Illustrated State-of-the-Art Capsules of the ISTH 2024 Congress.

Here, we present a series of illustrated capsules from the State of the Art (SOA) speakers at the 2024 International Society on Thrombosis and Haemostasis Congress in Bangkok, Thailand. This year's Congress marks the first time that the International Society on Thrombosis and Haemostasis has held its flagship scientific meeting in Southeast Asia and is the first to be organized by an international Planning Committee. The Bangkok program will feature innovative science and clinical updates from around the world, reflecting the diversity and multidisciplinary growth of our field. In these illustrated SOA capsules, you will find an exploration of novel models of thrombosis and bleeding and biomaterial discoveries that can trigger or block coagulation. Thromboinflammation is now understood to drive many disease states, and the SOA speakers cover cellular and coagulation responses to COVID-19 and other infections. The theme of crosstalk between coagulation and inflammation expands with capsules on protein S signaling, complement, and fibrinolytic inhibitors. Novel agents for hemophilia and thrombosis prevention are introduced. Challenging clinical conditions are also covered, such as inherited platelet disorders and antiphospholipid antibody syndrome. The scientific program in Bangkok will also showcase the work of clinicians and scientists from all parts of the world and chronicle real-world challenges. For example, 2 SOA capsules address the diagnosis and management of von Willebrand disease in low-income settings. Take some time to browse through these short illustrated reviews; we're sure that you'll be entertained, educated, and inspired to further explore the world of thrombosis and hemostasis.

TAM receptors in phagocytosis: Beyond the mere internalization of particles.

TYRO3, AXL, and MERTK constitute the TAM family of receptor tyrosine kinases, activated by their ligands GAS6 and PROS1. TAMs are necessary for adult homeostasis in the immune, nervous, reproductive, skeletal, and vascular systems. Among additional cellular functions employed by TAMs, phagocytosis is central for tissue health. TAM receptors are dominant in providing phagocytes with the molecular machinery necessary to engulf diverse targets, including apoptotic cells, myelin debris, and portions of live cells in a phosphatidylserine-dependent manner. Simultaneously, TAMs drive the release of anti-inflammatory and tissue repair molecules. Disruption of the TAM-driven phagocytic pathway has detrimental consequences, resulting in autoimmunity, male infertility, blindness, and disrupted vascular integrity, and which is thought to contribute to neurodegenerative diseases. Although structurally and functionally redundant, the TAM receptors and ligands underlie complex signaling cascades, of which several key aspects are yet to be elucidated. We discuss similarities and differences between TAMs and other phagocytic pathways, highlight future directions and how TAMs can be harnessed therapeutically to modulate phagocytosis.

Phagocytosis in the retina promotes local insulin production in the eye.

The retina is highly metabolically active, relying on glucose uptake and aerobic glycolysis. Situated in close contact to photoreceptors, a key function of cells in the retinal pigment epithelium (RPE) is phagocytosis of damaged photoreceptor outer segments (POS). Here we identify RPE as a local source of insulin in the eye that is stimulated by POS phagocytosis. We show that Ins2 messenger RNA and insulin protein are produced by RPE cells and that this production correlates with RPE phagocytosis of POS. Genetic deletion of phagocytic receptors ('loss of function') reduces Ins2, whereas increasing the levels of the phagocytic receptor MerTK ('gain of function') increases Ins2 production in male mice. Contrary to pancreas-derived systemic insulin, RPE-derived local insulin is stimulated during starvation, which also increases RPE phagocytosis. Global or RPE-specific Ins2 gene deletion decreases retinal glucose uptake in starved male mice, dysregulates retinal physiology, causes defects in phototransduction and exacerbates photoreceptor loss in a mouse model of retinitis pigmentosa. Collectively, these data identify RPE cells as a phagocytosis-induced local source of insulin in the retina, with the potential to influence retinal physiology and disease.

Regulation of bone homeostasis by MERTK and TYRO3.

The fine equilibrium of bone homeostasis is maintained by bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we show that TAM receptors MERTK and TYRO3 exert reciprocal effects in osteoblast biology: Osteoblast-targeted deletion of MERTK promotes increased bone mass in healthy mice and mice with cancer-induced bone loss, whereas knockout of TYRO3 in osteoblasts shows the opposite phenotype. Functionally, the interaction of MERTK with its ligand PROS1 negatively regulates osteoblast differentiation via inducing the VAV2-RHOA-ROCK axis leading to increased cell contractility and motility while TYRO3 antagonizes this effect. Consequently, pharmacologic MERTK blockade by the small molecule inhibitor R992 increases osteoblast numbers and bone formation in mice. Furthermore, R992 counteracts cancer-induced bone loss, reduces bone metastasis and prolongs survival in preclinical models of multiple myeloma, breast- and lung cancer. In summary, MERTK and TYRO3 represent potent regulators of bone homeostasis with cell-type specific functions and MERTK blockade represents an osteoanabolic therapy with implications in cancer and beyond.

TAM Signaling in the Nervous System.

Tyro3, Axl and Mertk are members of the TAM family of tyrosine kinase receptors. TAMs are activated by two structurally homologous ligands GAS6 and PROS1. TAM receptors and ligands are widely distributed and often co-expressed in the same cells allowing diverse functions across many systems including the immune, reproductive, vascular, and the developing as well as adult nervous systems. This review will focus specifically on TAM signaling in the nervous system, highlighting the essential roles this pathway fulfills in maintaining cell survival and homeostasis, cellular functions such as phagocytosis, immunity and tissue repair. Dysfunctional TAM signaling can cause complications in development, disruptions in homeostasis which can rouse autoimmunity, neuroinflammation and neurodegeneration. The development of therapeutics modulating TAM activities in the nervous system has great prospects, however, foremost we need a complete understanding of TAM signaling pathways.

Myeloid cell-derived PROS1 inhibits tumor metastasis by regulating inflammatory and immune responses via IL-10.

Stimulation of TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases promotes tumor progression through numerous cellular mechanisms. TAM cognate ligands GAS6 and PROS1 (for TYRO3 and MERTK) are secreted by host immune cells, an interaction which may support tumor progression. Here, we revealed an unexpected antimetastatic role for myeloid-derived PROS1: suppressing metastatic potential in lung and breast tumor models. Pros1 deletion in myeloid cells led to increased lung metastasis, independent of primary tumor infiltration. PROS1-cKO bone marrow-derived macrophages (BMDMs) led to elevated TNF-α, IL-6, Nos2, and IL-10 via modulation of the Socs3/NF-κB pathway. Conditioned medium from cKO BMDMs enhanced EMT, ERK, AKT, and STAT3 activation within tumor cells and promoted IL-10-dependent invasion and survival. Macrophages isolated from metastatic lungs modulated T cell proliferation and function, as well as expression of costimulatory molecules on DCs in a PROS1-dependent manner. Inhibition of MERTK kinase activity blocked PROS1-mediated suppression of TNF-α and IL-6 but not IL-10. Overall, using lung and breast cancer models, we identified the PROS1/MERTK axis within BMDMs as a potent regulator of adaptive immune responses with a potential to suppress metastatic seeding and revealed IL-10 regulation by PROS1 to deviate from that of TNF-α and IL-6.

Corrigendum: Macrophage-Derived Protein S Facilitates Apoptotic Polymorphonuclear Cell Clearance by Resolution Phase Macrophages and Supports Their Reprogramming.

[This corrects the article DOI: 10.3389/fimmu.2018.00358.].

TAM receptors, Phosphatidylserine, inflammation, and Cancer.

The numerous and diverse biological roles of Phosphatidylserine (PtdSer) are featured in this special issue. This review will focus on PtdSer as a cofactor required for stimulating TYRO3, AXL and MERTK - comprising the TAM family of receptor tyrosine kinases by their ligands Protein S (PROS1) and growth-arrest-specific 6 (GAS6) in inflammation and cancer. As PtdSer binding to TAMs is a requirement for their activation, the biological repertoire of PtdSer is now recognized to be broadened to include functions performed by TAMs. These include key homeostatic roles necessary for preserving a healthy steady state in different tissues, controlling inflammation and further additional roles in diseased states and cancer. The impact of PtdSer on inflammation and cancer through TAM signaling is a highly dynamic field of research. This review will focus on PtdSer as a necessary component of the TAM receptor-ligand complex, and for maximal TAM signaling. In particular, interactions between tumor cells and their immediate environment - the tumor microenvironment (TME) are highlighted, as both cancer cells and TME express TAMs and secrete their ligands, providing a nexus for a multifold of cross-signaling pathways which affects both immune cells and inflammation as well as tumor cell biology and growth. Here, we will highlight the current and emerging knowledge on the implications of PtdSer on TAM signaling, inflammation and cancer.

Inhibition of MERTK Promotes Suppression of Tumor Growth in BRAF Mutant and BRAF Wild-Type Melanoma.

Molecularly-targeted agents have improved outcomes for a subset of patients with BRAF-mutated melanoma, but treatment of resistant and BRAF wild-type tumors remains a challenge. The MERTK receptor tyrosine kinase is aberrantly expressed in melanoma and can contribute to oncogenic phenotypes. Here we report the effect of treatment with a MERTK-selective small molecule inhibitor, UNC2025, in preclinical models of melanoma. In melanoma cell lines, treatment with UNC2025 potently inhibited phosphorylation of MERTK and downstream signaling, induced cell death, and decreased colony formation. In patient-derived melanoma xenograft models, treatment with UNC2025 blocked or significantly reduced tumor growth. Importantly, UNC2025 had similar biochemical and functional effects in both BRAF-mutated and BRAF wild-type models and irrespective of NRAS mutational status, implicating MERTK inhibition as a potential therapeutic strategy in tumors that are not amenable to BRAF-targeting and for which there are limited treatment options. In BRAF-mutated cell lines, combined treatment with UNC2025 and the BRAF inhibitor vemurafenib provided effective inhibition of oncogenic signaling through ERK, AKT, and STAT6, increased induction of cell death, and decreased colony-forming potential. Similarly, in NRAS-mutated cell lines, addition of UNC2025 to cobimetinib therapy increased cell death and decreased colony-forming potential. In a BRAF-mutated patient-derived xenograft, treatment with combined UNC2025 and vemurafenib was well-tolerated and significantly decreased tumor growth compared with vemurafenib alone. These data support the use of UNC2025 for treatment of melanoma, irrespective of BRAF or NRAS mutational status, and suggest a role for MERTK and targeted combination therapy in BRAF and NRAS-mutated melanoma.

Multiple Regulatory Levels of Growth Arrest-Specific 6 in Mucosal Immunity Against an Oral Pathogen.

Growth arrest-specific 6 (GAS6) expressed by oral epithelial cells and dendritic cells (DCs) was shown to play a critical role in the maintenance of oral mucosal homeostasis. In this study, we demonstrate that the induction of pathogen-specific oral adaptive immune responses is abrogated in Gas6-/- mice. Further analysis revealed that GAS6 induces simultaneously both pro- and anti-inflammatory regulatory pathways upon infection. On one hand, GAS6 upregulates expression of adhesion molecules on blood vessels, facilitating extravasation of innate inflammatory cells to the oral mucosa. GAS6 also elevates expression of CCL19 and CCL21 chemokines and enhances migration of oral DCs to the lymph nodes. On the other hand, expression of pro-inflammatory molecules in the oral mucosa are downregulated by GAS6. Moreover, GAS6 inhibits DC maturation and reduces antigen presentation to T cells by DCs. These data suggest that GAS6 facilitates bi-directional trans-endothelial migration of inflammatory cells and DCs, whereas inhibiting mucosal activation and T-cell stimulation. Thus, the orchestrated complex activity of GAS6 enables the development of a rapid and yet restrained mucosal immunity to oral pathogens.

Cell-intrinsic regulation of murine epidermal Langerhans cells by protein S.

AXL, a member of the TYRO3, AXL, and MERTK (TAM) receptor tyrosine kinase family, has been shown to play a role in the differentiation and activation of epidermal Langerhans cells (LCs). Here, we demonstrate that growth arrest-specific 6 (GAS6) protein, the predominant ligand of AXL, has no impact on LC differentiation and homeostasis. We thus examined the role of protein S (PROS1), the other TAM ligand acting primarily via TYRO3 and MERTK, in LC function. Genetic ablation of PROS1 in keratinocytes resulted in a typical postnatal differentiation of LCs; however, a significant reduction in LC frequencies was observed in adult mice due to increased apoptosis. This was attributed to altered expression of cytokines involved in LC development and tissue homeostasis within keratinocytes. PROS1 was then excised in LysM+ cells to target LCs at early embryonic developmental stages, as well as in adult monocytes that also give rise to LCs. Differentiation and homeostasis of LCs derived from embryonic precursors was not affected following Pros1 ablation. However, differentiation of LCs from bone marrow (BM) precursors in vitro was accelerated, as was their capability to reconstitute epidermal LCs in vivo. These reveal an inhibitory role for PROS1 on BM-derived LCs. Collectively, this study highlights a cell-specific regulation of LC differentiation and homeostasis by TAM signaling.

Macrophage-Derived Protein S Facilitates Apoptotic Polymorphonuclear Cell Clearance by Resolution Phase Macrophages and Supports Their Reprogramming.

The complete resolution of inflammation requires the uptake of apoptotic polymorphonuclear cells (PMN) by local macrophages (efferocytosis) and the consequent reprogramming of the engulfing phagocytes to reparative and pro-resolving phenotypes. The tyrosine kinase receptors TYRO3, AXL, and MERTK (collectively named TAM) are fundamental mediators in regulating inflammatory responses and efferocytosis. Protein S (PROS1) is a ligand for all TAM receptors that mediates various aspects of their activity. However, the involvement of PROS1 in the resolution of inflammation is incompletely understood. Here, we report the upregulation of Pros1 in macrophages during the resolution of inflammation. Selective knockout of Pros1 in the myeloid lineage significantly downregulated macrophage pro-resolving properties. Hence, Pros1-deficient macrophages engulfed fewer apoptotic PMN remnants in vivo, and exogenous PROS1 rescued impaired efferocytosis ex vivo. Moreover, Pros1-deficient peritoneal macrophages secreted higher levels of the pro-inflammatory mediators TNFα and CCL3, while they secreted lower levels of the reparative/anti-inflammatory IL-10 following exposure to lipopolysaccharide in comparison to their WT counterparts. Moreover, Pros1-deficient macrophages expressed less of the anti-inflammatory/pro-resolving enzymes arginase-1 and 12/15-lipoxygenase and produced less of the specialized pro-resolving mediator resolvin D1. Altogether, our results suggest that macrophage-derived PROS1 is an important effector molecule in regulating the efferocytosis, maturation, and reprogramming of resolution phase macrophages, and imply that PROS1 could provide a new therapeutic target for inflammatory and fibrotic disorders.

Programming asynchronous replication in stem cells.

Many regions of the genome replicate asynchronously and are expressed monoallelically. It is thought that asynchronous replication may be involved in choosing one allele over the other, but little is known about how these patterns are established during development. We show that, unlike somatic cells, which replicate in a clonal manner, embryonic and adult stem cells are programmed to undergo switching, such that daughter cells with an early-replicating paternal allele are derived from mother cells that have a late-replicating paternal allele. Furthermore, using ground-state embryonic stem (ES) cells, we demonstrate that in the initial transition to asynchronous replication, it is always the paternal allele that is chosen to replicate early, suggesting that primary allelic choice is directed by preset gametic DNA markers. Taken together, these studies help define a basic general strategy for establishing allelic discrimination and generating allelic diversity throughout the organism.

TAM receptor signaling in development.

TYRO3, AXL and MERTK comprise the TAM family of receptor protein tyrosine kinases. Activated by their ligands, protein S (PROS1) and growth-arrest-specific 6 (GAS6), they mediate numerous cellular functions throughout development and adulthood. Expressed by a myriad of cell types and tissues, they have been implicated in homeostatic regulation of the immune, nervous, vascular, bone and reproductive systems. The loss-of-function of TAM signaling in adult tissues culminates in the destruction of tissue homeostasis and diseased states, while TAM gain-of-function in various tumors promotes cancer phenotypes. Combinatorial ligand-receptor interactions may elicit different molecular and cellular responses. Many of the TAM regulatory functions are essentially developmental, taking place both during embryogenesis and postnatally. This review highlights current knowledge on the role of TAM receptors and their ligands during these developmental processes in the immune, nervous, vascular and reproductive systems.

Protein S Negatively Regulates Neural Stem Cell Self-Renewal through Bmi-1 Signaling.

Revealing the molecular mechanisms underlying neural stem cell self-renewal is a major goal toward understanding adult brain homeostasis. The self-renewing potential of neural stem and progenitor cells (NSPCs) must be tightly regulated to maintain brain homeostasis. We recently reported the expression of Protein S (PROS1) in adult hippocampal NSPCs, and revealed its role in regulation of NSPC quiescence and neuronal differentiation. Here, we investigate the effect of PROS1 on NSPC self-renewal and show that genetic ablation of Pros1 in neural progenitors increased NSPC self-renewal by 50%. Mechanistically, we identified the upregulation of the polycomb complex protein Bmi-1 and repression of its downstream effectors p16Ink4a and p19Arf to promote NSPC self-renewal in Pros1-ablated cells. Rescuing Pros1 expression restores normal levels of Bmi-1 signaling, and reverts the proliferation and enhanced self-renewal phenotypes observed in Pros1-deleted cells. Our study identifies PROS1 as a novel negative regulator of NSPC self-renewal. We conclude PROS1 is instructive for NSPC differentiation by negatively regulating Bmi-1 signaling in adult and embryonic neural stem cells.

Protein S drives oral squamous cell carcinoma tumorigenicity through regulation of AXL.

The TAM family of proto-oncogenic receptor protein tyrosine kinases, comprising of TYRO3, AXL, and MERTK, is implicated in many human cancers. Their activation leads to cancer cell proliferation, enhanced migration, invasion, and drug resistance; however how TAMs are activated in cancers is less understood. We previously showed that Protein S (PROS1) is a ligand of the TAM receptors. Here we identify PROS1 as a mediator of Oral Squamous Cell Carcinoma (OSCC) in proliferation, cell survival and migration. We demonstrate that excess PROS1 induces OSCC proliferation and migration. Conversely, blocking endogenous PROS1 expression using shRNA significantly inhibits cell proliferation and migration in culture. This inhibition was rescued by the addition of purified PROS1. Moreover, PROS1 knockdown reduced anchorage-independent growth in-vitro, reduced tumor xenograft growth in nude mice and altered their differentiation profile. Mechanistically, we identify the downregulation of AXL transcripts and protein following PROS1 knockdown. Re-introducing PROS1 rescues AXL expression both at the protein and transcriptional levels. The anti-proliferative effect of the AXL inhibitor R428 was significantly reduced following PROS1 inhibition, indicating the functional significance of PROS1-mediated regulation of AXL in OSCC. Taken together, we identify PROS1 as a driver of OSCC tumor growth and a modulator of AXL expression. Our results point to PROS1 as a potential novel anti-cancer therapeutic target.

GAS6 is a key homeostatic immunological regulator of host-commensal interactions in the oral mucosa.

The oral epithelium contributes to innate immunity and oral mucosal homeostasis, which is critical for preventing local inflammation and the associated adverse systemic conditions. Nevertheless, the mechanisms by which the oral epithelium maintains homeostasis are poorly understood. Here, we studied the role of growth arrest specific 6 (GAS6), a ligand of the TYRO3-AXL-MERTK (TAM) receptor family, in regulating oral mucosal homeostasis. Expression of GAS6 was restricted to the outer layers of the oral epithelium. In contrast to protein S, the other TAM ligand, which was constitutively expressed postnatally, expression of GAS6 initiated only 3-4 wk after birth. Further analysis revealed that GAS6 expression was induced by the oral microbiota in a myeloid differentiation primary response gene 88 (MyD88)-dependent fashion. Mice lacking GAS6 presented higher levels of inflammatory cytokines, elevated frequencies of neutrophils, and up-regulated activity of enzymes, generating reactive nitrogen species. We also found an imbalance in Th17/Treg ratio known to control tissue homeostasis, as Gas6-deficient dendritic cells preferentially secreted IL-6 and induced Th17 cells. As a result of this immunological shift, a significant microbial dysbiosis was observed in Gas6-/- mice, because anaerobic bacteria largely expanded by using inflammatory byproducts for anaerobic respiration. Using chimeric mice, we found a critical role for GAS6 in epithelial cells in maintaining oral homeostasis, whereas its absence in hematopoietic cells synergized the level of dysbiosis. We thus propose GAS6 as a key immunological regulator of host-commensal interactions in the oral epithelium.

Protein S Regulates Neural Stem Cell Quiescence and Neurogenesis.

Neurons are continuously produced in brains of adult mammalian organisms throughout life-a process tightly regulated to ensure a balanced homeostasis. In the adult brain, quiescent Neural Stem Cells (NSCs) residing in distinct niches engage in proliferation, to self-renew and to give rise to differentiated neurons and astrocytes. The mechanisms governing the intricate regulation of NSC quiescence and neuronal differentiation are not completely understood. Here, we report the expression of Protein S (PROS1) in adult NSCs, and show that genetic ablation of Pros1 in neural progenitors increased hippocampal NSC proliferation by 47%. We show that PROS1 regulates the balance of NSC quiescence and proliferation, also affecting daughter cell fate. We identified the PROS1-dependent downregulation of Notch1 signaling to correlate with NSC exit from quiescence. Notch1 and Hes5 mRNA levels were rescued by reintroducing Pros1 into NCS or by supplementation with purified PROS1, suggesting the regulation of Notch pathway by PROS1. Although Pros1-ablated NSCs show multilineage differentiation, we observed a 36% decrease in neurogenesis, coupled with a similar increase in astrogenesis, suggesting PROS1 is instructive for neurogenesis, and plays a role in fate determination, also seen in aged mice. Rescue experiments indicate PROS1 is secreted by NSCs and functions by a NSC-endogenous mechanism. Our study identifies a duple role for PROS1 in stem-cell quiescence and as a pro-neurogenic factor, and highlights a unique segregation of increased stem cell proliferation from enhanced neuronal differentiation, providing important insight into the regulation and control of NSC quiescence and differentiation. Stem Cells 2017;35:679-693.

Retinal Phenotype following Combined Deletion of the Chemokine Receptor CCR2 and the Chemokine CX3CL1 in Mice.

PURPOSE: Conflicting data were reported with respect to the retinal phenotype of mice with dual perturbation of the CCL2 and CX3CR1 genes. We report the generation and retinal phenotype of mice with a reverse CCR2/CX3CL1 gene deficiency as a suggested model for age-related macular degeneration (AMD). METHODS: Crossing of single-deficient mice generated CCR2/CX3CL1 DKO mice. DKO mice were compared with age-matched C57BL6J mice. Evaluation included color fundus photographs, electroretinography (ERG), histology and morphometric analysis. Immunohistochemistry for CD11b in retinal cross-sections and retinal pigment epithelium (RPE)-choroid flat mounts was performed to assess microglia and macrophage recruitment. RESULTS: A minority of DKO mice showed yellowish subretinal deposits at 10 months. ERG recordings showed reduced cone sensitivity in young, but not older DKO mice. Compared to wild-type mice, DKO mice exhibited 11% reduction in the number of outer nuclear layer nuclei. Old DKO mice had an increased number of CD11b-positive cells across the retina, and on RPE-choroid flat mounts. CONCLUSIONS: In the absence of the rd8 allele, deficiency of CCR2 and CX3CL1 in mice leads to a mild form of retinal degeneration which is associated with the recruitment of macrophages, particularly to the subretinal space. This model enables to assess consequences of perturbed chemokine signaling, but it does not recapitulate cardinal AMD features.