No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
TAM receptors, Gas6, and protein S: Roles in inflammation and hemostasis
Abstract
TAM receptors (Tyro3, Axl and Mer) belong to a family of Receptor Tyrosine Kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein S. Protein S is more commonly known as an important co-factor for protein C, as well as a direct inhibitor of multiple coagulation factors. To our current knowledge the functions of Gas6 are limited to TAM receptor-activation. When activated, the TAM receptors have effects on primary hemostasis and coagulation, and they display an anti-inflammatory or a pro-inflammatory effect, depending on cell-type. To comprehend the effects that the TAM receptors and their ligands have on hemostasis and inflammation, we compare studies that report the different phenotypes displayed by mice with deficiencies in the genes of this receptor family and its ligands (protein S(+/-), Gas6(-/-), TAM(-/-), and variations to these). In this manner we aim to display which features are attributable to the different ligands. Because of the effects the TAM receptors have on hemostasis, inflammation and cancer growth, their modulation could make interesting therapeutic targets in thrombo-embolic disease, atherosclerosis, sepsis, autoimmune disease and cancer.
... Gas6 (growth arrest-specific gene 6) is a secreted 75 kDa glycoprotein, whose function is vitamin K-dependent, and is expressed in many cell types and tissues, such as those of the heart, lungs, stomach, kidney, pancreas, bone marrow, central nervous system, and gut [1][2][3][4][5][6][7][8]. In physiological conditions, plasma Gas6 levels are around 18-50 ng/mL, which are sufficient to ensure its homeostatic functions [4,9]. ...
... Gas6 (growth arrest-specific gene 6) is a secreted 75 kDa glycoprotein, whose function is vitamin K-dependent, and is expressed in many cell types and tissues, such as those of the heart, lungs, stomach, kidney, pancreas, bone marrow, central nervous system, and gut [1][2][3][4][5][6][7][8]. In physiological conditions, plasma Gas6 levels are around 18-50 ng/mL, which are sufficient to ensure its homeostatic functions [4,9]. Gas6 is known to be involved in regulating many biological processes, ranging from cell proliferation, adhesion and migration, to efferocytosis and apoptosis. ...
... The formation of such Gas6/sTAM complexes represents a useful protection mechanism, intended to limit ligand-mediated signaling and, thus, to prevent the accidental activation of downstream TAM signaling pathways in cells and tissues [14,15,[18][19][20][21]. 2 of 13 It is noteworthy that Gas6 shows different binding affinities to the different members of the TAM family: it binds with the strongest affinity to Axl (with a Kd in the nM range), and with the lowest affinity to MerTK, with a Kd in the µM range [22,23]. Despite the different binding affinities to TAM receptors, the downstream activation mechanism is similar: once the ligand is bound, the receptor dimerizes and its tyrosine kinase domains become activated, mediating various cellular responses, such as cell growth and proliferation, the regulation of apoptosis, as well as the modulation of vascular and inflammatory responses in a cell and tissue-dependent manner [4,15,18,20,24]. The different cellular responses triggered by the Gas6/TAM axis activation mainly rely on well-known intracellular signaling mediators such as the p38/MAPK, the PI3K/Akt, the ERK1/2 and the JAK/STAT pathways, which need to work in a coordinated manner to ensure homeostasis in the whole organism [25][26][27]. ...
Gas6 (growth arrest-specific gene 6) is a widely expressed vitamin K-dependent protein that is involved in many biological processes such as homeostatic regulation, inflammation and repair/fibrotic processes. It is known that it is the main ligand of TAMs, a tyrosine kinase receptor family of three members, namely MerTK, Tyro-3 and Axl, for which it displays the highest affinity. Gas6/TAM axis activation is known to be involved in modulating inflammatory responses as well as fibrotic evolution in many different pathological conditions. Due to the rapidly evolving COVID-19 pandemic, this review will focus on Gas6/TAM axis activation in SARS-CoV-2 infection, where de-regulated inflammatory responses and fibrosis represent a relevant feature of severe disease manifestation. Furthermore, this review will highlight the most recent scientific evidence supporting an unsuspected role of Axl as a SARS-CoV-2 infection driver, and the potential therapeutic advantages of the use of existing Axl inhibitors in COVID-19 management. From a physiological point of view, the Gas6/TAM axis plays a dual role, fostering the tissue repair processes or leading to organ damage and loss of function, depending on the prevalence of its anti-inflammatory or profibrotic properties. This review makes a strong case for further research focusing on the Gas6/TAM axis as a pharmacological target to manage different disease conditions, such as chronic fibrosis or COVID-19.
... Gas6 has the highest affinity to Axl among the TAM receptor [14]. Therefore, we hypothesize that the protective effect of rGas6 is mediated by its receptor Axl. ...
... Interestingly, we found that the concentration of Gas6 is much higher (about 90-fold) than that of Pros1 in the CNS, which is contrast with that in peripheral circulatory system (PCS). In PCS Gas6 has a much lower concentration than Pros1 [14]. This might suggest Gas6 has more important function than Pros1 in the CNS. ...
... n = 6, *p < 0.05, **p < 0.01, and ***p < 0.001 versus sham group; #p < 0.05 and ##p < 0.01, versus SAH + vehicle group; &p < 0.05, &&p < 0.01, and &&&p < 0.001 versus SAH + rGas6 group, one-way ANOVA not rPros1, alleviated BBB disruption and improved neurological function. This indicates that Gas6 functions as a ligand for all three TAM receptors, with the highest affinity for Axl; in contrast, Pros1 activates Tyro3 and Mer, but not Axl, similarly as demonstrated before [14]. To determine the specific receptor that mediated the beneficial effect of rGas6, TAM receptors inhibitor BMS-777706 or Axl siRNA-3 were co-administrated with rGas6. ...
Early brain injury (EBI) following subarachnoid hemorrhage (SAH) is characterized by rapid development of neuron apoptosis and dysregulated inflammatory response. Microglia efferocytosis plays a critical role in the clearance of apoptotic cells, attenuation of inflammation, and minimizing brain injury in various pathological conditions. Here, using a mouse SAH model, we aim to investigate whether microglia efferocytosis is involved in post-SAH inflammation and to determine the underlying signaling pathway. We hypothesized that TAM receptors and their ligands regulate this process. To prove our hypothesis, the expression and cellular location of TAM (Tyro3, Axl, and Mertk) receptors and their ligands growth arrest-specific 6 (Gas6) and Protein S (ProS1) were examined by PCR, western blots, and fluorescence immunostaining. Thirty minutes after SAH, mice received an intraventricular injection of recombinant Gas6 (rGas6) or recombinant ProS1 (rPros1) and underwent evaluations of inflammatory mediator expression, neurological deficits, and blood–brain barrier integrity at 24 h. Microglia efferocytosis of apoptotic neurons was analyzed in vivo and in vitro. The potential mechanism was determined by inhibiting or knocking down TAM receptors and Rac1 by specific inhibitors or siRNA. SAH induced upregulation of Axl and its ligand Gas6. The administration of rGas6 but not rPros1 promoted microglia efferocytosis, alleviated inflammation, and ameliorated SAH-induced BBB breakdown and neurological deficits. The beneficial effects of rGas6 were arrogated by inhibiting or knocking down Axl and Rac1. We concluded that rGas6 attenuated the development of early brain injury in mice after SAH by facilitating microglia efferocytosis and preventing inflammatory response, which is partly dependent on activation of Axl and Rac1.
... Additionally, miles of researches have focused on blood coagulation and inflammation to elucidate the close relationship between them. Molecules that affect blood coagulation usually affect inflammation, and vice versa [25][26][27]. The Receptor tyrosine kinases TAM family, protein S (PROS1), GLA domain, and C2 domain, binding to PtdSer, all participate in both blood coagulation and immune processes, which means a crucial role of Ptdser in the immune and blood coagulation system (Fig. 1). ...
... TAM-RTKs (hereafter referred to as TAM) are members of the RTKs family. They are expressed by many cells, such as Macrophages [74], Dendritic Cells [74,75], Antigen presenting cell [74], immature natural killer (NK) cells [76], Cerebellar Purkinje cells [77], Hippocampal dentate gyrus [77], Retinal pigment epithelium (RPE) cells [78], and Sertoli cells [79] and play a vital role in hemostasis and anti-inflammatory [25,80,81]. ...
... TAM is a single transmembrane receptor (the orange part in the figure). From N-terminal to C-terminal (from top to bottom in the figure), there are three parts:1, the extracellular domain, including two IG domains (IG1, IG2) and two FNIII domains; 2, one single TM; 3, the intracellular domain, including one conserved PTK domain, one autophosphorylation site, and one ITIM domain [23][24][25][26][27]. The intracellular PTK domain of TAM is the functional domain. ...
The negatively charged aminophospholipid, phosphatidylserine (PtdSer), is located in the inner leaflet of the plasma membrane in normal cells, and may be exposed to the outer leaflet under some immune and blood coagulation processes. Meanwhile, Ptdser exposed to apoptotic cells can be recognized and eliminated by various immune cells, whereas on the surface of activated platelets Ptdser interacts with coagulation factors prompting enhanced production of thrombin which significantly facilitates blood coagulation. In the case where PtdSer fails in exposure or mistakenly occurs, there are occurrences of certain immunological and haematological diseases, such as the Scott syndrome and Systemic lupus erythematosus. Besides, viruses (e.g., Human Immunodeficiency Virus (HIV), Ebola virus (EBOV)) can invade host cells through binding the exposed PtdSer. Most recently, the Corona Virus Disease 2019 (COVID-19) has been similarly linked to PtdSer or its receptors. Therefore, it is essential to comprehensively understand PtdSer and its functional characteristics. Therefore, this review summarizes Ptdser, its eversion mechanism; interaction mechanism, particularly with its immune receptors and coagulation factors; recognition sites; and its function in immune and blood processes. This review illustrates the potential aspects for the underlying pathogenic mechanism of PtdSer-related diseases, and the discovery of new therapeutic strategies as well.
... Using pathway enrichment analysis, we found that GFP effectively exerted a protective effect against gastric bleeding through multiple pathways, influencing signaling by interleukins, cytokine signaling in the immune system, signaling by receptor tyrosine kinases, platelet activation, and others. It is widely acknowledged that the inflammatory mechanism is closely correlated with hemorrhage and with the regulation of several interleukins (55,56). In particular, interleukin 4 and interleukin 13 can induce downstream anti-inflammatory responses and result in hematoma and healing (57). ...
... Wound repair is associated with hemostasis, and cytokine signaling is one of the well-known pathways for wound healing (58). In addition, receptor tyrosine kinases stimulate hemostasis by promoting platelet stabilization and modulating inflammation (56). ...
Background:
As documented in the Chinese Pharmacopoeia, Gardeniae fructus Praeparatus (GFP) can cool the blood during hemostasis and treat various internal hemorrhagic diseases. However, the underlying mechanisms are not yet well understood. This work was designed to decipher the possible mechanism by which GFP prevents hemorrhage. The integration of pharmacodynamics-based and bioinformatics-based methods provided evidence to support the clinical effects of GFP in treating bleeding.
Methods:
Using ultra-performance liquid chromatography (UPLC) analysis, we quantified the main active ingredients for a preliminary quality assessment of GFP. The pharmacology study was conducted to confirm the essential antihemorrhagic effects of GFP. A rat model of ethanol-induced gastric hemorrhage was established and was followed by intervention with GFP in low, middle, and high doses (4.5, 9, 18 g/kg). Gastric tissues were harvested for macroscopic and histological evaluation of lesions. The contents of thromboxane B2 (TXB2) and 6-keto-prostaglandin-F1α (6-keto-PGF1α) in the serum were determined. Additionally, network pharmacology was proposed to illuminate the potential mechanisms. Following the collection of GFP compositions, the compound- and hemorrhage-related targets were retrieved from public databases. The protein-protein interaction (PPI), gene ontology, pathways analysis, and molecular docking were performed for targets of GFP in gastrointestinal bleeding.
Results:
The study found ten main active ingredients that could be used for quality control of GFP. Importantly, the middle and high doses of GFP were found to promote the healing of gastric bleeding. The content of 6-keto-PGF1α was significantly degraded in the middle and high treated groups (P<0.05). The level of TXB2 was augmented by a middle (P<0.05) and high dose of GFP. Further, we constructed the network of candidate ingredients and hemorrhage-related targets. Pathway analysis predicted the mechanisms associated with interleukin 4 and interleukin 13 signaling and platelet activation. PPI analysis identified subnetworks with biological functions and also sifted hub targets that affected the antihemorrhagic progress. The candidate proteins had a good binding force with major components.
Conclusions:
GFP exhibits a promising effect in ameliorating bleeding, with the relevant molecular mechanisms possibly being related to the regulation of the immune system and platelet activation. Therefore, GFP can potentially exert a protective effect on gastrointestinal bleeding in clinic.
... To our knowledge, this is the first report correlating lung signaling to PE driven by Gas6/AXL interaction. The vitamin K-dependent protein Gas6 is a secreted protein expressed in the liver, kidneys, lungs, and placenta [29,30]. This protein has a high affinity ...
... To our knowledge, this is the first report correlating lung signaling to PE driven by Gas6/AXL interaction. The vitamin K-dependent protein Gas6 is a secreted protein expressed in the liver, kidneys, lungs, and placenta [29,30]. This protein has a high affinity for the transmembrane receptor tyrosine kinase (RTK) AXL. ...
Preeclampsia (PE) is an obstetric complication associated with significant health implications for the fetus and mother. Studies have shown a correlation between lung disease development and PE. Gas6 protein is expressed in the lung and placenta, and binds to the AXL Tyrosine kinase receptor. Recently, our laboratory utilized Gas6 to induce preeclamptic-like conditions in rats. Our objective was to determine the role of Gas6/AXL signaling in the maternal lung during PE development. Briefly, pregnant rats were divided into control, Gas6, or Gas6 + R428 (an AXL inhibitor). Immunofluorescence was performed to determine AXL expression. Bronchoalveolar lavage fluid (BALF) was procured for the assessment of inflammatory cell secretion. Western blot was performed to detect signaling molecules and ELISA determined inflammatory cytokines. We observed increased proteinuria and increased blood pressure in Gas6-treated animals. AXL was increased in the lungs of the treated animals and BALF fluid revealed elevated total protein abundance in Gas6 animals. Extracellular-signal regulated kinase (ERK) and protein kinase B (AKT) signaling in the lung appeared to be mediated by Gas6 as well as the secretion of inflammatory cytokines. We conclude that Gas6 signaling is capable of inducing PE and that this is associated with increased lung inflammation.
... Many of the coagulant proteins also have signaling roles that can impact hemostasis ( Figure 1B), for example thrombin (the active form of prothrombin) activation of platelets through platelet activated receptors [18]. Platelet activation is essential for wound repair because it results in the incorporation of platelets into the fibrin clot, and platelet activation is also mediated by the VKD protein Gas6 [19,20], which is not a coagulation protein. ...
... Many of the hemostatic VKD proteins are also expressed in extrahepatic tissues, where they have additional roles, for example protein S that impacts complement and phagocytosis, protein C that regulates inflammation and barrier function, and Gas6 that functions in growth control [19][20][21][22][23][24][25][26] (Figure 1B). ...
Vitamin K-dependent (VKD) proteins undergo an unusual post-translational modification, which is the conversion of specific Glu residues to carboxylated Glu (Gla). Gla generation is required for the activation of VKD proteins, and occurs in the endoplasmic reticulum during their secretion to either the cell surface or from the cell. The gamma-glutamyl carboxylase produces Gla using reduced vitamin K, which becomes oxygenated to vitamin K epoxide. Reduced vitamin K is then regenerated by a vitamin K oxidoreductase (VKORC1), and this interconversion of oxygenated and reduced vitamin K is referred to as the vitamin K cycle. Many of the VKD proteins support hemostasis, which is suppressed during therapy with warfarin that inhibits VKORC1 activity. VKD proteins also impact a broad range of physiologies beyond hemostasis, which includes regulation of calcification, apoptosis, complement, growth control, signal transduction and angiogenesis. The review covers the roles of VKD proteins, how they become activated, and how disruption of carboxylation can lead to disease. VKD proteins contain clusters of Gla residues that form a calcium-binding module important for activity, and carboxylase processivity allows the generation of multiple Glas. The review discusses how impaired carboxylase processivity results in the pseudoxanthoma elasticum-like disease.
... Genetic deletion of all three TAM receptors (Tyro3/Axl/Mertk triple knock-out in mice) is not embryonically lethal. However, the animals display various postnatal phenotypes associated with thromboembolic disease, atherosclerosis, sepsis, inflammatory and autoimmune diseases (25). These phenotypes may be partly explained by a reduced ability to clear apoptotic cells observed in several tissues in association with increased levels of pro-inflammatory cytokines (e.g., TNF-a, IL-6) (25,26). ...
... However, the animals display various postnatal phenotypes associated with thromboembolic disease, atherosclerosis, sepsis, inflammatory and autoimmune diseases (25). These phenotypes may be partly explained by a reduced ability to clear apoptotic cells observed in several tissues in association with increased levels of pro-inflammatory cytokines (e.g., TNF-a, IL-6) (25,26). Indeed, TAM receptors are expressed on phagocytic cells, and alteration of TAM-mediated phagocytosis perturbs the clearance of apoptotic cells. ...
The development and implementation of Immune Checkpoint Inhibitors (ICI) in clinical oncology have significantly improved the survival of a subset of cancer patients with metastatic disease previously considered uniformly lethal. However, the low response rates and the low number of patients with durable clinical responses remain major concerns and underscore the limited understanding of mechanisms regulating anti-tumor immunity and tumor immune resistance. There is an urgent unmet need for novel approaches to enhance the efficacy of ICI in the clinic, and for predictive tools that can accurately predict ICI responders based on the composition of their tumor microenvironment. The receptor tyrosine kinase (RTK) AXL has been associated with poor prognosis in numerous malignancies and the emergence of therapy resistance. AXL is a member of the TYRO3-AXL-MERTK (TAM) kinase family. Upon binding to its ligand GAS6, AXL regulates cell signaling cascades and cellular communication between various components of the tumor microenvironment, including cancer cells, endothelial cells, and immune cells. Converging evidence points to AXL as an attractive molecular target to overcome therapy resistance and immunosuppression, supported by the potential of AXL inhibitors to improve ICI efficacy. Here, we review the current literature on the prominent role of AXL in regulating cancer progression, with particular attention to its effects on anti-tumor immune response and resistance to ICI. We discuss future directions with the aim to understand better the complex role of AXL and TAM receptors in cancer and the potential value of this knowledge and targeted inhibition for the benefit of cancer patients.
... Growth arrest-specific protein 6 (GAS6) is a vitamin K-dependent protein, and is widely expressed in the central nervous system (van der Meer et al., 2014;Shafit-Zagardo et al., 2018). Axl is a member of the TAM (Tyro3, Axl and Mertk) family of receptor tyrosine kinases which are all activated by the ligand GAS6 (Graham et al., 2014). ...
... January 2022 | Volume 9 | Article 784035 GAS6 is a vitamin K-dependent protein, first identified in murine fibroblasts in 1988. The biological functions of GAS6 are mediated by the interaction with TAM receptors, including cell survival and proliferation, immune regulation and apoptosis (van der Meer et al., 2014). Notably, the neuroprotective role of GAS6/ Axl axis has been identified in many studies. ...
Ischemic stroke is characterized by insufficient blood supply to brain tissue and is associated with increased morbidity and mortality in adults worldwide. Growth arrest-specific protein 6 (GAS6) is a vitamin K-dependent protein and is widely expressed in the central nervous system. The biological functions of GAS6 are mediated by the interaction with TAM (Tyro3, Axl and Mertk) receptors, including cell survival and proliferation, immune regulation and apoptosis. Omentin-1, also known as intelectin-1 (ITLN-1), is a novel adipocytokine that is involved in a variety of biological events, such as insulin resistance, endothelial dysfunction, programmed cell death and metabolic disorders. Our previous study has found that omentin-1 act as a novel regulator of vascular and anti-apoptotic response in cerebral ischemia. However, the specific molecular mechanism of omentin-1’s protective effect on cerebral ischemia-reperfusion injury (IRI) is still unclear. First, the toxicity of recombinant human omentin-1 (rh-omentin) was assessed and a safe concentration was chosen for the next experiments. Then, rh-omentin exerted neuroprotection against hypoxia/reoxygenation (H/R) injury in N2a cells, indicated by increased cell viability, decreased LDH, ROS generation, and cell apoptotic rate. Furthermore, the similar protective effect was observed in omentin-1 overexpression cells constructed by lentivirus transfection. Rh-omentin could also inhibit H/R-induced apoptotic molecules, oxidative stress molecules, and GAS6/Axl signaling molecules which as evidence by increased omentin-1, GAS6, Axl, p-Axl, NQO1, HO-1, Nrf2, Bcl2 and decreased Bax expressions. However, GAS6 siRNA could reverse rh-omentin-induced neuroprotection and the levels of these molecules mentioned above. In conclusion, these findings suggest that omentin-1 treatment exerts neuroprotection against H/R injury partly via activating GAS6/Axl signaling at least. Therefore, these finding may favor omentin-1 a potential neuroprotective drug candidate to alleviate ischemia-reperfusion injury in clinic.
... GAS6 (Growth arrest-specific protein 6)-ligand for tyrosine-protein kinase receptors AXL, TYRO3, and MER, whose signaling is implicated in cell growth and survival, cell adhesion, and cell migration. GAS6 has important effects on hemostasis and inflammation [40]. Its deficiency affects various processes such as preventing apoptosis of endothelial cells during acidification, cytokine signaling, hepatic regeneration, gonadotropin-releasing hormone neuron survival and migration, platelet activation, or regulation of thrombotic responses. ...
In patients with acute myeloid leukemia (AML), malignant cells modify the properties of multipotent mesenchymal stromal cells (MSCs), reducing their ability to maintain normal hematopoiesis. The aim of this work was to elucidate the role of MSCs in supporting leukemia cells and the restoration of normal hematopoiesis by analyzing ex vivo MSC secretomes at the onset of AML and in remission. The study included MSCs obtained from the bone marrow of 13 AML patients and 21 healthy donors. The analysis of proteins contained in the MSCs-conditioned medium demonstrated that secretomes of patient MSCs differed little between the onset of AML and remission; pronounced differences were observed between MSC secretomes of AML patients and healthy donors. The onset of AML was accompanied by a decrease in the secretion of proteins related to ossification, transport, and immune response. In remission, but not at the onset, secretion of proteins responsible for cell adhesion, immune response, and complement was reduced compared to donors. We conclude that AML causes crucial and, to a large extent, irreversible changes in the secretome of bone marrow MSCs ex vivo. In remission, functions of MSCs remain impaired despite the absence of tumor cells and the formation of benign hematopoietic cells.
... Gas6 is a vitamin K-dependent glycoprotein known to be involved in many homeostatic functions, as well as in regulating inflammatory responses. In physiological conditions, plasma Gas6 levels are usually low, while they increase in the case of inflammation [122][123][124][125][126][127][128][129][130]. ...
Clinical knowledge about SARS-CoV-2 infection mechanisms and COVID-19 pathophysiology have enormously increased during the pandemic. Nevertheless, because of the great heterogeneity of disease manifestations, a precise patient stratification at admission is still difficult, thus rendering a rational allocation of limited medical resources as well as a tailored therapeutic approach challenging. To date, many hematologic biomarkers have been validated to support the early triage of SARS-CoV-2-positive patients and to monitor their disease progression. Among them, some indices have proven to be not only predictive parameters, but also direct or indirect pharmacological targets, thus allowing for a more tailored approach to single-patient symptoms, especially in those with severe progressive disease. While many blood test-derived parameters quickly entered routine clinical practice, other circulating biomarkers have been proposed by several researchers who have investigated their reliability in specific patient cohorts. Despite their usefulness in specific contexts as well as their potential interest as therapeutic targets, such experimental markers have not been implemented in routine clinical practice, mainly due to their higher costs and low availability in general hospital settings. This narrative review will present an overview of the most commonly adopted biomarkers in clinical practice and of the most promising ones emerging from specific population studies. Considering that each of the validated markers reflects a specific aspect of COVID-19 evolution, embedding new highly informative markers into routine clinical testing could help not only in early patient stratification, but also in guiding a timely and tailored method of therapeutic intervention.
... It has the highest binding affinity to AXL, followed by Tyro 3 and Mer. 5 AXL is a transmembrane protein, but the extracellular part of the AXL receptor tyrosine kinase can be shedded from cells, resulting in a soluble receptor (sAXL). Ligands of TAM receptors can be inactivated by binding to soluble receptors, known as decoy receptors, which are formed by shedding the extracellular domains of these receptors. ...
Background & Objective: Ischemic stroke is the leading cause of death and long-term disability worldwide. In patients with ischemic stroke, both cell loss and inflammation are observed. GAS6/sAXL signaling is effective in both inflammation and clearance of dead/dying cells. This study investigated the GAS6/sAXL pathway and its role in patients with acute stroke. Specifically, we evaluated whether GAS6/sAXL was associated with stroke severity and infarct volume. Methods: This study involved 53 patients with acute ischemic stroke (AIS) and 49 healthy controls. GAS6 and sAXL proteins were collected in the first 24 hours in the acute stroke. NIHSS scores, GCS, and demographic data of the patients at the time of admission to the hospital were recorded. The infarct area was calculated using cranial magnetic resonance imaging. Results: Mean age of the patients was 64±12 years, 60% were female. HDL was lower in AIS group (40.5±13.01 mg/dl) than in the control group (55.4±14.9 mg/ dl) (p<0.05). The GAS6 levels of patients with ischemic stroke (30.58 [1.58-162.33] ng/dL) were significantly lower than the control group (83.33 [10.71-181.96] ng/dL) (p < 0.001). There was a significant difference in the GAS6/sAXL ratio between the AIS (8.60 [0.55-48] ng/mL) and control groups (14.78 [1.82-53.71] ng/mL) (p < 0.001). The serum GAS6 level and MR infarct area was positively correlated (r = 0.381 p = 0.005). The GAS6/sAXL ratio was positively correlated with the NIHSS and infarct area (p = 0.004). The GAS6/sAXL ratio and GCS showed a negative correlation (p = 0.001). Conclusion: Plasma GAS6 levels were positively correlated with infarct size, and the GAS6/sAXL ratio was positively correlated with the NIHSS score and infarct area in patients with AIS. Plasma GAS levels and GAS6/sAXL ratio can be used as an indicator of severity of AIS.
... These nonstandard AAs are primarily present in living organisms though they are not found in proteins. The modified calcium-binding γ-carboxy glutamic acid is abundantly present in blood-clotting proteins such as prothrombin and collagen (Van Der Meer et al., 2014). ...
Macromolecules are large polymer molecules made up of several smaller molecules bonded together by covalent bonds. These macromolecules can be either naturally occurring or chemically manufactured counterparts. All groups of biological and synthetic macromolecules are elucidated in detail structurally in this chapter, providing a fundamental grasp of its macromolecular chemistry underpinning numerous phenomena of life and their roles. Proteins, nucleic acids, lipids, and carbohydrates are all macromolecules, composed of biopolymers, which are the basic building blocks of life. There are several molecular applications for synthetic polymers, such as biomedical and therapeutics, which are also discussed. Overall, in this chapter, we have dealt with chemistry and structural aspects, notably with diverse in silico, cutting-edge structural biology methodologies to nanotechnology, providing a greater grasp of macromolecular chemical structure, function, and its applications.
... The extracellular domain of these receptors can be proteolytically cleaved and released into the plasma in soluble forms to act as decoy receptors of GAS6, which reduces interaction between the ligand and receptor [6,7]. GAS6/TAM interaction has been implicated in platelet function regulation [8], cell growth [9], phagocytosis of apoptotic cells [10], and reduction of inflammatory response [11]. GAS6 has been regarded as a risk factor for adverse effects in many diseases, such as systemic lupus erythematosus [12,13], rheumatoid arthritis [14], liver cirrhosis [15] and venous thromboembolism [16,17]. ...
Background
Growth arrest-specific 6 (GAS6) is a vitamin K-dependent protein related to inflammation, fibrosis, as well as platelet function. Genetic ablation of GAS6 in mice protects against cardiac hypertrophy and dysfunction. Nonetheless, the association between plasma GAS6 levels and acute heart failure (AHF) patients is still unknown.
Methods
We measured plasma GAS6 concentrations in 1039 patients with AHF who were enrolled in the DRAGON-HF trial (NCT03727828). Mean follow-up of the study was 889 days. The primary endpoint is all-cause death.
Results
In total, there were 195 primary endpoints of all-cause death and 135 secondary endpoints of cardiovascular death during the mean follow-up duration of 889 days. The higher levels of GAS6 were associated with higher rates of all-cause and cardiovascular death (P < 0.05). Baseline plasma GAS6 levels were still strongly correlated with clinical outcomes in different models after adjustment for clinical factors and N-terminal pro-brain natriuretic peptide (NT-proBNP, P < 0.05). GAS6 could further distinguish the risks of clinical outcomes based on NT-proBNP measurement.
Conclusion
Elevated plasma GAS6 levels were associated with an increased risk of all-cause and cardiovascular death in patients with AHF.
Trial registration NCT03727828 (DRAGON-HF trial) clinicaltrials.gov
... The anticoagulant activity of PS is mediated either by direct interaction with procoagulant and their active form or indirect interaction with other anticoagulant proteins such as activated protein C (APC) and tissue factor pathway inhibitor (TFPI). In addition, PS also serves as an activating ligand for the TAM family of receptor tyrosine kinases (RTKs), particularly TYRO3 and MERTK [7]. Signaling through RTKs will limit the intensity and duration of immune response with at least two distinct mechanisms: (1) the inhibition of proinflammatory cytokines production and secretion and (2) enhancing the clearance of apoptotic cells by phagocytosis [8,9]. ...
Patients with protein S (PS) deficiency possibly have a higher risk of developing severe COVID-19 disease. Therefore, vaccination against SARS-CoV-2 infections is recommended for PS-deficient patients. However, there are limited data regarding the safety and immunogenicity of the currently available COVID-19 mRNA vaccine in PS-deficient patients. We report a case of monitoring the antibody response of a 40-year-old female diagnosed with PS deficiency and on warfarin treatment following a single dose of BNT162b2 mRNA vaccine. Antibody against the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (anti-S) was measured on days 7, 14, and 21 after vaccination. Seroconversion was detected on day 21 but was possibly lower than the anti-S level previously reported in healthy individuals after receiving the first dose of the BNT162b2 mRNA vaccine. There were no local and systemic events reported up to 7 days in this patient after vaccination. This case highlights that the administration of the BNT162b2 vaccine had a favourable safety profile, and the second dose of the vaccine is required to provide the optimal protection against SARS-CoV-2 infection in PS-deficient patients.
... PS shares structural similarities with its homolog GAS6, and both PS and GAS6 bind to TAM tyrosine kinase receptors (TYRO3, AXL, and MER receptors) that regulate various biological processes including cell survival and inflammation (7). Although structurally similar, PS and GAS6 have disparate binding affinities to individual TAM receptors (8,9), leading to divergent functions (10). ...
Podocyte injury and loss are key drivers of primary and secondary glomerular diseases, such as focal segmental glomerulosclerosis (FSGS) and diabetic kidney disease (DKD). We previously demonstrated the renoprotective role of protein S (PS) and its cognate tyrosine-protein kinase receptor, TYRO3, in models of FSGS and DKD and that their signaling exerts anti-apoptotic and anti-inflammatory effects to confer protection against podocyte loss. Among the three TAM receptors (TYRO3, AXL, and MER), only TYRO3 expression is largely restricted to podocytes, and glomerular TYRO3 mRNA expression negatively correlates with human glomerular disease progression. We, therefore, posited that the agonism PS-TYRO3 signaling could serve as a potential therapeutic approach to attenuate glomerular disease progression. As PS function is not limited to TYRO3-mediated signal transduction but includes its anticoagulant activity, we focused on the development of TYRO3 agonist as an optimal therapeutic approach to glomerular disease. Among the small molecule TYRO3 agonist compounds screened, compound-10 (C-10) showed a select activation of TYRO3 without any effects on AXL or MER. We also confirmed that C-10 directly binds to TYRO3, but not the other receptors. In vivo, C-10 attenuated proteinuria, glomerular injury, and podocyte loss in mouse models of adriamycin-induced nephropathy and db/db model of type 2 diabetes. Moreover, these renoprotective effects of C-10 are lost in Tyro3 knockout mice, indicating that C-10 is a select agonist of TYRO3 activity that mitigates podocyte injury and glomerular disease. Therefore, C-10, a novel TYRO3 agonist, could be potentially developed as a new therapy for glomerular disease.
... Platelet stabilization occurs after integrin activation, granule secretion, and platelet aggregation through platelet-to-platelet contact. Without this mechanism, platelet plugs disaggregate prematurely (van der Meer et al., 2014). ...
Background: Gastric cancer (GC) is the most common malignant tumor. Due to the lack of practical molecular markers, the prognosis of patients with advanced gastric cancer is still poor. A number of studies have confirmed that the coagulation system is closely related to tumor progression. Therefore, the purpose of this study was to construct a coagulation-related gene signature and prognostic model for GC by bioinformatics methods.
Methods: We downloaded the gene expression and clinical data of GC patients from the TCGA and GEO databases. In total, 216 coagulation-related genes (CRGs) were obtained from AmiGO 2. Weighted gene co-expression network analysis (WGCNA) was used to identify coagulation-related genes associated with the clinical features of GC. Last absolute shrinkage and selection operator (LASSO) Cox regression was utilized to shrink the relevant predictors of the coagulation system, and a Coag-Score prognostic model was constructed based on the coefficients. According to this risk model, GC patients were divided into high-risk and low-risk groups, and overall survival (OS) curves and receiver operating characteristic (ROC) curves were drawn in the training and validation sets, respectively. We also constructed nomograms for predicting 1-, 2-, and 3-year survival in GC patients. Single-sample gene set enrichment analysis (ssGSEA) was exploited to explore immune cells’ underlying mechanisms and correlations. The expression levels of coagulation-related genes were verified by real-time quantitative polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC).
Results: We identified seven CRGs employed to construct a Coag-Score risk model using WGCNA combined with LASSO regression. In both training and validation sets, GC patients in the high-risk group had worse OS than those in the low-risk group, and Coag-Score was identified as an independent predictor of OS, and the nomogram provided a quantitative method to predict the 1-, 2-, and 3-year survival rates of GC patients. Functional analysis showed that Coag-Score was mainly related to the MAPK signaling pathway, complement and coagulation cascades, angiogenesis, epithelial–mesenchymal transition (EMT), and KRAS signaling pathway. In addition, the high-risk group had a significantly higher infiltration enrichment score and was positively associated with immune checkpoint gene expression. Conclusion: Coagulation-related gene models provide new insights and targets for the diagnosis, prognosis prediction, and treatment management of GC patients.
... The Gas6/Axl signaling has been shown to play an important role in inflammation, homeostasis, autoimmune diseases, nervous system, reproductive system, vascular system, cancer, and associated glucose intolerance with metabolic disorders [15,16]. ...
Objectives
Metformin is the first choice for type 2 diabetes mellitus (T2DM) treatment in the guidelines and is used in combination with many drugs. Growth arrest-specific protein 6 (Gas6)/Axl signaling plays a role in many metabolic disorders. This study aims to investigate the effects of metformin and metformin-insulin combination used in patients with T2DM on Gas6, Axl, and soluble Axl (sAxl) levels.
Materials and methods
A total of 71 patients diagnosed with T2DM and 21 healthy subjects were divided into 4 groups control, diet and exercises recommended (DER), metformin, and metformin + insulin. Diabetic patients were treated with metformin only or with a metformin-insulin combination and monitored for six months. Gas6, Axl, and sAxl levels of subjects’ sera obtained from their baseline and post-therapeutic sixth month blood samples were measured by ELISA methods.
Results
Compared to baseline, the sixth month Gas6 and Axl levels of Metformin and Metformin + Insulin groups significantly decreased (p<0.05). However, there was no statistically significant difference in sAxl values for these two groups of patients.
Discussion
The use of metformin in diabetic patients may be beneficial for inhibiting the Gas6/Axl pathway. This study presents a new aspect of the pleiotropic effects of metformin. This study will be clinically useful for designing therapeutic approaches targeting Gas6/Axl.
... In humans, ProS1 circulates in the plasma at 350 nM concentration, of which 30%-40% exists in a free form whilst 60%-70% is in complex with the β-chain of C4b binding protein (C4BP), which precludes it from binding TAMs [8]. While Gas6 is expressed widely across many tissues and cell types [9], ProS1 is mainly synthesised by liver hepatocytes, although local tissue expression in different organs has also been detected. The TAM receptors possess in the extracellular (N-terminal) region a combination of two immunoglobulin (Ig)-like and two fibronectin type III domains, a single-pass transmembrane domain, and an intracellular region with intrinsic tyrosine kinase activity [10]. ...
The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the C-terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed in silico homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction.
... We further explored the mechanism by which PF induces SOCS3 expression. The Axl receptor is an upstream regulator of SOCS3 (van der Meer et al., 2014). As shown in Figures 6A,D, compared with the control group, PF significantly increased the phosphorylation of Axl in RAW264.7 and HUVEC. ...
Radiation enteritis is one of the main adverse effects of radiotherapy, presenting with a poorly understood etiology and limited options for therapy. Intestinal inflammation and ischemia are the core mechanisms of radiation enteritis. Suppressor of cytokine signaling 3 (SOCS3) is an endogenous “inflammation brake.” We hypothesized that paeoniflorin, a pinane monoterpene bitter glycoside, could increase SOCS3 expression to reduce inflammation and ischemia and improve enteritis in mice. Laser Doppler flowmetry was used to detect changes in intestinal blood flow. RAW264.7 and human umbilical vein endothelial cells were used to investigate the mechanism of action of paeoniflorin. It was observed that radiation caused high mortality, intestinal inflammatory responses, and low blood flow in mice. Paeoniflorin effectively alleviated intestinal atrophy, prevented thrombosis, improved radiation enteritis, and reduced mortality in mice undergoing radiotherapy. In addition, paeoniflorin increased the release of growth arrest-specific gene 6 (Gas6) and phosphorylation of the Axl receptor, subsequently inducing the expression of SOCS3 and inhibiting the expression of p-apoptosis signal-regulating kinase 1 and tissue factor in vivo and in vitro . Based on our findings, we suggest that paeoniflorin is potentially effective in alleviating radiation enteritis via the activation of the Gas6/Axl/SOCS3 axis and subsequent reduction in intestinal inflammation and ischemia.
... However, acquired resistance develops in most patients receiving these new therapies, leading to cancer progression and fatal outcomes [8,9]. An important mechanism of acquired resistance to anticancer treatment is the overexpression of the Axl receptor tyrosine kinase [10][11][12]. Increased expression and activation of Axl contribute to primary and acquired drug resistance by stimulating mesenchymal differentiation and survival of cancer cells and suppressing the host immune response [13][14][15][16]. Furthermore, Axl activation can also promote the expression of immune checkpoint molecules and tumor growth [17]. ...
The leading cause of death worldwide is cancer. Many reports have proved the beneficial effect of mushrooms in cancer. However, the precise mechanism is not completely clear. In the present study, we focused on the medicinal properties of biomolecules released by fairy ring-forming mushrooms. Fairy chemicals generally stimulate or inhibit the growth of surrounding vegetation. In the present study, we evaluated whether fairy chemicals (2-azahypoxanthine, 2-aza-8-oxohypoxanthine, and imidazole-4-carboxamide) exert anticancer activity by decreasing the expression of Axl and immune checkpoint molecules in melanoma cells. We used B16F10 melanoma cell lines and a melanoma xenograft model in the experiments. Treatment of melanoma xenograft with cisplatin combined with imidazole-4-carboxamide significantly decreased the tumor volume compared to untreated mice or mice treated cisplatin alone. In addition, mice treated with cisplatin and imidazole-4-carboxamide showed increased peritumoral infiltration of T cells compared to mice treated with cisplatin alone. In vitro studies showed that all fairy chemicals, including imidazole-4-carboxamide, inhibit the expression of immune checkpoint molecules and Axl compared to controls. Imidazole-4-carboxamide also significantly blocks the cisplatin-induced upregulation of PD-L1. These observations point to the fairy chemical imidazole-4-carboxamide as a promising coadjuvant therapy with cisplatin in patients with cancer.
... Mer is a membrane tyrosine kinase receptor that belongs to the TAM receptor family. These receptors have pleiotropic effects on inflammation and hemostasis together with their ligand Gas6 [31]. During inflammatory processes, the cleavage of the extracellular part of Mer by ADAM17 results in the release of sMer [32]. ...
Soluble tyrosine kinase receptor Mer (sMer) and its ligand Growth arrest-specific protein 6 (Gas6) are predictors of mortality in patients with sepsis. Our aim is to clarify whether their measurement at emergency department (ED) presentation is useful in risk stratification. We re-analyzed data from the Need-Speed trial, evaluating mortality and the presence of organ damage according to baseline levels of sMer and Gas6. 890 patients were eligible; no association with 7- and 30-day mortality was observed for both biomarkers (p > 0.05). sMer and Gas6 levels were significantly higher in acute kidney injury (AKI) patients compared to non-AKI ones (9.8 [4.1–17.8] vs. 7.9 [3.8–12.9] ng/mL and 34.8 [26.4–47.5] vs. 29.8 [22.1–41.6] ng/mL, respectively, for sMer and Gas6), and Gas6 also emerged as an independent AKI predictor (odds ratio (OR) 1.01 [1.00–1.02]). Both sMer and Gas6 independently predicted thrombocytopenia in sepsis patients not treated with anticoagulants (OR 1.01 [1.00–1.02] and 1.04 [1.02–1.06], respectively). Moreover, sMer was an independent predictor of both prothrombin time-international normalized ratio (PT-INR) > 1.4 (OR 1.03 [1.00–1.05]) and sepsis-induced coagulopathy (SIC) (OR 1.05 [1.02–1.07]). An early measurement of the sMer and Gas6 plasma concentration could not predict mortality. However, the biomarkers were associated with AKI, thrombocytopenia, PT-INR derangement and SIC, suggesting a role in predicting sepsis-related organ damage.
... Moreover, an increase in TGFβ1 following activation of MerTK on macrophages led to HSC activation with increased collagen production [45]. Interestingly, TAM receptors and their ligands, especially Protein S, regulate hemostasis (previously reviewed in detail [49]). Moreover, patients with liver cirrhosis display changes in hemostasis, leading to thrombotic or hemorrhagic events, which are associated with high morbidity and mortality [50]. ...
TAM receptors (Tyro3, Axl and MerTK) are a family of tyrosine kinase receptors that are expressed in a variety of cell populations, including liver parenchymal and non-parenchymal cells. These receptors are vital for immune homeostasis, as they regulate the innate immune response by suppressing inflammation via toll-like receptor inhibition and by promoting tissue resolution through efferocytosis. However, there is increasing evidence indicating that aberrant TAM receptor signaling may play a role in pathophysiological processes in the context of liver disease. This review will explore the roles of TAM receptors and their ligands in liver homeostasis as well as a variety of disease settings, including acute liver injury, steatosis, fibrosis, cirrhosis-associated immune dysfunction and hepatocellular carcinoma. A better understanding of our current knowledge of TAM receptors in liver disease may identify new opportunities for disease monitoring as well as novel therapeutic targets. Nonetheless, this review also aims to highlight areas where further research on TAM receptor biology in liver disease is required.
... Growing evidences indicate that CSE-induced oxidative stress and inflammation may be prevented by antioxidant molecules, such as alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C) [37], resveratrol [29], glutathione, melatonin, lipoic acid and Coenzyme Q 10 (CoQ 10 ) [38]. Ubiquinol (QH), the active form of CoQ 10 , and vitamin K play a fundamental role in vascular health in physiological conditions by protecting the endothelium from oxidative damage, inflammation and by ensuring the functionality of important vitamin K-dependent proteins (VKDP), mainly known so far for their functions in preventing vascular calcification (MG-and GLA-rich proteins) [39] although VKDP have also recently been associated with anti-inflammatory activities [40]. Hence, it is possible that QH and K vitamins could also counteract CSE-induced damage in endothelial cells. ...
The liver is the only solid organ capable of regenerating itself to regain 100% of its mass and function after liver injury and/or partial hepatectomy (PH). This exceptional property represents a therapeutic opportunity for severe liver disease patients. However, liver regeneration (LR) might fail due to poorly understood causes. Here, we have investigated the regulation of liver proteome and phosphoproteome at a short time after PH (9 h), to depict a detailed mechanistic background of the early LR phase. Furthermore, we analyzed the dynamic changes of the serum proteome and metabolome of healthy living donor liver transplant (LDLT) donors at different time points after surgery. The molecular profiles from both analyses were then correlated. Insulin and FXR-FGF15/19 signaling were stimulated in mouse liver after PH, leading to the activation of the main intermediary kinases (AKT and ERK). Besides, inhibition of the hippo pathway led to an increased expression of its target genes and of one of its intermediary proteins (14-3-3 protein), contributing to cell proliferation. In association with these processes, metabolic reprogramming coupled to enhanced mitochondrial activity cope for the energy and biosynthetic requirements of LR. In human serum of LDLT donors, we identified 56 proteins and 13 metabolites statistically differential which recapitulate some of the main cellular processes orchestrating LR in its early phase. These results provide mechanisms and protein mediators of LR that might prove useful for the follow-up of the regenerative process in the liver after PH as well as preventing the occurrence of complications associated with liver resection.
Neurodegeneration, a progressive disease due to the arrested development of atrophy and regression of neuron function, is the most common disorder people go through. The treatment of the disease is generally symptomatic, and after a certain time, the effectivity of the drug decreases. A less toxic compound, vitamin K, a lipophilic (fat-soluble) vitamin, shows some promising results. Generally, vitamin K is found in three different forms: phylloquinone (vitamin K1) from green vegetables, menaquinone (vitamin K2) from anaerobic bacteria, and synthetic analog menadione (vitamin K3). The biological activities of each form of vitamin K differ from each other. Vitamin K1 is mainly stored in the liver and has a more prominent role in forming coagulation proteins. On the other hand, vitamin K2 is widely dispersed throughout the human body. Here in this review, the therapeutic role of vitamin K in different disorders, mainly focusing the neurological disorders, including neuroinflammation, Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis, is discussed. As a limited number of laboratories are involved in understanding vitamin K as a neuroprotective agent, the review will encourage further exploration in validating the neuroprotective effects of vitamin K among the pharmacognostic community.Graphical abstract
Objectives:
TAM Receptors (TYRO3, AXL, and MerTK) and their ligands on tumor-associated macrophages are promising therapeutic targets for most solid cancers. However, in endometrial cancer, the most common invasive gynecologic malignancy, the TAM receptor-mediated activation pathway, its molecular mechanisms, and its pathophysiology are unknown. The goal of this research; to uncover the comprehensive genetic profile of TAM receptors and ligands in endometrial cancer.
Material and methods:
Mutation and expression profiles of the Uterine Corpus Endometrial Carcinoma (UCEC) cohort (n = 509) were obtained using bioinformatics tools providing data from The Cancer Genome Atlas (TCGA). PolyPhen-2 and SNAP tools were used to predict the oncogenic/pathogenic properties of the identified mutations for UCEC. STRING network analysis was performed to better understand the functional relationships of the mutant proteins in cellular processes. Furthermore to the mutation profile, gene expression and survival profiles were also determined. Finally, the correlation between target genes and macrophage infiltration was investigated using the tool TIMER.
Results:
A total of 229 mutations were detected in 6 genes, and 81 missense mutations are pathogenic. In the UCEC cohort, the expression level of MerTK, AXL, GAS6, and PROS1 was statistically significantly lower in the patient group, while the expression level of CD47 was higher in the patient group than in the healthy group (p < 0.01). Protein-protein interaction analysis identified target genes, SRC protein responsible for important cellular mechanisms such as cell proliferation, adhesion and migration, ITGB3, ITGAV and THSB1 proteins involved in endothelial mesenchymal transition and tumor metabolism reprogramming, and FOLR1 involved in DNA replication and damage repair.
Conclusion:
We believe that TAM receptors and their ligands may be attractive molecular targets for the treatment of endometrial carcinoma because they act as pleiotropic inhibitors of immune cells, effectively regulate phagocytic clearance of apoptotic cells, and make the tumor microenvironment a more suitable niche for the tumour.
A novel compound (1) along with two known compounds (2 and 3) were isolated from the culture broth of Chlorophyllum molybdites, and three known compounds (4–6) were isolated from its fruiting bodies. The planar structure of 1 was determined by the interpretation of spectroscopic data. By comparing the specific rotation of the compound with that of the analog compound, the absolute configuration of 1 was determined to be R. This is the first time that compounds 2–4 were isolated from a mushroom-forming fungus. Compound 2 showed significant inhibition activity against Axl and immune checkpoints (PD-L1, PD-L2). In the bioassay to examine growth inhibitory activity against the phytopathogenic bacteria Peptobacterium carotovorum, Clavibacter michiganensis and Burkholderia glumae, compounds 2 and 3 inhibited the growth of P. carotovorum and C. michiganensis. In the bioassay to examine plant growth regulatory activity, compounds 1–4 showed a significant regulatory activity on lettuce growth.
Purpose
To investigate whether polymorphisms of GAS6 and PROS1, which each encode protein ligands for a family of tyrosine kinase receptors, are associated with Behçet’s disease (BD) in a Japanese population.
Methods
We recruited 734 Japanese patients with BD and 1789 Japanese healthy controls. In all participants, we genotyped two single-nucleotide polymorphisms (SNPs) reportedly associated with BD: rs9577873 in GAS6 and rs4857037 in PROS1.
Results
We found that GAS6 rs9577873 was not significantly associated with BD. In contrast, PROS1 rs4857037, specifically the A allele, was associated with increased risk for BD. The A allele was also significantly associated with BD under additive and recessive genetic models. Expression analysis revealed that this allele was significantly associated with increased PROS1 expression.
Conclusions
Our findings suggest that increased PROS1 expression related to the A risk allele of rs4857037 affects tyrosine kinase receptor signaling, contributing to the development of BD.
Different types of muscle contraction can cause different damage to the musculature and differences in inflammatory responses. Acute increases in circulatory inflammation markers can influence the crosstalk between coagulation and fibrinolysis processes, increasing the risk of thrombus formation and detrimental cardiovascular events. The aim of this study was to analyze the effects of concentric and eccentric exercise on hemostasis markers, CRP, and the relationship between these variables. Eleven healthy subjects with a mean age of 25.4±2.8, non-smokers, with no history of cardiovascular disease and blood type O, randomly performed an isokinetic exercise protocol consisting of 75 concentric (CP) or eccentric (EP) contractions of knee extension, divided into 5 sets of 15 repetitions combined with 30-second rest. Blood samples for analysis of FVIII, VWF, t-PA, PAI-1, and CRP were collected pre, post, 24h and 48h after each protocol. Increased levels of CRP at 48h in EP vs CP (p=0.002), increased PAI-1 activity 48h in EP vs CP (p=0.044), and a reduction in t-PA at 48h when compared to post-protocol in both protocols (p=0.001). A correlation was found between CRP and PAI-1 at 48h of PE (r2=0.69; p=0.02). The present study showed that both eccentric and concentric exercise protocols increase the clotting process, albeit only the exercise performed eccentrically induces inhibition of fibrinolysis. This is possibly due to the increase in PAI-1 48h after the protocol, which correlates with the increase in inflammation as demonstrated by the CRP levels.
BACKGROUND: Systemic lupus erythematosus (SLE) is an immune disorder with alternating active and remission phases. Cardiovascular diseases and thrombosis are the major causes of mortality in SLE. The anticoagulant activity of Protein S (PS) is complemented by C4 binding protein (C4BP) and tissue factor pathway inhibitor (TFPI). AIM: This study aims to determine the extent of change in the levels of PS activity, C4BP, and total TFPI in active SLE in comparison to the SLE remission phase and their association with thrombosis during SLE flare. METHODS: The study included 180 Egyptian SLE patients who were classified into two groups: 100 SLE cases as the active group and 80 SLE cases as the remission group. The PS activity levels were processed on automated coagulation analyzers, whereas the C4BP and total TFPI levels were measured via enzyme-linked immunosorbent assay. RESULTS: The PS activity and C4BP levels were lower in the active SLE cases than in the remitted ones (p < 0.05). The levels of PS activity and C4BP were revealed to be independent predictors of SELENA-SLEDAI flare scores. In active SLE cases, the PS activity and C4BP levels were rated as excellent and fair classifiers of thrombotic risk in SLE flare, respectively. The total TFPI levels showed no association with SLE activity or its thrombotic consequences. CONCLUSIONS: The levels of PS activity and C4BP act as important biomarkers for SLE activity. Both can be implanted as predictive tools for thrombosis during activity.
Oxidative stress plays a key part in cardiovascular event. Growth arrest-specific gene 6 (GAS6) is a vitamin K-dependent ligand which has been shown to exert important effects in heart. The effects of GAS6 were evaluated against hydrogen peroxide (H2O2) ‑induced oxidative stress injury in HL-1 cardiomyocytes. A series of experimental methods were used to analyze the effects of GAS6 on cell viability, apoptosis, oxidative stress, mitochondrial function and AMPK/ACC signaling in H2O2‑injured HL-1 cells. In this study, we found that H2O2 reduced cell viability, increased apoptotic rate and intracellular reactive oxygen species (ROS). Meanwhile, H2O2 decreased the protein levels of GAS6, and increased the protein level of p-AMPK/AMPK, p-ACC/ACC. Then, we observed that overexpression of GAS6 significantly reduced cell death, manifested as increased cell viability, improved oxidative stress, apoptosis and upregulated the levels of GAS6, p-Axl/Axl, Nrf2, NQO1, HO-1, Bcl-2/Bax, PGC-1α, NRF1, TFAM, p-AMPK/AMPK, and p-ACC/ACC-related protein expression in HL-1 cells and H2O2‑injured cardiomyocytes. To further verify the results, we successfully constructed GAS6 lentiviral vectors, and found GAS6 shRNA partially reversed the above results. These data suggest that AMPK/ACC may be a downstream effector molecule in the antioxidant action of GAS6. In summary, our findings indicate that activation GAS6/Axl-AMPK signaling protects H2O2‑induced oxidative stress which is accompanied by the amelioration of oxidative stress, apoptosis, and mitochondrial function.
La protéine S (PS) est une protéine vitamine K-dépendante qui limite la génération de thrombine à plusieurs niveaux de la coagulation. En effet, la PS joue un rôle bien connu de cofacteur de la protéine C activée (PCa), en stimulant son activité dans l’inactivation des facteurs Va (FVa) et VIIIa (FVIIIa). La PS est également un cofacteur de l’inhibiteur de la voie du facteur tissulaire (TFPIα) dans l’inhibition directe du facteur Xa. Cependant, l’importance relative de ces activités de la PS n’est pas connue in vivo. Afin de mieux comprendre les fonctions de la PS in vitro et in vivo, nous avons généré des nanobodies (anticorps à domaine unique produits chez les camélidés) dirigés contre la PS. Ces nanobodies ont été identifiés grâce à des stratégies variées de phage-display réalisées sur une banque immune obtenue à partir d’un lama immunisé avec de la PS recombinante humaine (rhPS). Parmi 14 nanobodies anti-PS identifiés (PS001 à PS014), le nanobody PS003 se liait fortement à la rhPS et présentait un effet de potentialisation tout à fait inattendu de l’activité cofacteur de la PCa de la PS, dans un test fonctionnel plasmatique. Nous avons donc choisi de caractériser ce nanobody plus en détail. Pour augmenter l’affinité du nanobody PS003 pour la PS, nous avons généré une forme bivalente du nanobody PS003 (PS003biv), mais seul un très faible gain d’affinité pour la rhPS, en ELISA, a été obtenu (constantes de dissociation apparentes de 26.8 ± 2.7 nM et 13.8 ± 5.7 nM pour PS003 et PS003biv, respectivement). En ELISA, les deux nanobodies reconnaissent le domaine SHBG isolé de la PS mais pas Gas6, qui est une protéine hautement homologue à PS et qui possède également un domaine SHBG. Ce résultat suggère donc que les nanobodies PS003 et PS003biv sont spécifiques de la PS. PS003 et PS003biv potentialisent tous deux l'activité cofacteur de la PCa de la PS (de 40 % et 80 % respectivement) dans un test de coagulation plasmatique, et de façon intéressante le nanobody PS003biv présente un effet de potentialisation plus important. Aucun effet de potentialisation de ces nanobodies n'a été observé sur l'activité de cofacteur du TFPIα de la PS en système purifié. Nous avons ensuite évalué le potentiel effet anti-thrombotique de PS003biv dans un modèle murin de thrombose induite par du chlorure de fer au niveau de la microcirculation du mésentère. L'injection de PS003biv (IV 10 mg/kg) a prolongé le temps d'occlusion dans les veinules mésentériques par rapport à un nanobody contrôle et semble induire la formation de thrombi instables et très susceptibles de former des emboles. De façon intéressante, dans un modèle de saignement de type « tail-clip », les souris injectées avec PS003biv (IV 10 mg/kg) ont un temps de saignement et un volume de sang perdu normaux, comparativement à des souris non injectées. En conclusion, le nanobody PS003biv potentialise l'activité cofacteur de la PS sur la PCa et présente un effet anti-thrombotique in vivo, sans affecter l'hémostase physiologique. Ainsi, l’augmentation de cette activité de la PS avec PS003biv pourrait constituer une stratégie thérapeutique anti-thrombotique originale. À l’heure actuelle, le mécanisme d’action des nanobodies PS003 et PS003biv reste inconnu, probablement complexe, et pourrait faire intervenir un autre partenaire de la PS que la PCa. En effet, nous n’avons pu retrouver l’effet de potentialisation de PS003 et PS003biv observé dans le test de coagulation plasmatique dans un test d’inactivation du FVa ou du FVIIIa par la PCa en présence de PS. Les travaux réalisés au cours de cette thèse apportent également une preuve de concept que des nanobodies anti-PS modulant l’activité anticoagulante de la PS peuvent être identifiés. Ces travaux pourraient ouvrir la voie à des stratégies de sélection par phage-display visant à identifier des nanobodies bloquant spécifiquement l’activité cofacteur de la PCa et du TFPIα.
Background: In recent years, many studies have found that vitamin K is beneficial to wound healing. However, some research results seem to be in conflict. The purpose of this study was to evaluate the effect of vitamin K on wound healing.
Methods: We systematically and comprehensively searched the PubMed, Web of Science, Embase, Cochrane library, China National Knowledge Infrastructure (CNKI), VIP and Wanfang eletronic databases. We applied revman5.3 software to calculate the weighted mean difference (WMD) of 95% confidence interval (CI) of animal and cell groups to evaluate the effect of vitamin K on wound healing. Two researchers independently selected studies and used the Cochrane Collaboration tool to assess the risk of bias in the included studies. The overall quality of evidence was assessed using the Recommendation, Assessment, Development and Evaluation (GRADE) working group approch.
Results: Among the 1081 articles searched, 6 articles (16 studies in total) met the inclusion criteria. The results of quantitative analysis showed that vitamin K was beneficial to increase the wound healing rate in animal models [rat model: WMD = 27.45 (95% CI: 13.46, 41.44); p = 0.0001], but the opposite result was obtained in cell experiments [WMD = −33.84 (95% CI: −56.90, −10.79); p = 0.004].
Conclusion: This meta-analysis hits that vitamin K could affect the process of wound healing, especially in animal models. While we could not know the clear role at present, which requires larger scale research. In addition, the concentration and safe dose of vitamin K also deserve further study.
Endometritis (inflammation of the endometrial lining) is one of the most devastating reproductive diseases in dairy cattle, resulting in substantial production loss and causing more than $650 million in lost revenue annually in the US. We hypothesize that alternative polyadenylation (APA) sites serve as decisive sensors for endometrium health and disease in dairy cows. Endometrial cells collected from 18 cows with purulent vaginal discharge scored 0 to 2 were used for APA profiling with our whole transcriptome termini site sequencing (WTTS-seq) method. Overall, pathogens trigger hosts to use more differentially expressed APA (DE-APA), more intronic DE-APA, more DE-APA sites per gene and more DE-genes associated with inflammation. Host CD59 molecule (CD59), Fc fragment of IgG receptor IIa (FCGR2A), lymphocyte antigen 75 (LY75) and plasminogen (PLG) may serve as initial contacts or combats with pathogens on cell surface, followed by activation of nuclear receptor subfamily 1 group H member 4 (NR1H4) to regulate AXL receptor tyrosine kinase (AXL), FGR proto-oncogene, Src family tyrosine kinase (FGR), HCK proto-oncogene, Src family tyrosine kinase (HCK) and integrin subunit beta 2 (ITGB2) for anti-inflammation. This study is the first to show significance of cilium pathways in endometrium health and animal reproduction. MIR21 and MIR30A would be perfect antagonistic biomarkers for diagnosis of either inflammation or anti-inflammation. These novel findings will set precedent for future genomic studies to aid the dairy industry develop new strategies to reduce endometritis incidence and improve fertility.
Cardiovascular disease is currently the leading cause of death worldwide. Atherosclerosis is an important pathological basis of cardiovascular disease, and its early diagnosis is of great significance. Urine bears no need nor mechanism to be stable, so it accumulates many small changes and is therefore a good source of biomarkers in the early stages of disease. In this study, ApoE-/- mice were fed a high-fat diet for 5 months. Urine samples from the experimental group and control group (C57BL/6 mice fed a normal diet) were collected at seven time points. Proteomic analysis was used for comparison within the experimental group and for comparison between the experimental group and the control group. The results of the comparison within the experimental group showed a significant difference in the urinary proteome before and after a one-week high-fat diet, and several of the differential proteins have been reported to be associated with atherosclerosis and/or as biomarker candidates. The results of the comparison between the experimental group and the control group indicated that the biological processes enriched by the GO analysis of the differential proteins correspond to the progression of atherosclerosis. The differences in chemical modifications of urinary proteins have also been reported to be associated with the disease. This study demonstrates that urinary proteomics has the potential to sensitively monitor changes in the body and provides the possibility of identifying early biomarkers of atherosclerosis.
The anticoagulant protein S (PS) binds phospholipids with very high affinity, but PS interaction with lipoproteins and lipidrich atherosclerotic plaques remains still poorly defined. We investigated PS in plasma lipoproteins and in atherosclerotic plaques from ten patients undergoing endarterectomy. PS was detected by Western blotting after exposure of the necrotic core to liposomes and was found to maintain its ability to bind phosphatidylserine micelles. The amounts of PS bound to low/very low-density lipoproteins in patient’ plasmas were higher and more variable than those detected in healthy subjects. A direct correlation between bound PS and low-density lipoproteins (LDL), plasma levels was found only in patients (r=0.921, p<0.001), thereby leading to hypothesize that the PS-phospholipids binding may increase by oxidative processes of LDL in atherosclerotic patients. The presence of the PS into the necrotic core of atherosclerotic plaques and on the surface of lipoproteins, particularly the atherogenic LDL, suggests a LDL-based delivery of PS to the atherosclerotic plaques and emphasizes the deep link between plasma lipids and coagulation in cardiovascular diseases.
Efferocytosis is defined as the clearance of apoptotic cells (ACs) in physiological and pathological states and is performed by efferocytes, such as macrophages. Efferocytosis can lead to the resolution of inflammation and restore tissue homoeostasis; however, the mechanisms of efferocytosis in determining inflammation resolution are still not completely understood, and the effects of efferocytosis on other proresolving properties need to be explored and explained. In this review, the process of efferocytosis will be summarized briefly, and then these mechanisms and effects will be thoroughly discussed. In addition, the association between the mechanisms of efferocytosis in determining inflammation resolution and cardiovascular diseases will also be reviewed, as an understanding of this association may provide information on novel treatment targets.
Phylloquinone (vitamin K1) and menaquinones (vitamin K2 family) are essential for post-translational γ-carboxylation of a small number of proteins, including clotting factors. These modified proteins have now been implicated in diverse physiological and pathological processes including cancer. Vitamin K intake has been inversely associated with cancer incidence and mortality in observational studies. Newly discovered functions of vitamin K in cancer cells include activation of the steroid and xenobiotic receptor (SXR) and regulation of oxidative stress, apoptosis, and autophagy. We provide an update of vitamin K biology, non-canonical mechanisms of vitamin K actions, the potential functions of vitamin K-dependent proteins in cancer, and observational trials on vitamin K intake and cancer.
Background: Growth arrest-specific 6 (GAS6) is a biomarker related to inflammation, fibrosis, as well as platelet function. Knockout of GAS6 in mice subjected to pressure overloading protects against cardiac hypertrophy and dysfunction. Nonetheless, the association between plasma GAS6 levels and acute heart failure (AHF) in patients is still unknown.
Methods: We measured GAS6 concentrations in 1039 patients with AHF who were enrolled in the DRAGON-HF trial. We followed them and examine future clinical events.
Results: In total, there were 195 primary endpoints of all-cause mortality and 135 secondary endpoints of cardiovascular death during the median follow-up period of 889 days. The higher level of GAS6 was associated with higher rates of all-cause and cardiovascular mortality (P<0.05). Baseline plasma GAS6 levels were still strongly correlated with clinical outcomes in different models after adjustment for clinical factors and N-terminal pro-brain natriuretic peptide (NT-proBNP, P<0.05). GAS6 could further distinguish the risks of clinical outcomes based on the NT-proBNP measurement.
Conclusion: We demonstrated that elevated plasma GAS6 levels were associated with an increased risk of all-cause and cardiovascular death in patients with AHF.
Registration Number: NCT03727828 (DRAGON-HF trial)
https://clinicaltrials.gov/ct2/show/NCT03727828?term=NCT03727828&draw=2&rank=1.
Clearing amyloid-β (Aβ) through immunotherapy is one of the most promising therapeutic approaches to Alzheimer’s disease (AD). Although several monoclonal antibodies against Aβ have been shown to substantially reduce Aβ burden in patients with AD, their effects on improving cognitive function remain marginal. In addition, a significant portion of patients treated with Aβ-targeting antibodies experience brain edema and microhemorrhage associated with antibody-mediated Fc receptor activation in the brain. Here, we develop a phagocytosis inducer for Aβ consisting of a single-chain variable fragment of an Aβ-targeting monoclonal antibody fused with a truncated receptor binding domain of growth arrest-specific 6 (Gas6), a bridging molecule for the clearance of dead cells via TAM (TYRO3, AXL, and MERTK) receptors. This chimeric fusion protein (αAβ–Gas6) selectively eliminates Aβ plaques through TAM receptor-dependent phagocytosis without inducing NF-kB-mediated inflammatory responses or reactive gliosis. Furthermore, αAβ–Gas6 can induce synergistic clearance of Aβ by activating both microglial and astrocytic phagocytosis, resulting in better behavioral outcomes with substantially reduced synapse elimination and microhemorrhage in AD and cerebral amyloid angiopathy model mice compared with Aβ antibody treatment. Our results suggest that αAβ–Gas6 could be a novel immunotherapeutic agent for AD that overcomes the side effects of conventional antibody therapy.
Hepatic macrophages, the key cellular components of the liver, emerge as essential players in liver inflammation, tissue repair, and subsequent fibrosis, as well as tumorigenesis. Recently, the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl and MerTK, was found to be a pivotal modulator of macrophages. Activation of macrophage TAM receptor signaling promotes the efferocytosis of apoptotic cells and skews the polarization of macrophages. After briefly reviewing the mechanisms of TAM receptor signaling in macrophage polarization, we focus on their role in liver diseases from acute injury to chronic inflammation, fibrosis and then to tumorigenesis. Notably, macrophage TAM receptor signaling seems to be a two‐edged sword for liver diseases. On one hand, the activation of TAM receptor signaling inhibits inflammation and facilitates tissue repair during acute liver injury. On the other hand, continuous activation of the signaling contributes to the process of chronic inflammation into fibrosis and tumorigenesis by evoking hepatic stellate cells and inhibiting anti‐tumor immunity. Therefore, targeting macrophage TAM receptors and clarifying its downstream pathways will be exciting prospects for the precaution and treatment of liver diseases, particularly at different stages or statuses.
The gamma-glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout the multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M, S300F) whose combined heterozygosity causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation: complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX (fIX)) or calcification (Matrix Gla Protein (MGP)) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of VKD protein in the complex. The VKD protein in the complex with wild type carboxylase was carboxylated before challenge protein carboxylation occurred, and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time, and did not completely carboxylate fIX in the complex. S300F carboxylation was poor with both fIX and MGP. Additional studies analyzed fIX and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The V255M mutant generated more carboxylated peptide than wild type GGCX, however the peptides were partially carboxylated. Analysis of the V255M mutant in fIX-HEK293 cells lacking endogenous GGCX revealed poor fIX clotting activity. The studies show that disrupted processivity causes disease, and explain the defect in the patient. The kinetic analyses also suggest that disrupted processivity may occur in wild type carboxylase under some conditions, e.g. warfarin therapy or vitamin K deficiency.
Background:
Growth-arrest-specific protein 6 (Gas6) exerts nervous protective effects on acute brain injury. We endeavored to ascertain whether serum Gas6 concentrations are associated with severity, delayed cerebral ischemia (DCI) and prognosis following aneurysmal subarachnoid hemorrhage (aSAH).
Methods:
We measured serum Gas6 concentrations of 124 aSAH patients. The Hunt-Hess scale and modified Fisher grading scale were used to evaluate illness severity. Multivariate analysis was utilized to determine relationships between serum Gas6 concentrations and severity, DCI plus 90-day unfavorable outcome (Glasgow outcome scale score of 1-3).
Results:
Patients with unfavorable outcome or DCI had significantly higher serum Gas6 concentrations than other remainders (median, 35.0 vs 23.3 ng/ml; 36.1 vs 25.3 ng/ml; both P < 0.001). Serum Gas6 concentrations displayed independent correlations with Hunt-Hess scores (t = 5.518, P < 0.001) and modified Fisher scores (t = 3.531, P = 0.001). Serum Gas6 concentrations were independently associated with unfavorable outcome (OR: 1.125; 95% CI, 1.063-1.190; P = 0.014) and DCI (OR: 1.104; 95% CI, 1.041-1.170; P = 0.010) as well as exhibited AUCs of 0.786 (95% CI, 0.703-0.854) and 0.753 (95% CI, 0.668-0.826) for predicting unfavorable outcome and DCI respectively. Its discriminatory ability for risk of unfavorable outcome or DCI was similar to those of Hunt-Hess scores and modified Fisher scores (all P > 0.05).
Conclusions:
Serum Gas6 concentrations are independently associated with stroke severity and worse clinical outcome after aSAH, indicating serum Gas6 may be a potential prognostic biomarker for aSAH.
Reliable biomarkers allowing early patients’ stratification for the risk of adverse outcomes in COVID-19 are lacking. Gas6, together with its tyrosine kinase receptors named TAM, is involved in the regulation of immune homeostasis, fibrosis, and thrombosis. Our aim was to evaluate whether Gas6, sAxl, and sMerTK could represent early predictors of disease evolution either towards a negative (death or need of ICU admission) or a positive (discharge and/or clinical resolution within the first 14 days of hospitalization) outcome. To this purpose, between January and May 2021 (corresponding to third pandemic wave in Italy), 139 consecutive SARS-CoV-2 positive patients were enrolled in a prospective observational study. Plasma levels of these molecules were measured by ELISA at the time of hospitalization and after 7 and 14 days. We observed that higher plasma Gas6 concentrations at hospital admission were associated with a worsening in clinical conditions while lower sMerTK concentrations at baseline and after 7 days of hospitalization were associated with a more favorable outcome. At multivariate analysis, after correction for demographic and COVID-19 severity variables (NEWS2 and PiO2/FiO2), only Gas6 measured at baseline predicted an adverse prognosis with an odds ratio of 1.03 (C.I. 1.01-10.5). At ROC curve analysis, baseline Gas6 levels higher than 58.0 ng/ml predicted a severe disease evolution with 53.3% sensitivity and 77.6% specificity (area under the curve 0.653, p = 0.01 , likelihood ratio of 2.38, IQR: 1.46-3.87). Taken together, these results support the hypothesis that a dysregulation in the Gas6/TAM axis could play a relevant role in modulating the course of COVID-19 and suggest that plasma Gas6 may represent a promising prognostic laboratory parameter for this condition.
In the general population low‐grade inflammation has been established as a risk factor for all‐cause mortality. We hypothesized that an inflammatory milieu beyond the time of recovery from the surgical trauma could be associated with increased long‐term mortality in kidney transplant recipients (KTRs). This cohort study included 1044 KTRs. Median follow‐up time post‐engraftment was 10.3 years. Inflammation was assessed 10 weeks after transplantation by different composite inflammation scores based on 21 biomarkers. We constructed an overall inflammation score and five pathway‐specific inflammation scores (fibrogenesis, vascular inflammation, metabolic inflammation, growth/angiogenesis, leukocyte activation). Mortality was assessed with Cox regression models adjusted for traditional risk factors. A total of 312 (29.9%) patients died during the follow‐up period. Hazard ratio (HR) for death was 4.71 (95% CI:2.85‐7.81, p<0.001) for patients in the highest quartile of the overall inflammation score and HRs 2.35‐2.54 (95%CI:1.40‐3.96, 1.52‐4.22, p:0.001) for patients in the intermediate groups. The results were persistent when the score was analyzed as a continuous variable (HR 1.046, 95% CI:1.033‐1.056, p<0.001). All pathway‐specific analyses showed the same pattern with HRs ranging from 1.19 to 2.70. In conclusion, we found a strong and consistent association between low‐grade systemic inflammation 10 weeks after kidney transplantation and long‐term mortality.
In recent years, cancer therapies using immune checkpoint inhibitors (ICIs) have achieved meaningful success, with patients with advanced tumors presenting longer survival times and better quality of life. However, several patients still do not exhibit good clinical outcomes for ICI therapy due to low sensitivity. To solve this, researchers have focused on identifying the cellular and molecular mechanisms underlying resistance to ICI therapy. ICI therapy induces apoptosis, which is the most frequent regulated cell death (RCD) but lacks immunogenicity and is regarded as an “immune silent” cell death. Ferroptosis, a unique type of non-apoptotic-RCD, has been preliminarily identified as an immunogenic cell death (ICD), stimulating tumor-antigen-specific immune responses and augmenting anti-tumor immune effects. However, ferroptosis has rarely been used in clinical practice. Present evidence strongly supports that the interferon-γ signaling pathway is at the crossroads of ICI therapy and ferroptosis. TYRO3, a receptor tyrosine kinase, is highly expressed in tumors and can induce anti-programmed cell death (PD)-ligand 1/PD-1 therapy resistance by limiting tumoral ferroptosis. Therefore, in this review, we summarize the clinical practice and effects of ICI therapy in various cancers. We also provide an overview of ferroptosis and report the molecular connections between cancer cell ferroptosis and ICI therapy, and discuss the possibility to reverse ICI therapy resistance by inducing cancer cell ferroptosis.
Maternal immunotolerance toward the semi-allogeneic fetus is critical for normal pregnancy. As a secretory protein, growth arrest-specific factor 6 (GAS6) promotes cancer progression by inducing the conversion of tumour-associated macrophages to an immunosuppressive M2-like phenotype. However, little is known about whether GAS6 regulates decidual macrophages (dMφs) in early maternal-fetal interface. In this study, first-trimester decidual tissues were obtained from normal pregnant women undergoing elective terminations and patients with miscarriages. The expression of GAS6 and its receptors (AXL, TYRO3 and MERTK) in decidua and GAS6 secretion by decidual stromal cells (DSCs) was measured. Then, we investigated the effect of recombinant human GAS6 (rhGAS6) on dMφs isolated from normal pregnancy and THP-1 cells, and revealed the underlying mechanism. Both the expression of GAS6 in DSCs and MERTK in dMφs, in addition to GAS6 secretion by DSCs, was found to be significantly decreased in miscarriage patients compared to that in normal pregnancy. Additionally, we observed that rhGAS6 polarized dMφs and THP-1 cells toward an M2-like phenotype, as evidenced by the up-regulated CD163 expression. Moreover rhGAS6 enhanced the clearance of toxic cell-free hemoglobin by dMφs by up-regulating CD163 expression, and rhGAS6 also boosted cell proliferation of dMφs and THP-1 cells. Finally, we demonstrated that rhGAS6 stimulated CD163 expression and cell proliferation by activating the PI3K/Akt signaling pathway. Collectively, these findings suggest that GAS6-mediated dialogue between DSCs and dMφs is crucial for the establishment and maintenance of maternal-fetal immunotolerance, and decreased GAS6 secretion by DSCs may lead to the occurrence of miscarriage in first trimester.
The pathogenesis of sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) has not yet been fully elucidated. Growth arrest–specific 6 (Gas6) has marked effects on hemostasis and reduces inflammation through its interaction with receptor tyrosine kinases of the TAM family: Tyro3, Axl, and Mer. Here, we found that plasma concentrations of Gas6 and soluble Mer were greater in patients with severe sepsis or septic ALI/ARDS compared with those in normal healthy donors. To determine whether the Gas6-Mer axis was critical in the pathogenesis of ALI/ARDS, we investigated the effects of intravenous administration of the selective Mer inhibitor UNC2250 on lipopolysaccharide (LPS)–induced ALI in mouse models subjected to inhalation of LPS. UNC2250 markedly inhibited the infiltration into the lungs of neutrophils and monocytes with increased amounts of Gas6 and Mer proteins, severe lung damage, and increased amounts of reactive oxygen species (ROS) in LPS-induced ALI in mice. In human pulmonary aortic endothelial cells, LPS induced decreases in the amounts of endothelial nitric oxide synthase, thrombomodulin, and vascular endothelial–cadherin, which was blocked by treatment with UNC2250. UNC2250 also inhibited the LPS-dependent increases in cell proliferation and enhanced apoptosis in HL-60 cells, a human neutrophil–like cell line, and RAW264.7 cells, a mouse monocyte/macrophage cell line. These data provide insights into the potential multiple beneficial effects of the Mer inhibitor UNC2250 as a therapeutic reagent to treat inflammatory responses in ALI/ARDS.
Ongoing research to fight cancer, one of the dominant diseases of the 21st century has led to big progress especially when it comes to understanding the tumor growth and metastasis. This includes the discovery of the molecular mechanisms of tumor vascularization, which is critically required for establishment of tumor metastasis. Formation of new blood vessels is the first step in tumor vascularization. Therefore, understanding the molecular and cellular basis of tumor vascularization attracted a significant effort studying in biomedical research. The blood vessels for supplying tumor can be formed by sprouting from pre-existing vessels, a process called angiogenesis, or by vasculogenesis, that is de novo formation of blood vessels from not fully differentiated progenitor cell populations. Vasculogenic endothelial progenitor cells (EPCs) can either be activated from populations in the bone marrow reaching the pathological region via the circulation or they can be recruited from local reservoirs. Neovessel formation influences tumor progression, hence therapeutic response model systems of angiogenesis/vasculogenesis are necessary to study the underlying mechanisms. Although, initially the research in this area focused more on angiogenesis, it is now well understood that both angiogenesis and postnatal vasculogenesis contribute to neovessel formation in adult under both most pathological as well as physiological conditions. Studies in the last two decades demonstrate that in addition to the intimal layer of fully differentiated mature endothelial cells (ECs) and various smaller supplying vessels (vasa vasorum) that can serve as a source for new vessels by angiogenesis, especially the adventitia of large and medium size blood vessels harbors various vascular wall-resident stem and progenitor cells (VW-SPCs) populations that serve as a source for new vessels by postnatal vasculogenesis. However, little is known about the potential role of VW-SPCs in tumor vascularization. To this end, the present work started first to establish a modified aortic ring assay (ARA) using mouse aorta in order to study the contribution of vascular adventitia-resident VW-SPCs to neovascularization in general and in presence of tumor cells. ARA is already established an ex vivo model for neovascularization allows to study the morphogenetic events of complex new vessel formation that includes all layers of mature blood vessels, a significant advantage over the assays that employ monolayer endothelial cell cultures. Moreover, in contrast to assays employing endothelial cells monocultures, both angiogenic and vasculogenic events take place during new vessel formation in ARA although the exact contribution of these two processes to new vessel formation cannot be easily distinguished in conventional ARA. Thus, in this study, a modified protocol for the ARA (mdARA) was established by either removing or keeping the aortic adventitia in place. The mdARA allows to distinguish the role of VW-SPCs from those of other aortic layers. The present data show that angiogenic sprouting from mature aortic endothelium was markedly delayed when the adventitial layer was removed. Furthermore, the network between the capillary-like sprouts was significantly reduced in absence of aortic adventitia. Moreover, the stabilization of new sprouts by assembling the NG2+ pericyte-like cells that enwrapped the endothelial sprouts from the outside was improved when the adventitial layer remained in place. Next, mimicking the tumor-vessel adventitia-interaction, multicellular tumor spheroids (MCTS) and aortic rings (ARs) with or without adventitia of C57BL/6-Tg (UBC-GFP) mice were confronted within the collagen gel and cultured ex vivo. This 3D model enabled analysis of the mobilization, migration and capillary-like sprouts formation by VW-SPCs within tumor-vessel wall-interface in comparison to tumor-free side of the ARs. Interestingly, while MCTS preferred the uptake of single vascular adventitia-derived cells, neural spheroids were directly penetrated by capillary-like structures that were sprouted from the aortic adventitia. In summary, the model established in this work allows to study new vessel formation by both postnatal vasculogenesis and angiogenesis under same conditions. It can be applied in various mouse models including reporter mouse models, e.g. Cxcr1 CreER+/mTmG+/- mice, in which GFP-marked macrophages of the vessel wall were directly observed as they mobilized from their niche and migrated into collagen gel. Another benefit of the model is that it can be used for testing different factors such as small molecules, growth factors, cytokines, and drugs with both pro- and anti-angiogenic/vasculogenic effects.
The levels of total, free, and bound protein S and C4BP were determined using enzyme-linked immunosorbent assays (ELISAs) in plasma samples (8 males and 8 females) that were individually subjected to immunoadsorption studies in which “free protein S” was defined as that not adsorbed by anti-C4BP antibody-Sepharose column and “free C4BP” as that not adsorbed by anti-protein S antibody-Sepharose. Bound species were calculated as the difference between total and free species. Free protein S (131 nmol/L) averaged 38% of total protein S (346 nmol/L) and free C4BP (37 nmol/L) averaged 14% of total C4BP (264 nmol/L) in plasma. There was an excellent correlation between bound protein S and bound C4BP with 1:1 molar stoichiometry and a good correlation between free protein S antigen and protein S anticoagulant activity. It appears that free protein S is a necessary consequence of the molar excess of protein S over C4BP. C4BP beta chain antigen levels, measured using a new ELISA, averaged 218 nmol/L, a value indistinguishable from the molar concentrations of bound protein S (215 nmol/L) and bound C4BP (227 nmol/L). The free C4BP beta chain antigen was only 4 nmol/L compared with 131 nmol/L free protein S. These results suggest that free C4BP in plasma predominantly lacks the beta chain, that almost all C4BP capable of binding protein S is associated with protein S, and that the plasma levels of oligomeric forms of C4BP containing a beta chain (alpha 7 beta and alpha 6 beta) that bind protein S directly and stoichiometrically regulate free protein S levels.
Gas6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. Gas6 null (-/-) mice are protected from lethal venous and arterial thromboembolism through platelet signalling defects induced only by 5 µM ADP and 10 µM of the thromboxane analogue, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raise the possibility that Gas6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that Gas6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/reconstitution experiments generating mice with selective ablations of Gas6 from either the hematopoietic or non-hematopoietic compartments demonstrate an approximately equal contribution by Gas6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of Gas6(-/-) mice as compared to WT. In vitro, thrombin-induced tissue factor expression was reduced in Gas6(-/-) endothelial cells as compared to WT endothelium. Taken together, these results demonstrate that vascular Gas6 contribute to thrombus formation in vivo and can be explained by the ability of Gas6 to promote tissue factor expression and activity. These findings support the notion that vascular wall derived Gas6 may play a pathophysiologic role in venous thromboembolism.
The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6-/- mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6
-/- mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis.
Protein S is a vitamin K-dependent glycoprotein, which besides its anti-coagulant function, acts as an agonist for the tyrosine kinase receptors Tyro3, Axl, and Mer. The endothelium expresses Tyro3, Axl, and Mer and produces protein S. The interaction of protein S with endothelial cells and particularly its effects on angiogenesis have not yet been analysed. Here we show that human protein S, at circulating concentrations, inhibited vascular endothelial growth factor (VEGF) receptor 2-dependent vascularization of Matrigel plugs in vivo and the capacity of endothelial cells to form capillary-like networks in vitro as well as VEGF-A-induced endothelial migration and proliferation. Furthermore, protein S inhibited VEGF-A-induced endothelial VEGFR2 phosphorylation and activation of mitogen activated kinase-Erk1/2 and Akt. Protein S activated the tyrosine phosphatase SHP2 and the SHP2 inhibitor, NSC 87877, reversed the observed inhibition of VEGF-A-induced endothelial proliferation. Using siRNA directed against Tyro3, Axl and Mer, we demonstrate that protein S-mediated SHP2 activation and inhibition of VEGF-A-stimulated proliferation were mediated by Mer. Our report provides the first evidence for the existence of a protein S/Mer/SHP2 axis which inhibits VEGFR2 signaling and regulates endothelial function and points to a role for protein S as an endogenous angiogenesis inhibitor.
The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation.
Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis.
WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays.
Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals.
These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.
The Gas6/Axl pathway has been increasingly implicated in regeneration and tissue repair and, recently, in the control of innate immunity. In liver, we have demonstrated that Gas6 and its receptor Axl are expressed in macrophages, progenitor cells, and myofibroblasts and that Gas6 deficiency reduced inflammation and myofibroblast activation, causing delayed liver repair in response to acute injury. All these data suggest a role of Gas6/Axl signaling in pathogenesis of chronic liver diseases. In the present study, we address the role of Gas6 in steatohepatitis and progression to liver fibrosis using Gas6-deficient mice fed a choline-deficient ethionine-supplemented diet (CDE) or receiving a chronic carbon tetrachloride (CCl(4)) treatment. Gas6 deficiency attenuated hepatic steatosis by limiting CDE-induced downregulation of genes involved in β-oxidation observed in wild-type animals. Moreover, Gas6-deficient mice displayed reduction of hepatic inflammation, revealed by limited F4/80-positive macrophage infiltration, decreased expression of IL-1β, TNF-α, lymphotoxin-β, and monocyte chemotactic protein-1, and attenuated hepatic progenitor cell response to CDE diet. Gas6 deficiency reduced CDE-induced fibrogenesis and hepatic myofibroblast activation and decreased expression of TGF-β and collagen 1 mRNAs. After chronic CCl(4) injury, Gas6-deficient mice also exhibited reduced liver fibrosis as a consequence of defective macrophage recruitment compared with wild-type animals. We conclude that improvement of steatohepatitis and fibrosis in Gas6(-/-) mice is linked to an inhibition of the inflammatory response that controls lipid metabolism and myofibroblast activation. This study highlights the deleterious effect of Gas6 in the progression of steatosis to steatohepatitis and fibrosis.
Tubby and tubby-like protein 1 (Tulp1) are newly identified phagocytosis ligands to facilitate retinal pigment epithelium (RPE) and macrophage phagocytosis. Both proteins without classical signal peptide have been demonstrated with unconventional secretion. Here, we characterized them as novel MerTK ligands to facilitate phagocytosis. Tulp1 interacts with Tyro3, Axl and MerTK of the TAM receptor tyrosine kinase subfamily, whereas tubby binds only to MerTK. Excessive soluble MerTK extracellular domain blocked tubby- or Tulp1-mediated phagocytosis. Both ligands induced MerTK activation with receptor phosphorylation and signalling cascade, including non-muscle myosin II redistribution and co-localization with phagosomes. Tubby and Tulp1 are bridging molecules with their N-terminal region as MerTK-binding domain and C-terminal region as phagocytosis prey-binding domain (PPBD). Five minimal phagocytic determinants (MPDs) of K/R(X)(1-2)KKK in Tulp1 N-terminus were defined as essential motifs for MerTK binding, receptor phosphorylation and phagocytosis. PPBD was mapped to the highly conserved 54 amino acids at the C-terminal end of tubby and Tulp1. These data suggest that tubby and Tulp1 are novel bridging molecules to facilitate phagocytosis through MerTK.
The TAM (tyro 3, axl, mer) kinases are key regulators of innate immunity and are important in the phagocytosis of apoptotic cells. Gas6 and protein S are ligands for these TAM kinases and bind to phosphatidyl serine residues exposed during apoptosis. In animal models, absence of TAM kinases is associated with lupus-like disease. To test whether human systemic lupus erythematosus (SLE) patients might have deficient levels of TAM ligands, we measured Gas 6 and protein S levels in SLE.
107 SLE patients were recruited. Of these, 45 SLE patients were matched age, gender and ethnicity with normal controls (NC). Gas6 and free protein S were measured with sandwich enzyme linked immunosorbent assays (ELISAs).
Overall, the plasma concentrations of Gas6 and free protein S were not different between 45 SLE patients and 45 NC. In SLE patients, the levels of free protein S were positively correlated with age (r = 0.2405, P = 0.0126), however those of Gas6 were not. There was no correlation between the concentrations of Gas6 and free protein S in individuals. Levels of free protein S were significantly lower in SLE patients with a history of serositis, neurologic disorder, hematologic disorder and immunologic disorder. Gas6 levels were elevated in patients with a history of neurologic disorder. The SLE patients with anti-Sm or anti-cardiolipin IgG showed lower free protein S levels. Circulating free protein S was positively correlated with complement component 3 (C3) (r = 0.3858, P < 0.0001) and complement component 4 (C4) (r = 0.4275, P < 0.0001). In the patients with active BILAG hematologic involvement, the levels of free protein S were lower and those of Gas6 were higher.
In SLE, free protein S was decreased in patients with certain types of clinical history and disease activity. Levels of free protein S were strongly correlated with C3 and C4 levels. Gas6 levels in SLE patients differed little from levels in NC, but they were elevated in the small numbers of patients with a history of neurological disease. The correlation of decreased protein S levels with lupus disease activity is consistent with a role for the TAM receptors in scavenging apoptotic cells and controlling inflammation. Protein S appears more important functionally in SLE patients than Gas6 in this regard.
The peritoneal cavity is recognized as an important site for autoreactive B cells prior to their transit to other immune tissues; however, little is known of the genes that may regulate this process. Mice lacking the receptor tyrosine kinase, Mertk, display a lupus-like autoimmune phenotype with splenomegaly and high autoantibodies titers. In this study, we investigate whether Mertk regulates the composition of peritoneal cells that favor an autoimmune phenotype. We found an increase in the number of macrophages, dendritic cells (DCs), plasmacytoid DCs, T cells, and B cells in the peritoneal cavity of mertk-/- mice when compared with wild-type mice. This disparity in cell numbers was not due to changes in cell proliferation or cell death. In adoptive transfer experiments, we showed an increase in migration of labeled donor cells into the mertk-/- peritoneal cavity. In addition, bone marrow chimeric mice showed hematopoietic-derived factors were also critical for T cell migration. Consistent with this migration and the increase in the number of cells, we identified elevated expression of CXCL9, its receptor CXCR3, and IL-7R on peritoneal cells from mertk-/- mice. To corroborate the migratory function of CXCR3 on cells, the depletion of CXCR3 donor cells significantly reduced the number of adoptively transferred cells that entered into the peritoneum of mertk-/- mice. This control of peritoneal cells numbers correlated with autoantibody production and was exclusively attributed to Mertk because mice lacking other family members, Axl or Tyro 3, did not display dysregulation in peritoneal cell numbers or the autoimmune phenotype.
The anticoagulant factor protein S (PS) has direct cellular activities. Lack of PS in mice causes lethal coagulopathy, ischemic/thrombotic injuries, vascular dysgenesis, and blood-brain barrier (BBB) disruption with intracerebral hemorrhages. Thus, we hypothesized that PS maintains and/or enhances the BBB integrity. Using a BBB model with human brain endothelial cells, we show PS inhibits time- and dose-dependently (half maximal effective concentration [EC(50)] = 27 +/- 3 nM) oxygen/glucose deprivation-induced BBB breakdown, as demonstrated by measurements of the transmonolayer electrical resistance, permeability of endothelial monolayers to dextran (40 kDa), and rearrangement of F-actin toward the cortical cytoskeletal ring. Using Tyro-3, Axl, and Mer (TAM) receptor, tyrosine kinase silencing through RNA interference, specific N-terminus-blocking antibodies, Tyro3 phosphorylation, and Tyro3-, Axl- and Mer-deficient mouse brain endothelial cells, we show that Tyro3 mediates PS vasculoprotection. After Tyro3 ligation, PS activated sphingosine 1-phosphate receptor (S1P(1)), resulting in Rac1-dependent BBB protection. Using 2-photon in vivo imaging, we show that PS blocks postischemic BBB disruption in Tyro3(+/+), Axl(-/-), and Mer(-/-) mice, but not in Tyro3(-/-) mice or Tyro3(+/+) mice receiving low-dose W146, a S1P(1)-specific antagonist. Our findings indicate that PS protects the BBB integrity via Tyro3 and S1P(1), suggesting potentially novel treatments for neurovascular dysfunction resulting from hypoxic/ischemic BBB damage.
Growth arrest-specific gene 6 (Gas6) is expressed in antigen-presenting cells and endothelial cells (ECs) but not in T cells. When wild-type (WT) or Gas6(-/-) mice received allogeneic non-T cell-depleted bone marrow cells, hepatic graft-versus-host disease (GVHD) was alleviated in Gas6(-/-) recipients regardless of donor genotype, but not in WT recipients. T-cell infiltration was more prominent and diffuse in WT than in Gas6(-/-) recipients' liver. When mice received 0.5 x 10(6) allogeneic T cells with T cell-depleted allogeneic bone marrow, clinical signs indicated that GVHD was less severe in Gas6(-/-) than in WT recipients, as shown by a significant improvement of the survival and reduced liver GVHD. These data demonstrate that donor cells were not involved in the protection mechanism. In addition, lack of Gas6 in antigen-presenting cells did not affect WT or Gas6(-/-) T-cell proliferation. We therefore assessed the response of WT or Gas6(-/-) ECs to tumor necrosis factor-alpha. Lymphocyte transmigration was less extensive through Gas6(-/-) than WT ECs and was not accompanied by increases in adhesion molecule levels. Thus, the lack of Gas6 in ECs impaired donor T-cell transmigration into the liver, providing a rationale for considering Gas6 pathway as a potential nonimmunosuppressive target to minimize GVHD in patients receiving allogeneic hematopoietic stem cell transplantation.
Protein S (ProS) is a blood anticoagulant encoded by the Pros1 gene, and ProS deficiencies are associated with venous thrombosis, stroke, and autoimmunity. These associations notwithstanding, the relative risk that reduced ProS expression confers in different disease settings has been difficult to assess without an animal model. We have now described a mouse model of ProS deficiency and shown that all Pros1-/- mice die in utero,from a fulminant coagulopathy and associated hemorrhages. Although ProS is known to act as a cofactor for activated Protein C (aPC), plasma from Pros1+/- heterozygous mice exhibited accelerated thrombin generation independent of aPC, and Pros1 mutants displayed defects in vessel development and function not seen in mice lacking protein C. Similar vascular defects appeared in mice in which Pros1 was conditionally deleted in vascular smooth muscle cells. Mutants in which Pros1 was deleted specifically in hepatocytes, which are thought to be the major source of ProS in the blood, were viable as adults and displayed less-severe coagulopathy without vascular dysgenesis. Finally, analysis of mutants in which Pros1 was deleted in endothelial cells indicated that these cells make a substantial contribution to circulating ProS. These results demonstrate that ProS is a pleiotropic anticoagulant with aPC-independent activities and highlight new roles for ProS in vascular development and homeostasis.
Many receptors that are employed for the engulfment of apoptotic cells are also used for the recognition and phagocytosis of bacteria. Tyro3, Axl, and Mertk (TAM) are important in the phagocytosis of apoptotic cells by macrophages. Animals lacking these receptors are hypersensitive to bacterial products. In this report, we examine whether the TAM receptors are involved in the phagocytosis of bacteria. We found that macrophages lacking Mertk, Axl, Tyro3 or all three receptors were equally efficient in the phagocytosis of Gram-negative E. coli. Similarly, the phagocytosis of E. coli and Gram-positive S. aureus bioparticles by macrophages lacking TAM receptors was equal to wild-type. In addition, we found that Mertk did not play a role in killing of extracellular E. coli or the replication status of intracellular Francisella tularensis. Thus, while TAM receptors may regulate signal transduction to bacterial components, they are not essential for the phagocytosis and killing of bacteria.
Protein S (PS) is an important natural anticoagulant with potentially multiple biologic functions. To investigate further the role of PS in vivo, we generated Pros(+/-) heterozygous mice. In the null (-) allele, the Pros exons 3 to 7 have been excised through conditional gene targeting. Pros(+/-) mice did not present any signs of spontaneous thrombosis and had reduced PS plasma levels and activated protein C cofactor activity in plasma coagulation and thrombin generation assays. Tissue factor pathway inhibitor cofactor activity of PS could not be demonstrated. Heterozygous Pros(+/-) mice exhibited a notable thrombotic phenotype in vivo when challenged in a tissue factor-induced thromboembolism model. No viable Pros(-/-) mice were obtained through mating of Pros(+/-) parents. Most E17.5 Pros(-/-) embryos were found dead with severe intracranial hemorrhages and most likely presented consumptive coagulopathy, as demonstrated by intravascular and interstitial fibrin deposition and an increased number of megakaryocytes in the liver, suggesting peripheral thrombocytopenia. A few E17.5 Pros(-/-) embryos had less severe phenotype, indicating that life-threatening manifestations might occur between E17.5 and the full term. Thus, similar to human phenotypes, mild heterozygous PS deficiency in mice was associated with a thrombotic phenotype, whereas total homozygous deficiency in PS was incompatible with life.
The levels of total, free, and bound protein S and C4BP were determined using enzyme-linked immunosorbent assays (ELISAs) in plasma samples (8 males and 8 females) that were individually subjected to immunoadsorption studies in which "free protein S" was defined as that not adsorbed by anti-C4BP antibody-Sepharose column and "free C4BP" as that not adsorbed by anti-protein S antibody-Sepharose. Bound species were calculated as the difference between total and free species. Free protein S (131 nmol/L) averaged 38% of total protein S (346 nmol/L) and free C4BP (37 nmol/L) averaged 14% of total C4BP (264 nmol/L) in plasma. There was an excellent correlation between bound protein S and bound C4BP with 1:1 molar stoichiometry and a good correlation between free protein S antigen and protein S anticoagulant activity. It appears that free protein S is a necessary consequence of the molar excess of protein S over C4BP. C4BP beta chain antigen levels, measured using a new ELISA, averaged 218 nmol/L, a value indistinguishable from the molar concentrations of bound protein S (215 nmol/L) and bound C4BP (227 nmol/L). The free C4BP beta chain antigen was only 4 nmol/L compared with 131 nmol/L free protein S. These results suggest that free C4BP in plasma predominantly lacks the beta chain, that almost all C4BP capable of binding protein S is associated with protein S, and that the plasma levels of oligomeric forms of C4BP containing a beta chain (alpha 7 beta and alpha 6 beta) that bind protein S directly and stoichiometrically regulate free protein S levels.
Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients
with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine
kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a
140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of
the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein
is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain,
as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of
receptor tyrosine kinases.
Protein S is a cofactor of activated protein C; together they function as a regulator of blood coagulation. A human liver cDNA library constructed in bacteriophage lambda gt11 was screened with DNA fragments from a full-length bovine cDNA clone encoding protein S. Several cDNA clones were isolated and sequenced. The combined cDNA sequences encoded the mature protein and 15 residues of the leader sequence when compared to bovine protein S. Human protein S is a single-chain protein consisting of 635 amino acids with 82% homology to bovine protein S. After an NH2-terminal gamma-carboxyglutamic acid-containing region, there is a short region with thrombin-sensitive bond(s), followed by a region with four repeat sequences that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the COOH-terminal portion of human protein S does not show any resemblance to serine proteases.
A family with a history of severe recurrent venous thromboembolic disease was studied to determine if a plasma protein deficiency could account for observed disease. Protein S levels in plasma were determined immunologically using the Laurell rocket technique. The propositus, his mother, his aunt, and his cousin who were clinically affected had 17% to 65% of the control levels of protein S antigen (normal range, 71% to 147%). Since three of these patients were receiving oral anticoagulant therapy, the ratios of protein S to prothrombin, factor X, and protein C in these patients were compared with values for a group of orally anticoagulated controls. These results suggested that protein S is half-normal in all family members with thrombotic disease. Other proteins known to be associated with familial thrombotic disease, including antithrombin III, plasminogen, fibrinogen, and protein C, were normal. Because plasma protein S serves as a cofactor for the anticoagulant activity of activated protein C and because protein C deficiency is associated with recurrent thrombotic disease, it is suggested that recurrent thrombotic disease in this family is the result of an inherited deficiency of protein S.
Protein S, a recently described vitamin K-dependent plasma protein, is shown to exist in two forms in plasma--free protein and in complex with C4b-binding protein. C4b-binding protein is involved in the regulation of the rate of complement activation. A major proportion of C4b-binding protein in plasma is in complex with protein S. The complex is a major and previously unrecognized component of the group of plasma proteins that adsorbs to barium citrate. The complex dissociates in the presence of NaDodSO4, indicating that C4b-binding protein and protein S are held together by noncovalent bonds. Uncomplexed C4b-binding protein was purified from the supernatant after barium citrate adsorption. On NaDodSO4/polyacrylamide gels without reduction, it appeared to have a slightly faster migration rate than the C4b-binding protein dissociated from the complex with protein S. After reduction, the subunits of the two forms of C4b-binding protein appeared to have identical molecular weights. Furthermore, there is an equilibrium between free and bound protein S in plasma. The role of protein S in the complex is unknown.
A family with a history of severe recurrent venous thromboembolic disease was studied to determine if a plasma protein deficiency could account for observed disease. Protein S levels in plasma were determined immunologically using the Laurell rocket technique. The propositus, his mother, his aunt, and his cousin who were clinically affected had 17% to 65% of the control levels of protein S antigen (normal range, 71% to 147%). Since three of these patients were receiving oral anticoagulant therapy, the ratios of protein S to prothrombin, factor X, and protein C in these patients were compared with values for a group of orally anticoagulated controls. These results suggested that protein S is half-normal in all family members with thrombotic disease. Other proteins known to be associated with familial thrombotic disease, including antithrombin III, plasminogen, fibrinogen, and protein C, were normal. Because plasma protein S serves as a cofactor for the anticoagulant activity of activated protein C and because protein C deficiency is associated with recurrent thrombotic disease, it is suggested that recurrent thrombotic disease in this family is the result of an inherited deficiency of protein S.
Protein-S is a vitamin K (Vit K)-dependent protein synthesized by hepatocytes, megakaryocytes, and endothelial cells and plays an important role in the regulation of hemostasis. Two cases of free protein-S congenital deficiency were recently reported to be associated with osteopenia. We hypothesized that this osteopenia could be the result of a bone deficit of protein-S synthesized by bone cells. Using enzyme-linked immunoassay, immunocytochemistry, immunoblotting, and immunoprecipitation after labeling with [35S]methionine, we have shown that this protein is secreted by three human osteosarcoma cell lines and by human adult osteoblast-like cells. In addition, protein-S was present in protein extracts of human bone matrix. Protein-S secreted by MG 63 cells increased linearly from 1-7 days of culture, was biologically active, and was regulated by warfarin, as previously described for the other cell types secreting protein-S. Vit K had no direct effect on protein-S secretion or activity, but could overcome ...
Rse, Axl, and c-Mer comprise a family of cell adhesion molecule-related tyrosine kinase receptors. Human Gas6 was recently shown to act as a ligand for both human Rse (Godowski et al., 1995) and human Axl (Varnum et al., 1995). Gas6 contains an NH2-terminal Gla domain followed by four epidermal growth factor-like repeats and tandem globular (G) domains, The G domains are related to those found in sex hormone-binding globulin and to those utilized by laminin and agrin for binding to the dystroglycan complex. A series of Gas6 variants were tested for their ability to bind to Rse and Axl. The Gla domain and epidermal growth factor-like repeats were not required for receptor binding, as deletion variants of Gas6 which lacked these domains bound to the extracellular domains of both Rse and Axl, A deletion variant of Gas6 containing just the G domain region was shown to activate Rse phosphorylation. These results provide evidence that G domains can act as signaling molecules by activating transmembrane receptor tyrosine kinases. Furthermore, they provide a structural link between the activation of cell adhesion related receptors and the control of cell growth and differentiation by the G domain-containing superfamily of proteins.
C4BP beta is one of the two polypeptides that in humans compose the plasma glycoprotein C4b-binding protein (C4BP). C4BP beta binds the anticoagulant vitamin K-dependent protein S. Two, nonmutually exclusive, roles have been proposed for the C4BP-protein S interaction. It has been suggested to play a role in the control of the protein C anticoagulatory pathway. In addition, it may serve an important role in localizing C4BP to the surface of injured or activated cells. While the physiological significance of C4BP-protein S interaction is unclear, it has clinical relevance because elevated plasma levels of C4BP are associated with increased risk for thromboembolic disorders in humans, due to an inactivation of the protein C anticoagulatory pathway. Using a human C4BP beta cDNA probe, we have isolated and characterized a genomic DNA fragment that includes the murine C4BPB gene. Murine C4BPB is a single-copy gene that maps close to the C4BPA gene in chromosome 1. It contains two exons homologous to the exons coding for the SCR-1 and SCR-2 repeats of the human C4BP beta polypeptide chain. Sequence analysis of the C4BPB exons in the Mus musculus inbred strains CBA, Balb/c, and C57BL/6, in pen-bred Swiss mice, and in Mus spretus demonstrated the presence of two in-phase stop codons that are incompatible with the expression of a functional C4BP beta polypeptide. Thus, the characterization of the murine C4BPB gene documents the peculiar situation of a single-copy gene that is functional in humans but has become a pseudogene in the mouse. Interestingly, the loss of a functional C4BPB gene is a relatively recent event in the evolution of the mouse. In addition, our data indicate that this genetic change has been fixed in the mouse population, suggesting that those individual lacking the C4BP beta polypeptide were conferred with some kind of selective advantage. (C) 1994 Academic Press, Inc.
THE Axl receptor tyrosine kinase was identified as a protein encoded by
a transforming gene from primary human myeloid leukaemia cells by
DNA-mediated transformation of NIH 3T3 cells1-3. Axl is the
founding member of a family of related receptors that includes
Eyk4, encoded by a chicken proto-oncogene originally
described as a retroviral transforming gene, and c-Mer5,
encoded by a human protooncogene expressed in neoplastic B- and T-cell
lines. The transforming activity of Axl demonstrates that the receptor
can drive cellular proliferation. The function of Axl in non-transformed
cells and tissues is unknown, but may involve the stimulation of cell
proliferation in response to an appropriate signal, namely a ligand that
activates the receptor. We report here the purification of an Axl
stimulatory factor, and its identification as the product of
growth-arrest-specific gene 6 (ref. 6). This is, to our knowledge, the
first description of a ligand for the Axl family of receptors.
Sepsis is an acute inflammatory condition that can result in multiple organ failure and acute lung injury (ALI). Growth arrest-specific protein 6 (Gas6) is a broad regulator of the innate immune response involved with the NF-κB signaling pathway. We hypothesized that Gas6 could have a protective role in attenuating the severity of ALI and sepsis. Male mice were subjected to sepsis by cecal ligation and puncture (CLP) after which recombinant murine Gas6 (rmGas6; 5 μg/mouse) or normal saline (vehicle) was administered intravenously. Blood and lung tissues were collected at 20 h after CLP for various measurements. Treatment with rmGas6 significantly reduced serum levels of the injury markers AST, ALT and LDH as well as proinflammatory cytokines IL-6 and IL-17, compared to the vehicle group (P<0.05). The parenchyma of the lungs damaged by CLP was attenuated by rmGas6 treatment. Lung mRNA levels of TNF-α, IL-1β, IL-6, IL-17 and MIP-2 were decreased by 60%, 86%, 82%, 93% and 82%, respectively, with rmGas6 treatment as determined by real time RT-PCR (P<0.05). The degradation of IκB-α induced by CLP in the lungs was inhibited by rmGas6 treatment. The number of neutrophils and myeloperoxidase activity in the lungs were significantly reduced in the rmGas6 group. Moreover, rmGas6 reduced the in-vitro migration of differentiated human promyelocytic HL60 cells by 64%. Finally, the 10-day survival rate of mice subjected to CLP was increased from 31% in the vehicle group to 67% in the rmGas6 group (P<0.05). Thus, Gas6 has potential to be developed as a novel therapeutic agent to treat patients with sepsis and acute lung injury.