Plasma Hypercoagulability and Hypofibrinolysis Are Associated with Increased Markers of Inflammation in Pediatric VTE: Findings from the Kids-DOTT Trial Biobank

Background: The crosstalk between the immune and coagulation systems has been identified as a key driver for the development of venous thromboembolism (VTE) in preclinical models, and in limited adult studies. However, data investigating the association between plasma markers of inflammation and coagulation in pediatric VTE are scarce.

Aim: To investigate the association between global measures of plasma coagulative and fibrinolytic function with inflammatory markers in plasma of children with provoked VTE.

Methods: We analyzed banked plasma biospecimens collected from patients enrolled in the Kids-DOTT trial (NCT00687882), a multinational randomized controlled trial investigating duration of anticoagulation therapy in patients <21 years of age with a first episode of provoked VTE. Plasma samples were collected at 6 weeks and 3 months post-VTE diagnosis. The Clot Formation and Lysis (CloFAL) assay and the modified mini-euglobulin clot lysis assay (ECLA) were performed to measure overall coagulative (i.e.; CloFAL area under the curve [AUC]), and fibrinolytic functions (i.e.; modified mini-ECLA clot lysis time ratio [CLTR]) in plasma. Markers of inflammation including interleukins [IL] -1β, IL-6, and IL-8, C-reactive protein (CRP), and serum amyloid A (SAA) were measured using the MesoScale Discovery assay. Descriptive statistics were used to summarize demographic characteristics. Kendall Tau Rank correlation coefficient was used to assess the strength and direction of association between inflammatory markers and measures of hypercoagulability and hypofibrinolysis across timepoints (6-week and 3-month) and age groups (neonates <30 days old, children 30 days - <13 years, and adolescents 13 years - <21 years).

Results: A total of 169 plasma specimens from 85 participants (n=85, 6-weeks; n=84, 3-months) were analyzed. Median (IQR) age was 13.87(5.12-16.38) years, 53% were female. Levels of IL-6, CRP and SAA positively associated with increased plasma coagulability as measured by the CloFAL AUC, with stronger associations observed at the 6-week timepoint (IL-6 Ƭ_ƅ= 0.43 p= <.0001, CRP Ƭ_ƅ= 0.439 p= <.0001, and SAA Ƭ_ƅ= 0.382 p= <.0001), compared to the 3-month timepoint (IL-6 Ƭ_ƅ= 0.311 p= <.0001, CRP Ƭ_ƅ= 0.422 p= <.0001, and SAA Ƭ_ƅ= 0.36 p= <.0001). Similarly, levels of IL-6, CRP and SAA showed significant association with measures of impaired fibrinolysis (modified mini-ECLA CLTR) at the 6-week (IL-6 Ƭ_ƅ= 0.411 p= <.0001, CRP Ƭ_ƅ= 0.424 p= <.0001, and SAA Ƭ_ƅ= 0.326 p= <.0001) and 3-month timepoints (IL-6 Ƭ_ƅ= 0.263 p= .0004, CRP Ƭ_ƅ= 0.445 p= <.0001, and SAA Ƭ_ƅ= 0.291 p= .0001). When analyzed by age group, IL-6, CRP and SAA were positively associated with increased plasma coagulability in children (IL-6 Ƭ_ƅ= 0.498 p= <.0001, CRP Ƭ_ƅ= 0.477 p= <.0001, and SAA Ƭ_ƅ= 0.456 p= <.0001) and adolescents (IL-6 Ƭ_ƅ= 0.363 p= <.0001, CRP Ƭ_ƅ= 0.442 p= <.0001, and SAA Ƭ_ƅ= 0.341 p= <.0001), but not in neonates (IL-6 Ƭ_ƅ= 0.183 p= 0.323, CRP Ƭ_ƅ= 0.35 p= 0.059, and SAA Ƭ_ƅ= 0.317 p= 0.087). Both CRP and SAA were associated with impaired fibrinolysis in all three age groups (neonates: CRP Ƭ_ƅ= 0.475 p= 0.011, SAA Ƭ_ƅ= 0.39 p= 0.037; children: CRP Ƭ_ƅ= 0.461 p= <.0001, SAA Ƭ_ƅ= 0.381 p= <.0001; adolescents: CRP Ƭ_ƅ= 0.463 p= <.0001, SAA Ƭ_ƅ= 0.308 p= <.0001), while IL-6 was significantly associated with impaired fibrinolysis in children (Ƭ_ƅ= 0.389 p= <.0001) and adolescents (Ƭ_ƅ= 0.364 p= <.0001) but not neonates (Ƭ_ƅ= 0.186 p= 0.32).

Conclusions: Increased levels of IL-6, CRP, and SAA are associated with plasma measures of increased coagulability and hypofibrinolysis during the acute and subacute post-VTE period in children <21 years of age presenting with provoked VTE, implicating the role of thromboinflammation in the development of thrombosis in this population.