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GDF-15 is associated with thrombus burden in patients with deep venous thrombosis
Journal Pre-proof GDF-15 is associated with thrombus burden in patients with deep venous thrombosis
Wei Liang, Fen Wei, Chao Yang, Fen Xie, Xin-Xin Shuai, Min Wang, Miao Yu PII:
S0049-3848(20)30031-1
DOI:
https://doi.org/10.1016/j.thromres.2020.01.022
Reference:
TR 7581
To appear in:
Thrombosis Research
Received date:
24 November 2019
Revised date:
4 January 2020
Accepted date:
19 January 2020
Please cite this article as: W. Liang, F. Wei, C. Yang, et al., GDF-15 is associated with thrombus burden in patients with deep venous thrombosis, Thrombosis Research (2018), https://doi.org/10.1016/j.thromres.2020.01.022
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© 2018 Published by Elsevier.
Journal Pre-proof GDF-15 is associated with thrombus burden in patients with deep venous thrombosis Wei Liang *, 1, Fen Wei*, 1, Chao Yang#, 1, Fen Xie#, Xin-Xin Shuai*, Min Wang*, 2, Miao Yu *, 2 * Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China. # Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
1 Joint first authors: The first three authors contributed equally to the study.
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2 Corresponding authors: Miao Yu: Department of Cardiology, Union Hospital, Tongji Medical
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College, Huazhong University of Science and Technology, Wuhan, 430022, China. Tel: +862785726005. Fax: +862785727340. E-mail address: [email protected]. Min Wang:
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Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science
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and Technology, Wuhan, 430022, China. E-mail address: [email protected].
Journal Pre-proof Abstract Introduction: Growth differentiation factor-15 (GDF-15) has been identified as a predictor in cardiovascular diseases and acute pulmonary embolism. However, the association of GDF-15 and deep venous thrombosis (DVT) remains unclear. This study aimed to investigate levels of GDF-15 in patients with DVT, and determine its association with the thrombus burden. Materials and Methods: 72 newly diagnosed DVT patients and 30 healthy volunteers were enrolled, and the levels of plasma GDF-15 were detected. To explore the relationship between GDF-15 and thrombus severity, we analyzed the thrombus burden and the association with
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pulmonary embolism of DVT patients. In vitro, the effect of GDF-15 on platelet aggregation and
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thrombin/antithrombin activity were investigated.
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Results: We found that the mean levels of plasma GDF-15 in DVT patients were significantly higher than those in healthy controls (1448.78 ± 61.98 pg/ml VS 805.70 ± 112.95 pg/ml, P<0.001).
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Furthermore, GDF-15 showed an increase with more venous segments with thrombus (P<0.001),
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and the patients with higher levels of GDF-15 and more thrombus segments showed higher scores of Wells-PE and Geneva and increased incidence of pulmonary embolism (P<0.05). In vitro, we
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confirmed that GDF-15 significantly reduced platelet aggregation induced by ADP and the effect was concentration-dependent (P<0.001). However, GDF-15 showed no direct effect on thrombin
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and anti-thrombin activity.
Conclusions: Increased GDF-15 level was associated with more thrombus severity of DVT patients and GDF-15 could inhibit platelet aggregation induced by ADP in vitro. These findings suggest that GDF-15 might not only be an indicator for thrombus severity but also be a potential treatment target in DVT.
Keywords: GDF-15; deep venous thrombosis; thrombus burden
Abbreviations: GDF-15, growth differentiation factor-15; DVT, deep venous thrombosis; PE, pulmonary embolism; PRP, platelet-rich plasma; PPP, platelet-poor plasma; CTRL, control; ADP, adenosine diphosphate; PT, prothrombin time; APTT, activated partial thromboplastin time; TT, thrombin time.
Journal Pre-proof Introduction Deep venous thrombosis (DVT) is one of the most common peripheral vascular diseases in the worldwide. It is closely related to pulmonary embolism (PE), which leads to death in a substantial fraction of patients every year [1]. Recently, evidence has been accumulating that the factors influencing DVT formation are not only restricted to the coagulation system alone, but also the immune system is closely associated with formation of thrombosis [2]. Growth differentiation factor-15 (GDF-15) is a member of the transforming growth factor-β cytokine superfamily, which is known for its role in cell growth, differentiation, apoptosis, and
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inflammation [3, 4]. GDF-15 is weakly secreted in most tissues under physiological conditions,
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but strongly produced during inflammation [5]. Therefore, GDF-15 has been identified as a
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predictor for cardiovascular events, heart failure, and all-cause mortality in cardiovascular diseases, independently from traditional biomarkers such as cardiac troponins, natriuretic peptides,
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and C-reactive protein [6-9]. Recently, preliminary data have also suggested that GDF-15 may be
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valuable for stratifying risk of complications in patients with acute pulmonary embolism（PE） [10, 11]. However, the association of GDF-15 and DVT remains unclear.
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Therefore, this study aimed to investigate levels of GDF-15 in patients with DVT, and
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determine its association with the thrombus burden. Materials and Methods Patients
A total of 72 DVT patients diagnosed with lower extremity venous color Doppler ultrasonic in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were randomly recruited for this study. The ultrasound screening protocol included all deep and superficial lower extremity veins, including iliac vein, femoral vein, popliteal vein and tibial vein, with bilateral lower-extremity compression, followed by color and spectral Doppler ultrasound evaluation of filling and flow patterns. Patients with previously known DVT or signs of chronic DVT on Doppler ultrasound screening were excluded. Patients with any other acute or chronic inflammation-related diseases were excluded. None of the patients had been treated with antiinflammatory, antiplatelet, or anti-coagulant drug. Additionally, 30 aged and sex-matched healthy
Journal Pre-proof volunteers were enrolled as the controls in the study. Written informed consent was obtained from each patient and the study was conducted in accordance with the guidelines of the Declaration of Helsinki and its amendments. This research was approved by the Ethics Committee of Tongji Medical College at Huazhong University of Science and Technology. The clinical data of patients were collected. Blood samples were obtained from the patients and the healthy volunteers in the recumbent position and a fasting state on the next morning before any treatment, with a 21-gauge needle for clean antecubital venipuncture. Each sample of 2 ml was collected into vacutainer tube containing 3.2% sodium citrate. The blood samples were
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centrifuged at 2,500 g for 15 minutes and the plasma was stored at -80°C until analysis.
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Enzyme-linked immunosorbent assay
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Plasma levels of GDF-15 were detected with human GDF-15 enzyme-linked immunosorbent assay (ELISA) kit (RayBiotech, United States) according to the manufacturers’ instructions. The
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sensitivity of GDF-15 detection was 2 pg/mL. All samples were detected in triplicate.
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Platelet aggregation assay
Blood was obtained from the DVT patients and collected into vacutainer tube containing 3.2%
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sodium citrate. Platelet-rich plasma (PRP) was separated by centrifugation (100 g, 10 minutes, 20℃). The remaining blood sample was further centrifuged at 2000 g for 15 minutes to obtain
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platelet-poor plasma (PPP). Platelet aggregation was measured using a turbidimetric aggregation monitoring device (AggRAM, Helena Laboratories, USA). PPP was used to set the 100% aggregation. PRP was pre-warmed to 37°C in a resting state and then incubated with 10, 20, or 50 ng/mL GDF-15 (Peprotech, USA) for 10 minutes respectively. PRP treated without GDF-15 was designated as CTRL. Subsequently, 3μM adenosine diphosphate (ADP, Helena Laboratories, USA) or 10μg/ml collagen (Helena Laboratories, USA) was added to the plasma to induce aggregation. Platelet aggregability was evaluated by the maximal percent of platelet aggregation. The activity of coagulation factor and antithrombin assay The PPP was gathered from patients with DVT, and incubated with 10, 20, or 50 ng/mL GDF-15 (Peprotech, USA) for 10 minutes at 37°C in a resting state respectively. The change of coagulation and anticoagulation function was detected with Stago® automated haemostasis analyser according to the manufacturer’s instructions.
Journal Pre-proof Statistical analysis Data were shown as the mean ± SEM. IBM SPSS Statistics (version 22) was used for all statistical analyses. 2-tailed Student’s t test (two groups) or one-way ANOVA (more than two groups) was performed for the mean comparison. Fisher's exact test was performed for the categoric variables of clinical characteristics. P<0.05 was considered statistically significant. Results 1. GDF-15 level was increased in DVT patients
Compared with healthy controls, the DVT patients showed significant increased GDF-15
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levels (805.70 ± 112.95 pg/ml VS 1448.78 ± 61.98 pg/ml, P<0.001). There were no
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differences in age (P=0.107) and sex (P=0.515) between healthy controls and DVT patients.
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2. GDF-15 was associated with thrombus burden and the incidence of PE The deep lower extremity veins were divided into four segments as iliac vein, femoral vein,
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popliteal vein and tibial vein, and the number of vein segments involved in DVT patients could be
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an indicator of thrombus burden [12, 13]. GDF-15 levels were compared against number of segments involved in DVT, and the results are presented in Table 1. GDF-15 showed an increase
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with more venous segments with thrombus (Figure 1). Table 1 The levels of GDF-15 according to number of vein segments involved n/N
Mean±SEM (pg/ml)
1
18/72
1128.13±130.55
2
12/72
1190.04±151.80
3
22/72
1659.06±98.72
4
20/72
1661.28±67.23
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No. of segments with thrombus
Figure 1 The levels of GDF-15 in DVT patients with different numbers of vein segments involved.
Journal Pre-proof Pulmonary artery CTA was performed on 34 patients (47%) according to the decision of the clinicians, and 7 of them were combinated with PE. Lankeit M et al have reported that the upper limit of normal for GDF-15 level is 1200 pg/ml [10]. According to the number of segments involved in DVT (Table 1), the average values of GDF-15 in patients with No. of segments 1 and 2 were lower than 1200 pg/ml, and those in patients with No. of segments 3 and 4 were higher than 1200pg/ml. Therefore, we divided the 34 patients received pulmonary artery CTA according to number of vein segments involved in DVT, and their baseline characteristics were presented in Table 2. We found that patients with No. of segments 3-4 group had higher Wells-PE / Geneva
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scores and incidence of PE than those with No. of segments 1-2 group (P<0.05).
P value
55.62±3.96
58.19±2.71
0.596
9
9
0.172
23.31±0.46
23.13±0.79
23.41±0.59
0.779
5
1
4
0.627
10
3
7
0.704
9
5
4
0.254
57.21±2.23
Sex, male
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Body mass index, kg/m²
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Diabetes
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Age, years
Hypertension
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Smoking
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No. of segments 3-4 n=21
Clinical characteristics
No. of segments 1-2 n=13
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ALL n=34
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Table 2 Baseline characteristics of patients received pulmonary artery CTA
Malignant disease
6
2
4
1.000
Recent surgery or immobilization
12
4
8
0.727
Combinated with PE
7
0
7
0.029
Wells-PE
5.84±0.32
4.62±0.49
6.60±0.34
0.002
Geneva
8.65±0.47
7.46±0.67
9.38±0.60
0.047
D-Dimer,ug/ml
5.23±1.12
4.24±2.06
5.84±1.32
0.496
PT,s
15.16±0.43
15.12±0.72
15.20±0.55
0.935
APTT,s
48.83±1.99
47.55±3.11
46.39±2.65
0.783
234.35±11.66
220.77±18.78
242.76±14.93
0.368
Scores
Biomarkers
PLT,10^9/L
Journal Pre-proof 3. GDF-15 prevented platelet aggregation induced by ADP To investigate the direct role of GDF-15 in platelet aggregation, we prepared the platelet suspensions in DVT patients and healthy controls, and performed the experiment of platelet aggregation in vitro. Compared with CTRL, the ratio of platelet aggregation administrated with 10, 20, or 50 ng/mL GDF-15 was calculated. We confirmed that GDF-15 significantly reduced platelet aggregation induced by ADP both in DVT patients and healthy controls. Moreover, the effect of GDF-15 on platelet aggregation induced by ADP was concentration-dependent. However, it showed that GDF-15 had no effect on platelet aggregation induced by collagen in DVT patients
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and healthy controls in vitro. (Figure 2)
Figure 2 The effect of GDF-15 on platelet aggregation induced by ADP and collagen
Journal Pre-proof (A) The representative picture for platelet aggregation treated with CTRL or different concentrations of GDF-15 induced by ADP (3μM) in DVT patients and healthy controls. (B) The representative picture for platelet aggregation treated with CTRL or different concentrations of GDF-15 induced by collagen (10μg/ml) in DVT patients and healthy controls. (C) The results of statistical analysis on the ratio of platelet aggregation treated with CTRL or different concentrations of GDF-15 induced by ADP in DVT patients and healthy controls, n=6, *: P<0.01 vs CTRL, #: P<0.01 vs 10ng/ml GDF-15 group, δ: P<0.01 vs 20ng/ml GDF-15 group. (D) The results of statistical analysis on the ratio of platelet aggregation treated with CTRL or different concentrations of GDF-15 induced by collagen in DVT patients and healthy controls, n=6, P>0.05. 4. GDF-15 did not influence thrombin and anti-thrombin activity
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To detect the direct effect of GDF-15 on thrombin and anti-thrombin activity, we prepared the
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PPP in DVT patients in vitro and pre-incubated with 10, 20, or 50 ng/mL GDF-15. It showed that GDF-15 had no effect on prothrombin time (PT), activated partial thromboplastin time (APTT),
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thrombin time (TT), fibrinogen level, and the activity of thrombin and anti-thrombin (Table 3).
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Table 3 The effect of GDF-15 on thrombin and anti-thrombin activity
20ng/ml
PT,s
12.77±0.43
13.03±0.39
13.17±0.47
13.00±0.45
0.930
APTT,s
39.33±0.74
40.20±0.55
40.13±0.44
39.63±0.64
0.708
TT,s
17.33±0.62
17.77±0.58
17.50±0.61
17.47±0.43
0.955
2.68±0.31
2.55±0.39
2.44±0.40
2.41±0.38
0.956
99.00±3.79
95.00±2.65
93.67±2.96
88.00±4.04
0.228
Antithrombin activity,% 84.67±6.94 78.33±4.41 Data were shown as the mean ± SEM, n=6.
73.33±3.38
69.67±4.91
0.256
Discussion
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Thrombin activity,%
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Fibrinogen level, g/L
GDF-15
10ng/ml
50ng/ml
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CTRL
P value
According to the Virchow triad, DVT formation has traditionally been thought to be induced by blood stagnancy, blood hypercoagulability, and venous endothelial injury [14]. However, more and more studies suggest that inflammation is a common pathway through which various risk factors trigger DVT formation. In support of this, several inflammatory cytokines such as interleukin (IL)-6, TNF-α, and IL-8 were shown to be increased in DVT patients [15-17]. Our previous studies further indicated that IL-17A and IL-9 could promote DVT formation by facilitating platelet activation and aggregation [18-20]. Unfortunately, these inflammatory cytokines have not been applied to clinical diagnosis and therapy probably due to the low concentrations in DVT patients. In this study, we revealed for the first time that the levels of GDF-
Journal Pre-proof 15 were significantly increased in DVT patients with high concentrations. To explore the relationship between GDF-15 and thrombus severity, we analyzed the thrombus burden of DVT patients. The results showed that GDF-15 was increased with more venous segments of the lower extremity, suggesting that GDF-15 might be associated with thrombus severity in DVT. DVT severity is also associated with the occurrence of PE. Previous studies have verified higher levels of GDF-15 related to the severity of PE [10], but there is no evidence that it is associated with the occurrence of PE. In this study, we found that the patients with higher levels of GDF-15 and more thrombus segments showed higher scores of Wells-PE and Geneva for
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thrombus burden and PE occurrence in patients with DVT.
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predicting PE and increased incidence of PE. Therefore, GDF-15 might be a predictor for the
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Subsequently, we aim to find out whether the increased GDF-15 was involved in the thrombus forming within the vein. Thus, the effect of GDF-15 on platelet aggregation and
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thrombin/antithrombin activity were investigated. Platelets play a pivotal role in the initiation of
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venous thrombosis [21]. Rossaint et al showed that GDF-15 could reduce platelet aggregation by ADP, thrombin, or U46619 (thromboxane A2 analog) probably through inhibiting integrin
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activation in normal human and mice [22]. However, Giuseppe et al revealed that GDF-15 might be associated with overall global platelet hyperactivity in the whole blood from healthy controls
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[23]. These results appeared to be contradictory. In this study, we tested the effect of GDF-15 on platelet aggregation once again, and found that GDF-15 inhibited platelet aggregation induced by ADP not only in normal population but also in patients with DVT. In agreement with the study of Rossaint et al, both of us isolated platelet in vitro. It might be more reliable because we eliminated the interference of other components in the whole blood for platelet aggregation assay. However, we also found that GDF-15 had no effect on platelet aggregation induced by collagen in vitro. Previous studies have confirmed that ADP binds to two platelet purinergic receptors P2Y1 and P2Y12 to induce platelet aggregation by suppressing cAMP signaling pathway [24]. Differently, collagen promotes platelet adhesion and aggregation by binding to two major collagen receptors, GPVI and integrin α2β1, which lead to the activation of Syk-PI3K/PLCγ2 pathway [25]. GDF-15 promoted EGFR signaling which could enhance activation of cAMP [26, 27], but there is no evidence that GDF-15 could affect Syk-PI3K/PLCγ2 pathway according to the present reports.
Journal Pre-proof Therefore, we speculated that GDF-15 might inhibit ADP-induced platelet aggregation by enhancing cAMP response. More researches need to be carried out such as screening the specific receptor of GDF-15 on platelet and the signaling pathway in inhibiting platelet aggregation. The recent research has revealed that there is a weak positive association of GDF-15 with endogenous thrombin potential (ETP) in patients with atrial fibrillation [28]. However, the direct role of GDF-15 on thrombin activity remains unclear. In this study, we prove that GDF-15 did not influence thrombin activity in vitro. In agreement with the previous report that GDF-15 showed no association with thrombin-activatable fibrinolysis inhibitor (TAFI) and tPA [28], we also failed to
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find a significant influence of GDF-15 on anti-thrombin activity and fibrinogen level.
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In this study, we reported that increased GDF-15 level was associated with more venous
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segments of the lower extremity in DVT patients. GDF-15 could inhibit platelet aggregation induced by ADP with not influence on thrombin and antithrombin activity in vitro. It might be
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surprising since the increased GDF-15 indicate elevated thrombus severity conflicting with
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reduced platelet aggregation induced by ADP. In fact, it is similar to the mechanism of B-type natriuretic peptide (BNP) in heart failure. BNP is secreted mainly by cardiomyocytes reacted to a
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volume overload of ventricle. Increased BNP indicates elevated severity and worse prognosis of heart failure. At the same time, treating with recombinant human BNP can significantly improve
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the condition of patients with heart failure [29]. According to previous studies, GDF-15 was abundantly expressed in cardiomyocytes, macrophages, vascular smooth muscle cell and endothelial cells responded to tissue injury, anoxia and proinflammatory cytokines including interleukin-1β (IL-1β) and TNF-α [30-34]. Venous endothelial injury and inflammation are common pathways through which the risk factors trigger DVT formation. Therefore, we speculate that vascular endothelial cell injury and inflammation promote prothrombotic alterations, which induce the increased GDF-15 secretion and exert its anti-inflammatory and anti-platelet effects. Moreover, GDF-15-/- mice showed an accelerated thrombus formation with enhance platelet aggregation and neutrophil recruitment [22, 35]. Thus, GDF-15 might not only be an indicator for thrombus severity but also be a potential treatment target in DVT. There were some noted limitations in our study. Further clinical studies may also be to look into the relationship between GDF-15 and the prognosis of disease such as DVT recurrence or
Journal Pre-proof pulmonary embolism prediction in clinical follow-up study. Another direction for further studies could be conducted on the receptor expression of GDF-15 on platelet and the signaling pathway in inhibiting platelet aggregation. And repletion of GDF-15 in DVT mice would further clarify its potential effect in the treatment of thrombotic disease. Conflict of Interest Statement None. Funding This work was supported by the National Natural Science Foundation of China (81873497 to MY,
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and 81600317 to XXS).
Di Nisio M, van Es N, Buller HR. Deep vein thrombosis and pulmonary embolism.
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[1]
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[31] Schlittenhardt D, Schober A, Strelau J, Bonaterra GA, Schmiedt W, Unsicker K, Metz J, Kinscherf R. Involvement of growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in oxLDL-induced apoptosis of human macrophages in vitro and in arteriosclerotic lesions. CELL TISSUE RES 2004;318:325-33. [32] Ding Q, Mracek T, Gonzalez-Muniesa P, Kos K, Wilding J, Trayhurn P, Bing C. Identification of macrophage inhibitory cytokine-1 in adipose tissue and its secretion as an adipokine by human adipocytes. ENDOCRINOLOGY 2009;150:1688-96. [33] Ferrari N, Pfeffer U, Dell'Eva R, Ambrosini C, Noonan DM, Albini A. The transforming growth factor-beta family members bone morphogenetic protein-2 and macrophage inhibitory cytokine-1 as mediators of the antiangiogenic activity of N-(4-hydroxyphenyl)retinamide. CLIN CANCER RES 2005;11:4610-9. [34] Kempf T, Eden M, Strelau J, Naguib M, Willenbockel C, Tongers J, Heineke J, Kotlarz D,
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Journal Pre-proof Highlights Plasma GDF-15 were significantly increased in DVT patients with high concentrations.
Increased GDF-15 level was associated with thrombus severity of DVT.
GDF-15 could inhibit ADP-induced platelet aggregation.
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Wei Liang, Fen Wei, Chao Yang, Fen Xie, Xin-Xin Shuai, Min Wang, Miao Yu PII:
S0049-3848(20)30031-1
DOI:
https://doi.org/10.1016/j.thromres.2020.01.022
Reference:
TR 7581
To appear in:
Thrombosis Research
Received date:
24 November 2019
Revised date:
4 January 2020
Accepted date:
19 January 2020
Please cite this article as: W. Liang, F. Wei, C. Yang, et al., GDF-15 is associated with thrombus burden in patients with deep venous thrombosis, Thrombosis Research (2018), https://doi.org/10.1016/j.thromres.2020.01.022
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2018 Published by Elsevier.
Journal Pre-proof GDF-15 is associated with thrombus burden in patients with deep venous thrombosis Wei Liang *, 1, Fen Wei*, 1, Chao Yang#, 1, Fen Xie#, Xin-Xin Shuai*, Min Wang*, 2, Miao Yu *, 2 * Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China. # Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
1 Joint first authors: The first three authors contributed equally to the study.
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2 Corresponding authors: Miao Yu: Department of Cardiology, Union Hospital, Tongji Medical
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College, Huazhong University of Science and Technology, Wuhan, 430022, China. Tel: +862785726005. Fax: +862785727340. E-mail address: [email protected]. Min Wang:
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Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science
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and Technology, Wuhan, 430022, China. E-mail address: [email protected].
Journal Pre-proof Abstract Introduction: Growth differentiation factor-15 (GDF-15) has been identified as a predictor in cardiovascular diseases and acute pulmonary embolism. However, the association of GDF-15 and deep venous thrombosis (DVT) remains unclear. This study aimed to investigate levels of GDF-15 in patients with DVT, and determine its association with the thrombus burden. Materials and Methods: 72 newly diagnosed DVT patients and 30 healthy volunteers were enrolled, and the levels of plasma GDF-15 were detected. To explore the relationship between GDF-15 and thrombus severity, we analyzed the thrombus burden and the association with
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pulmonary embolism of DVT patients. In vitro, the effect of GDF-15 on platelet aggregation and
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thrombin/antithrombin activity were investigated.
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Results: We found that the mean levels of plasma GDF-15 in DVT patients were significantly higher than those in healthy controls (1448.78 ± 61.98 pg/ml VS 805.70 ± 112.95 pg/ml, P<0.001).
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Furthermore, GDF-15 showed an increase with more venous segments with thrombus (P<0.001),
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and the patients with higher levels of GDF-15 and more thrombus segments showed higher scores of Wells-PE and Geneva and increased incidence of pulmonary embolism (P<0.05). In vitro, we
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confirmed that GDF-15 significantly reduced platelet aggregation induced by ADP and the effect was concentration-dependent (P<0.001). However, GDF-15 showed no direct effect on thrombin
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and anti-thrombin activity.
Conclusions: Increased GDF-15 level was associated with more thrombus severity of DVT patients and GDF-15 could inhibit platelet aggregation induced by ADP in vitro. These findings suggest that GDF-15 might not only be an indicator for thrombus severity but also be a potential treatment target in DVT.
Keywords: GDF-15; deep venous thrombosis; thrombus burden
Abbreviations: GDF-15, growth differentiation factor-15; DVT, deep venous thrombosis; PE, pulmonary embolism; PRP, platelet-rich plasma; PPP, platelet-poor plasma; CTRL, control; ADP, adenosine diphosphate; PT, prothrombin time; APTT, activated partial thromboplastin time; TT, thrombin time.
Journal Pre-proof Introduction Deep venous thrombosis (DVT) is one of the most common peripheral vascular diseases in the worldwide. It is closely related to pulmonary embolism (PE), which leads to death in a substantial fraction of patients every year [1]. Recently, evidence has been accumulating that the factors influencing DVT formation are not only restricted to the coagulation system alone, but also the immune system is closely associated with formation of thrombosis [2]. Growth differentiation factor-15 (GDF-15) is a member of the transforming growth factor-β cytokine superfamily, which is known for its role in cell growth, differentiation, apoptosis, and
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inflammation [3, 4]. GDF-15 is weakly secreted in most tissues under physiological conditions,
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but strongly produced during inflammation [5]. Therefore, GDF-15 has been identified as a
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predictor for cardiovascular events, heart failure, and all-cause mortality in cardiovascular diseases, independently from traditional biomarkers such as cardiac troponins, natriuretic peptides,
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and C-reactive protein [6-9]. Recently, preliminary data have also suggested that GDF-15 may be
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valuable for stratifying risk of complications in patients with acute pulmonary embolism（PE） [10, 11]. However, the association of GDF-15 and DVT remains unclear.
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Therefore, this study aimed to investigate levels of GDF-15 in patients with DVT, and
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determine its association with the thrombus burden. Materials and Methods Patients
A total of 72 DVT patients diagnosed with lower extremity venous color Doppler ultrasonic in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were randomly recruited for this study. The ultrasound screening protocol included all deep and superficial lower extremity veins, including iliac vein, femoral vein, popliteal vein and tibial vein, with bilateral lower-extremity compression, followed by color and spectral Doppler ultrasound evaluation of filling and flow patterns. Patients with previously known DVT or signs of chronic DVT on Doppler ultrasound screening were excluded. Patients with any other acute or chronic inflammation-related diseases were excluded. None of the patients had been treated with antiinflammatory, antiplatelet, or anti-coagulant drug. Additionally, 30 aged and sex-matched healthy
Journal Pre-proof volunteers were enrolled as the controls in the study. Written informed consent was obtained from each patient and the study was conducted in accordance with the guidelines of the Declaration of Helsinki and its amendments. This research was approved by the Ethics Committee of Tongji Medical College at Huazhong University of Science and Technology. The clinical data of patients were collected. Blood samples were obtained from the patients and the healthy volunteers in the recumbent position and a fasting state on the next morning before any treatment, with a 21-gauge needle for clean antecubital venipuncture. Each sample of 2 ml was collected into vacutainer tube containing 3.2% sodium citrate. The blood samples were
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centrifuged at 2,500 g for 15 minutes and the plasma was stored at -80°C until analysis.
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Enzyme-linked immunosorbent assay
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Plasma levels of GDF-15 were detected with human GDF-15 enzyme-linked immunosorbent assay (ELISA) kit (RayBiotech, United States) according to the manufacturers’ instructions. The
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sensitivity of GDF-15 detection was 2 pg/mL. All samples were detected in triplicate.
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Platelet aggregation assay
Blood was obtained from the DVT patients and collected into vacutainer tube containing 3.2%
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sodium citrate. Platelet-rich plasma (PRP) was separated by centrifugation (100 g, 10 minutes, 20℃). The remaining blood sample was further centrifuged at 2000 g for 15 minutes to obtain
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platelet-poor plasma (PPP). Platelet aggregation was measured using a turbidimetric aggregation monitoring device (AggRAM, Helena Laboratories, USA). PPP was used to set the 100% aggregation. PRP was pre-warmed to 37°C in a resting state and then incubated with 10, 20, or 50 ng/mL GDF-15 (Peprotech, USA) for 10 minutes respectively. PRP treated without GDF-15 was designated as CTRL. Subsequently, 3μM adenosine diphosphate (ADP, Helena Laboratories, USA) or 10μg/ml collagen (Helena Laboratories, USA) was added to the plasma to induce aggregation. Platelet aggregability was evaluated by the maximal percent of platelet aggregation. The activity of coagulation factor and antithrombin assay The PPP was gathered from patients with DVT, and incubated with 10, 20, or 50 ng/mL GDF-15 (Peprotech, USA) for 10 minutes at 37°C in a resting state respectively. The change of coagulation and anticoagulation function was detected with Stago® automated haemostasis analyser according to the manufacturer’s instructions.
Journal Pre-proof Statistical analysis Data were shown as the mean ± SEM. IBM SPSS Statistics (version 22) was used for all statistical analyses. 2-tailed Student’s t test (two groups) or one-way ANOVA (more than two groups) was performed for the mean comparison. Fisher's exact test was performed for the categoric variables of clinical characteristics. P<0.05 was considered statistically significant. Results 1. GDF-15 level was increased in DVT patients
Compared with healthy controls, the DVT patients showed significant increased GDF-15
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levels (805.70 ± 112.95 pg/ml VS 1448.78 ± 61.98 pg/ml, P<0.001). There were no
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differences in age (P=0.107) and sex (P=0.515) between healthy controls and DVT patients.
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2. GDF-15 was associated with thrombus burden and the incidence of PE The deep lower extremity veins were divided into four segments as iliac vein, femoral vein,
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popliteal vein and tibial vein, and the number of vein segments involved in DVT patients could be
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an indicator of thrombus burden [12, 13]. GDF-15 levels were compared against number of segments involved in DVT, and the results are presented in Table 1. GDF-15 showed an increase
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with more venous segments with thrombus (Figure 1). Table 1 The levels of GDF-15 according to number of vein segments involved n/N
Mean±SEM (pg/ml)
1
18/72
1128.13±130.55
2
12/72
1190.04±151.80
3
22/72
1659.06±98.72
4
20/72
1661.28±67.23
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No. of segments with thrombus
Figure 1 The levels of GDF-15 in DVT patients with different numbers of vein segments involved.
Journal Pre-proof Pulmonary artery CTA was performed on 34 patients (47%) according to the decision of the clinicians, and 7 of them were combinated with PE. Lankeit M et al have reported that the upper limit of normal for GDF-15 level is 1200 pg/ml [10]. According to the number of segments involved in DVT (Table 1), the average values of GDF-15 in patients with No. of segments 1 and 2 were lower than 1200 pg/ml, and those in patients with No. of segments 3 and 4 were higher than 1200pg/ml. Therefore, we divided the 34 patients received pulmonary artery CTA according to number of vein segments involved in DVT, and their baseline characteristics were presented in Table 2. We found that patients with No. of segments 3-4 group had higher Wells-PE / Geneva
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scores and incidence of PE than those with No. of segments 1-2 group (P<0.05).
P value
55.62±3.96
58.19±2.71
0.596
9
9
0.172
23.31±0.46
23.13±0.79
23.41±0.59
0.779
5
1
4
0.627
10
3
7
0.704
9
5
4
0.254
57.21±2.23
Sex, male
18
Body mass index, kg/m²
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Diabetes
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Age, years
Hypertension
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Smoking
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No. of segments 3-4 n=21
Clinical characteristics
No. of segments 1-2 n=13
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ALL n=34
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Table 2 Baseline characteristics of patients received pulmonary artery CTA
Malignant disease
6
2
4
1.000
Recent surgery or immobilization
12
4
8
0.727
Combinated with PE
7
0
7
0.029
Wells-PE
5.84±0.32
4.62±0.49
6.60±0.34
0.002
Geneva
8.65±0.47
7.46±0.67
9.38±0.60
0.047
D-Dimer,ug/ml
5.23±1.12
4.24±2.06
5.84±1.32
0.496
PT,s
15.16±0.43
15.12±0.72
15.20±0.55
0.935
APTT,s
48.83±1.99
47.55±3.11
46.39±2.65
0.783
234.35±11.66
220.77±18.78
242.76±14.93
0.368
Scores
Biomarkers
PLT,10^9/L
Journal Pre-proof 3. GDF-15 prevented platelet aggregation induced by ADP To investigate the direct role of GDF-15 in platelet aggregation, we prepared the platelet suspensions in DVT patients and healthy controls, and performed the experiment of platelet aggregation in vitro. Compared with CTRL, the ratio of platelet aggregation administrated with 10, 20, or 50 ng/mL GDF-15 was calculated. We confirmed that GDF-15 significantly reduced platelet aggregation induced by ADP both in DVT patients and healthy controls. Moreover, the effect of GDF-15 on platelet aggregation induced by ADP was concentration-dependent. However, it showed that GDF-15 had no effect on platelet aggregation induced by collagen in DVT patients
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and healthy controls in vitro. (Figure 2)
Figure 2 The effect of GDF-15 on platelet aggregation induced by ADP and collagen
Journal Pre-proof (A) The representative picture for platelet aggregation treated with CTRL or different concentrations of GDF-15 induced by ADP (3μM) in DVT patients and healthy controls. (B) The representative picture for platelet aggregation treated with CTRL or different concentrations of GDF-15 induced by collagen (10μg/ml) in DVT patients and healthy controls. (C) The results of statistical analysis on the ratio of platelet aggregation treated with CTRL or different concentrations of GDF-15 induced by ADP in DVT patients and healthy controls, n=6, *: P<0.01 vs CTRL, #: P<0.01 vs 10ng/ml GDF-15 group, δ: P<0.01 vs 20ng/ml GDF-15 group. (D) The results of statistical analysis on the ratio of platelet aggregation treated with CTRL or different concentrations of GDF-15 induced by collagen in DVT patients and healthy controls, n=6, P>0.05. 4. GDF-15 did not influence thrombin and anti-thrombin activity
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To detect the direct effect of GDF-15 on thrombin and anti-thrombin activity, we prepared the
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PPP in DVT patients in vitro and pre-incubated with 10, 20, or 50 ng/mL GDF-15. It showed that GDF-15 had no effect on prothrombin time (PT), activated partial thromboplastin time (APTT),
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thrombin time (TT), fibrinogen level, and the activity of thrombin and anti-thrombin (Table 3).
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Table 3 The effect of GDF-15 on thrombin and anti-thrombin activity
20ng/ml
PT,s
12.77±0.43
13.03±0.39
13.17±0.47
13.00±0.45
0.930
APTT,s
39.33±0.74
40.20±0.55
40.13±0.44
39.63±0.64
0.708
TT,s
17.33±0.62
17.77±0.58
17.50±0.61
17.47±0.43
0.955
2.68±0.31
2.55±0.39
2.44±0.40
2.41±0.38
0.956
99.00±3.79
95.00±2.65
93.67±2.96
88.00±4.04
0.228
Antithrombin activity,% 84.67±6.94 78.33±4.41 Data were shown as the mean ± SEM, n=6.
73.33±3.38
69.67±4.91
0.256
Discussion
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Thrombin activity,%
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Fibrinogen level, g/L
GDF-15
10ng/ml
50ng/ml
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CTRL
P value
According to the Virchow triad, DVT formation has traditionally been thought to be induced by blood stagnancy, blood hypercoagulability, and venous endothelial injury [14]. However, more and more studies suggest that inflammation is a common pathway through which various risk factors trigger DVT formation. In support of this, several inflammatory cytokines such as interleukin (IL)-6, TNF-α, and IL-8 were shown to be increased in DVT patients [15-17]. Our previous studies further indicated that IL-17A and IL-9 could promote DVT formation by facilitating platelet activation and aggregation [18-20]. Unfortunately, these inflammatory cytokines have not been applied to clinical diagnosis and therapy probably due to the low concentrations in DVT patients. In this study, we revealed for the first time that the levels of GDF-
Journal Pre-proof 15 were significantly increased in DVT patients with high concentrations. To explore the relationship between GDF-15 and thrombus severity, we analyzed the thrombus burden of DVT patients. The results showed that GDF-15 was increased with more venous segments of the lower extremity, suggesting that GDF-15 might be associated with thrombus severity in DVT. DVT severity is also associated with the occurrence of PE. Previous studies have verified higher levels of GDF-15 related to the severity of PE [10], but there is no evidence that it is associated with the occurrence of PE. In this study, we found that the patients with higher levels of GDF-15 and more thrombus segments showed higher scores of Wells-PE and Geneva for
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thrombus burden and PE occurrence in patients with DVT.
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predicting PE and increased incidence of PE. Therefore, GDF-15 might be a predictor for the
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Subsequently, we aim to find out whether the increased GDF-15 was involved in the thrombus forming within the vein. Thus, the effect of GDF-15 on platelet aggregation and
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thrombin/antithrombin activity were investigated. Platelets play a pivotal role in the initiation of
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venous thrombosis [21]. Rossaint et al showed that GDF-15 could reduce platelet aggregation by ADP, thrombin, or U46619 (thromboxane A2 analog) probably through inhibiting integrin
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activation in normal human and mice [22]. However, Giuseppe et al revealed that GDF-15 might be associated with overall global platelet hyperactivity in the whole blood from healthy controls
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[23]. These results appeared to be contradictory. In this study, we tested the effect of GDF-15 on platelet aggregation once again, and found that GDF-15 inhibited platelet aggregation induced by ADP not only in normal population but also in patients with DVT. In agreement with the study of Rossaint et al, both of us isolated platelet in vitro. It might be more reliable because we eliminated the interference of other components in the whole blood for platelet aggregation assay. However, we also found that GDF-15 had no effect on platelet aggregation induced by collagen in vitro. Previous studies have confirmed that ADP binds to two platelet purinergic receptors P2Y1 and P2Y12 to induce platelet aggregation by suppressing cAMP signaling pathway [24]. Differently, collagen promotes platelet adhesion and aggregation by binding to two major collagen receptors, GPVI and integrin α2β1, which lead to the activation of Syk-PI3K/PLCγ2 pathway [25]. GDF-15 promoted EGFR signaling which could enhance activation of cAMP [26, 27], but there is no evidence that GDF-15 could affect Syk-PI3K/PLCγ2 pathway according to the present reports.
Journal Pre-proof Therefore, we speculated that GDF-15 might inhibit ADP-induced platelet aggregation by enhancing cAMP response. More researches need to be carried out such as screening the specific receptor of GDF-15 on platelet and the signaling pathway in inhibiting platelet aggregation. The recent research has revealed that there is a weak positive association of GDF-15 with endogenous thrombin potential (ETP) in patients with atrial fibrillation [28]. However, the direct role of GDF-15 on thrombin activity remains unclear. In this study, we prove that GDF-15 did not influence thrombin activity in vitro. In agreement with the previous report that GDF-15 showed no association with thrombin-activatable fibrinolysis inhibitor (TAFI) and tPA [28], we also failed to
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find a significant influence of GDF-15 on anti-thrombin activity and fibrinogen level.
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In this study, we reported that increased GDF-15 level was associated with more venous
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segments of the lower extremity in DVT patients. GDF-15 could inhibit platelet aggregation induced by ADP with not influence on thrombin and antithrombin activity in vitro. It might be
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surprising since the increased GDF-15 indicate elevated thrombus severity conflicting with
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reduced platelet aggregation induced by ADP. In fact, it is similar to the mechanism of B-type natriuretic peptide (BNP) in heart failure. BNP is secreted mainly by cardiomyocytes reacted to a
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volume overload of ventricle. Increased BNP indicates elevated severity and worse prognosis of heart failure. At the same time, treating with recombinant human BNP can significantly improve
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the condition of patients with heart failure [29]. According to previous studies, GDF-15 was abundantly expressed in cardiomyocytes, macrophages, vascular smooth muscle cell and endothelial cells responded to tissue injury, anoxia and proinflammatory cytokines including interleukin-1β (IL-1β) and TNF-α [30-34]. Venous endothelial injury and inflammation are common pathways through which the risk factors trigger DVT formation. Therefore, we speculate that vascular endothelial cell injury and inflammation promote prothrombotic alterations, which induce the increased GDF-15 secretion and exert its anti-inflammatory and anti-platelet effects. Moreover, GDF-15-/- mice showed an accelerated thrombus formation with enhance platelet aggregation and neutrophil recruitment [22, 35]. Thus, GDF-15 might not only be an indicator for thrombus severity but also be a potential treatment target in DVT. There were some noted limitations in our study. Further clinical studies may also be to look into the relationship between GDF-15 and the prognosis of disease such as DVT recurrence or
Journal Pre-proof pulmonary embolism prediction in clinical follow-up study. Another direction for further studies could be conducted on the receptor expression of GDF-15 on platelet and the signaling pathway in inhibiting platelet aggregation. And repletion of GDF-15 in DVT mice would further clarify its potential effect in the treatment of thrombotic disease. Conflict of Interest Statement None. Funding This work was supported by the National Natural Science Foundation of China (81873497 to MY,
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and 81600317 to XXS).
Di Nisio M, van Es N, Buller HR. Deep vein thrombosis and pulmonary embolism.
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[1]
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GDF-15 could inhibit ADP-induced platelet aggregation.
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