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www.eje-online.org © 2017 European Society of EndocrinologyPrinted in Great Britain

Published by Bioscientifica Ltd.DOI: 10.1530/EJE-16-0835

Contact system activation and high thrombin generation in hyperthyroidismNamhee Kim1, Ja-Yoon Gu1, Hyun Ju Yoo1, Se Eun Han2, Young Il Kim2, Il Sung Nam-Goong2, Eun Sook Kim2 and Hyun Kyung Kim1

1Department of Laboratory Medicine and Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea and 2Department of Internal Medicine, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea

Abstract

Background: Hyperthyroidism is associated with increased thrombotic risk. As contact system activation through

formation of neutrophil extracellular traps (NET) has emerged as an important trigger of thrombosis, we hypothesized

that the contact system is activated along with active NET formation in hyperthyroidism and that their markers

correlate with disease severity.

Subjects and methods: In 61 patients with hyperthyroidism and 40 normal controls, the levels of coagulation factors

(fibrinogen, and factor VII, VIII, IX, XI and XII), D-dimer, thrombin generation assay (TGA) markers, NET formation

markers (histone–DNA complex, double-stranded DNA and neutrophil elastase) and contact system markers (activated

factor XII (XIIa), high-molecular-weight kininogen (HMWK), prekallikrein and bradykinin) were measured.

Results: Patients with hyperthyroidism showed higher levels of fibrinogen (median (interquartile range), 315 (280–344)

vs 262 (223–300), P = 0.001), D-dimer (103.8 (64.8–151.5) vs 50.7 (37.4–76.0), P < 0.001), peak thrombin (131.9 (102.2–

159.4) vs 31.6 (14.8–83.7), P < 0.001) and endogenous thrombin potential (649 (538–736) vs 367 (197–1147), P = 0.021)

in TGA with 1 pM tissue factor, neutrophil elastase (1.10 (0.39–2.18) vs 0.23 (0.20–0.35), P < 0.001), factor XIIa (66.9

(52.8–87.0) vs 73.0 (57.1–86.6), P < 0.001), HMWK (6.11 (4.95–7.98) vs 3.83 (2.60–5.68), P < 0.001), prekallikrein (2.15

(1.00–6.36) vs 1.41 (0.63–2.22), P = 0.026) and bradykinin (152.4 (137.6–180.4) vs 118.3 (97.1–137.9), P < 0.001) than did

normal controls. In age- and sex-adjusted logistic regression analysis, fibrinogen, factor VIII, IX and XIIa, D-dimer, peak

thrombin, neutrophil elastase, HMWK and bradykinin showed significant odds ratios representing hyperthyroidism’s

contribution to coagulation and contact system activation. Free T4 was significantly correlated with factors VIII and IX,

D-dimer, double-stranded DNA and bradykinin.

Conclusion: This study demonstrated that contact system activation and abundant NET formation occurred in the high

thrombin generation state in hyperthyroidism and were correlated with free T4 level.

Introduction

Hyperthyroidism is associated with increased thrombotic risk (1). It has been suggested that hypercoagulability associated with hyperthyroidism is the consequence of direct effects of thyroid hormones on the hepatic synthesis of coagulation factors (2). Recent reports have suggested contact system activation as a new mechanism of thrombosis (3, 4). Until now, there have been no

reports on whether the contact system is activated in hyperthyroidism.

In the contact system, negatively charged surfaces can activate factor XII; activated factor XII (XIIa) converts prekallikrein to α-kallikrein, which cleaves high-molecular-weight kininogen (HMWK) to produce bradykinin (3). Factor XIIa subsequently activates factor

Correspondence should be addressed to E S Kim or H K Kim Email endo10@daum.net or lukekhk@snu.ac.kr

European Journal of Endocrinology (2017) 176, 583–589

www.eje-online.org © 2017 European Society of Endocrinology

176:5 583–589N Kim and others Contact system activation in hyperthyroidism

176:5

10.1530/EJE-16-0835

Clinical Study

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XI and the intrinsic coagulation pathway. Recent studies have revealed that neutrophil extracellular traps (NET), which consist mainly of the DNA–histone complex and neutrophil elastase, can directly activate factor XII (5, 6). Considering that thyroid hormone induces reactive oxygen species (ROS) production in neutrophils and ROS can induce NET formation (7, 8), it is plausible that NET may be actively formed in hyperthyroidism. NET can activate the contact system (3, 5). Therefore, NET components can be expected to activate the contact system, eventually promoting thrombosis.

Thrombin generation assay (TGA) has been introduced as a global coagulation assay to assess hypocoagulability or hypercoagulability (9, 10). TGA measures the amount of thrombin generated over the reaction time in test plasma stimulated with various concentrations of tissue factor (TF). It reflects thrombotic tendency (9, 11).

This study investigated whether patients with hyperthyroidism show hypercoagulability as assessed by TGA together with assays for individual coagulation factors. In addition, to investigate whether NET-induced contact system activation occurs in hyperthyroidism, we measured several markers of NET formation and contact system activation.

Subjects and methods

Study population

This study was approved by the Institutional Review Board of Ulsan University Hospital (UUH-IRB-12-078), and signed informed consents were obtained from all subjects. A total of 61 patients with hyperthyroidism and 40 age- and sex-matched healthy controls were enrolled. Patients were recruited when they showed untreated biochemically hyperthyroid (free T4 >1.7 ng/dL and/or T3 >1.7 ng/mL and/or thyroid-stimulating hormone (TSH) <0.4 IU/mL) and/or had a clinical history suggestive of hyperthyroidism such as weight loss, heat intolerance, sweating, palpitations and nervousness. The recruited patients were confirmed to be suffering from hyperthyroidism by medical history, physical examination and laboratory results. Patients who were on antithyroid drugs within the past 3 months were excluded.

Peripheral blood was collected into commercially available tubes (serum separation and sodium citrate tubes). Within 1 h of blood collection, serum or plasma was separated by centrifugation of whole blood at 1550 g for 15 min. The aliquots were stored at −80°C.

Coagulation assays

Fibrinogen, D-dimer and coagulation factors (VII, VIII, IX, XI and XII) were measured on an automated coagulation analyzer (ACL TOP, Beckman Coulter, Fullerton, CA, USA). Fibrinogen and D-dimer were measured using the HemosIL Fibrinogen-C XL reagent and HemosIL D-Dimer HS (Instrumentation Laboratory SpA, Milan, Italy) respectively. Coagulation factors were tested using a PT-based clotting assay with the HemosIL RecombiPlasTin reagent and an aPTT-based clotting assay using the SynthASil reagent (Instrumentation Laboratory, Bedford, MA, USA).

Thrombin generation assay

Thrombin generation was measured with a Fluoroskan Ascent Fluorometer (Thermo Lab Systems, Helsinki, Finland) according to a previously described method (10). Briefly, 80 μL plasma were mixed with 20 μL of reagents containing TF (final concentration, 1 or 5 pmol/L) and phospholipid. Then, 20 μL of a fluorogenic substrate with CaCl2 was added. The amount of thrombin generated was determined by using the Thrombinoscope software (Diagnostica Stago, Asnières, France). The output curves describe the initiation, propagation and termination phases of thrombin generation. Lag time, peak thrombin concentration and endogenous thrombin potential (ETP) were calculated from the curves.

Markers of contact system activation (factor XIIa, HMWK, PK and bradykinin)

Factor XIIa activity was measured using a chromogenic method with a CoaChrom Factor XIIa test kit (CoaChrom Diagnostica, Maria Enzersdorf, Austria). HMWK and PK were measured with ELISA kits from Cloud-Clone Corp. (Houston, TX, USA), and bradykinin with an ELISA kit from Abcam.

Markers of NET (histone–DNA complex, double-stranded DNA and neutrophil elastase)

The plasma level of the histone–DNA complex was quantified with a Cell Death Detection ELISA kit (Roche Diagnostics). The double-stranded DNA (dsDNA) level was measured using the Quant-iT PicoGreen dsDNA reagent (Molecular Probes) and a microplate fluorometer (Fluoroskan Ascent, Thermo Fisher Scientific). Neutrophil

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elastase level was measured using a Human PMN Elastase Platinum ELISA kit (eBioscience, Vienna, Austria).

Thyroid hormones

Levels of free T4 and TSH were measured with commercially available assays (Elecsys and cobas e immunoassay analyzers; Roche Diagnostics).

Statistical analysis

Data were compared using the Mann–Whitney U test (continuous variables) and the chi-square test (categorical variables). To investigate the contribution extent of hyperthyroidism to coagulation and contact system markers, age- and sex-adjusted logistic regression analyses were performed. Spearman’s rank correlation was used to evaluate the relationship with various tested markers with hormone levels. We arbitrarily divided patients into 3 groups according to free T4 levels (<2.5, 2.5–3.5 and >3.5 ng/dL), and then levels of coagulation and

contact activation markers were compared by using Kruskal–Wallis tests. Two-sided P value of <0.05 was considered significant. All statistical analyses were carried out with SPSS, version 21.0 (SPSS) and Microsoft Excel (Microsoft Corporation).

Results

Hormone and antibody level in hyperthyroid patients

The median levels of free T4, TSH and T3 in recruited hyperthyroid patients were 2.29 ng/dL (interquartile range: 1.62–3.61; reference range: 0.80–1.70), 0.008 IU/mL (interquartile range: 0.006–0.011; reference range: 0.400–4.700) and 2.34 ng/mL (interquartile range: 1.28–4.14; reference range: 0.60–1.70) respectively. Positive rates of thyroid autoantibodies were 77.0% for antithyroid peroxidase antibody, 54.1% for anti-thyroglobulin antibody and 96.7% for thyroid-stimulating immunoglobulin. The causes of hyperthyroidism were Graves’ disease (n = 59) and autoimmune thyroiditis (n = 2).

Table 1 Mean values of the tested markers in patients and normal controls. Data are shown as the median (interquartile range)

for continuous variables and as number (percentage) for categorical variables. Mann–Whitney U test was used to compare

median values and chi-square test to compare positivity.

Patients (n = 61) Control (n = 40) P value

Gender, female (n) 41 (67.2%) 23 (57.5%) 0.400Age (years) 43 (28–52) 35 (32–39) 0.085Fibrinogen (mg/dL) 315 (280–344) 262 (223–300) <0.001Factor VII (%) 91.8 (77.0–103.7) 85.0 (76.1–96.8) 0.327Factor VIII (%) 123.5 (98.7–140.7) 81.8 (67.4–94.3) <0.001Factor IX (%) 118.3 (103.5–129.9) 100.9 (94.1–111.0) 0.001Factor XI (%) 116.5 (99.4–132.6) 108.2 (95.4–120.1) 0.066Factor XII (%) 66.9 (52.8–87.0) 73.0 (57.1–86.6) 0.805D-dimer (ng/mL) 103.8 (64.8–151.5) 50.7 (37.4–76.0) <0.001Markers of TGA with 1 pM TF Lag time (min) 7.67 (6.67–8.63) 8.00 (6.67–9.50) 0.195 Peak thrombin (nM) 131.9 (102.2–159.4) 31.6 (14.8–83.7) <0.001 ETP (nM min) 649 (538–736) 367 (197–1147) 0.021Markers of TGA with 5 pM TF Lag time (min) 4.61 (4.00–5.00) 4.00 (3.67–5.00) 0.080 Peak thrombin (nM) 62.29 (50.46–74.38) 21.36 (14.76–55.11) <0.001 ETP (nM min) 665 (535–742) 362 (234–985) 0.030Markers of neutrophil extracellular traps Histone–DNA complex (ng/mL) 36 (23–73) 30 (21–55) 0.204 dsDNA (ng/mL) 77.16 (65.44–86.62) 74.87 (69.64–80.35) 0.857 Neutrophil elastase (ng/mL) 110 (39–218) 23 (20–35) <0.001Markers of contact system activation Factor XIIa (U/L) 29.4 (250.6–33.1) 25.2 (23.2–26.9) <0.001 HMWK (ng/mL) 6.11 (4.95–7.98) 3.83 (2.60–5.68) <0.001 PK (ng/mL) 2.15 (1.00–6.36) 1.41 (0.63–2.22) 0.026 Bradykinin (pg/mL) 152.4 (137.6–180.4) 118.3 (97.1–137.9) <0.001

dsDNA, double-stranded DNA; ETP, endogenous thrombin potential; HMWK, high-molecular-weight-kininogen; PK, prekallikrein; TF, tissue factor; TGA, thrombin generation assay.

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Hypercoagulability, NET formation and contact system activation in hyperthyroidism

Patients with hyperthyroidism showed significantly higher levels of fibrinogen, factor VIII, factor IX and D-dimer than did normal controls (Table 1). In TGA with 1 pM or 5 pM TF, the levels of peak thrombin and ETP were also significantly higher in patients than those in normal controls. The markers of contact system activation (factor XIIa, HMWK, PK and bradykinin) were significantly higher in patients than those in normal controls. Among NET markers, the neutrophil elastase level was significantly higher in patients than that in normal controls.

The results of logistic regression analyses were performed to assess the contribution extent of hyperthyroidism to coagulation and contact system markers (Table 2). The odds ratio of fibrinogen was 1.016 (95% confidence interval: 1.007–1.024), meaning that when fibrinogen level increased by 1 mg/dL, possibility of being hyperthyroidism increased by 1.016-fold. The odd ratios of factors VIII and IX, D-dimer and peak

thrombin in TGA with 1 pM and 5 pM TF were also significant, suggesting hyperthyroidism contributes to the coagulation markers. Those markers of contact system activation (factor XIIa, HMWK and bradykinin) and NET (neutrophil elastase) also showed significant.

Correlations of hypercoagulability, NET formation and contact system activation with hyperthyroidism severity

We used serum levels of thyroid hormones (TSH and free T4) as indicators of hyperthyroidism severity. TSH levels were negatively correlated with factor VIII and bradykinin (Table  3). Interestingly, free T4 levels were positively correlated with factor VIII and IX, D-dimer, bradykinin and dsDNA. Factor VII was negatively correlated with free T4. We arbitrarily divided patients into 3 groups according to free T4 levels (<2.5, 2.5–3.5 and >3.5 ng/dL). Median levels of fibrinogen, factor VIII, D-dimer and dsDNA gradually increased with increasing free T4 levels (Fig. 1).

Table 2 Logistic regression analysis for prediction of the

levels of contribution to hyperthyroidism.

Odds ratio (95% CI) P value

Fibrinogen (mg/dL) 1.016 (1.007–1.024) <0.001Factor VII (%) 0.996 (0.985–1.008) 0.554Factor VIII (%) 1.060 (1.035–1.086) <0.001Factor IX (%) 1.028 (1.006–1.050) 0.011Factor XI (%) 1.018 (0.999–1.039) 0.068Factor XII (%) 0.998 (0.982–1.015) 0.813D-dimer (ng/mL) 1.017 (1.007–1.027) 0.001Markers of TGA with 1 pM TF

Lag time (min) 0.933 (0.802–1.087) 0.374Peak thrombin (nM) 1.025 (1.015–1.036) <0.001ETP (nM min) 1.000 (0.999–1.001) 0.665

Markers of TGA with 5 pM TF Lag time (min) 1.350 (0.821–2.218) 0.207Peak thrombin (nM) 1.026 (1.009–1.043) 0.002ETP (nM min) 1.000 (0.999–1.001) 0.931

Markers of neutrophil extracellular traps

Histone–DNA complex (ng/mL)

1.007 (0.999–1.016) 0.106

dsDNA (ng/mL) 1.015 (0.984–1.047) 0.347Neutrophil elastase (ng/mL) 6.597 (2.190–19.874) 0.001

Markers of contact system activation

Factor XIIa (U/L) 1.177 (1.064–1.301) 0.001HMWK (ng/mL) 1.740 (1.336–2.266) <0.001PK (ng/mL) 1.057 (0.959–1.166) 0.255Bradykinin (pg/mL) 1.024 (1.010–1.037) <0.001

CI, confidence interval; dsDNA, double-stranded DNA; ETP, endogenous thrombin potential; HMWK, high-molecular-weight kininogen; PK, prekallikrein; TF, tissue factor; TGA, thrombin generation assay.

Table 3 Correlation of thyroid-stimulating hormone (TSH)

and free T4 with tested parameters. Values are presented as

correlation coefficients.

TSH Free T4

Age (years) 0.057 0.046Fibrinogen (mg/dL) 0.022 0.246Factor VII (%) 0.099 −0.274*Factor VIII (%) −0.366* 0.477*Factor IX (%) −0.201 0.263*Factor XI (%) −0.093 0.195Factor XII (%) 0.049 −0.146D-dimer (ng/mL) −0.142 0.272*Markers of TGA with 1 pM TF Lag time (min) 0.057 −0.021 Peak thrombin (nM) 0.006 0.109 ETP (nM min) 0.088 0.067Markers of TGA with 5 pM TF Lag time (min) 0.129 −0.077 Peak thrombin (nM) −0.023 0.180 ETP (nM min) −0.001 0.029Markers of neutrophil extracellular traps Histone–DNA complex (ng/mL) −0.058 0.094 dsDNA (ng/mL) 0.034 0.381* Neutrophil elastase (ng/mL) 0.067 0.157Markers of contact system activation Factor XIIa (U/L) −0.230 0.042 HMWK (ng/mL) −0.018 0.001 PK (ng/mL) 0.039 0.021 Bradykinin (pg/mL) −0.294* 0.282*

*P < 0.05.dsDNA, double-stranded DNA; ETP, endogenous thrombin potential; HMWK, high-molecular-weight kininogen; PK, prekallikrein; TF, tissue factor; TGA, thrombin generation assay.

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Discussion

This study demonstrated that patients with hyperthyroidism showed hypercoagulability as assessed by TGA together with assays for individual coagulation factors. Of note, NET formation was elevated and contact system was activated in hyperthyroidism. Moreover, some markers of NET and contact system activation were significantly correlated with hyperthyroidism severity.

When a neutrophil is stimulated by bacterial pathogens, it releases the DNA–histone complex and neutrophil elastase into the extracellular space, referred to as NET (5, 6). Any stimuli that induce ROS production in neutrophils can also promote NET formation. As thyroid hormones can induce ROS production in neutrophils (7), NET formation can be expected to increase in hyperthyroidism. Indeed, we observed high levels of neutrophil elastase in our patients with hyperthyroidism.

The NET components, including DNA and histone, activate factor XII in the contact system (3, 5). Subsequently, factor XIIa activates the factor XI downstream pathway, which finally results in fibrin formation. Therefore, NET formation can increase thrombotic tendency. In our patients, the levels of the markers of contact system activation were increased along with hypercoagulability (assessed by TGA), suggesting that

contact system activation may induce hypercoagulability in hyperthyroidism.

Recent animal and human studies have demonstrated that blockers of contact system activation, including factor XII inhibitor, exert an antithrombotic effect (12, 13). As our results show contact system activation in hyperthyroidism, contact system inhibition would be potential antithrombotic therapy for thrombotic patients with hyperthyroidism.

Among markers of contact system activation, PK and HMWK are zymogens. As factor XIIa cleaves PK to kallikrein and HMWK to bradykinin, contact system activation is expected to decrease both PK and HMWK and increase both kallikrein and bradykinin (14). However, because PK and HMWK are acute-phase reactants (15, 16), their levels can be elevated in hyperthyroidism through active hepatic synthesis. Indeed, the circulating levels of PK and HMWK as well as factor XIIa and bradykinin were elevated in our hyperthyroidism patients.

TGA helps to elucidate coagulation status in various clinical conditions. In TGA with both 1 pM and 5 pM TF, we observed high peak thrombin and ETP levels in hyperthyroidism, confirming the presence of hypercoagulability in hyperthyroidism. Moreover, we observed high levels of fibrinogen and factor VIII, IX and XI in hyperthyroidism, which are considered to contribute to hypercoagulability (1, 2). Consistent with our results, an increased hepatic synthesis of coagulation factors has been reported in patients with hyperthyroidism (2, 17).

As expected, coagulation factors and the results of TGA contributed to hyperthyroidism significantly. Some markers of NET and contact system activation also significantly contributed to hyperthyroidism. These findings suggest that hypercoagulability, NET formation and contact system activation are characteristic features of hyperthyroidism.

Although determination of TSH levels is the most reliable test for screening for thyroid disease, if hyperthyroidism is strongly suspected, both free T4 and TSH tests are recommended (18, 19, 20). Measurement of free T4 level is still important in many patients as only the free form may be available for uptake into cells and for interaction with nuclear receptors (21). In our study, free T4 levels showed significant correlations with the levels of factor VIII and IX, D-dimer, dsDNA and bradykinin. When patients were divided into 3 groups according to their free T4 levels, the levels of fibrinogen, factor VIII, D-dimer and dsDNA gradually increased with increasing free T4 level. These findings mean that hyperthyroidism severity correlates with hypercoagulability, NET

Figure 1

Median levels of fibrinogen (A), factor VIII (B), D-dimer (C),

and dsDNA (D) in patients with hyperthyroidism who had

different free T4 levels. Boxplots display the median,

interquartile range (IQR), outliers (○, >1.5 × IQR) and extremes

(▼, >3 × IQR). dsDNA, double-stranded DNA.

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formation and contact system activation. Although the exact mechanisms of NET formation and contact system activation in hyperthyroidism remain to be elucidated, our findings implicate high thyroid hormone level as a potential cause of NET formation and subsequent contact system activation. Consistent with our data, there has been a report that suggested correlation of thyroid hormone with coagulation proteins (22).

Our study has several limitations. First, it was a cross-sectional case–control study. Therefore, to investigate the exact causes of hypercoagulability, NET formation and contact system activation in hyperthyroidism, prospective studies are required in future. Second, due to a small sample size, we used non-parametric statistical methods, which are usually less powerful than corresponding tests designed for use on data that come from a specific distribution. Therefore, some results, such as the negative correlation between the levels of factor VII and free T4, were ambiguous and difficult to interpret. Further studies with large populations are necessary to confirm our results. Third, we did not access von Willebrand factor and anticoagulant factors such as antithrombin and fibrinolytic factors. This study just focused on the potential mechanism of hypercoagulability through NET formation and contact system activation.

In summary, we demonstrated, for the first time, that patients with hyperthyroidism have abundant NET formation and contact system activation as well as hypercoagulability. Hypercoagulability in hyperthyroidism may be caused by NET-induced contact activation as well as hepatic synthesis of coagulation factors, although the exact mechanism is still unresolved. A strategy to block contact system activation may be beneficial to manage thrombosis in hypercoagulable patients with hyperthyroidism.

Declaration of interestThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

FundingThis work (2016R1A2B4015571) was supported by Mid-career Researcher Program through NRF grant funded by the Korea government (MSIP).

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Received 9 October 2016Revised version received 17 January 2017Accepted 30 January 2017

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