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