Abstract Oxidative stress and iron accumulation-induced ferroptosis occurs in injured vascular cells and can promote thrombogenesis. Transferrin receptor 1 (encoded by the TFRC gene) is an initial element involved in iron transport and ferroptosis and is highly expressed in injured vascular tissues, but its role in thrombosis has not been determined. To explore the potential mechanism and therapeutic effect of TFRC on thrombogenesis, a DVT model of femoral veins (FVs) was established in rats, and weighted correlation network analysis (WGCNA) was used to identify TFRC as a hub protein that is associated with thrombus formation. TFRC was knocked down by adeno-associated virus (AAV) or lentivirus transduction in FVs or human umbilical vein endothelial cells (HUVECs), respectively. Thrombus characteristics and ferroptosis biomarkers were evaluated. Colocalization analysis, molecular docking and coimmunoprecipitation (co-IP) were used to evaluate protein interactions. Tissue-specific TFRC knockdown alleviated iron overload and redox stress, thereby preventing ferroptosis in injured FVs. Loss of TFRC in injured veins could alleviate thrombogenesis, reduce thrombus size and attenuate hypercoagulability. The protein level of thrombospondin-1 (THBS1) was increased in DVT tissues, and silencing TFRC decreased the protein level of THBS1. In vitro experiments further showed that TFRC and THBS1 were sensitive to erastin-induced ferroptosis and that TFRC knockdown reversed this effect. TFRC can interact with THBS1 in the domain spanning from TSR1-2 to TSR1-3 of THBS1. Amino acid sites, including GLN320 of TFRC and ASP502 of THBS1, could be potential pharmacological targets. Erastin induced ferroptosis affected extracellular THBS1 levels and weakened the interaction between TFRC and THBS1 both in vivo and in vitro, and promoted the interaction between THBS1 and CD47. This study revealed a linked relationship between venous ferroptosis and coagulation cascades. Controlling TFRC and ferroptosis in endothelial cells can be an efficient approach for preventing and treating thrombogenesis. Keywords: Transferrin receptor 1, Vascular injury, Ferroptosis, Thrombogenesis, Thrombospondin 1 Graphical abstract Image 1 [35]Open in a new tab Abbreviation AAV Adeno-Associated Virus CD36 Collagen type I receptor CD47 Integrin-associated protein co-IP Coimmunoprecipitation COX2 Cyclooxygenase 2 CSA Cross-Sectional Area DFOM Deferoxamine Mesylate DHE Dihydroethidium DVT Deep Vein Thrombus FVs Femoral Veins GPX4 Glutathione peroxidase 4 GSH Glutathione GSSG Oxidized glutathione H2DCFDA 2′,7′-Dichlorodihydrofluorescein diacetate HUVECs Human Umbilical Vein Endothelial Cells ROS Reactive Oxygen Species MDA Malondialdehyde MMP Mitochondrial Membrane Potential PI Propidium Iodide PPI Protein‒Protein Interaction TF Tissue Factor TFRC Transferrin receptor 1 THBS1 Thrombospondin 1 TSA Tyramide Signal Amplification vWF von Willebrand Factor WGCNA Weighted Correlation Network Analysis 1. Introduction In clinical medical practice, trauma to deep venous tissues can readily result in deep vein thrombus (DVT), necessitating advanced pharmacological management strategies. Discovering potential mechanism of vascular injury and thrombogenesis is important to the treatment and drug intervention of DVT. Traumatic injury is a major cause of tissue redox stress, hypoxia, and ischemia reperfusion, factors above can induce cell death. Vascular cells in thrombosis can undergo various types of cell death, previous studies have shown that high iron levels are associated with thrombosis, suggesting the important role of iron homeostasis in thrombotic events [[36]1]. Ferroptosis is induced by the overload of iron and closely related to vascular injury [[37]2]. Iron levels in mechanical injured vascular tissue can be significantly increased, as proven in previous studies [[38]3]. Excess free divalent iron can exacerbate oxidative stress. Researchers created an iron load model in mice after long-term administration of dextran iron and found that iron accumulation significantly accelerated thrombosis after vascular injury and increased vascular oxidative stress [[39]4]. The formation of DVT accompanied by a reactive oxygen species (ROS) burst has been extensively reported in previous studies [[40][5], [41][6], [42][7], [43][8]]. ROS further react with polyunsaturated fatty acids on the cell membrane, causing ferroptosis [[44]9]. Our previous work showed that ferroptosis plays a positive role in the formation of thrombosis induced by vascular mechanical injury, and the use of ferroptosis inhibitors can reverse this process [[45]3]. Taken together, these findings suggest that mediating iron homeostasis in injured vessels may be an effective treatment strategy for thrombosis. In cells, transferrin receptor 1 (TFRC) regulates iron transport, storage, and other processes [[46]10]. TFRC is not only an initial marker and promoter of ferroptosis but is also a diagnostic biomarker of DVT [[47]11]. Several studies have shown that TFRC is highly expressed in DVT patients and animal models [[48]3,[49]11]. Furthermore, in early work, ferroptosis which was characterized by high TFRC expression, was identified through previous experiments [[50]12,[51]13]. What could TFRC regulate in thrombosis? There were few clues left in previous studies. Fortunately, a protein‒protein interaction network was built based on our previous proteomics analysis. Thrombospondin 1 (THBS1), a well-known adhesive glycoprotein that is involved in thrombosis, was identified as an interactive partner with TFRC [[52]3,[53]14]. These two proteins showed high and sustained co-expression in injured venous tissues. In this study, we examined the role of TFRC in DVT and explored the potential mechanism in vivo and in vitro. This is the first study on the effect of TFRC on thrombosis. We formulated the following hypotheses: (i) TFRC is highly expressed after vascular trauma and induces iron accumulation, causing ferroptosis; (ii) knocking down TFRC can ameliorate ferroptosis and thrombogenesis in rats and rescue erastin-induced ferroptosis in human umbilical vein endothelial cells (HUVECs); and (iii) an interaction exists between TFRC and THBS1, and controlling the expression of TFRC can affect the expression of THBS1. Our verification of these hypotheses provides experimental evidence and offers new insights into the pathophysiology and pharmacological intervention of thrombosis. 2. Materials and methods 2.1. Animals and experimental setup Eighty male Sprague Dawley rats (specific pathogen-free, average weight of 110 ± 22 g) were purchased from the Experimental Animal Center of Xi'an Jiaotong University. The rats were housed in individually ventilated cages under standard conditions. All the experimental procedures were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University. All the experiments were performed according to the UK Animals (Scientific Procedures) Act. All the rats were allowed to adapt to the environment for two weeks. The animal experiments included three parts ([54]Fig. S1A). Part 1: Twenty-four rats were divided into two groups: the sham and DVT groups (n = 12 for each group). Rats in the sham groups underwent only a sham operation, and rats in the DVT groups underwent DVT model establishment. Sham operation procedure: Anesthesia was induced and maintained via inhalation of 4 % and 1 % isoflurane. The femoral arteries, veins and nerves were separated via blunt dissection after the inner skin of the bilateral groin was incised. Both bilateral femoral veins were separated. The continuous suture method was used to close the incision. The DVT model was established according to a previously described method with simple modifications ([55]Fig. 1A) [[56]3,[57]15]. Briefly, anesthesia was induced and maintained via inhalation of 4 % and 1 % isoflurane. The femoral arteries, veins and nerves were separated via blunt dissection after the inner skin of the bilateral groin was incised. The FVs were crushed and blocked by fastening one tooth buckle of the 12.5 mm mosquito clamp at three positions for 3 s to cause venous injury. The model was established on both bilateral FVs. Severely clamped-crushed samples (in which both the media and adventitia were crushed) exhibited signs of hemorrhage or vein rupture and were excluded from the comparative analyses. For the rats that underwent DVT surgery, the injured limb was immobilized with a plaster bandage for 24 h. Fig. 1. [58]Fig. 1 [59]Open in a new tab ROS bursts and iron accumulation in injured femoral veins. (A) DVT establishment procedure, including skin preparation, surgical field exposure, vein, artery and nerve separation, clamp damage, gypsum immobilization and other steps. (B) Gross observation of the femoral veins. (C) Bleeding time of the tail vein (n = 6). The values are expressed as the mean ± SD of replicates. “**” (P < 0.01) indicates a significant difference according to the Mann‒Whitney U test. (D) Thrombosis rate. (E) Proportion of Grade 1 and Grade 2 thrombi. (E) Tissue pathological changes (n = 3); all the tissue samples were cross-sectioned, and the scale bar indicates 100 and 50 μm at low and high magnification, respectively. Endothelial cells (shown by the blue arrow), red blood cells (purple arrow), platelet trabeculae (black arrow), vascular smooth muscle cells (green arrow) and inflammatory cells (gray arrow) are labeled. (F) Perls staining (n = 3) shows the iron contents in vascular tissues (black arrows). The scale bar in the Perls staining images is 100 and 10 μm at low and high magnification, respectively. All the samples were cross-sectioned. (G) Quantitative measurement of tissue iron concentrations; n = 8. Values are expressed as the mean ± SD of replicates, and “****” (P < 0.0001) indicates a significant difference according to the unpaired t-test. (I) DHE fluorescence was used to detect ROS; n = 3. All the samples were cross-sectioned. The scale bar indicates 100 μm. Treatment with 0.3 % H[2]O[2] was used as a positive control. (J) Quantitative measurement of tissue ROS levels; n = 5. The values are expressed as the mean ± SD of replicates, and statistical analysis was performed with the Mann‒Whitney U test. “**” (P < 0.01) indicates a significant difference. (For interpretation of the references to color in this