The prevalence of obesity in children has tripled over the last three decades. Obesity in children is associated with childhood type 2 diabetes mellitus (T2DM) and increased cardiovascular risk. In adults both obesity and T2DM are associated with cardiovascular disease and alterations in blood that reflect an ongoing risk for thrombotic events and atherogenesis. Tissue factor (TF, FIII, F3) is the physiological initiating mechanism for blood coagulation and is both prothrombotic and proinflammatory (Tilley & Mackman, 2006). Adipose tissue is a site of TF production in response to leptin and proinflammatory stimuli and, in mouse models of obesity, the gene encoding TF (F3) is upregulated (Faber et al, 2009). Studies in humans indicate that there is blood borne TF, which is functional, participates in thrombogenesis and is elevated in prothrombotic states (Key et al, 1998; Sambola et al, 2003; Vaidyula et al, 2006). We have reported increased circulating TF procoagulant activity (TF-PCA) in T2DM patients (Boden et al, 2007) and in healthy nondiabetic adults during hyperglycemia and hyperinsulinemia (Vaidyula et al, 2006; Boden et al, 2007). In adults, obesity is associated with a procoagulant state with elevated plasma factor VII coagulant activity (FVII:C), FVIII:C, fibrinogen and plasminogen activator inhibitor (PAI-1, SERPINE1) (Mertens & Van Gaal, 2002; Faber et al, 2009). Some of these proteins are also elevated in childhood obesity (Sudi et al, 2001). To our knowledge, circulating levels of membrane-bound TF have not been studied in childhood obesity. We tested the hypothesis that obese children have higher levels of circulating TF-PCA and of plasma markers of coagulation, fibrinolysis, and endothelial perturbation compared to healthy-weight children.
Childhood obesity is rapidly increasing in prevalence. We compared circulating membrane-bound tissue factor (FIII, F3) procoagulant activity (TF-PCA) and plasma markers of coagulation, fibrinolysis and endothelial dysfunction in 21 obese (10·1 ± 1·5 years, mean ± standard deviation) and 22 healthy weight children (9·9 ± 1·6 years), classified by Body Mass Index (BMI). TF-PCA and factor VII coagulant activity (FVII:C), plasminogen activator inhibitor (PAI-1, SERPINE1) and soluble vascular cell adhesion molecule 1 (sVCAM1) were higher in obese children. BMI correlated positively with TF-PCA, FVII:C, and PAI-1. Childhood obesity is associated with a procoagulant state and endothelial dysfunction. Studies are needed to assess whether weight reduction reverses these abnormalities.
Materials and methods
Forty-seven children (aged 8–12 years) were classified according to on Body Mass Index (BMI) percentile (CDC growth curves) as underweight (≤5th percentile; n = 1), healthy weight (>5–84·9th percentile; n = 22), overweight (≥85th–94·9th percentile; n = 3) and obese (≥95th percentile; n = 21) (Dean et al, 1996). We compared the findings in 21 obese children and 22 healthy weight children (Table 1). Children with any medical conditions or taking any medications were excluded. The study was approved by the Institutional Review Board and informed consent and assent were obtained from all parents and children.
Blood collection and plasma preparation
Blood (9 ml) was drawn into 1 ml of 3·8% sodium citrate by venipuncture. Two aliquots (1 ml) were removed for TF-PCA assay; the remainder was centrifuged (20 min, 1590 g) to obtain plasma. A plasma aliquot was centrifuged (13 000 g, 2 min) to remove residual cells prior to microparticle assay.
Whole blood TF-PCA
TF-PCA was measured in whole blood cell lysates with a two-stage clotting assay using recombinant activated FVII (FVIIa; American Diagnostica Inc., Stamford, CT, USA) and factor X (Haematologic Technologies Inc., Essex Junction, VT, USA) (Key et al, 1998; Vaidyula et al, 2006). This assay measures cell-bound and microparticle-associated TF.
TF (FIII), PAI-1, tissue plasminogen activator (tPA) and von Willebrand factor antigen (VWF:Ag) were determined by enzyme-linked immunosorbent assay (ELISA) (American Diagnostica Inc.). FVIII:C and FVII:C and fibrinogen were measured by standard clotting assays (Vaidyula et al, 2006); FVIIa activity was measured by a clotting assay (Diagnostica Stago, Parsippany, NJ) using soluble TF. Thrombin-antithrombin complexes (TAT) (Enzygnost; Dade Behring, Marburg, Germany) and sVCAM1 (Diaclone, Besancon Cedex, France) were measured by ELISAs. Procoagulant activity of microparticles in plasma was determined by a thrombin generation assay (Zymuphen; Hyphen Biomed, Neuville-sur-Oise, France).
Differences in demographic factors between the two groups were evaluated with Student t-test. The Satterthwaite approximation for P-values was applied when the equal variance assumption was suspect. Pearson chi-square tests were used to compare categorical variables, and the extension of the Fisher exact test was utilized when assumptions underlying the chi-square test were violated. Differences between groups with respect to biomarkers were evaluated with Wilcoxon rank-sums tests. Spearman correlation coefficients, using data from all 47 participants, were used to assess the relationships between BMI and the biomarkers and among biomarkers. A P value < 0·05 was considered statistically significant.
Circulating membrane-bound TF-PCA was significantly higher in obese children than in healthy weight children (Table 1). Plasma FVII:C was significantly higher in the obese than in the healthy weight group, while plasma TF antigen, FVIIa, FVIII, fibrinogen and TAT were not different between the groups (Table 1). PAI-1 was significantly higher in the obese children; tPA antigen levels were slightly but not significantly higher in obese children. Endothelial markers VCAM1 and VWF:Ag were higher in obese than in healthy weight children, but did not reach statistical significance. Using all 47 subjects, BMI was significantly and positively correlated with TF-PCA, FVII:C, and PAI-1 (Fig. 1) with suggestive positive correlations between BMI and sVCAM1 (r = 0·27, P = 0·07), fibrinogen (r = 0·27, P = 0·06) and VWF:Ag (r = 0·26, P = 0·08).
Plasma sVCAM1 correlated positively with TF-PCA (r = 0·36, P = 0·02) and FVII:C (r = 0·35, P = 0·02) (Table S1), suggesting that TF-FVII driven thrombin generation contributes to endothelial perturbation and total TF-PCA. Plasma tPA (also endothelial-derived) correlated with FVIIa (r = 0·42, P < 0·01), and may reflect the same mechanism. PAI-1 levels correlated with circulating microparticles (r = 0·30, P = 0·04), which is interesting because PAI-1 promotes endothelial microparticle formation (Brodsky et al, 2002). Plasma FVIII (r = −0·42, P < 0·01) and VWF:Ag (r = −0·37, P = 0·01) correlated inversely with microparticles, possibly related to their removal from plasma via binding to microparticles.
In this study, circulating TF-PCA levels were higher in obese than in healthy–weight children (Table 1) and correlated with BMI (Fig. 1). Strikingly, the levels in obese children are comparable to those reported in adults with T2DM (Boden et al, 2007). Circulating TF plays a crucial role in coagulation and thrombosis (Key et al, 1998; Tilley & Mackman, 2006). There is enhanced thrombus formation when blood with elevated circulating TF levels is perfused over a collagen-coated surface (Sambola et al, 2003). Our findings suggest that, even in young children, obesity is associated with a potential prothrombotic state.
Obesity, TF, and monocytes are closely linked. The monocyte, which is the macrophage precursor, is a major source of TF in circulation, and macrophage and adipocyte functions converge in obesity. Macrophages infiltrate adipose tissue and both cells produce proinflammatory cytokines; fat tissue from obese individuals has a gene expression profile suggestive of macrophages. Obesity upregulates F3 expression in genetically obese mice and in lean mice insulin induces F3 mRNA in several tissues, which is relevant because obesity is associated with hyperinsulinemia (Faber et al, 2009). Overall, these studies point to a cross-talk between obesity, inflammation and coagulation, with obesity promoting inflammation and coagulation (Zhang & Lawrence, 2011). Moreover, TF triggers signalling through protease activated receptor-2 (PAR2) and recent studies (Badeanlou et al, 2011) implicate TF directly in the development of diet-induced obesity and insulin resistance. They showed that TF-VIIa-PAR2 signalling of adipocytes crucially regulates weight gain and, in adipose tissue, macrophages promote inflammation and insulin resistance. TF blockade has been proposed as a new therapeutic approach targeting obesity. Based on our findings, circulating TF may serve as a biomarker for these regulatory interactions in children.
In this study, plasma FVII:C was higher in obese than in healthy-weight children and correlated with BMI (Fig. 1). Plasma FVII:C is elevated in adults with metabolic syndrome, diabetes mellitus and obesity and is associated with increased risk for acute cardiovascular events (Mertens & Van Gaal, 2002; Green et al, 2010). The increased plasma FVII may contribute to the enhanced thrombin generation reported in the blood of obese children. In healthy young adults, plasma FVII levels have been positively associated with increased carotid intimal-media thickness (indicating subclinical atherosclerosis), and this association was modified by BMI (Green et al, 2010). Even in children, BMI was correlated with carotid intimal-media thickness (Beauloye et al, 2007) and in our studies, plasma FVII:C and TF-PCA correlated positively with BMI.
PAI-1 levels were higher in obese children and correlated with BMI. Elevated PAI-1 levels reflect endothelial dysfunction and are associated with increased cardiovascular risk (Mertens & Van Gaal, 2002). PAI-1 release is stimulated by insulin and chronic inflammation and the Serpine1 gene is upregulated in genetically obese mice (Mertens & Van Gaal, 2002). Together, the elevated TF-PCA, FVII and PAI-1 levels indicate that childhood obesity is a procoagulant state.
Endothelial surface markers are upregulated by cytokines and inflammation, and increased levels reflect endothelial dysfunction. Obese children had elevated sVCAM1, which correlated with TF-PCA. Our findings assume importance because elevated endothelial markers have predicted subsequent development of T2DM and cardiovascular events (Meigs et al, 2004).
In summary, we found that childhood obesity was associated with changes suggesting a procoagulant and proatherogenic state with concomitant endothelial dysfunction. Although children studied were mostly African American or Hispanic we are unaware of racial differences in these biomarkers in children; we believe the findings apply to other groups also. Larger studies are needed in children to define whether weight reduction can reverse these abnormalities and impact clinical consequences.
The excellent secretarial assistance of Ms. Denise Tierney is acknowledged.
Seed grant from Temple University and NIH R01 HL-073367.
AKR and GDF designed the research. AKR, AS and GDF wrote and edited the paper. AS and JG performed the measurements. EK analysed the data. TAM, TN and SVV recruited the subjects and processed blood samples.