A Rise in Plasma Coagulability during the First 3 Months Following Provoked Venous Thromboembolism Is Associated with the Development of Post-Thrombotic Syndrome in Children and Young Adults: Analysis of the Kids-DOTT Multicenter Trial-Derived Biobank

Background:Knowledge of prognostic factors for the development of post-thrombotic syndrome (PTS) is limited, particularly in the pediatric population. Elevated plasma levels of D-dimer and FVIII, both markers of coagulation activation and inflammation, are associated with an increased risk of adverse thrombosis outcomes, including PTS, in children. Similarly, increased levels of inflammatory cytokines, such as interleukin-6 (IL-6), have been associated with venous endothelial dysfunction and fibrosis, suggesting a potential role in the development of PTS. Given the strong interplay between inflammation and coagulation, the aims of this study were to: (1)Investigate a hypothesized association between increased levels of plasma cytokines during the first 3 months post-diagnosis of deep venous thrombosis (DVT) with the development of PTS in patients <21 years old; and (2) to investigate an association between increased plasma coagulability during the first 3 months post-DVT diagnosis with the development of PTS.

Methods:We used banked plasma biospecimens and masked clinical data from an ongoing NHLBI-sponsored multinational multicenter trial of VTE treatment in patients <21 years old (the Kids-DOTT trial, NCT00687882). All patients with an extremity DVT who underwent PTS assessment at 1 year were included in this analysis. The Clot Formation and Lysis (CloFAL) spectrophotometric assay was performed, as previously described, on banked plasma samples obtained at 6 weeks and 3 months post-VTE diagnosis. All samples were pre-treated with heparinase prior to assay. Assay measurements included maximal amplitude of the CloFAL waveform (MA), time to maximal amplitude (T1), and area under the curve at 60 minutes, indexed to that of the pooled normal plasma standard (AUC60). Levels of IL-6, IL-8, IL-12/IL-23p40, IL-17, vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, interferon (IFN)-γ and C-reactive protein (CRP) were measured via V-Plex kit (Meso Scale Discovery, Rockville, MD). For each patient, the change in each laboratory parameter between the 3 months and 6 weeks post-VTE time points was calculated. The diagnosis of PTS was made at the one year follow-up assessment using the Manco-Johnson Instrument. Descriptive statistics were used to summarize data on patient and VTE characteristics as well as PTS outcome. Putative prognostic factors for PTS (age group, complete thrombus occlusion, CloFAL parameters, and inflammatory cytokines) were evaluated via univariate logistic regression, using odds ratios (OR) and 95% confidence intervals (CI). Variables with P-values <0.1 in univariate analyses were pre-specified for inclusion in an adjusted (multivariate) regression analysis. The blind was maintained in the Kids-DOTT trial throughout data transfer and analysis.

Results: A total of 80 patients were included in the present analysis. Median age was 12.7 years (range 0.04 - 20.8 years). Patient and VTE characteristics at baseline, as well as PTS outcomes, are summarized in Table 1. PTS developed in 34% of patients by the 1 year post-VTE follow up visit. Univariate logistic regression analysis identified change in CloFAL AUC60 (OR=1.46, 95%CI=1.02-2.09; P=0.036) and complete thrombus occlusion at 6 weeks post-VTE (OR=3.29, 95%CI=0.93-11.6; P=0.064) as putative prognostic factors for PTS (Table 2). After adjustment in multivariate regression, change in CloFAL AUC60 remained the only possibly-significant (P=0.06) independent prognostic factor for PTS. Specifically, for each 1 A.U. increase in CloFAL AUC from the 6 weeks to 3 months post-VTE diagnosis, the odds of PTS increased by more than 40% (OR=1.43, 95%CI=0.99-2.08; Table 3). Changes in none of the measured inflammatory cytokines were prognostic of PTS.

Conclusions: PTS developed in 34% of patients <21 years old enrolled in enrolled in an ongoing multicenter trial of provoked DVT treatment. A rise in plasma coagulability during the first 3 months post-DVT diagnosis, as measured by increase in AUC60 in the CloFAL assay is a candidate prognostic factor for PTS in patients <21 years old with provoked DVT. Future work should seek to investigate the mechanisms of increased plasma coagulability following a course of anticoagulation therapy in young VTE patients, and to substantiate findings of its potential prognostic significance for the development of PTS.

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