Immunological role of Gas6/TAM signaling in hemostasis and thrombosis.

The immune system is an emerging regulator of hemostasis and thrombosis. The concept of immunothrombosis redefines the relationship between coagulation and immunomodulation, and the Gas6/Tyro3-Axl-MerTK (TAM) signaling pathway builds the bridge across them. During coagulation, Gas6/TAM signaling pathway not only activates platelets, but also promotes thrombosis through endothelial cells and vascular smooth muscle cells involved in inflammatory responses. Thrombosis appears to be a common result of a Gas6/TAM signaling pathway-mediated immune dysregulation. TAM TK and its ligands have been found to be involved in coagulation through the PI3K/AKT or JAK/STAT pathway in various systemic diseases, providing new perspectives in the understanding of immunothrombosis. Gas6/TAM signaling pathway serves as a breakthrough target for novel therapeutic strategies to improve disease management. Many preclinical and clinical studies of TAM receptor inhibitors are in process, confirming the pivotal role of Gas6/TAM signaling pathway in immunothrombosis. Therapeutics targeting the TAM receptor show potential both in anticoagulation management and immunotherapy. Here, we review the immunological functions of the Gas6/TAM signaling pathway in coagulation and its multiple mechanisms in diseases identified to date, and discuss the new clinical strategies that may generated by these roles.

Platelets: Orchestrators of immunity in host defense and beyond.

Platelets prevent blood loss during vascular injury and contribute to thrombus formation in cardiovascular disease. Beyond these classical roles, platelets are critical for the host immune response. They guard the vasculature against pathogens via specialized receptors, intracellular signaling cascades, and effector functions. Platelets also skew inflammatory responses by instructing innate immune cells, support adaptive immunosurveillance, and influence antibody production and T cell polarization. Concomitantly, platelets contribute to tissue reconstitution and maintain vascular function after inflammatory challenges. However, dysregulated activation of these multitalented cells exacerbates immunopathology with ensuing microvascular clotting, excessive inflammation, and elevated risk of macrovascular thrombosis. This dichotomy underscores the critical importance of precisely defining and potentially modulating platelet function in immunity.

Noncriteria antiphospholipid antibodies in antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombotic manifestations and/or obstetric complications in patients with persistently positive antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies, but only lupus anticoagulant, anticardiolipin (aCL), and antibeta2-glycoprotein I antibodies (aß2GPI) IgG or IgM are included as laboratory classification criteria. Seronegative APS patients are usually defined as patients with the clinical symptoms of APS but who test negative for aPL. The negativity to classic aPL criteria does not exclude the presence of other aPL. Several noncriteria aPL have been identified. Some noncriteria aPL are well studied, such as IgA aCL and aß2GPI, the antiphosphatidylserine-prothrombin (aPS/PT) antibodies, and the antibodies against the domain I of beta2-glycoprotein I (aDI), both latter groups receiving more attention for their role in thrombotic events and pregnancy complications. Other noncriteria aPL that have been studied are antibodies against annexin V, prothrombin, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, vimentin-cardiolipin complex, anti-protein S/protein C. Measurement of some of these noncriteria aPL (aPS/PT, aDI) is useful in the laboratory work-out of APS in specific situations. We have to differentiate between patients who are positive for noncriteria aPL only, and patients who have both criteria and noncriteria aPL to enable us to study their role in the diagnosis or risk stratification of APS. The research on noncriteria aPL is continually developing as the clinical relevance of these antibodies is not yet fully clarified.

The clinical relevance of different antiphospholipid antibody profiles in pediatric rheumatology patients.

BACKGROUND: The clinical relevance of different antiphospholipid antibody (aPL) profiles, including low level anticardiolipin (aCL) and anti-ß2-glycoprotein-I (aß2GPI) antibodies, is ill-defined in the pediatric population. Our purpose is to describe the demographic, clinical, and laboratory characteristics of aPL positive pediatric patients based on different aPL profiles. FINDINGS: In this single center retrospective cohort study, based on the screening of our pediatric (age ≤ 18) rheumatology electronic medical records (2016-2022), we identified patients who had at least one "positive" aPL (lupus anticoagulant [LA], aCL IgG/M, or aß2GPI IgG/M) result. Patients were grouped into high- (LA positive and/or aCL/aß2GPI IgG/M > 40U [ELISA]) and low-risk (LA negative and aCL/aß2GPI IgG/M 20-39U) aPL profiles; those with persistently positive aPL were descriptively analyzed for demographic and clinical characteristics. Of 57 included patients, 34 (59%) had initial high- and 23 (40%) had initial low-risk profiles. Based on subsequent aPL results available in 42/57 (74%) patients, 25/27 (93%) in the high-, and 7/15 (47%) in the low-risk groups remained still positive. Of these 32 patients with persistently positive aPL, moderate-to-large vessel or microvascular thrombosis occurred in nine (28%) patients with high-risk and in none with low-risk aPL profiles; non-thrombotic aPL-related manifestations were reported in 15 (47%) patients with persistent aPL positivity. CONCLUSION: An initial high-risk aPL profile was persistent in approximately 90% of our cohort, a third of whom had thrombosis, and half had non-thrombotic aPL manifestations. Our results underscore the need for a large-scale effort to better characterize aPL-related manifestations in pediatric patients with persistent high-risk aPL-profiles.

Therapeutic strategies in FcγIIA receptor-dependent thrombosis and thromboinflammation as seen in heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT).

INTRODUCTION: Fcγ-receptors (FcγR) are membrane receptors expressed on a variety of immune cells, specialized in recognition of the Fc part of immunoglobulin G (IgG) antibodies. FcγRIIA-dependent platelet activation in platelet factor 4 (PF4) antibody-related disorders have gained major attention, when these antibodies were identified as the cause of the adverse vaccination event termed vaccine-induced immune thrombocytopenia and thrombosis (VITT) during the COVID-19 vaccination campaign. With the recognition of anti-PF4 antibodies as cause for severe spontaneous and sometimes recurrent thromboses independent of vaccination, their clinical relevance extended far beyond heparin-induced thrombocytopenia (HIT) and VITT. AREAS COVERED: Patients developing these disorders show life-threatening thromboses, and the outcome is highly dependent on effective treatment. This narrative literature review summarizes treatment options for HIT and VITT that are currently available for clinical application and provides the perspective toward new developments. EXPERT OPINION: Nearly all these novel approaches are based on in vitro, preclinical observations, or case reports with only limited implementation in clinical practice. The therapeutic potential of these approaches still needs to be proven in larger cohort studies to ensure treatment efficacy and long-term patient safety.

Monocyte-Mediated Thrombosis Linked to Circulating Tissue Factor and Immune Paralysis in COVID-19.

BACKGROUND: SARS-CoV-2 infections cause COVID-19 and are associated with inflammation, coagulopathy, and high incidence of thrombosis. Myeloid cells help coordinate the initial immune response in COVID-19. Although we appreciate that myeloid cells lie at the nexus of inflammation and thrombosis, the mechanisms that unite the two in COVID-19 remain largely unknown. METHODS: In this study, we used systems biology approaches including proteomics, transcriptomics, and mass cytometry to define the circulating proteome and circulating immune cell phenotypes in subjects with COVID-19. RESULTS: In a cohort of subjects with COVID-19 (n=35), circulating markers of inflammation (CCL23 [C-C motif chemokine ligand 23] and IL [interleukin]-6) and vascular dysfunction (ACE2 [angiotensin-converting enzyme 2] and TF [tissue factor]) were elevated in subjects with severe compared with mild COVID-19. Additionally, although the total white blood cell counts were similar between COVID-19 groups, CD14+ (cluster of differentiation) monocytes from subjects with severe COVID-19 expressed more TF. At baseline, transcriptomics demonstrated increased IL-6, CCL3, ACOD1 (aconitate decarboxylase 1), C5AR1 (complement component 5a receptor), C5AR2, and TF in subjects with severe COVID-19 compared with controls. Using stress transcriptomics, we found that circulating immune cells from subjects with severe COVID-19 had evidence of profound immune paralysis with greatly reduced transcriptional activation and release of inflammatory markers in response to TLR (Toll-like receptor) activation. Finally, sera from subjects with severe (but not mild) COVID-19 activated human monocytes and induced TF expression. CONCLUSIONS: Taken together, these observations further elucidate the pathological mechanisms that underlie immune dysfunction and coagulation abnormalities in COVID-19, contributing to our growing understanding of SARS-CoV-2 infections that could also be leveraged to develop novel diagnostic and therapeutic strategies.

Deciphering the clinical significance of longitudinal antiphospholipid antibody titers.

In antiphospholipid syndrome (APS), the risk of clinical manifestations increases with higher titers of antiphospholipid antibodies (aPL). Despite the adoption of aPL titers in the classification approach to aPL-positive subjects, the value of longitudinal monitoring of those titers in the follow-up is still debated, being well studied only in systemic lupus erythematosus (SLE). The literature suggests that the rate of aPL positivity decreases during follow-up in primary APS, estimating that seroconversion occurs in between 8.9 and 59% of patients over time. Negativisation of aPL occurs more frequently in asymptomatic aPL carriers than in patients with full-blown APS as well as in subjects with single aPL positivity or low aPL antibody titers. In patients with SLE, aPL typically behave fluctuating from positive to negative and back again in the course of follow-up. The few studies assessing the longitudinal course of aPL positivity with no associated systemic connective tissue disease reported a progressive decrement of aPL titers over time, in particular of antibodies against ß2 glycoprotein I (antiß2GPI) and cardiolipin (aCL) of IgG isotype. After a thrombotic event, aPL titers tend to decrease, as emerged from cohorts of both primary and secondary APS. Hydroxychloroquine has been identified as the most effective pharmacological agent to reduce aPL titers, with multiple studies demonstrating a parallel reduction in thrombosis rate. This review addresses available evidence on the significance of aPL titer fluctuation from clinical, therapeutic and pathogenic perspectives.

Antagonistic Roles of Human Platelet Integrin αIIbß3 and Chemokines in Regulating Neutrophil Activation and Fate on Arterial Thrombi Under Flow.

BACKGROUND: Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches. METHODS: Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbß3. RESULTS: We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [N-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca2+]i rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide. CONCLUSIONS: Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.

CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation.

BACKGROUND: Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We incubated SARS-CoV-2 with neutrophils in the presence or absence of platelets to observe NET formation. We further isolated extracellular vesicles from COVID-19 patients' sera (COVID-19-EVs) to examine their ability to induce NET formation. RESULTS: We demonstrated that antagonistic mAbs against anti-CLEC5A mAb and anti-TLR2 mAb can inhibit COVID-19-EVs-induced NET formation, and generated clec5a-/-/tlr2-/- mice to confirm the critical roles of CLEC5A and TLR2 in SARS-CoV-2-induced lung inflammation in vivo. We found that virus-free extracellular COVID-19 EVs induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A) and TLR2. Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation was attenuated dramatically in clec5a-/-/tlr2-/- mice. CONCLUSIONS: This study demonstrates that SARS-CoV-2-activated platelets produce EVs to enhance thromboinflammation via CLEC5A and TLR2, and highlight the importance of CLEC5A and TLR2 as therapeutic targets to reduce the risk of ARDS in COVID-19 patients.

Neutrophils, Cancer and Thrombosis: The New Bermuda Triangle in Cancer Research.

Spontaneous venous thrombosis is often the first clinical sign of cancer, and it is linked to a worsened survival rate. Traditionally, tumor-cell induced platelet activation has been the main actor studied in cancer-associated-thrombosis. However, platelet involvement alone does not seem to be sufficient to explain this heightened pro-thrombotic state. Neutrophils are emerging as key players in both thrombus generation and cancer progression. Neutrophils can impact thrombosis through the release of pro-inflammatory cytokines and expression of molecules like P-selectin and Tissue Factor (TF) on their membrane and on neutrophil-derived microvesicles. Their role in cancer progression is evidenced by the fact that patients with high blood-neutrophil counts have a worsened prognosis. Tumors can attract neutrophils to the cancer site via pro-inflammatory cytokine secretions and induce a switch to pro-tumoral (or N2) neutrophils, which support metastatic spread and have an immunosuppressive role. They can also expel their nuclear contents to entrap pathogens forming Neutrophil Extracellular Traps (NETs) and can also capture coagulation factors, enhancing the thrombus formation. These NETs are also known to have pro-tumoral effects by supporting the metastatic process. Here, we strived to do a comprehensive literature review of the role of neutrophils as drivers of both cancer-associated thrombosis (CAT) and cancer progression.

Covid-19 and Liver Injury: Role of Inflammatory Endotheliopathy, Platelet Dysfunction, and Thrombosis.

Liver injury, characterized predominantly by elevated aspartate aminotransferase and alanine aminotransferase, is a common feature of coronavirus disease 2019 (COVID-19) symptoms caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Additionally, SARS-CoV-2 infection is associated with acute-on-chronic liver failure in patients with cirrhosis and has a notably elevated mortality in patients with alcohol-related liver disease compared to other etiologies. Direct viral infection of the liver with SARS-CoV-2 remains controversial, and alternative pathophysiologic explanations for its hepatic effects are an area of active investigation. In this review, we discuss the effects of SARS-CoV-2 and the inflammatory environment it creates on endothelial cells and platelets more generally and then with a hepatic focus. In doing this, we present vascular inflammation and thrombosis as a potential mechanism of liver injury and liver-related complications in COVID-19.

Intestinal Abnormalities in Patients With SARS-CoV-2 Infection: Histopathologic Changes Reflect Mechanisms of Disease.

Approximately 20% of patients with symptomatic syndrome-associated coronavirus-2 (SARS-CoV-2) infection have gastrointestinal bleeding and/or diarrhea. Most are managed without endoscopic evaluation because the risk of practitioner infection outweighs the value of biopsy analysis unless symptoms are life-threatening. As a result, much of what is known about the gastrointestinal manifestations of coronavirus disease-2019 (COVID-19) has been gleaned from surgical and autopsy cases that suffer from extensive ischemic injury and/or poor preservation. There are no detailed reports describing any other gastrointestinal effects of SARS-CoV-2 even though >3,000,000 people have died from COVID-19 worldwide. The purpose of this study is to report the intestinal findings related to SARS-CoV-2 infection by way of a small case series including one with evidence of direct viral cytopathic effect and 2 with secondary injury attributed to viral infection. Infection can be confirmed by immunohistochemical stains directed against SARS-CoV-2 spike protein, in situ hybridization for spike protein-encoding RNA, and ultrastructural visualization of viruses within the epithelium. It induces cytoplasmic blebs and tufted epithelial cells without inflammation and may not cause symptoms. In contrast, SARS-CoV-2 infection can cause gastrointestinal symptoms after the virus is no longer detected, reflecting systemic activation of cytokine and complement cascades rather than direct viral injury. Reversible mucosal ischemia features microvascular injury with hemorrhage, small vessel thrombosis, and platelet-rich thrombi. Systemic cytokine elaboration and dysbiosis likely explain epithelial cell injury that accompanies diarrheal symptoms. These observations are consistent with clinical and in vitro data and contribute to our understanding of the protean manifestations of COVID-19.

Recognizing Vaccine-Induced Immune Thrombotic Thrombocytopenia.

OBJECTIVES: Vaccine-induced immune thrombotic thrombocytopenia is an unexpected consequence of the coronavirus disease 2019 pandemic era. We reviewed the pathogenesis, clinical presentation, diagnosis, and treatment of this rare side effect. DATA SOURCES: Online search of published medical literature through PubMed, Scopus, Web of Science, and Google Scholar using the terms "COVID-19," "vaccine," "thrombosis" was performed. STUDY SELECTION: Articles were chosen for inclusion based on their relevance to coronavirus disease 2019, vaccine, and thrombosis. DATA SYNTHESIS: Vaccine-induced immune thrombotic thrombocytopenia manifests most often as unusual thromboses (cerebral venous sinus thrombosis, splanchnic vein thrombosis) but sometimes also "usual" thromboses (arterial stroke, pulmonary embolism, deep-vein thrombosis), with oftentimes severe thrombocytopenia, that becomes clinically evident 5-30 days after adenovirus-vectored coronavirus disease 2019 vaccine administration. Most patients have disseminated intravascular coagulation. These features are the result of vaccine-triggered formation of anti-platelet factor 4 immunoglobulin G that activate platelets, clinically mimicking autoimmune heparin-induced thrombocytopenia. Early recognition based on thrombosis (sometimes, hemorrhage), thrombocytopenia, and d-dimer elevation within the day 5-30 postvaccine "window" is important given treatment with high-dose IV immunoglobulin plus nonheparin anticoagulation. CONCLUSIONS: Vaccine-induced immune thrombotic thrombocytopenia is a serious complication of vaccination that is not feasible to anticipate or prevent. When the patient presents with sustained headache, neurologic symptoms/signs, abdominal pain, dyspnea, or limb pain/swelling beginning 5-30 days post vaccination, platelet count and d-dimer must be measured, and imaging for thrombosis performed. Confirmation of vaccine-induced immune thrombotic thrombocytopenia diagnosis should be ordered (platelet factor 4/polyanion enzyme-linked immunosorbent assay; platelet factor 4-enhanced platelet activation testing) as treatment is initiated (nonheparin anticoagulation, IV immunoglobulin).

Characteristics of purified anti-ß2GPI IgG N-glycosylation associate with thrombotic, obstetric and catastrophic antiphospholipid syndrome.

OBJECTIVE: Anti-ß-2 glycoprotein I (anti-ß2GPI) antibodies, defined as primary pathogenic antibody in antiphospholipid syndrome (APS). It has been reported that IgG Fc N-glycosylation affects IgG effector, we aim to investigate the association of Fc glycosylation profiles of purified anti-ß2GP1 IgG with clinical features of APS. METHODS: We purify anti-ß2GPI IgG and total IgG from 82 APS patients including nine catastrophic antiphospholipid syndrome (CAPS) patients, as well as total IgG from 103 healthy controls to quantitatively analyse all detectable Fc N-glycanforms of all IgG subclasses with Multiple Reaction Monitoring (MRM) method based on UPLC-ESI-QqQ mass spectrometry. RESULTS: Both purified anti-ß2GPI IgG and APS total IgG showed altered N-glycan profiles when compared with healthy control (HC) IgG. Anti-ß2GPI IgG presented with lower galactosylation, increased bisection and core fucosylation compared with APS total IgG and HC IgG. We found higher galactosylation of aß2GPI IgG2 in thrombotic APS compared with the obstetric APS, and lower galactosylation of aß2GPI IgG2 associated with late pregnancy morbidity. Moreover, low galactosylation of all anti-ß2GPI IgG subclasses, increased bisection and core fucosylation of anti-ß2GPI IgG1/2 were strongly associated with CAPS and triple positivity of antiphospholipid antibodies (aPLs). CONCLUSION: We comprehensively characterize the N-Glycans landscape of both anti-ß2GP1 and total IgG in APS. Altered N-glycan profiles of anti-ß2GPI IgG enables enabled the antibodies with proinflammatory properties. Furthermore, we associated levels of IgG Fc-glycosylation with clinical features antiphospholipid syndrome. These findings could increase our understanding of anti-ß2GPI antibody mediated mechanisms in APS and be used to develop diagnostics and new target treatments.

A Review of the Impact of Neutrophils and Neutrophil Extracellular Traps (NETs) on the Development of Aortic Aneurysms in Animal and Human Studies.

The pathogenesis of the aortic aneurysm (AA) includes several mechanisms, such as chronic sterile inflammation and homeostasis imbalance, with arteriosclerosis, hemodynamic forces, and genetic factors. In addition to the roles of these processes in the development of AA, neutrophilic activity may play a pivotal role (mostly in inflammation and thrombus formation). Neutrophils, which play a crucial role in innate immunity, can release neutrophil extracellular traps (NETs), one of the mechanisms against fighting pathogens, beside phagocytosis and degranulation. NETs are structures composed of nuclear elements (eg, chromatin and modified histones) and granular and cytoplasmic components, which can lead to inflammation and coagulation changes. In addition, the exacerbation of NETosis (the process of NET formation) can be noticed in vascular diseases, including in the development of AA and myocardial infarction and in diabetes, hypertension, and COPD, which are the risk factors of the presence of AA. The discharge of NETs, which are extracellular materials formed by citrullinated histones (Cit-H), cell-free DNA fibers (cf-DNA), and granular and cytoplasmic molecules, is a newly identified method of neutrophil activation that can be activated by endogenous inflammatory stimuli, which contribute to AA development. Cit-H and cf-DNA can be used as biomarkers of AA growth. By understanding the neutrophilic influence of NET release, a new pathway of screening AA growth (by measurement of biomarkers of NETosis) and pharmacological assessment (by repression of NET formation) can be developed. This review summarizes the current knowledge about the influence of NETs on AA growth in human and animal studies.