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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 6  |  Issue : 3  |  Page : 115-118

Elevated levels of pro-coagulant microvesicles in children in-steady state sickle cell disease


1 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia
2 Department of Pathology, College of Medicine, King Khalid University, Abha, Saudi Arabia
3 Department of Internal Medicine, Hematology and Oncology Unit, Aseer Central Hospital, Ministry of Health, Abha, Saudi Arabia

Date of Web Publication18-Sep-2015

Correspondence Address:
Hassan A Hamali
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, P.O. Box 9060, Abha 61413
Saudi Arabia
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Source of Support: Nil., Conflict of Interest: There are no conflicts of interest.


DOI: 10.4103/1658-5127.165650

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  Abstract 

Introduction: Sickle cell disease (SCD) is an inherited genetic disorder characterized by various complications, including thrombosis. Increased levels of circulating microvesicles (MVs) and tissue factor (TF)- bearing MVs have been reported in SCD. Objectives: The present study compares the levels of circulating MVs and TF- bearing MVs in steady state SCD children with age- and gender- matched healthy controls using an indirect ELISA. Citrated whole blood was collected from 54 SCD patients homozygous for sickle haemoglobin (HbSS) (aged from 2 to 12 years-old) and 34 healthy controls. Results: SCD patients showed significantly higher levels of MVs in their plasma as compared to the controls (P = 0.0095). Although the TF activity on MVs was low in both groups, there was a significant difference between them P <0.05). A strong correlation between the level of MVs and TF-MVs in the patient group was also noted. Conclusion: This suggests their involvement in the hypercoagulable state in the study group of patients. Further studies are recommended to elucidate the functional activity of MVs and TF-MVs, as well as the size and origin of MVs in the plasma.

Keywords: Hypercoagulable, microvesicles, microvesicles, sickle cell disease, tissue factor-bearing


How to cite this article:
Hamali HA, Elhussein OG, Jamil A, Hussain S, Alshraim M, Alshehri A. Elevated levels of pro-coagulant microvesicles in children in-steady state sickle cell disease. J Appl Hematol 2015;6:115-8

How to cite this URL:
Hamali HA, Elhussein OG, Jamil A, Hussain S, Alshraim M, Alshehri A. Elevated levels of pro-coagulant microvesicles in children in-steady state sickle cell disease. J Appl Hematol [serial online] 2015 [cited 2018 Jan 16];6:115-8. Available from: http://www.jahjournal.org/text.asp?2015/6/3/115/165650


  Introduction Top


Sickle cell disease (SCD) is a common genetic disorder observed throughout the kingdom of Saudi Arabia.[1] The pathogenesis of SCD is attributed to a substitution of glutamic acid to valine at the sixth position of the β-globin protein, which results in the production of an abnormal hemoglobin S.[2] SCD is considered as a "hypercoagulable" condition, characterized by chronic activation of the coagulation system. Platelets and endothelial cells were also known to contribute to the hypercoagulability condition, which lead to the high incidence of thrombotic events in the SCD.[3],[4],[5],[6]

One of the important factors reported to drive SCD into the hypercoagulable state is the increased levels of microvesicles (MVs)[7] and tissue factor (TF) bearing MVs.[8],[9] MVs are procoagulant due to the presence of the negatively charged phospholipids (mainly phosphatidylserine [PS]) on their surface), which increased by the presence of TF.[10] MVs have been reported in many diseases and also associated with venous thrombosis.[11],[12] Procoagulant MVs has the ability to support the binding of coagulation complexes,[13],[14] which accelerates the formation of thrombin in vivo.[15] Although the level of MVs is well studied in sickle cell patients in many countries of the world, this is thefirst study designed and conducted to investigate the levels of MVs and TF-bearing MV in steady state children with SCD in the Southwestern area, Aseer region, of Saudi Arabia. Therefore, this study aimed at measuring the levels of MVs and TF-bearing MVs in steady state children with SCD compared to age and gender matched healthy controls (HCs).


  Materials and Methods Top


Patients and Blood Collection

A total number of 54 SCD children aging from 2–12 years old homozygous for sickle haemoglobin (HbSS) were recruited from sickle cell clinics at the Aseer Central Hospital, Al-Mjardah and Mahyl Central hospitals. The selected patients were in steady state at the time of blood collection and with no history of blood transfusion over the last 6 weeks. These patients were on hydroxyurea. Another 34 age and gender matched healthy children were also recruited from home rotation in Abha city as control group. These children in the control group were selected on the basis of absence of personal or family history of SCD or any other hemoglobin disorders. Routine hematological investigation on whole blood analysis and peripheral blood smear (PBS) confirmed these, children having no history of anemia.

Whole blood was collected in ethylenediaminetetraacetic acid (EDTA) and sodium citrate anticoagulant tubes, (IMPROVE, Guangzhou, Improve Medical Instruments Co., Ltd., China) from both the groups of children. Blood samples were collected under strict standard laboratory guidelines to avoid the activation of platelets. The study was approved by King Khalid University Research Ethical Committee (#2012/04/05) and all participants' guardians provided informed consent. The study was carried out according to the Declaration of Helsinki.

Microvesicles Isolation

Platelet-free plasma (PFP) was obtained by two-step separation of citrated whole blood at 1800 g for 30 min at 22°C, followed by a sharp spin of the PFP at 13,000 g for 2 min. The supernatant was immediately stored at −80°C for later MV and TF-bearing MV analysis, as previously described elsewhere with some modification.[12]

Whole Blood Count and Blood Film Preparation

EDTA anticoagulant tubes were used for whole blood count and blood film preparation. Whole blood count was obtained using an automated cell counter (Sysemx-500xs, Germany). Various parameters were obtained, including red cell, white cell, and platelet counts. Thin blood smears were prepared from EDTA tubes of the patients and control groups. Leishman stain was used according to the manufacturer's guidelines (Bayer diagnostics, Hema-Tek, Hamilton). All PBS were assessed by hematologist.

Measurement of Microvesicles and Tissue Factor-bearing Microvesicles in the Plasma by ELISA

Plasma procoagulant MV levels and TF-bearing MV levels were measured using Zymuphen MP-activity kit (Aniara Diagnostica LLC, OH, USA) and Zymuphen MP-TF ELISA kit (Aniara Diagnostica LLC, OH, USA) according to the manufacturer's instructions (Hyphen Biomed), respectively. The optical density was measured using ELISA plate reader (Stat fax-2100 Awareness Technology Inc., Palm city, FL, USA).

Statistics

Statistical analysis was performed with GraphPad Prism software version 4.0 for Windows (GraphPad Software Inc., San Diego, CA, USA). Unless otherwise stated, results are presented as a mean ± standard deviation. Unpaired t-test was used for hematological and MVs analysis. Chi-squared test was used for demographic data analysis. P values were considered statistically significant if <0.05.


  Results Top


Demographic and Hematological Characteristics

Fifty-four patients with SCD in steady state and thirty-four age and sex matched controls were evaluated. The demographic data and hematological values are shown in [Table 1]. The results did not shows significant differences in the age and sex among the cohort. However, the hematological values showed significantly lower red blood cells (RBCs), hemoglobin, and haematocrit values in the SCD patients compared to the controls, which indicates the presence of the anemic condition. Patients with SCD were found to have significantly lower hemoglobin (8.8 ± 1.8 vs. 12.7 ± 0.7 g/dl, P < 0.001), RBCs count (3.5 ± 0.8 vs. 4.9 ± 0.3 × 106/µl, P < 0.001), and hematocrit (26.3 ± 5.0 vs. 37.1 ± 2.4%, P < 0.001). Whereas the platelet and white blood cell counts were significantly higher in SCD patients compared to the controls (419 ± 176 vs.334 ± 68.0 × 109/L, P < 0.05) and (11.0 ± 5.1 vs. 9.2 ± 3.1 × 109/L, P < 0.05), respectively. The examination of the PBSs showed normocytic normochromic with variable degrees of polychromasia and aniso-poikilocytosis in SCD patients. Sickle cells and sickle-like cells were prominent in all examined PBSs. In addition, target cells, nucleated RBCs and  Howell-Jolly bodies More Details were occasionally seen. The PBSs of HCs showed normal RBC morphology (data not shown).
Table 1: Demographic and hematological values of SCD patients and HCs

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Elevated Circulating Levels of Microvesicles and Tissue Factor-bearing Microvesicles in Patients

SCD patients had significantly higher levels (2.5-fold increase) of MVs in their plasma compared to the control group (31.0 ± 3.6 vs. 16.4 ± 4.16 nM; P = 0.0095; [Table 2]). Although the level of TF-bearing MVs were low in both groups, SCD patients had significantly higher levels of TF-bearing MVs in their plasma than controls (patients vs. controls, 0.83 ± 0.39 vs. 0.48 ± 0.21 pg/ml; P < 0.05; [Table 2]).
Table 2: Circulating procoagulant MVs and TF-bearing MVs. MVs and TF-bearing MVs Levels in the plasma from SCD patients (n=53) and HC (n=29)

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Correlation of MVs TF-bearing MVs and Hematological Values in SCD Patients' Plasma

MV levels positively correlated with the levels of TF-bearing MVs in the plasma of SCD patients (r = 0.436; P = 0.0003; [Figure 1]). MVs and TF-bearing MVs were not correlated with either haemoglobin concentrations or RBCs count (data not shown). There was no observable correlation between MV levels and TF – MVs in the control group.
Figure 1: Correlation between procoagulant microvesicles and tissue factor-bearing microvesicles. Microvesicles is correlated with tissue factor-microvesicles level in the sickle cell disease patients (r=0.436; P =0.0003)

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  Discussion Top


SCD is a complex pathophysiological disorder characterized by chronic changes of the hemostatic system and a known hypercoagulable condition.[3],[4],[5] Procoagulant cell-derived MVs are one of the factors that contribute to the hypercoagulability condition and have been proposed to be associated with an increased risk of thrombosis.[16] MVs thrombogenicity comes from the negatively charged phospholipids on their outer membrane surface, which is a platform for the assembly of coagulation factors to support thrombin generation in vivo and in vitro.[10],[15] Previous studies have proposed that MVs and thrombin generation are associated with thrombotic diseases and have been suggested as predictors for the occurrence and recurrence of thrombosis.[11],[17] Elevated levels of cell-derived MVs are seen in many pathological conditions and considered as potential biomarkers reflecting prothrombotic states.[12],[15]

The current study is thefirst to measure MV levels in children with steady state SCD in Aseer region of Saudi Arabia. This study shows increased levels of MVs (2.5-fold increase) in the plasma of SCD patients compared to the matched HCs. This finding is in line with previous published studies.[8],[14],[16],[18] Levels of circulating RBC-derived MVs are up to 6-fold increase in sickle cell anemia patients than controls.[8],[14] Although this study did not determine the origin of MVs in the study cohorts, it is assumed that circulatory MVs in the patients were mainly derived from RBCs due to the sickling/desickling process in vivo[8],[14] In addition, the MV levels in the study cohort were higher than the control, a finding that is likely to have been underestimated as the patients in the study were receiving hydroxyurea treatment. Nébor et al.[19] found that SCD patients treated with hydroxyurea had lower levels of circulatory MVs than untreated SCD patients. In addition, the level of PS exposure on the surface of platelet and RBCs is reduced compared to untreated SCD patients due to the effect of hydroxyurea.[19],[20] Furthermore, it has been assumed that infants with SCD (with elevated levels of HbF) have higher MV levels than older children.[7]

TF is another procoagulant factor that contributes to the hypercoagulable condition.[21] TF is normally sequestrated from blood circulation but can be found either bound to MVs or soluble in the circulation.[8],[21] In this study, the levels of TF-bearing MVs in the plasma from patients with SCD are significantly higher than in the matched HCs. The findings in this study are consistent with previous studies that have reported the presence of TF-MVs in vivo in SCD.[8],[9] In SCD, FVII/FVIIa are found in the circulation on the surface of circulating endothelial cells, which might contribute to the chronic activation of the coagulation system.[9] On the other hand, no correlation was observed between the levels of MVs or TF-bearing MVs with hematological parameters such as Hgb and RBC count, which could be explained by the treatment, hydoxryurea, the patients were on at the time of blood collection.


  Conclusion Top


The data present in this study clearly demonstrate the presence of procoagulant MVs and TF-bearing MVs in the plasma of SCD patients, as they supported more thrombin generation compared to the matched HCs. We found an increase in the circulatory procoagulant MVs and TF-bearing MVs during steady state, which might reflect cell activation and a contribution to the increased risk of thrombotic complication in SCD patients. Further studies to elucidate the functional activity of MVs and TF-bearing MVs, and size and origin of MVs in the plasma will throw light on the clinical importance of the MV-TF in the SCD.

Financial Support and Sponsorship

This work was supported by the program of research and researcher, Deanship of Scientific research, King Khalid University grant #KKU_S272_33.

Conflicts of Interest

There are no conflicts of interest.

 
  References Top

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Solovey A, Gui L, Key NS, Hebbel RP. Tissue factor expression by endothelial cells in sickle cell anemia. J Clin Invest 1998;101:1899-904.  Back to cited text no. 9
    
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Bidot L, Jy W, Bidot C Jr, Jimenez JJ, Fontana V, Horstman LL, et al. Microparticle-mediated thrombin generation assay: Increased activity in patients with recurrent thrombosis. J Thromb Haemost 2008;6:913-9.  Back to cited text no. 11
    
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Burton JO, Hamali HA, Singh R, Abbasian N, Parsons R, Patel AK, et al. Elevated levels of procoagulant plasma microvesicles in dialysis patients. PLoS One 2013;8:e72663.  Back to cited text no. 12
    
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Sinauridze EI, Kireev DA, Popenko NY, Pichugin AV, Panteleev MA, Krymskaya OV, et al. Platelet microparticle membranes have 50-to 100-fold higher specific procoagulant activity than activated platelets. Thromb Haemost 2007;97:425-34.  Back to cited text no. 13
    
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van Beers EJ, Schaap MC, Berckmans RJ, Nieuwland R, Sturk A, van Doormaal FF, et al. Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease. Haematologica 2009;94:1513-9.  Back to cited text no. 14
    
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Berckmans RJ, Nieuwland R, Böing AN, Romijn FP, Hack CE, Sturk A. Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost 2001;85:639-46.  Back to cited text no. 15
    
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Tantawy AA, Adly AA, Ismail EA, Habeeb NM, Farouk A. Circulating platelet and erythrocyte microparticles in young children and adolescents with sickle cell disease: Relation to cardiovascular complications. Platelets 2013;24:605-14.  Back to cited text no. 16
    
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Wood BL, Gibson DF, Tait JF. Increased erythrocyte phosphatidylserine exposure in sickle cell disease: Flow-cytometric measurement and clinical associations. Blood 1996;88:1873-80.  Back to cited text no. 18
    
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Nébor D, Romana M, Santiago R, Vachiery N, Picot J, Broquere C, et al. Fetal hemoglobin and hydroxycarbamide moduate both plasma concentration and cellular origin of circulating microparticles in sickle cell anemia children. Haematologica 2013;98:862-7.  Back to cited text no. 19
    
20.
Covas DT, de Lucena Angulo I, Vianna Bonini Palma P, Zago MA. Effects of hydroxyurea on the membrane of erythrocytes and platelets in sickle cell anemia. Haematologica 2004;89:273-80.  Back to cited text no. 20
    
21.
Giesen PL, Rauch U, Bohrmann B, Kling D, Roqué M, Fallon JT, et al. Blood-borne tissue factor: Another view of thrombosis. Proc Natl Acad Sci U S A 1999;96:2311-5.  Back to cited text no. 21
    


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