IgA Anti-ß2-Glycoprotein I Antibodies as Markers of Thrombosis and Severity in COVID-19 Patients.

Patients with COVID-19 may develop a hypercoagulable state due to tissue and endothelial injury, produced by an unbalanced immune response. Therefore, an increased number of thromboembolic events has been reported in these patients. The aim of this study is to investigate the presence of antiphospholipid antibodies (aPL) in COVID-19 patients, their role in the development of thrombosis and their relationship with the severity of the disease. In this retrospective study, serum samples from 159 COVID-19 patients and 80 healthy donors were analysed for the presence of aPL. A total of 29 patients (18.2%) and 14 healthy donors (17.5%) were positive for aPL. Nineteen COVID-19 patients (12%) but no healthy donor presented a positive percentage of the IgA isotype aPL. IgA anti-ß2-glycoprotein I antibodies (anti-ß2GPI) were the most frequent type (6.3%) in patients but was not detected in any healthy donor. The positivity of this antibody was found to be significantly elevated in patients with thromboembolic events (25% vs. 5%, p = 0.029); in fact, patients with positive IgA anti-ß2GPI had an incidence of thrombosis over six times higher than those who had normal antibody concentrations [OR (CI 95%) of 6.67 (1.5-30.2), p = 0.014]. Additionally, patients with moderate-severe disease presented a higher aPL positivity than patients with mild disease according to the Brescia (p = 0.029) and CURB-65 (p = 0.011) severity scales. A multivariate analysis showed that positivity for IgA anti-ß2GPI is significantly associated with disease severity measured by CURB-65 [OR (CI 95%) 17.8 (1.7-187), p = 0.0016]. In conclusion, COVID-19 patients have a significantly higher positive percentage of the IgA isotype aPL than healthy donors. IgA anti-ß2GPI antibodies were the most frequently detected aPL in COVID-19 patients and were associated with thrombosis and severe COVID-19 and are thus proposed as a possible marker to identify high-risk patients.

Neutrophil glucose flux as a therapeutic target in antiphospholipid syndrome.

Neutrophil hyperactivity and neutrophil extracellular trap release (NETosis) appear to play important roles in the pathogenesis of the thromboinflammatory autoimmune disease known as antiphospholipid syndrome (APS). The understanding of neutrophil metabolism has advanced tremendously in the past decade, and accumulating evidence suggests that a variety of metabolic pathways guide neutrophil activities in health and disease. Our previous work characterizing the transcriptome of APS neutrophils revealed that genes related to glycolysis, glycogenolysis, and the pentose phosphate pathway (PPP) were significantly upregulated. Here, we found that neutrophils from patients with APS used glycolysis more avidly than neutrophils from people in the healthy control group, especially when the neutrophils were from patients with APS with a history of microvascular disease. In vitro, inhibiting either glycolysis or the PPP tempered phorbol myristate acetate- and APS IgG-induced NETosis, but not NETosis triggered by a calcium ionophore. In mice, inhibiting either glycolysis or the PPP reduced neutrophil reactive oxygen species production and suppressed APS IgG-induced NETosis ex vivo. When APS-associated thrombosis was evaluated in mice, inhibiting either glycolysis or the PPP markedly suppressed thrombosis and circulating NET remnants. In summary, these data identify a potential role for restraining neutrophil glucose flux in the treatment of APS.

Antiphospholipid antibodies and antiphospholipid syndrome.

Individuals with antiphospholipid syndrome (APS) have antibodies directed against phospholipid-binding proteins (aPL). The condition is most associated with an increased risk of thromboembolism and obstetric complications. The 2023 classification criteria for APS include six clinical domains (venous thromboembolism, arterial thrombosis, microvascular events, obstetric events, cardiac valve, thrombocytopaenia) and two laboratory domains (lupus anticoagulant, and anti-cardiolipin or anti-ß2-glycoprotein-I antibodies). Diagnosis and treatment of APS are specialist tasks and are summarised in this review.

The convergent model of coagulation.

It is increasingly apparent that the pathologic interplay between coagulation and innate immunity, ie, immunothrombosis, forms the common basis of many challenges across the boundaries of specialized medicine and cannot be fully explained by the conventional concepts of cascade and cell-based coagulation. To improve our understanding of coagulation, we propose a model of coagulation that converges with inflammation and innate immune activation as a unified response toward vascular injury. Evolutionarily integral to the convergent response are damage-associated molecular patterns, which are released as a consequence of injury. Damage-associated molecular patterns facilitate diverse interactions within and between systems, not only to complement and reinforce cell-based clot formation but also to steer the response toward clot resolution and wound healing. By extending coagulation beyond its current boundaries, the convergent model aims to deliver novel diagnostics and therapeutics for contemporary and unexpected challenges across medicine, as exposed by COVID-19 and vaccine-induced immune thrombotic thrombocytopenia.

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.

Unraveling the Intricate Web: Complement Activation Shapes the Pathogenesis of Sepsis-Induced Coagulopathy.

BACKGROUND: Sepsis-associated coagulopathy specifically refers to widespread systemic coagulation activation accompanied by a high risk of hemorrhage and organ damage, which in severe cases manifests as disseminated intravascular coagulation (DIC), or even develops into multiple organ dysfunction syndrome (MODS). The complement system and the coagulation system as the main columns of innate immunity and hemostasis, respectively, undergo substantial activation after sepsis. SUMMARY: Dysfunction of the complement, coagulation/fibrinolytic cascades caused by sepsis leads to "thromboinflammation," which ultimately amplifies the systemic inflammatory response and accelerates the development of MODS. Recent studies have revealed that massive activation of the complement system exacerbates sepsis-induced coagulation and even results in DIC, which suggests that inhibition of complement activation may have therapeutic potential in the treatment of septic coagulopathy. KEY MESSAGES: Sepsis-associated thrombosis involves the upregulation or activation of procoagulant factors, down-regulation or inactivation of anticoagulant factors, and impairment of the fibrinolytic mechanism. This review aims to summarize the latest literature and analyze the underlying molecular mechanisms of the activation of the complement system on the abnormal coagulation cascades in sepsis.

Autoantibodies against complement proteins in patients with antiphospholipid syndrome: Prevalence and clinical associations.

Seventy-seven patients with antiphospholipid syndrome were tested for autoantibodies against C1q, C3, FB, FH, and C4bp. Fifty-seven patients had at least one anti-complement antibody. IgM anti-FH positivity was associated with thrombosis when anti-C3 and anti-FB were, negatively or positively, associated with various noncriteria manifestations of antiphospholipid syndrome.

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.

Laboratory approach for vaccine-induced thrombotic thrombocytopenia diagnosis in the Netherlands.

BACKGROUND AND OBJECTIVES: Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare adverse effect characterized by thrombocytopenia and thrombosis occurring after COVID-19 vaccination. VITT pathophysiology is not fully unravelled but shows similarities to heparin-induced thrombocytopenia (HIT). HIT is characterized by the presence of antibodies against platelet factor 4 (PF4)/heparin complex, which can activate platelets in an FcγRIIa-dependent manner, whereas IgG-antibodies directed against PF4 play an important role in VITT. MATERIALS AND METHODS: We characterized all clinically suspected VITT cases in the Netherlands from a diagnostic perspective and hypothesized that patients who developed both thrombocytopenia and thrombosis display underlying mechanisms similar to those in HIT. We conducted an anti-PF4 ELISA and a functional PF4-induced platelet activation assay (PIPAA) with and without blocking the platelet-FcγRIIa and found positivity in both tests, suggesting VITT with mechanisms similar to those in VITT. RESULTS: We identified 65 patients with both thrombocytopenia and thrombosis among 275 clinically suspected VITT cases. Of these 65 patients, 14 (22%) tested positive for anti-PF4 and PF4-dependent platelet activation. The essential role of platelet-FcγRIIa in VITT with mechanisms similar to those in HIT was evident, as platelet activation was inhibited by an FcγRIIa-blocking antibody in all 14 patients. CONCLUSION: Our study shows that only a small proportion of clinically suspected VITT patients with thrombocytopenia and thrombosis have anti-PF4-inducing, FcɣRIIa-dependent platelet activation, suggesting an HIT-like pathophysiology. This leaves the possibility for the presence of another type of pathophysiology ('non-HIT like') leading to VITT. More research on pathophysiology is warranted to improve the diagnostic algorithm and to identify novel therapeutic and preventive strategies.

A meta-analysis to assess the risk of bleeding and thrombosis following chimeric antigen receptor T-cell therapy: Communication from the ISTH SSC Subcommittee on Hemostasis and Malignancy.

BACKGROUND: Chimeric antigen receptor T-cell (CAR T-cell) therapy is increasingly utilized for treatment of hematologic malignancies. Hematologic toxicities including thrombosis and bleeding complications have been reported. Accurate estimates for thrombotic and bleeding outcomes are lacking. OBJECTIVES: We performed a systematic review and meta-analysis in patients who received CAR T-cell therapy for an underlying hematologic malignancy with the objective to: a) assess the thrombosis and bleeding risk associated with CAR T-cell therapy, b) assess the impact of CRS and ICANS on the risks of thrombosis and bleeding, and c) assess the safety of anticoagulant or antiplatelet use in the period following treatment with CAR T-cell therapy. METHODS: We searched MEDLINE, EMBASE, and Cochrane CENTRAL up to February 2022 for studies reporting thrombotic or bleeding outcomes in patients receiving CAR T-cell therapy. Pooled event rates were calculated using a random-effects model. We performed subgroup analyses stratified by follow-up duration, CAR T-cell target antigen, and underlying hematologic malignancy. RESULTS: We included 47 studies with a total of 7040 patients. High heterogeneity between studies precluded reporting of overall pooled rates of thrombotic and bleeding events. In studies with follow-up duration of ≤6 months, the pooled incidence of venous thrombotic events was 2.4% (95% CI, 1.4%-3.4%; I2 = 0%) per patient-month, whereas the rate was 0.1% (95% CI, 0%-0.1%; I2 = 0%) per patient-month for studies with longer follow-up periods (>6 months). The pooled incidences of any bleeding events per patient-month in studies with follow-up duration of ≤6 months and >6 months were 1.9% (95% CI, 0.6%-3.1%; I2 = 78%) and 0.3% (95% CI: 0%-0.8%, I2 = 40%), respectively. Secondary analyses by CAR T-cell target antigen, underlying malignancy, and primary outcome of the studies did not reveal significant differences in the rates of thromboembolism, any bleeding events, or major bleeding events. CONCLUSION: The risk of both thrombosis and bleeding following CAR T-cell therapy appears to be highest in the initial months following infusion.

Predominance of M2 macrophages in organized thrombi in chronic thromboembolic pulmonary hypertension patients.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a debilitating disease characterized by thrombotic occlusion of pulmonary arteries and vasculopathy, leading to increased pulmonary vascular resistance and progressive right-sided heart failure. Thrombotic lesions in CTEPH contain CD68+ macrophages, and increasing evidence supports their role in disease pathogenesis. Macrophages are classically divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which are involved in wound healing and tissue repair. Currently, the phenotype of macrophages and their localization within thrombotic lesions of CTEPH are largely unknown. In our study, we subclassified thrombotic lesions of CTEPH patients into developing fresh thrombi (FT) and organized thrombi (OT), based on the degree of fibrosis and remodeling. We used multiplex immunofluorescence histology to identify immune cell infiltrates in thrombotic lesions of CPTEH patients. Utilizing software-assisted cell detection and quantification, increased proportions of macrophages were observed in immune cell infiltrates of OT lesions, compared with FT. Strikingly, the proportions with a CD206+INOS- M2 phenotype were significantly higher in OT than in FT, which mainly contained unpolarized macrophages. Taken together, we observed a shift from unpolarized macrophages in FT toward an expanded population of M2 macrophages in OT, indicating a dynamic role of macrophages during CTEPH pathogenesis.

Prothrombotic autoantibodies targeting platelet factor 4/polyanion are associated with pediatric cerebral malaria.

BACKGROUNDFeatures of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM.METHODSPlasma from children with uncomplicated malaria (UM) (n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-platelet factor 4/polyanion (anti-PF4/P), anti-phospholipid, anti-phosphatidylserine, anti-myeloperoxidase, anti-proteinase 3, anti-dsDNA, anti-ß-2-glycoprotein I, and anti-cardiolipin. Plasma samples from individuals with nonmalarial coma (NMC) (n = 49) and healthy controls (HCs) (n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses.RESULTSMedian anti-PF4/P and anti-PS IgG levels were elevated in individuals with malaria infection relative to levels in HCs (P < 0.001) and patients with NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in children with CM (median = 0.27, IQR: 0.19-0.41) compared with those with UM (median = 0.19, IQR: 0.14-0.22, P < 0.0001). Anti-PS IgG levels did not differ between patients with UM and those with CM (P = 0.39). When patients with CM were stratified by malaria retinopathy (Ret) status, the levels of anti-PF4/P IgG correlated negatively with the peripheral platelet count in patients with Ret+ CM (Spearman's rho [Rs] = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02-275, P = 0.048). Plasma from patients with CM induced greater platelet activation in an ex vivo assay relative to plasma from patients with UM (P = 0.02), and the observed platelet activation was associated with anti-PF4/P IgG levels (Rs= 0.293, P = 0.035).CONCLUSIONSThrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.

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.

Mutual regulation of CD4+ T cells and intravascular fibrin in infections.

Innate myeloid cells especially neutrophils and their extracellular traps are known to promote intravascular coagulation and thrombosis formation in infections and various other conditions. Innate myeloid cell-dependent fibrin formation can support systemic immunity while its dysregulation enhances the severity of infectious diseases. Less is known about the immune mechanisms preventing dysregulation of fibrin homeostasis in infection. During experimental systemic infections local fibrin deposits in the liver microcirculation cause rapid arrest of CD4+ T cells. Arrested T-helper cells mostly represent Th17 cells that partially originate from the small intestine. Intravascular fibrin deposits activate mouse and human CD4+ T cells which can be mediated by direct fibrin-CD4+ T-cell interactions. Activated CD4+ T cells suppress fibrin deposition and microvascular thrombosis by directly counteracting coagulation activation by neutrophils and classical monocytes. T-cell activation, which is initially triggered by IL-12p40- and MHC-II-dependent mechanisms, enhances intravascular fibrinolysis via LFA-1. Moreover, CD4+ T cells disfavor the association of the thrombin-activatable fibrinolysis inhibitor (TAFI) with fibrin whereby fibrin deposition is increased by TAFI in the absence but not in the presence of T cells. In human infections thrombosis development is inversely related to microvascular levels of CD4+ T cells. Thus, fibrin promotes LFA-1-dependent T-helper cell activation in infections which drives a negative feedback cycle that rapidly restricts intravascular fibrin and thrombosis development.

The role of leukocytes in myeloproliferative neoplasm thromboinflammation.

Classic myeloproliferative neoplasms lacking the Philadelphia chromosome are stem cell disorders characterized by the proliferation of myeloid cells in the bone marrow and increased counts of peripheral blood cells. The occurrence of thrombotic events is a common complication in myeloproliferative neoplasms. The heightened levels of cytokines play a substantial role in the morbidity and mortality of these patients, establishing a persistent proinflammatory condition that culminates in thrombosis. The etiology of thrombosis remains intricate and multifaceted, involving blood cells and endothelial dysfunction, the inflammatory state, and the coagulation cascade, leading to hypercoagulability. Leukocytes play a pivotal role in the thromboinflammatory process of myeloproliferative neoplasms by releasing various proinflammatory and prothrombotic factors as well as interacting with other cells, which contributes to the amplification of the clotting cascade and subsequent thrombosis. The correlation between increased leukocyte counts and thrombotic risk has been established. However, there is a need for an accurate biomarker to assess leukocyte activation. Lastly, tailored treatments to address the thrombotic risk in myeloproliferative neoplasms are needed. Therefore, this review aims to summarize the potential mechanisms of leukocyte involvement in myeloproliferative neoplasm thromboinflammation, propose potential biomarkers for leukocyte activation, and discuss promising treatment options for controlling myeloproliferative neoplasm thromboinflammation.