All data were expressed as the median (range). Two-sided P < 0.05 was considered statistically significant. Data analysis was done with SPSS (version 17.0, SPSS Inc., IBM corporation, USA).
| Results|| |
A total of 26 children were recruited during the study period. Baseline demographics and blood investigations recorded were analyzed. Children with JIA were divided into two groups. Group I was JIA children with active disease (n = 12) and Group 2 was JIA children with inactive disease (n = 14) as assessed by Wallace criteria.
The median age of JIA patients was 9.5 years in active disease group and inactive disease group was 10 years. Between JIA groups, there were no differences in age, sex distribution, disease duration, or types of JIA [Table 1]. Out of 12 children with active disease, 6 (50%) had oligoarticular JIA followed by 4 (34%) had systemic arthritis and 1 (8%) each of polyarticular RF + and polyarticular RF-JIA. Out of 14 children with inactive disease, 6 (43%) had systemic arthritis followed by 3 (22%) each for enthesitis-related arthritis and oligoarticular JIA followed by 2 of polyarticular RF + JIA [Table 1].
|Table 1: Demographic and clinical parameters of Juvenile idiopathic arthritis patients|
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Platelet counts were higher in JIA patients, especially those with active disease. The number of PMPs was higher in JIA patients with active disease than in controls. Patients with active JIA had more PMPs than did patients with inactive JIA, but this difference was not statistically significant (P = 0.06) [Table 2] and [Figure 1].
|Figure 1: Number of platelet microparticles (PMPs) in children with active and inactive disease Horizontal lines show the medians; boxes show the interquartile ranges; vertical lines show the high and low values|
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Receiver-operating characteristic curve analysis was applied to determine the best cutoff value of serum PMPs in distinguishing active from inactive JIA. The area under the curve was 0.744, and the optimum cutoff level was 24501 cells/μL [Figure 2].
|Figure 2: Receiver-operating characteristic curve analysis showing the diagnostic performance of platelet microparticles for discriminating children with active juvenile idiopathic arthritis from children with inactive juvenile idiopathic arthritis disease activity area under the curve for platelet microparticles = 0.744|
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The PMP count did not correlate with the platelet levels, CRP level, or the ESR. There was no significant correlation between PMPs and platelets level in JIA children with active disease (P = 0.21 and r = 0.389). There was no significant correlation between PMPs and CRP levels in JIA children with active disease (P = 0.75 and r = 0.102). There was negative correlation between PMP and ESR levels in JIA children with active disease (P = 0.56 and r = −0.186).
| Discussion|| |
This is a cross-sectional study done in the pediatric population, which demonstrates that PMP levels are higher in JIA children, but its correlation with disease activity was not statistically proven in our study. Most of the studies on the role of platelet MPs in autoimmune disease were on the older population. The role of PMPs in inflammation is further supported by the observation that they can activate neutrophils. Moreover, PMP formation can be initiated by complement factors, which probably also play a role in rheumatic disease.
There was no significant difference in platelet levels between the two groups studied, although there was a trend toward higher platelet counts in patients with active JIA than in children patients with inactive JIA. This contrasts with earlier studies that demonstrated elevated platelet counts in patients with active disease. Perhaps, the number of participants in the present study was too small to allow detection of significant differences. A higher number of PMPs per platelet rather than a higher platelet count, or differences in their production rate and/or clearance, may also be responsible for the higher PMP numbers in patients with active JIA.
In 2002, Knijff-Dutmer et al. demonstrated that elevated levels of platelet MPs are associated with disease activity in RA. This cross-sectional study carried out among 19 RA patients and 10 HCs states that level of platelet MPs is significantly elevated in active RA patients when compared to inactive patients and HCs. This result confirms the hypothesis that PMPs are involved in the inflammatory process of RA. Their results were similar to the present study. As our results, this study also did not show any positive correlation between PMP level in JIA children with active disease and ESR, CRP, or platelets.
Sellam et al. measured plasma levels of total, platelet, and leukocyte MPs by prothrombinase capture assay and flow cytometry in 43 patients with progressive systemic sclerosis (pSS), 20 with SLE, and 24 with RA and in 44 HCs. They concluded that plasma MP level is elevated in pSS, as well as in SLE and RA, hence could be used as a biomarker reflecting systemic cell activation. In 2012, Sari et al. evaluated the profiles of EMP and platelet microparticles (PMP) in men with AS and healthy individuals. They also aimed to determine whether MP correlate with disease activity, function, and spinal mobility indices. Correlation analysis revealed no correlation with Bath AS Disease Activity Index, Bath AS Functional Index, or Bath AS Metrology Index. CRP was significantly correlated with PMP and CD42a−/CD31 + EMP (P < 0.05). As our cohort had very few numbers of children with ERA, such comparison was not done.
Michael et al., demonstrated higher levels of Annexin-V + MPs in both plasma and synovial fluid of RA versus osteoarthritis patients, suggest a role for MPs in the pathogenesis of RA. Rodríguez-Carrio et al. analyzed MPs counts in RA patients. Absolute MP number was increased in RA patients compared with HC and positively correlated with traditional cardiovascular risk (CVR) factors, similar to that of CVR patients. In addition, frequency of the different MP subsets was different in RA patients and significantly associated with disease features. In our study, we were able to demonstrate that PMPs correlate with active disease.
We consider that heterogeneity of JIA is a major limitation for our study. Ideally, serial samples in same children would have been done at follow-up. Comparisons based on sequential sampling may have provided additional information on the accuracy of these markers. Our study was performed in a relatively small number of patients in a single unit. A multicenter study would be necessary to confirm these results. We also did not have normal levels of PMPs in healthy children which could be used as a control group.
Each subtype of JIA is characterized by a different mode of presentation, disease course and outcome. It might be one of the difficult challenges of the study in a pediatric population of JIA. In our study, there was difference in the types of JIA in both comparing groups, which might have an impact on the results, thus making the comparison between different studies difficult and emphasizing the need further researches.
| Conclusion|| |
We demonstrate that the level of circulating PMPs is significantly elevated in children with active JIA compared to inactive JIA. However, since the level of PMPs increases in children with active disease, the interest of using PMP levels for monitoring disease activity will be useful for physician. Additional studies of function are required to understand the involvement of PMPs in signaling pathways of remote cellular cross-talk in autoimmune diseases. Increased PMPs are seen in children with JIA and are associated with disease activity. Their role in the inflammatory processes needs to be further elucidated.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Sims PJ, Faioni EM, Wiedmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor va and express prothrombinase activity. J Biol Chem 1988;263:18205-12.
VanWijk MJ, VanBavel E, Sturk A, Nieuwland R. Microparticles in cardiovascular diseases. Cardiovasc Res 2003;59:277-87.
Holme PA, Orvim U, Hamers MJ, Solum NO, Brosstad FR, Barstad RM, et al.
Shear-induced platelet activation and platelet microparticle formation at blood flow conditions as in arteries with a severe stenosis. Arterioscler Thromb Vasc Biol 1997;17:646-53.
Aatonen M, Grönholm M, Siljander PR. Platelet-derived microvesicles: Multitalented participants in intercellular communication. Semin Thromb Hemost 2012;38:102-13.
Miao D, Ma TT, Chen M, Zhao MH. Platelets release proinflammatory microparticles in anti-neutrophil cytoplasmic antibody-associated vasculitis. Rheumatology (Oxford) 2019. pii: kez044.
Lin MK, Farewell V, Vadas P, Bookman AA, Keystone EC, Pruzanski W. Secretory phospholipase A2 as an index of disease activity in rheumatoid arthritis. Prospective double blind study of 212 patients. J Rheumatol 1996;23:1162-6.
Hilliquin P, Guinot P, Chermat-Izard V, Puechal X, Menkes CJ. Treatment of rheumatoid arthritis with platelet activating factor antagonist BN 50730. J Rheumatol 1995;22:1651-4.
Barry OP, Kazanietz MG, Praticò D, FitzGerald GA. Arachidonic acid in platelet microparticles up-regulates cyclooxygenase-2-dependent prostaglandin formation via a protein kinase C/mitogen-activated protein kinase-dependent pathway. J Biol Chem 1999;274:7545-56.
Littler AJ, Buckley CD, Wordsworth P, Collins I, Martinson J, Simmons DL. A distinct profile of six soluble adhesion molecules (ICAM-1, ICAM-3, VCAM-1, E-selectin, L-selectin and P-selectin) in rheumatoid arthritis. Br J Rheumatol 1997;36:164-9.
Consolaro A, Ruperto N, Bazso A, Pistorio A, Magni-Manzoni S, Filocamo G, et al.
Development and validation of a composite disease activity score for juvenile idiopathic arthritis. Arthritis Rheum 2009;61:658-66.
Ringold S, Seidel KD, Koepsell TD, Wallace CA. Inactive disease in polyarticular juvenile idiopathic arthritis: Current patterns and associations. Rheumatology (Oxford) 2009;48:972-7.
Angeles-Han ST, Pelajo CF, Vogler LB, Rouster-Stevens K, Kennedy C, Ponder L, et al.
Risk markers of juvenile idiopathic arthritis-associated uveitis in the childhood arthritis and rheumatology research alliance (CARRA) registry. J Rheumatol 2013;40:2088-96.
Duurland CL, Wedderburn LR. Current developments in the use of biomarkers for juvenile idiopathic arthritis. Curr Rheumatol Rep 2014;16:406.
Schulze zur Wiesch A, Foell D, Frosch M, Vogl T, Sorg C, Roth J, et al.
Myeloid related proteins MRP8/MRP14 may predict disease flares in juvenile idiopathic arthritis. Clin Exp Rheumatol 2004;22:368-73.
Gerss J, Roth J, Holzinger D, Ruperto N, Wittkowski H, Frosch M, et al.
Phagocyte-specific S100 proteins and high-sensitivity C reactive protein as biomarkers for a risk-adapted treatment to maintain remission in juvenile idiopathic arthritis: A comparative study. Ann Rheum Dis 2012;71:1991-7.
Sari I, Bozkaya G, Kirbiyik H, Alacacioglu A, Ates H, Sop G, et al.
Evaluation of circulating endothelial and platelet microparticles in men with ankylosing spondylitis. J Rheumatol 2012;39:594-9.
Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J, et al.
International league of associations for rheumatology classification of juvenile idiopathic arthritis: Second revision, Edmonton, 2001. J Rheumatol 2004;31:390-2.
Wallace CA, Ruperto N, Giannini E; Childhood Arthritis and Rheumatology Research Alliance, Pediatric Rheumatology International Trials Organization, Pediatric Rheumatology Collaborative Study Group. Preliminary criteria for clinical remission for select categories of juvenile idiopathic arthritis. J Rheumatol 2004;31:2290-4.
György B, Szabó TG, Turiák L, Wright M, Herczeg P, Lédeczi Z, et al.
Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases. PLoS One 2012;7:e49726.
Jy W, Mao WW, Horstman L, Tao J, Ahn YS. Platelet microparticles bind, activate and aggregate neutrophils in vitro
. Blood Cells Mol Dis 1995;21:217-31.
Farr M, Scott DL, Constable TJ, Hawker RJ, Hawkins CF, Stuart J. Thrombocytosis of active rheumatoid disease. Ann Rheum Dis 1983;42:545-9.
Knijff-Dutmer EA, Koerts J, Nieuwland R, Kalsbeek-Batenburg EM, van de Laar MA. Elevated levels of platelet microparticles are associated with disease activity in rheumatoid arthritis. Arthritis Rheum 2002;46:1498-503.
Sellam J, Proulle V, Jüngel A, Ittah M, Miceli Richard C, Gottenberg JE, et al.
Increased levels of circulating microparticles in primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity. Arthritis Res Ther 2009;11:R156.
Michael BN, Misra DP, Chengappa KG, Negi VS. Relevance of elevated microparticles in peripheral blood and synovial fluid of patients with rheumatoid arthritis. Indian J Rheumatol 2018;13:222-8. [Full text]
Rodríguez-Carrio J, Alperi-López M, López P, Alonso-Castro S, Carro-Esteban SR, Ballina-García FJ, et al.
Altered profile of circulating microparticles in rheumatoid arthritis patients. Clin Sci (Lond) 2015;128:437-48.
[Figure 1], [Figure 2]
[Table 1], [Table 2]