Introduction

Trauma-induced coagulopathy (TIC) is a dynamic and complex coagulation dysfunction, characterized by hypocoagulability in the early hours, resulting in hemorrhagic shock, and hypercoagulability following the hypocoagulable state, resulting in venous thromboembolism and multiple organ failure [1]. As a result, TIC is regarded as an independent risk factor for poor prognosis among trauma patients [2,3,4,5]. Most studies on TIC mainly focus on hypocoagulability, however, 22.2–85.1% of trauma patients within days of injury develop trauma-induced hypercoagulopathy (TIH). TIH raises the risk of thrombotic events and mortality by 2–4 times [3, 6, 7]. Hence, discovering the potential biomarkers of TIH is essential for medical practice [4].

Routine coagulation tests, such as prothrombin time (PT), activated partial prothrombin time (APTT), D-dimers (DD) and fibrinogen (or fibrin) degradation products (FDPs), are suggested as the diagnostic indicators of TIH. However, these are not widely accepted diagnostic criteria of TIH due to the insensitivity [4]. Lymphatic vessels, as the second circulatory pathway, drain approximately 10% of the interstitial fluid into the lymphatic system and form lymph [8]. At the end of lymphatic draining pathways, most lymph is returned to the circulatory system through the thoracic duct. In contrast to blood vessels, lymphatic vessels play a vital role in macromolecule transport, lipid metabolism and waste clearance [9,10,11,12,13]. Previous research also demonstrates that lymph is not a plasma ultrafiltrate and presents some unique substances [14]. Therefore, to compensate for the shortcomings of serum and plasma, lymph might be a potential and valuable source of novel biomarkers for TIH.

Our previous investigation found that lymphatic drainage insufficiency was caused by a significant amount of lymphatic platelet thrombosis (LPT) blocking lymphatic vessels on the first day post -traumatic fracture [14]. Approximately 155 proteins were identified as uniquely existing in the lymph, including extracellular matrix-related proteins, actin cytoskeleton reorganization markers, and pancreatic proteins [14]. In our study, O88767 (Parkinson disease protein 7 homolog, Park7), P05197 (Elongation factor 2, Eef2), P07824 (Arginase-1, Arg1), P11232 (Thioredoxin, Txn), P30152 (Neutrophil gelatinase-associated lipocalin, Lcn2), P31044 (Phosphatidylethanolamine-binding protein 1, Pebp1), Q01129 (Decorin, Dcn), Q63716 (Peroxiredoxin-1, Prdx1), P07943 (Aldo-keto reductase family 1 member B1, Akr1b1) are the up-regulated proteins enriched in top 5 GO terms of BP (Fig. 1B). Monika’s proteomic data [14] suggests that wounded patients’ lymph, rather than plasma, may include Park7, Pebp1, and Prdx1. PARK7 is abundantly expressed in the brain, skeletal muscle, and adrenal gland (BioProject: PRJNA280600) and regulates mitochondrial dysfunction and oxidative stress [24, 25]. PEBP1, a tiny scaffold protein, inhibits protein kinase cascades and promotes ferroptosis cell death by binding with 15-lipoxygenases (15-LO) to produce hydroperoxy-PE [26]. Prdx1 is an enzyme with several functions, including oxidative defense, aging, inflammation, redox signaling, cell cycle, and carcinogenesis [27]. This finding revealed that Park7, Pebp1, and Prdx1 in collected lymph were possible diagnostic markers of injured individuals.

TIH is caused by a complex interaction of numerous processes, involving vascular endothelial injury, platelet hyperactivity, excessive release of procoagulants, hyperfibrinogenemia, anticoagulant pathways impairment, and fibrinolysis shutdown [4]. Immunoregulatory platelet dysfunction is the main pathological mechanism of TIH, despite the platelet count is at a normal level [1, 28]. On the one hand, injury-induced platelet activation promotes platelets to bind with leukocytes, forms platelet-leukocyte aggregates and activates innate immune response [29, 30]. On the other hand, activated neutrophils and macrophages release extracellular traps to simulate platelet aggregation and thrombin formation [1]. Therefore, we assumed that thrombolysis therapy reduced not just TIH but also excessive immune response. Figure 2C shows that the lymph of the CLO group had much lower levels of pro-inflammatory and immune-associated proteins, as confirmed by the top 5 GO terms of BP. Except for Q5WRG2 (Angiogenin, Ang) and Q6P6T1 (Complement C1s subcomponent, C1s) were previously reported to be presented in both plasma and lymph after trauma [14], we discovered following new protein molecules of lymph, including P50115 (Protein S100-A8, S100a8), P52925 (High mobility group protein B2, Hmgb2), F7FP65 (Retinoic acid receptor responder protein 2, Rarres2), D3ZWD6 (Complement C8 alpha chain, C8a), P01836 (Ig kappa chain C region, A allele), P00697 (Lysozyme C-1, Lyz1), G3V9C7 (Histone H2B, Hist1h2bk), M0R485 (Peptidoglycan recognition protein 2, Pglyrp2), P55314 (Complement component C8 beta chain, C8b), Q91YB6 (Complement inhibitory factor H, Cfh), D3ZPI8 (Complement C8 gamma chain, C8g). Although these down-regulated proteins are linked to inflammatory and immunological responses, it’s unclear if they’re just found in lymph fluid. Hence, more study is needed to identify particular lymph biomarkers following TIH or TIH plus thrombotic treatment.

Insight of lymph metabolome

The metabolomic researches of trauma-induced hemorrhagic shock are continuously reported [31,32,33,34,35], while the metabolomic changes of TIH are still little known. We depicted the lymph metabolomics of TIH and the potential pharmaceutical effect of antithrombotic therapy.

Compared to the sham group, homocystine, the up-regulated metabolite in the lymph of the VEH group, is enriched in cysteine and methionine metabolism (Fig. 3B). Homocystine is synthesized via transmethylation of methionine and enzymatic reaction [36]. Homocystine is regarded as an independent risk factor for thrombotic disorders and cardiovascular disease [37,38,39]. In addition, cysteine and methionine metabolism is also significantly increased in the plasma metabolome of injured animals and patients [31,32,33,34,35]. This result indicates that homocystine of lymph and plasma might be a potential biomarker of TIH.

Lysophosphatidylcholine is the main active component of oxidized low-density lipoprotein and can expand inflammation and exacerbate diseases by inducing the migration of lymphocytes and macrophages to produce pro-inflammatory cytokines [40,41,42,43]. Koji et al. detected lysophosphatidylcholine significantly increased in the mesenteric lymph on the model of trauma-induced hemorrhagic shock [43]. To our surprise, compared to the sham group, lysophosphatidylcholine is the down-regulated metabolite in the lymph of the VEH group and enriched in choline metabolism in cancer and glycerophospholipid metabolism (Fig. 3C). This result is opposite to that of previous research probably due to different animal models. Additionally, compared to the VEH group, lysophosphatidylcholine was found to significantly increase in the lymph of the CLO group (Fig. 4B). We inferred that (1) the sham, VEH, and CLO groups induced lysophosphatidylcholine generation due to operation; (2) In VEH group, lysophosphatidylcholine was accumulated in the injured sites and failed to be transported into thoracic duct due to the blockage of lymphatic vessels by lymphatic platelet thrombosis; (3) In CLO group, unblocked lymphatic vessels drained increased lysophosphatidylcholine of injured sites into thoracic duct.

Cholic acid is the main component of bile acids in the human body and has versatile roles in maintaining bile acid homeostasis, alleviating metabolic inflammation, and protecting neural injury [44]. Compared to the VEH group, cholic acid, enriched in bile secretion and primary bile acid biosynthesis, is significantly up-regulated in the CLO group. This result suggests that lymphatic platelet thrombolysis not only alleviates TIH but also improves systematical pathology.

Docosahexaenoic acid is an acknowledged neuroprotective and anti-inflammatory agent with multiple functions of alleviating endoplasmic reticulum and oxidative stress and regulating autophagy [45,46,47,48]. N1-Methyl-2-pyridone-5-carboxamide is one of the major metabolites of nicotinamide and is elevated in renal failure, vascular inflammation, chronic ulcerative colitis, and asthma [49,50,51,52]. Compared to the VEH group, docosahexaenoic acid, and N1-Methyl-2-pyridone-5-carboxamide are decreased in the CLO group (Fig. 4C). This is probably because antithrombotic therapy improves lymphatic transport of docosahexaenoic acid and N1-Methyl-2-pyridone-5-carboxamide, thus inhibiting their accumulation at fracture sides and decreasing oxidative response and nicotinamide metabolism.

Isoleucine is a kind of branchedchain and an essential amino acid for humans and animals [53]. Isoleucine plays diverse roles in physiological functions and metabolic pathways, including maintaining the growth and development of animals, enhancing immunity, regulating glucose transportation, and stimulating protein synthesis [53,54,55,56]. Testosterone is synthesized and secreted by testicular Leydig cells and the adrenal cortex and is regulated by the hypothalamic-pituitary-gonadal axis [57]. Testosterone plays irreplaceable roles in the growth and development of the human body, maintaining musculoskeletal homeostasis and regulating post-traumatic stress disorder [58,59,60]. Although little literature reports their direct relationships with trauma,  the information above seems to imply that higher levels of isoleucine and testosterone are beneficial for patients with trauma. However, the data of lymph metabolomics indicated that the levels of isoleucine and testosterone in the CLO group were significantly lower than VEH group. Therefore, general analysis suggests that (1) thrombolysis therapy is good for relieving TIH, circulatory system disorder, and inflammatory and oxidative response; (2) but might lead to side effects, such as metabolic and endocrine disorders. In a word, these DEMs above are potential and sensitive biomarkers of TIH patients with antithrombotic therapy.

Limitations and outlook

TIH is a dynamic, sequential pathophysiological process. Our work focused on a single time point of TIH to evaluate the constitutive alterations of lymph using integrated proteome and metabolome. (2) To distinguish the different and unique biomarkers between blood-derived and lymph-derived samples, a time course co-analysis of plasma and lymph muti-omics in trauma patients is mandatory. (3) Because obtaining lymph is challenging, the previous collection of lymph in trauma patients is under the help of anesthesiologists and intensive care physicians. Multidisciplinary collaboration in clinical trials of trauma patients is required to investigate the relationship between the possible biomarkers and the prognosis of TIH in order to validate the sensitivity and specificity of screening lymph biomarkers in this study. (4) Given that men have a higher risk of traumatic fractures and TIH [61,62,63,64], we only employed young male rats in this experiment. To increase the study’s relevance and effect, both genders are suggested to be incorporated into the study design.