Statistical analysis
Quantitative variables were described using mean and standard deviation, and the Wilcoxon paired test was used to compare these variables. Correlation analyses between quantitative variables were carried out using Pearson’s ”R” correlation coefficient, while the Mann-Whitney U test was utilized for qualitative variables. In this study, statistically significant differences were defined as those with a probability of error of less than 5% (p<0.05). Data analysis was conducted using the SAS software version 9.4 (SAS Institute Inc. Cari, NC).
Results (413)
Thirty paediatric patients with spongiform VM who underwent a therapeutic procedure under sedation between February 2019 and March 2023 at the Vascular Anomalies Unit of the Hospital Universitario La Paz were enrolled. The baseline patient characteristics are presented in Table 1. The malformations corresponded to spongiform lesions predominantly located in the upper extremities. Ninety percent of patients experienced episodes of pain, although only 43.6% required pharmacological treatment during their evolution. Thirteen patients had previously undergone sclerotherapy procedures, with an average of 3.4 procedures per patient. Ethoxysclerol was the most commonly used sclerosing agent.
Table 2 lists the analytical parameters of the 30 patients. At the time of analysis, the patients were free of clinically relevant symptomatology and had not recently used drugs that could alter coagulation parameters. Thirty patients (75%) had fibrinogen levels above 500 ng/mL, which is the cut-off point used in deep vein thrombosis diagnostic and treatment algorithms , and only one patient had elevated fibrinogen levels. All other parameters were within normal range.
Table 3 displays the results of the statistical comparisons between extralesional and intralesional samples and the reference values. The A10 and MCF values of the EXTEM test and MCF values of the INTEM test in intralesional blood were found to differ from Oswald’s reference values , whereas those from extralesional blood where within the reference range. The results of the EXTEM test showed significant differences between the intralesional and extralesional blood samples in the clot formation process (A5 and A10). When the cohort was analysed based on the D-dimer cut-off point (500 ng/mL), patients with values ​​lower than the cut-off point (25%) did not exhibit significant changes, whereas patients with a D-dimer level > 500 ng/mL (75%) showed statistically significant differences in clot formation in both EXTEM and INTEM between the intralesional and extralesional blood samples. Table 4 and figure 1 show the results of the modified fibrinolysis phase analysis.
SDC-1 exhibited significant variability in the different settings in which it was measured. In the cohort of 15 patients, we observed a substantial decrease in SDC-1 levels within the endothelial lumen (30.6 ng/mL) compared with the levels in extralesional samples (34.9 ng/mL) and controls (35.6 ng/mL).
Possible correlations between D-dimer levels and parameters that have traditionally been associated with the ILC phenomenon (sex, phleboliths, location), together with significant analytical parameters of ROTEM® and SDC-1 levels, were analysed. No significant correlation was detected, except for a strong correlation between the intralesional and extralesional SDC-1 levels (correlation coefficient, 0.954).
Discussion (740)
Coagulation and fibrinolysis processes are intricate, and while some of the proteins and enzymes involved can be measured, these values do not accurately reflect the dynamic functionality of the systems at play. Recent research has relied on biochemical models of HUVEC and analyses of extralesional blood samples. Nätynki et al. demonstrated that mutations in TIE-2 produced alterations in the plasminogen-plasmin system. However, studies on patients with extralesional blood samples have been inconclusive .
Rotational thromboelastometry, or ROTEM®, is an instrument that enables the rapid and straightforward evaluation of coagulation dynamics. Despite its prior utilisation , we used it in the present study to analyse and compare intralesional blood samples from VM with extralesional blood samples from the same patient. In our cohort, we observed a loss of firmness of the intralesional clot, probably caused by hyperfibrinolysis.
During the initial phase, we observed modifications in intralesional blood values relative to the age-specific reference ranges of Oswald et al. . Specifically, the A10 (46.7 and 48 s) and MFC (51.4 and 51 s) parameters were below the normal range in both the EXTEM (extrinsic pathway) and INTEM (intrinsic pathway). Although there were no reference values for A5, they were significantly lower than those for the extralesional samples. These findings suggest that the intralesional clot does not reach its maximum potential relative to the reference values and extralesional samples. Moreover, this effect is more pronounced in patients with higher levels of D-dimer (> 500 ng/ml), significant alterations were observed in both INTEM and EXTEM.
The onset of coagulation, dependent on coagulation factors and anticoagulants is reflected in the CT and CFT variables, was normal in the analyses. The firmness phase, which is dependent on platelets and fibrinogen and was assessed in the extralesional samples, also showed normal values. Aronniemi et al, performed extralesional analyzes in a cohort of patients who predominantly presented a venous endothelium, but also lymphatic and capillary endothelium. According to our analysis, they identified a decrease in the proteins responsible for stabilising the clot in the final phase whereas 54.8% had antithrombin levels above their range . This anticoagulant molecule binds to heparan sulfate of SDC-1, and alterations in the anchoring molecule can cause alterations in the coagulation system .
VMs are characterised by a supposed hyperfibrinolytic component, as they exhibit elevated D-dimer levels and the presence of phleboliths . Viscoelastic studies allow the analysis of the fibrinolysis phase; however, they require a long time and lose their clinical usefulness. To address this issue, we have added recombinant tPA to accelerate fibrinolytic process. Under these circumstances, we detected hyperfibrinolysis in patients with VM. The alterations in the coagulation system that occur in VM are partially reflected in the intralesional samples because, as demonstrated in Figure 1, the hyperfibrinolytic potential is not fully transferred to the extralesional samples.
Endothelial dysfunction, resulting from modifications in the composition of the glycocalyx, is a key factor in certain pathologies with a high incidence and may act as a prothrombotic factor . In our study, we identified a significant decrease in SDC-1 within the endothelial lumen compared to extralesional samples.In the case of a quiescent and unaltered endothelium, SDC-1 levels should be similar, but in line with the hypothesis of Redondo et al., there is a persistent process of seeking quiescence, which could disrupt angiogenic and haemostatic regulatory systems .
ROTEM® enables the identification of diverse coagulation patterns in venous, lymphatic, and mixed endotheliums. In addition, we will be able to perform early detection of susceptible patients and the implementation of individualized prevention measures based on their coagulation pattern. Furthermore, ROTEM® facilitates the monitoring of anticoagulant treatments and evaluation of the effectiveness of therapies such as sclerotherapy. SDC-1 is the first molecule to show a strong correlation between intralesional and extralesional levels in VMs. This glycoprotein has been proposed as a possible biomarker in different scenarios . In our study, SDC-1 may serve as a novel endothelial biomarker, indicating the stability of the ecosystem and enabling more precise monitoring of treatment, as compared to D-dimer levels. This study has several limitations: Since we did not perform a genetic analysis of the malformations in which blood samples were obtained, we do not know the venous endothelial behaviour under different mutational scenarios, although most of the mutations identified in spongiform VMs are in the TIE-2 gene . Lastly, the control group recruited was of adults, given the difficulty in finding paediatric samples.
Conclusions (96)
The interaction between the endothelium and abnormal venous environment plays a crucial role in determining the clinical and prognostic outcomes of VM. Our findings suggest that endothelial dysfunction of genetic origin can impair basic cellular systems, particularly the coagulation system, which exhibits a notable reduction in clot quality due to excessive fibrinolysis. Syndecan-1 has the potential to serve as a sensor of endothelial well-being, given its strong correlation between intralesional and extralesional levels, which may lead to the identification of a range of values that pose no risk of clinical onset or progression of the lesion.
Conflict of Interest Disclosures (includes financial disclosures): The authors have no conflicts of interest to disclose.
Funding/Support: No funding was secured for this study.
Role of Funder/Sponsor (if any): None
Clinical Trial Registration (if any): None
Acknowledgements: None