Increased Levels of Endothelial Dysfunction Markers and Matrix Metalloproteinases in Patients with Severe Post-Thrombotic Syndrome
Authors: Bittar Luis Fernando , Orsi Fernanda Andrade , Zapponi Kiara Cristina Senger , Mazetto Bruna Moraes , De Paula Erich Vinicius , Annichino-Bizzacchi Joyce Maria 
Published: October 2019

Abstract
Introduction: Post-thrombotic syndrome (PTS) is a common and chronic complication of deep venous thrombosis (DVT) of the lower limbs, which is present in 20% to 50% of patients, and is associated with a high morbidity, low quality of life and significant cost to the healthcare system. Understanding the mechanisms involved in the pathophysiology of PTS may improve the strategies towards disease prevention and treatment. Thus, the aim of the study was to evaluate blood levels of markers of coagulation, inflammation, endothelial dysfunction and matrix metalloproeinases in patients with different grades of PTS severity.
Methods: Between January 2012 and May 2015, 31 out 600 consecutive patients with a history of DVT treated at the Hemostasis and Thrombosis Clinic - University of Campinas - Brazil, presented severe PTS and were selected for this study. The presence of PTS was confirmed and classified by Villalta scale. For each patient with severe PTS included in the study, we also included a patient with mild PTS, a patient without PTS, matched by age, gender and time from last DVT, and a healthy control volunteer matched by age and gender. The coagulation markers included FVIII plasma levels evaluated by one-stage clotting assay, and D-dimer by immunoturbidimetric assay. Multiplex analysis was used to determine parameters related to inflammation (IL-1β, IL-6, IL-8, IL-10 and IL-13, MCP-1, TNF-α, RANTES), endothelial dysfunction (thrombomodulin, endothelin-1, sP-selectin, sE-selectin, sICAM-1, sVCAM-1), angiogenic and cell proliferation (EGF, VEGF-C, VEGF-D, TGF-β, PDGF-AA, PDGF-AB/BB) and matrix metalloproteinases and their inhibitors (MMP-1 MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-12, MMP-13, TIMP-1, TIMP-2). The inflammatory marker C-reactive protein (CRP) was determined by nephelometry.
Results: Thirty-one patients with severe PTS were included: 8 male / 23 female, the median age was 51 years and the median time since the last DVT episode occurred was 51 months before the day of the enrollment for the study. The distribution of the groups was similar according to gender, age, ABO blood group, and the time since the last thrombotic episode occurred.
Patients with DVT had higher FVIII and D-dimer levels when compared to controls, but there was no difference according to PTS severity.
CRP was the only inflammatory marker increased in patients with DVT when compared to controls (0.33 vs. 0.15 mg/L; p = 0.01), and in patients with severe PTS when compared to mild and no PTS (0.40 vs. 0.31 and 0.26 mg/dL; respectively; p=0.04). Regarding endothelial dysfunction markers, our results showed a significant increase of sICAM-1 and thrombomodulin in DVT patients when compared to controls (53.6 vs. 45.5 ng/mL; p≤0,001; 2.16 vs.1.81 ng/mL, p=0.04, respectively), but there was no difference according to the PTS severity. Patients with severe PTS showed increased sE-selectin when compared to patients without PTS (74.74 vs. 68.71 ng/mL; p=0.02). Patients with severe PTS showed increased MMP-7 (8.8 vs. 7.2 ng/mL; p=0.04) and MMP-10 levels (509.3 vs. 394.6 pg/mL; p = 0.04) when compared to patients without PTS. Furthermore, patients with severe PTS showed decreased MMP-9 levels (101.5 vs. 146.4 ng/mL; p=0.04) when compared to mild PTS. There was no difference in the parameters associated to angiogenesis (VEGF, endotelin-1, EGF) or cell proliferation (TGF-β, PDGF-AA, PDGF-AB/BB) between the groups.
Discussion and Conclusions: The results may support the association of coagulometric, inflammatory and endothelial processes in severe PTS, in a well-defined cohort of patients with severe PTS. Previous studies in animal models have shown that matrix metalloproteinases have an important role in the resolution of venous thrombus, and this process leads to changes in vein wall. Our results are consistent with this finding, once we observed increased blood levels of MMPs and markers of endothelial dysfunction/activation in patients with DVT, especially those with severe PTS. We speculate that matrix metalloproteinases can play an important role in the development of severe PTS, culminating in a chronic endothelial dysfunction of the affected limb.

Disclosures
No relevant conflicts of interest to declare.