Human Lymphatic Fluid Supports Coagulation Driven Primarily By the Extrinsic Pathway

The lymphatic system is a network of vessels, tissues, and organs whose main function is to maintain fluid balance. Lymphatic vessels form a unidirectional conduit system that traffics fluid derived from cell exudate and blood capillary leakage back to the bloodstream via the subclavian veins. This exchange introduces the potential for proteins from the blood to infiltrate the lymphatic system. Accordingly, previous analyses of lymphatic fluid, albeit limited, have established the presence of coagulation factors. Depending on their composition and concentration, these coagulation factors could present a risk for thrombosis in lymphatic fluid, especially when concurrent with other pathological conditions. For instance, although reports of thrombosis within the lymphatic system are rare, disruptions of blood flow, like those in cardiac conditions, can cause lymphedema and contribute to a hypercoagulable state. However, the current body of research lacks a comprehensive evaluation of coagulation within lymphatic fluid, thereby leaving our understanding of lymphatic thrombosis largely undefined. Here we evaluated coagulation in thoracic duct lymphatic fluid collected from pediatric patients at the Children's Hospital of Philadelphia. Coagulation of lymphatic fluid samples was evaluated using a thrombin generation assays (TGAs) as well as prothrombin time (PT) and activated partial thromboplastin time (aPTT) clotting assays. We also characterized antigen concentrations of major clotting factors using ELISAs. In TGAs initiated with low tissue factor (TF; 0.1 pM), most lymphatic fluid from patient samples (n= 52) had thrombin generation (TG) profiles comparable to pooled normal plasma (PNP). Average peak thrombin (IIa) and endogenous thrombin potential (ETP) were around 75% of PNP at 166.2 +/- 17.27 nM and 3185.7 +/- 314.3 nM*min, respectively. Lag times of lymphatic samples were slightly prolonged with an average of 14.1 +/- 1.9 min compared to PNP (10.2 +/- 1.4 min). Consistent with these results, lymphatic fluid samples also demonstrated appreciable clotting activity in PT assays, with most samples having a twofold increase in clot time in comparison to PNP (31.7 +/- 4.3 sec lymph vs. 13.6 +/- 0.6 sec PNP). In contrast to TF-initiated TGAs, initiation via the intrinsic pathway using a dilute aPTT reagent generally produced a weak TG profile (peak IIa: 86.9 +/- 15.4 nM, ETP: 2474.2 +/- 303.8 nM*min, lag time: 22.4 +/- 2.2 min) compared to PNP (peak IIa: 281.2 +/- 20.2 nM, ETP: 4195.58 +/- 279.1 nM*min, lag time: 21.25 +/- 3.75). These lymphatic fluid samples also had markedly prolonged aPTT clotting times compared to both PNP (32.6 +/- 0.4 sec) and FVIII deficient plasmas (96.1 +/- 3.0 sec). These findings suggest concentrations of one or more clotting factors involved in the intrinsic pathway may be low in lymphatic fluid compared to normal plasma. Coagulation assays of lymphatic fluid were supported by measurement of several key coagulation factors. ELISA data for FVIII revealed levels at 30% of PNP concentrations, which is consistent with antigen levels observed in moderate hemophilia A plasma (a FVIII deficiency with 5-40% of PNP antigen levels). Fibrinogen levels were also notably low at 30% of plasma concentrations. In contrast, FV antigen concentrations were just below the normal range of 50-150% antigen level at 43% while lymphatic FIX and FX antigen were above 50%, suggesting that none of these factors is limiting for coagulation in lymphatic fluid. Because lymphatic endothelial cells are known to express thrombomodulin, we also tested lymphatic samples for evidence of a functional Protein C (PC) pathway. The addition of soluble thrombomodulin to lymphatic fluid in TF-initiated TGAs produced a robust anti-thrombogenic effect comparable to a PNP control, confirming the presence of functional PC anticoagulant activity in lymphatic fluid. These data support further investigation of other anticoagulant regulators (e.g. antithrombin, tissue factor pathway inhibitor) in lymphatic fluid. Together these data confirm that human lymphatic fluid supports thrombin generation and clot formation in vitro, and this procoagulant activity is primarily TF-dependent. Further analysis of additional components of the pro- and anticoagulant pathways will provide a more complete understanding of coagulation in the lymphatic system and its potential to contribute to lymphatic thrombosis.