The relation between soluble endothelial protein C receptor and factor VIII levels and FVIII/sEPCR index in healthy infants / Saglikli sut cocuklarinda cozunur endotelyal protein C reseptoru ve faktor VIII duzeyleri arasinda iliski ve FVIII/sEPCR indeksi.
Subject: Protein C
Enzyme-linked immunosorbent assay
Blood coagulation factor VIII
Endothelium
Authors: Orhon, Filiz Simsek
Egin, Yonca
Ulukol, Betul
Baskan, Sevgi
Akar, Neiat
Pub Date: 03/01/2011
Publication: Name: Turkish Journal of Hematology Publisher: Aves Yayincilik Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 Aves Yayincilik ISSN: 1300-7777
Issue: Date: March, 2011 Source Volume: 28 Source Issue: 1
Product: Product Code: 2831133 Antihemophiliac Factor NAICS Code: 325414 Biological Product (except Diagnostic) Manufacturing SIC Code: 2836 Biological products exc. diagnostic
Accession Number: 305562771
Full Text: Abstract

Objective: Both soluble endothelial protein C receptor (sEPCR) and factor VIII (FVIII) seem to be potential mediators in thrombotic and inflammatory states. The aim of the present study was to determine the relation between plasma sEPCR and FVIII levels in a group of healthy Turkish infants.

Materials and Methods: The study population consisted of 50 healthy infants aged 6 months (Group 1, n=23) and 12 months (Group 2, n=27) having no acute or chronic infection and/or disease. sEPCR levels and FVIII levels were measured by ELISA and one stage factor assay method, respectively.

Results: The sEPCR levels of the infants aged 6 months were found higher than those of the infants aged 12 months (p<0.001). There was a correlation between sEPCR and FVIII levels of the infants in Group 1 (6-month-old infants) (r= 0.678, p <0.001). FVIII/sEPCR index was 0.73 [+ or -] 0.3 and 1.0 [+ or -] 0.5 in Group 1 and Group 2, respectively (p = 0.027). A correlation between infant age and FVIII/sEPCR index was found (r=0.312, p=0.027).

Conclusion: The FVIII/sEPCR index in healthy infants reflects the physiological condition of this population. The finding showing a positive relationship between sEPCR and FVIII levels suggests a possible interaction between these mediators in healthy infants aged six months. (Turk J Hematol 2011; 28: 27-32)

Key words: Soluble endothelial protein C receptor (sEPCR), factor VIII, healthy infants, thrombosis

Received: March 01, 2010

Accepted: June 29, 2010

Ozet

Amag: Cozunur endotelyal protein C reseptoru (sEPCR) ve faktor VIII (FVIII) trombotik ve inflamatuvar durumlarda potansiyel arabilesenler olarak gorulmektedir, Bu cahsmamn amaci; bir grup saghkh sut cocu-gunda plazma sEPCR ve FVIII duzeyleri arasmdaki iliskiyi tanimlamaktir.

Yontem ve GeresIer: (Cahsma grubunu herhangi bir akut ya da kronik hastahgi ve/veya enfeksiyonu olma-yan, saghkli 6 ayhk (Grup 1, n=23) ve 12 aylik (Grup 2, n=27) cocuklar olusturmaktadir. sEPCR duzeyleri ve FVIII duzeyleri sirasiyla; ELISA ve one stage factor metodu ile cahsilmistir.

Bulgular: Alti aykk gocuklann sEPCR duzeyleri oniki ayhk olanlardan daha yuksek bulunmustur (p<0.001). Grup l'i olusturan 6 ayhk cocuklarda sEPCR ve FVIII duzeyleri arasmda bir korelasyon vardir (r=0.678, p<0.001). FVIII/sEPCR indeksi Grup l'de 0.73 [+ or -] 0.3 ve Grup 2'de 1.0 [+ or -] 0.5 olarak bulunmustur (p=0.027). Cocugun yasi ve FVIII/sEPCR indeksi arasmda bir korelasyon saptanmistir (r=0.312, p=0.027). Sonuc: Cahsmada saghkli gocuklarda kullanilan FVIII/sEPCR indeksi bu populasyonun fizyolojik durumunu yansitmaktadir. Alti aylik cjocuklarda sEPCR ile FVIII arasindaki pozitif iliski bu yas grubundaki cocuklarda bu mediatorler arasinda muhtemel bir etkilesim oldugunu gosterebilir. (Turk J Hematol 2011; 28:27-32)

Anahtar kelimeler: sEPCR, Factor VIII, saghkli sut cocuklan, tromboz

Gelis tarihi: 1 Mart 2010

Kabul tarihi: 29 Haziran 2010

Introduction

The protein C anticoagulant pathway is critical to both regulation of the blood coagulation process and control of the innate inflammatory response and some of its associated downstream pathologies (1), (2). The endothelial protein C receptor (EPCR) plays an important role in this pathway (3). EPCR is preferentially expressed by endothelial cells of large blood vessels and increases the rate of protein C activation by the thrombin/thrombomodulin complex (3), (4). Increased levels of a soluble form of EPCR (sEPCR) in plasma lead to dysfunction of the EPCR-mediated coagulation (5). sEPCR levels increase in conditions associated with considerable thrombin production such as vasculitis, sepsis and systemic lupus erythematosus (6), (7).

Factor VIII (FVIII) is a plasma glycoprotein in the coagulation cascade, and it is the cofactor of factor IXa in the activation of factor X. FVIIIa can be inactivated by activated protein C (1), (2). Previous studies have shown that the coagulant activity of FVIII is increased as an acute phase reaction in thrombosis (8), (9). Elevated plasma levels of FVIII are associated with an increased risk of venous and arterial thrombosis (10), (11). Possible mechanisms thought to be associated with elevated FVIII are the enhancement of thrombin formation or the induction of acquired activated protein C resistance, but the molecular mechanisms that underlie elevated FVIII are still not clear.

Although in vivo physiological mechanisms and their importance are still unknown, both EPCR and FVIIII seem to be potential mediators in thrombotic and inflammatory states because of their roles in the protein C anticoagulant pathway. Thus, the determination of the association between sEPCR and FVIII may provide new knowledge about the pathogenesis of thrombotic and inflammatory conditions. The aim of this study, therefore, was to determine the relation between plasma sEPCR and FVIII levels in a group of healthy Turkish infants.

Materials and Methods

This study was conducted at the Department of Pediatrics, Divisions of Pediatric Molecular Genetics and Social Pediatrics. Ethics approval was obtained from the Ethics Committee of the School of Medicine. The study population consisted of healthy infants aged 6 and 12 months who were admitted for well-child visits. They had no acute or chronic infection and/or disease. The written informed consent to participate was obtained from the parents of all subjects.

Peripheral blood samples were collected from the subjects into tubes containing 1 ml 0.109 M trisodium citrate. Plasma was obtained by centrifugation at 2500xg for 10 minutes (min) at room temperature. Plasma specimens obtained were maintained until the date of measurement at 20[+ or -]5[degrees]C for 4 hours (h), at 2-8[degrees]C for 24 h, and at -20[degrees]C for 1 month. Soluble EPCR levels were determined in plasma by using sEPCR Asserachrom enzyme-linked immunosorbent assay (ELISA) kits from Diagnostica Stago (Asnieres, France), according to the manufacturer's instructions. Factor VIII levels were measured concomitantly with one stage factor assay method and FVIII-absent plasma (Sigma Diagnostica Inc, St. Louis, MO).

Statistical analysis

Statistical analysis was performed using the SPSS 11.5. Descriptive analysis summarizing the characteristics and the levels of sEPCR and FVIII is presented. Since the plasma sEPCR and FVIII measurements were not normally distributed, nonparametric tests were conducted to compare these parameters. The Mann-Whitney U test was used to compare sEPCR and FVIII levels between the groups. Nonparametric correlations between sEPCR and FVIII levels were evaluated using the Spearman correlation test. FVIII/sEPCR index was calculated in the groups. The Student t test was used to compare FVIII/sEPCR index between the groups, and the Pearson correlation test was used to evaluate the correlation between this index and age.

Results

Totally, 50 healthy infants were studied. These infants were divided into two groups according to age. Group 1 consisted of 23 infants aged 6 months (9 boys and 14 girls), and Group 2 consisted of 27 infants aged 12 months (13 boys and 14 girls).

Table 1 shows the sEPCR and FVIII levels of the infants. There was a significant difference between Group 1 and Group 2 with respect to sEPCR levels (p<0.001), with the sEPCR levels of infants aged 6 months found to be significantly higher than those of the infants aged 12 months. There was no difference between the groups with respect to the FVIII levels. Concerning all infants and groups, there was no statistical difference between boys and girls in sEPCR or FVIII levels.

In the correlation analysis, there was a correlation between sEPCR and FVIII levels of the infants in Group 1 (aged 6 months) (r=0.678, p<0.001) (Figure 1). However, no correlation was present between the sEPCR and FVIII levels in Group 2 (aged 12 months) (r=-0.251, p=0.206).

[FIGURE 1 OMITTED]

With respect to all infants, the mean FVIII/sEPCR index was found as 0.88 (median: 0.85, SE: 0.06, min: 0.3, max: 3.1). There was a statistically significant difference in the FVIII/sEPCR index between the groups (p=0.027; 0.73[+ or -]0.3 and 1.0[+ or -]0.5 for Group 1 and Group 2, respectively). In the correlation analysis, there was a correlation between the infant age and the FVIII/sEPCR index (r= 0.312, p=0.027).

Discussion

This is the first study in the literature to examine the relationship between plasma sEPCR and FVIII levels in healthy infants as an indicator of the physiological condition. This study also highlights the relation between infant age and the FVIII/sEPCR index.

Protein C activation is regulated by numerous mediators in the coagulation process, and activated protein C has a variety of anti-inflammatory activities (1). Increased levels of sEPCR in plasma lead to dysfunction of the EPCR-mediated coagulation (5). sEPCR levels vary among healthy subjects, and the bimodal distribution has been reported several times (12-14). A recent study showed a negative relationship between sEPCR levels and an individual's age; that is, sEPCR levels of healthy children were found to be higher than those of healthy adults (14). The levels of sEPCR and FVIII reflect the physiological condition of these mediators in this healthy population; however, the physiological importance and influence of sEPCR levels in vivo and the relation to FVIII are unknown.

Increased plasma sEPCR levels were found associated with an increased risk of venous thrombosis and thrombin generation (12), (15). Further, it was reported that increased FVIII levels are an important risk factor for venous and arterial thrombosis (11), (16). Yururer et al. (17) showed a negative relationship between sEPCR and FVIII levels in a group of pediatric stroke patients. They suggested that this association makes each of them regulate the action of the other, and may play a role in the stroke pathophysiology. Under the physiological circumstances, we found that the sEPCR levels of infants aged 6 months were significantly higher than of infants aged 12 months. We also found a positive relationship between sEPCR and FVIII levels in the 6-month-old healthy infants, but not in the 12-month-old infants. These findings, which indicate the physiological state in healthy infants, may reflect the interactions among the mediators in the coagulation system over the first months of life. Thrombin is a multifunctional serine protease generated at the site of vascular injury and has a key role in blood coagulation. Cellular effects of thrombin are mediated by protease-activated receptors (PARs), members of the G protein-coupled receptors. Their expression is low in contractile vascular smooth muscle cells (SMC), but becomes markedly upregulated upon injury (18). We suggest that the overexpression of PARs or the presence of unknown mechanisms during the early stage of life may play a role in the tendency towards thrombotic conditions. We may speculate that the positive relation between sEPCR and FVIII levels in 6-month-old Infants may be associated with the enhancement of thrombin formation and probably with the overexpression of PARs. Further studies are needed to evaluate the associations among the mediators in the coagulation system in a variety of healthy groups, including newborns, infants, children, and adults, under physiological circumstances.

As the first in the literature, we proposed the ratio of FVIII/sEPCR as an index of the physiological status of the coagulation system. We found a correlation between infant age and the FVIII/sEPCR index in this healthy group. In our study population, this index reflects the physiological conditions regarding the coagulation pathway process under normal conditions. Although the in vivo importance of this index is unknown, determining the difference in this index between physiological and pathological conditions may confirm the potential interactions between the mediators in the coagulation process. We consider the possibility of using this index in further studies in pathologic states in which the pathogenesis involves thrombotic and inflammatory processes.

In this study, we aimed primarily to determine the actual physiological condition in healthy infants. Because of the potential tendency to thrombotic states over the first months of life and the possible risk of complexity in our findings, the younger infants were not included in the study. On the other hand, the interactions among the mediators in the coagulation system over the first month of life need to be determined, in order to clarify the detailed mechanisms under the tendency of thrombosis. Therefore, further studies with the aim of assessing the relations among the mediators, such as EPCR, FVIII, other factors, PARs, or other receptors, may be conducted in healthy infants in the first month of life.

Various genetic and environmental conditions have been discussed as risk factors for thrombotic events in adult and pediatric patients (19). Functional polymorphisms in the EPCR gene may increase/decrease the risk of thrombosis, especially in carriers of prothrombotic mutations (20). Familial clustering of high factor VIII levels in patients with venous and arterial thromboembolism and retinal artery occlusion were reported in previous studies (21-23). These findings have pointed to genetic influences on these mediator levels. It is conceivable that the genetic polymorphisms of the FVIII and/or EPCR genes are linked with high FVIII and/or sEPCR levels in patients with thrombosis. The evaluation of these polymorphisms may be an important clinical indicator for determination of the thrombosis risk in a healthy population. In a recent study, Ay et al. (24) found no evidence of an association between observed single nucleotide polymorphisms in exons of the FVIII gene and high thrombosis levels. The findings in the literature seem to be contradictory about the polymorphisms in the genes of the mediators regarding the protein C system, therefore, further detailed studies are needed to assess these polymorphisms to determine their roles in the pathogenesis of thrombosis.

In conclusion, this is the first study in the literature to examine the relationship between plasma sEPCR and FVIII levels in healthy infants and also to determine the ratio of FVIII/sEPCR as an index. Both sEPCR and FVIIII seem to be potential mediators in thrombotic and inflammatory states. The levels of sEPCR and FVIII in our study group reflect the physiological levels in this healthy population. We found a positive relationship between sEPCR and FVIII levels in 6-month-old healthy infants, but not in 12-month-old infants. This may reflect the interactions among the mediators in the coagulation system over the first months of life under physiological circumstances. We also found a correlation between infant age and the FVIII/sEPCR index in this healthy group. Although the in vivo importance of this index is unknown, we consider the possibility of using this index in further studies in pathologic states in which the pathogenesis involves thrombotic and inflammatory processes. On the other hand, future studies are needed to evaluate the physiological associations among the mediators, such as sEPCR and FVIII, in the coagulation system in a variety of healthy groups, including newborns, infants, children, and adults.

Conflict of interest statement

None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Acknowledgement:

This study was supported by the Ankara University Research Fund.

References

(1.) Esmon CT. Inflammation and the activated protein C anticoagulant pathway. Semin Thromb Hemost 2006;32:49-60.

(2.) Griffin JH, Fernandez JA, Gale AJ, Mosnier LO. Activated protein C. J Thromb Haemost 2007;5:73-80.

(3.) Fukodome K, Esmon CT. Identification, cloning and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994;269:26486-91.

(4.) Regan LM, Stearns-Kurosawa DJ, Kurosawa S, Mollica J, Fukodome K, Esmon CT. The endothelial protein C receptor. J Biol Chem 1996;271:17499-503.

(5.) Esmon CT. The protein C pathway. Chest 2003;124:265-325.

(6.) Kurosawa S, Stearns-Kurosawa DJ, Carson CW, D'Angelo A, Della Vale P, Esmon CT. Plasma levels of endothelial cell protein C receptor are elevated in patients with sepsis and systemic lupus erythematosus: lack of correlation with thrombomodulin suggests involvement of different pathological processes. Blood 1998;91:725-7.

(7.) Boomsma MM, Stearns-Kurosawa DJ, Stegeman CA, Raschi E, Meroni PL, Kurosawa 5, Tervaert JW. Plasma levels of soluble endothelial cell protein C receptor in patients with Wegener's granulomatosis. Clin Exp Immunol 2002;128:187-94.

(8.) O'Donnell J, Mumford AD, Manning RA, Laffan M. Elevation of FVIII: C in venous thromboembolism is persistent and independent of the acute phase response. Thromb Haemost 2000;83:10-3.

(9.) Kamphuisen PW, Eikenboom JC, Bertina RM. Elevated FVIII levels and the risk of thrombosis. Arterioscler Thromb Vasc Biol 2001;21:731-8.

(10.) Rosendaal FR. High levels of factor VIII and venous thrombosis. Thromb Haemost 2000;83:1-2.

(11.) Kurekci AE, Gokce H, Akar N. Factor VIII levels in children with thrombosis. Pediatr Int 2003;45:159-62.

(12.) Stearns-Kurosawa DJ, Swindle K, D'Angelo A, Della Vale P, Fattorini A, Caron N, Grimaux M, Woodhams B, Kurosawa S. Plasma levels of endothelial protein C receptor respond to anticoagulant treatment. Blood 2002;99:526-30.

(13.) Stearns-Kurosawa DJ, Burgin C, Parker D, Comp P, Kurosawa S. Bimodal distribution of soluble endothelial protein C receptor levels in healthy subjects. J Thromb Haemost 2003;4:855-6.

(14.) Orhon FS, Ergun H, Egin Y, Ulukol B, Baskan S, Akar N. Soluble endothelial protein C receptor levels in healthy population. J Thromb Thrombolysis 2010;29:46-51.

(15.) Saposnik B, Reny JL, Gaussem I), Emmerich J, Aiach M, Gandrilel S. A haplotype of the EPCR gene is associated with increased plasma levels of sEPCR and is a candidate risk factor for thrombosis. Blood 2004;103:1311-8.

(16.) Bugnicourt JM, Roussel B, Tramier B, Lamy C, Godefroy 0. Cerebral venous thrombosis and plasma concentrations of factor VIII and von Willebrand factor: a case control study. J Neurol Neurosurg Psychiatry 2007;78:699-701.

(17.) Yururer D, Teber S, Deda G, Egin Y, Akar N. The relation between cytokines, soluble endothelial protein C receptor, and factor VIII levels in Turkish pediatric stroke patients. Clin Appl Thromb Hemost 2009;15:545-51.

(18.) Martorell L, Martinez-Gonzalez J, Rodriguez C, Gentile M, Calvayrac 0, Badimon L. Thrombin and protease-activated receptors (PARs) in atherothrombosis. Thromb Haemost 2008;99:305-15.

(19.) Esmon CT. Basic mechanisms and pathogenesis of venous thrombosis. Blood Rev 2009;23:225-9.

(20.) Medina P, Navarro S, Estelles A, Espana F. Polymorphisms in the endothelial protein C receptor gene and throm-bophilia. Thromb Haemost 2007;98:564-9.

(21.) Schambeck CM, Hinney K, Haubitz I, Mansouri Taleghani B, Aahler D, Keller F. Familial clustering of high factor VIII levels in patients with venous thromboembolism. Arterioscler Thromb Vasc Biol 2001;21:289-92.

(22.) Bank I, Libourel EJ, Middeldorp S, Hamulyak K, Van Pampus ECM, Koopman MMW, Prins MH, Van Der Meer J, Willer HR. Elevated levels of FVIII:C within families are associated with an increased risk for venous and arterial thrombosis. J Thromb Haemost 2004;3:79-84.

(23.) Akar N, Gokce H. High levels of FVIII and FIX in a pedi-atric patient with retinal artery occlusion. Pediatr Hematol Oncol 2002;19:593-5.

(24.) Ay M, Dolek B, Erdem G, Devecioglu 0, Gozukirmizi N. Is there any correlation between the elevated plasma levels and gene variations of factor VIII in Turkish thrombosis patients? Clin Appl Thromb Hemost 2011;17:46-50.

Address for Correspondence: Assoc. Prof. Filiz Simsek Orhon, Department of Pediatrics, Division of Social Pediatrics, School of Medicine, Ankara University, 06100 Ankara, Turkey Phone: +90 312 595 72 02 E-mail: simsekfiliz@hotmail.com

Filiz Simsek Orhon (1), Yonca Egin (2), Betul Ulukol (1), Sevgi Baskan (1), Nejat Akar (2)

(1) Department of Pediatrics, Division of Social Pediatrics, School of Medicine, Ankara University, Ankara, Turkey

(2) Department of Pediatrics, Division of Pediatric Molecular Genetics, School of Medicine, Ankara University, Ankara, Turkey

doi:10.5152/tjh.2011.02
Table 1. sEPCR and FVIII concentrations according to groups

                      All infants      Group 1             Group 2
                           (n=50)       (n=23)              (n=27)

sEPCR
(ng/ml)

            Mean            145.4        175.1               120.0
            [+ or -]     [+ or -]     [+ or -]            [+ or -]
            SE               10.7         17.9                10.8

            Median          112.0        130.0               102.0

            Min-Max    67.0-346.0   95.0-346.0          67.0-280.0

            95% CP    123.8-166.9  137.9-212.3          97.8-142.3

         p                                          <
         *                                      0.001

FVIII       Mean            109.6        116.1               104.1
(U/dl)      [+ or -]     [+ or -]     [+ or -]            [+ or -]
            SE                6.2         11.8                 5.4

            Median          100.5        103.9                96.6

            Min-Max    42.0-284.0   42.0-284.0          72.0-209.0

            95% CI     97.2-122.0   91.6-140.6          92.9-115.2
            (1)

         P                                          >
         *                                       0.05
Gale Copyright: Copyright 2011 Gale, Cengage Learning. All rights reserved.