The imbalance between von Willebrand factor and ADAMTS13 and the risk of venous thromboembolism

Introduction

Background

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that performs two essential functions in hemostasis: it mediates platelet adhesion at sites of vascular injury under shear stress conditions, and it is the carrier protein for procoagulant factor VIII (FVIII) in the circulation (1). VWF is synthesized by endothelial cells and megakaryocytes, and stored in the Weibel-Palade bodies of endothelial cells or in the α-granules of platelets (1). These organelles contain molecules that are larger than the normal circulating forms, known as unusually large (UL) VWF (2-4).

The multimeric size of VWF is a critical determinant for its biological activity. Larger molecules weight forms have greater potential for platelet interaction (5) and bind avidly to the extracellular matrix (6). These ULVWF multimers are cleaved into smaller molecules with lower prothrombotic activity by ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats), which is primarily synthesized and released from hepatic stellate cells and endothelial cells (7).

Rationale and knowledge gap

The importance of these proteins in hemostasis is exemplified by von Willebrand disease, the most common inherited bleeding disorder due to deficiency and/or abnormality of VWF (1), and by thrombotic thrombocytopenic purpura (TTP), an occlusive thrombotic microangiopathy associated with severe familial or acquired ADAMTS13 deficiency (4).

While low levels of VWF might lead to bleeding, increased levels have been associated to thrombosis, including myocardial infarction (8,9), ischemic stroke (10,11) and venous thrombosis (12,13). ADAMTS13 that regulates the multimeric size of VWF, and therefore is able to modulate its functional activity, has also been linked to arterial thrombosis (14-18), albeit with some controversy (19,20). With respect to venous thrombosis, a recent study has demonstrated an excess of rare and low-frequency coding single nucleotide variants (SNVs) of ADAMTS13 gene in patients with venous thrombosis when compared to controls (21).

Objective

Given the key role of ADAMTS13, VWF and FVIII in hemostasis and their close biological relationship, and the still limited data on the influence of ADAMTS13 on venous thrombotic disease, we aimed to investigate in the same population the effect of these proteins on venous thromboembolism (VTE) risk. We present this article in accordance with the STROBE reporting checklist (available at https://jlpm.amegroups.org/article/view/10.21037/jlpm-23-87/rc).

Methods

Study population

Between January 2007 and July 2011, 358 patients were consecutively admitted to a single anticoagulation clinic at Federal University of Sao Paulo, in Sao Paulo, Brazil, with a first objectively confirmed VTE event. The period of study inclusion for all subjects corresponded to the date of blood sampling (between January 2010 and June 2012). We included only patients with deep vein thrombosis (DVT) of the leg or pulmonary embolism (PE) with or without DVT (PE ± DVT). DVT was confirmed by compression ultrasonography and PE by helical computed tomography or combined ventilation/perfusion-scan. Patients were treated with oral anticoagulant (OAC) for at least 3 months, and inclusion occurred at least 1 month after stopping anticoagulation and ≥6 months after the thrombotic event. The period between VTE and sample collection was determined in accordance with the literature and aimed to reduce the interference of the inflammatory process with coagulation proteins (22,23).

Patients younger than 18 years (n=9) and older than 70 years (n=55), and with VTE at unusual sites (n=58) were excluded. Among the remaining 236 patients, we excluded those with known medical history of cancer (n=49), liver and renal failure (n=6), cardiovascular disease (n=17) and other conditions characterized by autoimmune/chronic inflammatory disease (n=22), such as systemic connective tissue disease, vasculitis and autoimmune hemolytic anemia. We further excluded patients still on anticoagulation (n=33) during the inclusion period due to various reasons (e.g., chronic thromboembolic pulmonary hypertension, recurrence after the VTE first event, deficiency of natural anticoagulants, lupus anticoagulant and prior history of recurrent thrombophlebitis). Finally, 33 patients did not participate in the study because of refusal or loss to follow-up. Therefore, 76 of the initially 358 VTE patients were included.

Controls were comprised of 96 individuals, submitted to a standardized questionnaire, with no personal history of thromboembolic events, recruited from acquaintances or partners of patients. Controls had no biological relationship with the patients and with each other, and were matched to patients by gender and age (±5 years at study inclusion). The other exclusion criteria for controls were the same as for patients, and in both groups pregnant or postpartum women at blood sampling were excluded.

Patients and controls were from the same geographic region and had similar ethnic backgrounds. It is important address that the Brazilian population is constituted of different ethnic backgrounds, an admixture of Caucasians, Africans, and Native Americans (24), and both patients and controls of the present study reflect Brazilian racial admixture.

Blood sampling

After an overnight fasting of 12 h, venous blood was collected with minimum stasis in 3.2% sodium citrate for the determination of hemostatic factors, in ethylenediaminetetraacetic acid (EDTA) for the determination of ABO blood group phenotype and in tubes without additives for C-reactive protein (CRP) determination. Within 30 minutes, all samples were centrifuged for 15 minutes at 2,000 ×g at room temperature and stored at −80 ℃ until use.

Materials

Commercial enzyme-linked immunosorbent assays (ELISAs) were used to measure VWF antigen (VWF:Ag) (StagoAsserachrom VWF:Ag, Diagnostica Stago, Asnièressur Seine, France) and ADAMTS13 antigen (Technozym ADAMTS13 Antigen ELISA, Technoclone, Vienna, Austria) in accordance with the manufacturer’s protocol. FVIII coagulant activity (FVIII:C) was determined on a Sysmex CA-560 analyzer using FVIII-deficient plasma. High-sensitivity CRP (hs-CRP) levels were measured on a Cobas 6.000 analyzer using an immunoturbidimetric method. In the present study, the results of ADAMTS13 antigen, VWF:Ag, and FVIII:C were obtained in µg/mL, percentage and percentage; respectively. The conversion into international units/mL (IU/mL) corresponds to 1 IU/mL = 1 µg/mL = 100% to standardize comparisons between studies (25).

Statistical analyses

Logistic regression model was used to calculate odds ratios (ORs) and their 95% confidence intervals (95% CI) as an estimate of the relative risk of VTE. All regression analysis was adjusted for gender and age at study inclusion. Further adjustment was made for potential confounders: ABO blood group (dichotomized as O and non-O groups) and hs-CRP (analyzed as a continuous variable).

High VWF:Ag (>150%) and FVIII:C (>150%) were defined by plasma levels of these hemostatic factors exceeding the 88^th and 94^th percentiles of the controls, respectively. To define low ADAMTS13 antigen levels we used the 10^th percentile of the controls (≤0.64 µg/mL). To calculate VTE risk, levels of VWF:Ag ≤88^th percentile, FVIII:C ≤94^th percentile and ADAMTS13 >P10^th percentile were used as reference categories. To investigate the association between VTE and the exposure to both high VWF:Ag and low ADAMTS13 antigen levels, the group with VWF:Ag ≤88^th percentile and ADAMTS13 >10^th percentile was used as the reference category. The selection of these percentiles was based on the Andersson et al. (18) in which the joint effect of high VWF:Ag and low ADAMTS13 supports previous findings suggesting that the ratio between VWF and ADAMTS13 in the same individual might be important as a risk for arterial thrombosis. There were 52 patients (68%) and 79 controls (82.5%) with VWF levels ≤P88^th and ADAMTS-13 >P10^th (reference category) versus 8 patients (10.5%) and 3 controls (3%) with high VWF and low ADAMTS-13 levels. This distribution resulted in a significant increase in the risk of VTE (OR =4.05; 95% CI: 1.03–15.98), which practically did not change with adjustment for age and sex (Table 1) and subsequently for CRP and fibrinogen (OR 4.29; 95% CI: 1.04–17.63). Further adjustment for elevated FVIII levels (>P94^th) attenuated the risk (OR 3.45; 95% CI: 0.81–14.72).

It is important to highlight that with the exception of the reference category (VWF ≤P88^th and ADAMTS-13 >P10^th), there were few individuals in the other categories, which widened the 95% CI and resulted in a less accurate calculation for VTE risk estimates.

Continuous variables were presented as median and 25^th–75^th percentile. The significance of difference in medians among independent samples was assessed by Mann-Whitney or Kruskal-Wallis tests. The chi-squared test was used to compare categorical variables among groups. Correlations between continuous parameters were calculated according to the Spearman’s rank correlation coefficient (r_s). A two-tailed P<0.05 was considered statistically significant. Statistical analyses were performed with Statistical Package for the Social Sciences, version 18.0.

Ethical consideration

The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by institutional ethics committee of Universidade Federal de São Paulo/Hospital São Paulo (No. CEP 1857/09) and informed consent was taken from all the patients.

Results

The casuistic was composed of 76 VTE patients and 96 controls and the characteristics of the study population are presented in Table 2.

The majority of patients were women with a median age of 42 years at the thrombotic event. The proportion of patients with DVT in compassion to those with PE ± DVT was almost 2:1.

In controls, non-O blood group subjects when compared to those with O blood group had higher median levels of VWF:Ag (P=0.001) and FVIII:C (P=0.003) but not of ADAMTS13 antigen (P=0.62). As expected, the correlation between VWF:Ag and FVIII:C levels was positive and strong (r_s=0.789, P<0.001) among controls. On the other hand, ADAMTS13 antigen levels correlated negatively and weakly with levels of VWF:Ag (r_s=−0.213, P=0.04) and FVIII:C (r_s=−0.251, P=0.01).

Figure 1 shows the distribution of VWF:Ag (Figure 1A), FVIII:C (Figure 1B) and ADAMTS13 antigen (Figure 1C) levels in patients and in controls. As expected, median (25^th–75^th percentile) VWF:Ag levels were higher in patients 116% (86–154%) as compared to controls 97% (76–130%), P=0.02. Median FVIII:C levels were also higher in patients 108% (94–123%) when compared to controls 100% (88–117%), P=0.03. However, there was no significant difference in median ADAMTS13 antigen levels between patients 0.91 µg/mL (0.78–1.08 µg/mL) and controls 0.93 µg/mL (0.79–1.22 µg/mL), P=0.18.

Figure 1 Plasma levels of Von Willebrand factor antigen, factor VIII and ADAMTS13 in patients with venous thromboembolism and in control subjects. (A) VWF:Ag. (B) FVIII:C. (C) ADAMTS13 antigen. Data are presented as median and the 25^th and 75^th percentiles. VWF:Ag, von Willebrand factor antigen; FVIII:C, factor VIII coagulant activity.

Table 1 shows the risk of VTE in relation to VWF antigen, FVIII and ADAMTS13 antigen plasma levels. The high levels of VWF:Ag and FVIII:C increased VTE risk and there was no substantial change in risk estimates after adjustment for hs-CRP and ABO blood group for VWF:Ag (OR 2.84, 95% CI: 1.18–6.83) and for FVIII:C (OR 2.90, 95% CI: 1.03–8.21). No significant association was observed between VTE and low ADAMTS13 antigen levels.

Next, we investigated ADAMTS13 antigen levels in relation to its substrate levels. For this purpose, VWF:Ag levels were grouped into different categories based on quartiles and the 88^th percentile of VWF:Ag measured in controls. As demonstrated in Table 3, median levels of ADAMTS13 antigen were calculate for each category of VWF:Ag in patients and in controls. Median ADAMTS13 antigen levels were similar in the first three quartiles of VWF:Ag in patients (P=0.96) and in controls (P=0.78). However, median ADAMTS13 antigen levels in the upper quartile of VWF:Ag were lower as compared to the median levels corresponding to the first three quartiles in patients (0.82 µg/mL, 0.55–1.00 versus 0.95 µg/mL, 0.81–1.11 µg/mL, P=0.02) and in controls (0.85 µg/mL, 0.67–1.02 versus 1.00 µg/mL, 0.80–1.25 µg/mL, P=0.06). Of note, there was a further decrease in ADAMTS13 antigen among subjects with high levels of VWF:Ag (>88^th percentile of controls). Their median protease levels were lower when compared to the levels of subjects with VWF:Ag ≤88^th percentile, in both patients (P=0.001) and controls (P=0.051).

Patients and controls were then categorized into four groups as depicted in Table 4. Eight patients (10.5%) and 3 controls (3.1%) had both high VWF:Ag levels and low ADAMTS13 antigen levels, which resulted in a 4-fold increased risk of VTE as compared to the reference category. Risk estimates did not substantially change with subsequent adjustment for hs-CRP and ABO blood group (OR 4.20, 95% CI: 1.02–17.32) but were attenuated when high FVIII:C levels entered in the regression model (OR 3.39, 95% CI: 0.79–14.55). Table 4 shows that when high VWF:Ag levels are combined with low ADAMTS13 antigen levels, the thrombosis risk was almost 2 times higher as compared to the risk conferred by the exposure of only high VWF:Ag.

In patients, the median time between the occurrence of VTE and blood collection was 14 months, with a range of 6 to 51 months. No correlation was found between time since the VTE event and levels of VWF (r_s=0.085, P=0.47), FVIII (r_s=0.013, P=0.91) and ADAMTS13 (r_s=−0.098, P=0.40).

Discussion

Key findings

In the present study, we investigated the relationship between levels of VWF:Ag and ADAMTS13 antigen in VTE patients and controls, and we found that elevated levels of VWF were associated with reduced levels of ADAMTS13. Additionally, subjects with high VWF and low ADAMTS13 had a 4-fold increased risk of VTE as compared to those with neither exposure.

Besides TTP, other pathological conditions have been associated with a reduction in ADAMTS13 levels such as liver cirrhosis (26,27), renal failure on hemodialysis (26), cancer (28), inflammatory bowel disease (27), systemic connective tissue disease (29), severe sepsis (30) and disseminated intravascular coagulation (31). In this case-control, some of the above-mentioned diseases were among our exclusion criteria to minimize the number of co-morbidities that might be possible confounders when investigating the association between ADAMTS13 levels and VTE. It is important to address that ADAMTS13 levels in those pathological conditions are not reduced to the extremely low values observed in TTP.

Comparison with similar researches

Given the epidemiological studies that associate VWF with arterial thrombosis (8-11) and the mice models that show its critical role in arterial thrombus formation (32-34), it is reasonable to consider that ADAMTS13 might be associated with thrombosis in vessels other than microcirculation. Indeed, plasma levels of ADAMTS13 were significantly lower in patients with acute myocardial infarction when compared to controls (14,15). In case-control studies in which patients were distant from the acute event, reduced levels of ADAMTS13 conferred an increased risk of both myocardial infarction (16-18) and ischemic stroke (18). In line with epidemiological research, the infusion of recombinant human ADAMTS13 after inducing focal cerebral or myocardial ischemia in mice models provided a protective effect by reducing infarct size (33,35).

Explanation of findings

In the present case-control composed of subjects without the main diseases that could affect either the occurrence of VTE or levels of VWF:Ag, FVIII:C and ADAMTS13, thrombosis risk increased approximately 3-fold in the presence of high VWF:Ag and FVIII:C levels. In view of epidemiological (12,13) and experimental (36,37) data that link VWF to venous thrombosis, it would be biologically plausible to also explore the role of its cleaving protease in venous thrombosis. In this study we found decreased levels of ADAMTS13 antigen in both VTE patients and control subjects with elevated VWF:Ag. ADAMTS13 levels were similar in the first three quartiles of VWF, ranging from 0.94 to 1.01 µg/mL, which were close to the levels described in normal human plasma (approximately 1 µg/mL) (38). However, median ADAMTS13 levels were lower in the highest quartile of VWF as compared to the median levels of the first three quartiles, and had a further decrease when VWF levels were raised above the 88^th percentile of the controls. Even though the imbalance between VWF and ADAMTS13 seemed to be more evident in patients, it was also observed in controls, indicating that the negative association between the two proteins might be a physiological phenomenon. Our results are consistent with prior studies involving healthy subjects where elevated levels of VWF antigen induced by the infusion of desmopressin were associated with a decrease in ADAMTS13 activity (39,40). Our results also are supported by findings of lower ADAMTS13 antigen during acute-phase reactions, such as inflammatory bowel disease, liver cirrhosis or after surgery, known to be associated with high levels of VWF (27).

The biological mechanisms underlying the imbalance between ADAMTS13 and VWF are not fully understood. One hypothesis is that ADAMTS13 antigen is exhausted by the excess of the substrate in the vascular endothelial cells. The rationale for this hypothesis is based on in vitro findings where under experimental flowing conditions ULVWF multimers secreted on the endothelial surface were rapidly cleaved by ADAMTS13 (41). Inflammation might be another plausible mechanism, since pro-inflammatory cytokines in vitro were shown to stimulate the release of ULVWF multimers from endothelial cells (42) and to inhibit the synthesis of ADAMTS13 (43). However, we consider unlikely that the imbalance between these two proteins was merely the result of an inflammatory state related to the post thrombotic period, as no correlation was found between time since the VTE event and levels of VWF and ADAMTS13. Moreover, in controls without thrombosis, ADAMTS13 also tended to be lower upon raised levels of VWF.

Recent studies point to the importance of evaluating the combined effect of VWF and ADAMTS 13 in various clinical conditions (25,44) and although we could not find a significant association between ADAMTS13 levels and VTE, when ADAMTS13 and its substrate were jointly analyzed, subjects with high levels of VWF and low levels of ADAMTS13 had a 4-fold increased risk of VTE when compared to those with neither exposure, and almost 2 times more chance to have VTE when compared to those with only high levels of VWF. Low levels of ADAMTS13 without the exposure to high levels of VWF had no effect on thrombosis risk (OR =1). This result might indicate that the present study was underpowered to detect a likely weak effect of ADAMTS13 on VTE risk. On the other hand, our finding might suggest that an imbalance between these two highly interconnected proteins rather than an independent role of ADAMTS13 would be more relevant in determining venous thrombosis risk. In the present study, the combined effect of high VWF and low ADAMTS13 on VTE risk was attenuated after adjustment for FVIII. It is important to point out that due to the current number of patients and controls, it is not possible to draw conclusion whether FVIII might act as a mediator on the association of VWF and its protease with VTE, and further studies with larger sample size are needed.

Of note, Lotta et al. (21) reported an association between rare and low-frequency coding SNVs of the ADAMTS13 gene and DVT. The authors also observed that patients with rare coding SNVs in ADAMTS13 gene had lower plasma levels of the protease when compared to those without them. In view of these findings, it is interesting to speculate on the existence of a genetic mechanism that could explain, at least partially, our results of an increased frequency of VTE patients as compared to controls with low levels of ADAMTS13 antigen and high levels of VWF:Ag.

Strengths and limitations

This study has limitations. Given the fact that blood samples were collected after the thrombotic event, hemostatic factors levels might have been affected by the acute-phase reaction of the post thrombotic period. However, in our study hemostatic factors were obtained at least 6 months after VTE, and by this time the acute-phase reaction is expected to be substantially attenuated (22). Moreover, we found no significant correlation between time since VTE event and levels of hemostatic factors. Another limitation was the sample size that might have impacted our results that presented wide 95% CI when data were stratified, such as the association of high VWF:Ag and low ADAMTS13 antigen with VTE and its attenuation with subsequent adjustment for FVIII:C. Therefore, our findings concerning the relationship between VWF-ADAMTS13 axis and VTE warrant further investigation. Finally, we excluded patients with known history of some of the main comorbidities that might be confounders on the association between VWF-ADAMTS13 axis and VTE, as renal failure treated with dialysis (26,45), cancer (28,46) and systemic connective tissue disease (29,47). Thus, the selection criteria of patients might have limited the application of our results to other less selective VTE population.

Implications and actions needed

Our findings are in line with two previous case-control studies on arterial thrombosis that demonstrated that the risk of having both high VWF and low ADAMTS13 was increased in comparison to neither exposure and to the exposure of only high VWF (17,18). Furthermore, our results linking high VWF and low ADAMTS13 with VTE along with the reported association of rare coding SNVs of the ADAMTS13 gene with DVT (21) reinforce the hypothesis of a biologically plausible role of VWF-ADAMTS13 axis in the pathophysiology of venous thrombosis.

Conclusions

Our data indicate that elevated VWF:Ag levels are associated with a reduction in ADAMTS13 antigen levels in both VTE patients and control subjects and an imbalance between these two highly interconnected proteins, as reflected by a combination of high VWF:Ag and low ADAMTS13 antigen levels, might affect thrombosis risk. Further studies are warranted to clarify the potential effect of the VWF-ADAMTS13 axis on VTE risk.

Acknowledgments

Funding: This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo under No. 05/56799-0).

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jlpm.amegroups.org/article/view/10.21037/jlpm-23-87/rc

Data Sharing Statement: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-23-87/dss

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-23-87/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.org/article/view/10.21037/jlpm-23-87/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by institutional ethics committee of Universidade Federal de São Paulo/Hospital São Paulo (No. CEP 1857/09) and informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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