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Heparin‐induced thrombocytopenia (HIT) is a serious adverse reaction to heparin. Other HIT‐like syndromes are increasingly recognised, mediated by antibodies binding to platelet factor 4, with or without identifiable polyanions. The history of heparin exposure is atypical for classical HIT and standard HIT laboratory tests may be negative. This manuscript describes subtypes of HIT‐like syndromes and highlights practical tips for diagnosis and therapy. [ABSTRACT FROM AUTHOR]

Heparin-induced thrombocytopenia (HIT) is an autoimmune disorder caused by antibodies against platelet factor 4 (PF4) and heparin complexes. Rapid immunoassays (IAs) for detection of these antibodies mark a milestone in HIT diagnosis, despite a higher false-positive rate compared with functional platelet-activation assays. However, combining different rapid IAs may help to improve their diagnostic specificity. Here, we compared the individual performance of the latex immunoturbidimetric assay (LIA; HemosIL HIT-Ab [PF4-H]; sensitivity 91.7%, specificity 68.4%) and chemiluminescence immunoassay (CLIA; HemosIL AcuStarHIT-Ab [PF4-H]; sensitivity 92.4%, specificity 85.8%) with their combined performance using two unique diagnostic algorithms in a single prospective cohort of suspected HIT patients. Using the simultaneous algorithm adapted from Warkentin et al, the combined LIA–CLIA had a sensitivity of 99.0% and specificity of 64.3%. The sequential algorithm adapted from Rittener-Ruff et al was applied in two theoretical scenarios to reflect real-world circumstances in diagnostic laboratories where access to clinical information is limited: (1) assuming all patients had an intermediate 4Ts score and (2) assuming all patients had a high 4Ts score. This algorithm correctly predicted HIT in 94.5% (high 4Ts) and 96.0% (intermediate 4Ts) and excluded HIT in 82.6% (high 4Ts) and 80.1% (intermediate 4Ts) of patients in either scenario, respectively. Although both combined algorithms improved diagnostic performance of individual IAs, the simultaneous algorithm showed fewer false predictions (7.9%) than the sequential algorithm (intermediate 4Ts: 37.6% and high 4Ts: 41.5%) and proved more practical as it does not rely on physician evaluations. Our findings highlight the importance of accounting for clinician and interlaboratory variability when evaluating diagnostic tests for HIT. [ABSTRACT FROM AUTHOR]

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Anti‐platelet factor 4 immunothrombotic syndromes comprise a group of disorders that include heparin‐induced thrombocytopenia and vaccine‐induced immune thrombocytopenia and thrombosis. These are highly prothrombotic, immunological disorders characterised by specific clinical and pathological criteria which include thrombocytopenia and thrombosis. While they are predominantly triggered by heparin and the adenoviral vector vaccines, respectively, other provoking factors have been described, as well as spontaneous forms. The unexplained co‐occurrence of thrombocytopenia with thrombosis should raise suspicion and prompt testing. This nutshell review discusses the pathophysiology, presenting features and diagnostic criteria for these conditions. [ABSTRACT FROM AUTHOR]

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but severe complication following vaccination with adenovirus vector-based COVID-19 vaccines. Antibodies directed against platelet factor 4 (PF4) are thought to be responsible for platelet activation and subsequent thromboembolic events in these patients. Since a single vaccination does not lead to sufficient immunization, subsequent vaccinations against COVID-19 have been recommended. However, concerns exist regarding the possible development of a new thromboembolic episode after subsequent vaccinations in VITT patients. Methods: We prospectively analyzed follow-up data from four VITT patients (three women and one man; median age, 44 years [range, 22 to 62 years]) who subsequently received additional COVID-19 vaccines. Platelet counts, anti-PF4/heparin antibody level measurements, and a functional platelet activation assay were performed at each follow-up visit. Additionally, we conducted a literature review and summarized similar reports on the outcome of subsequent vaccinations in patients with VITT. Results: The patients had developed thrombocytopenia and thrombosis 4 to 17 days after the first vaccination with ChAdOx1 nCoV-19. The optical densities (ODs) of anti-PF4/heparin antibodies decreased with time, and three out of four patients tested negative within 4 months. One patient remained positive even after 10 months post first vaccination. All four patients received an mRNA-based vaccine as a second vaccination against SARS-CoV-2. No significant drop in platelet count or new thromboembolic complications were observed during follow-up. We identified seven publications reporting subsequent COVID-19 vaccination in VITT patients. None of the patients developed thrombocytopenia or thrombosis after the subsequent vaccination. Conclusion: Subsequent vaccination with an mRNA vaccine appears to be safe in VITT patients. [ABSTRACT FROM AUTHOR]

Thrombosis after severe acute respiratory syndrome coronavirus 2 vaccination is a serious complication in patients with a thrombophilic predisposition. Herein, we present a 17-year-old female who had underlying antiphospholipid syndrome (APS) and developed deep vein thrombosis (DVT) 6 months after her second BNT162b2 vaccine dose. Although she had no family history of thrombosis, she had previously developed DVT at 6 years of age, with thrombus formation in the right common iliac vein and the inferior vena cava, along with concomitant left pulmonary infarction. The patient had received anticoagulant therapy for 6 years after DVT onset, with subsequent treatment cessation for 5 years without recurrence. She received the BNT162b2 vaccine at 17 years of age, 1 week before a routine outpatient visit. Platelet factor 4 elevation was detected 14 days after the first vaccination, persisting for 5 months without thrombotic symptoms. Six months after the second vaccine dose, the DVT recurred and was treated with a direct oral anticoagulant. The vaccine was hypothesized to exacerbate the patient's APS by activating coagulation. Platelet factor 4 levels may indicate coagulation status. When patients predisposed to thrombosis are vaccinated, coagulation status and platelet activation markers should be monitored to prevent DVT development. [ABSTRACT FROM AUTHOR]

Platelet factors regulate wound healing and can signal from the blood to the brain1,2. However, whether platelet factors modulate cognition, a highly valued and central manifestation of brain function, is unknown. Here we show that systemic platelet factor 4 (PF4) permeates the brain and enhances cognition. We found that, in mice, peripheral administration of klotho, a longevity and cognition-enhancing protein3-7, increased the levels of multiple platelet factors in plasma, including PF4. A pharmacologic intervention that inhibits platelet activation blocked klotho-mediated cognitive enhancement, indicating that klotho may require platelets to enhance cognition. To directly test the effects of platelet factors on the brain, we treated mice with vehicle or systemic PF4. In young mice, PF4 enhanced synaptic plasticity and cognition. In old mice, PF4 decreased cognitive deficits and restored aging-induced increases of select factors associated with cognitive performance in the hippocampus. The effects of klotho on cognition were still present in mice lacking PF4, suggesting this platelet factor is sufficient to enhance cognition but not necessary for the effects of klotho-and that other unidentified factors probably contribute. Augmenting platelet factors, possible messengers of klotho, may enhance cognition in the young brain and decrease cognitive deficits in the aging brain.

There is growing evidence that an active lifestyle can benefit brain health, delay the onset of neurodegenerative diseases and aging, and improve mood and cognitive function. Results seem to show that exercise-inspired or exercise-like drugs such as AICAR, metformin, GW501516, resveratrol, epicatechin, transfer plasma, platelet factor 4 and selenium effectively reduce excitotoxicity and mitochondrial dysfunction and improve neurodegenerative disease, angiogenesis and neurogenesis. The beneficial effects of exercise on the brain probably cannot be replaced by a single pill. Despite new discoveries, it is unlikely that a single molecule will ever be able to replicate all the benefits of exercise. Scientists developing drugs that mimic the effects of exercise do not aim to replace regular exercise throughout a person's life, but rather to use it in specific circumstances. However, not everyone is able to play sports due to age-related illnesses, injuries or frailty. The long-term goal is to understand how exercise increases neurogenesis and to try to develop new ways to mimic exercise-induced neurogenesis in people who cannot exercise. Identification of cellular targets activated by exercise could lead to the development of new compounds that can, to some extent, mimic the systemic and central effects of exercise. This review focuses on putative exercise mimetics such as AICAR, resveratrol, metformin, epicatechin, GW501516, transfer plasma, platelet factor 4 and selenium on brain health, neurodegenerative diseases and aging. [ABSTRACT FROM AUTHOR]

Thrombosis after severe acute respiratory syndrome coronavirus 2 vaccination is a serious complication in patients with a thrombophilic predisposition. Herein, we present a 17-year-old female who had underlying antiphospholipid syndrome (APS) and developed deep vein thrombosis (DVT) 6 months after her second BNT162b2 vaccine dose. Although she had no family history of thrombosis, she had previously developed DVT at 6 years of age, with thrombus formation in the right common iliac vein and the inferior vena cava, along with concomitant left pulmonary infarction. The patient had received anticoagulant therapy for 6 years after DVT onset, with subsequent treatment cessation for 5 years without recurrence. She received the BNT162b2 vaccine at 17 years of age, 1 week before a routine outpatient visit. Platelet factor 4 elevation was detected 14 days after the first vaccination, persisting for 5 months without thrombotic symptoms. Six months after the second vaccine dose, the DVT recurred and was treated with a direct oral anticoagulant. The vaccine was hypothesized to exacerbate the patient’s APS by activating coagulation. Platelet factor 4 levels may indicate coagulation status. When patients predisposed to thrombosis are vaccinated, coagulation status and platelet activation markers should be monitored to prevent DVT development.

Abstract Background Respiratory syncytial virus (RSV)-induced lung inflammation is one of the main causes of hospitalization and easily causes disruption of intestinal homeostasis in infants, thereby resulting in a negative impact on their development. However, the current clinical drugs are not satisfactory. Zedoary turmeric oil injection (ZTOI), a patented traditional Chinese medicine (TCM), has been used for clinical management of inflammatory diseases. However, its in vivo efficacy against RSV-induced lung inflammation and the underlying mechanism remain unclear. Purpose The present study was designed to confirm the in vivo efficacy of ZTOI against lung inflammation and intestinal disorders in RSV-infected young mice and to explore the potential mechanism. Study design and methods Lung inflammation was induced by RSV, and cytokine antibody arrays were used to clarify the effectiveness of ZTOI in RSV pneumonia. Subsequently, key therapeutic targets of ZTOI against RSV pneumonia were identified through multi-factor detection and further confirmed. The potential therapeutic material basis of ZTOI in target tissues was determined by non-target mass spectrometry. After confirming that the pharmacological substances of ZTOI can reach the intestine, we used 16S rRNA-sequencing technology to study the effect of ZTOI on the intestinal bacteria. Results In the RSV-induced mouse lung inflammation model, ZTOI significantly reduced the levels of serum myeloperoxidase, serum amyloid A, C-reactive protein, and thymic stromal lymphoprotein; inhibited the mRNA expression of IL-10 and IL-6; and decreased pathological changes in the lungs. Immunofluorescence and qPCR experiments showed that ZTOI reduced RSV load in the lungs. According to cytokine antibody arrays, platelet factor 4 (PF4), a weak chemotactic factor mainly synthesized by megakaryocytes, showed a concentration-dependent change in lung tissues affected by ZTOI, which could be the key target for ZTOI to exert anti-inflammatory effects. Additionally, sesquiterpenes were enriched in the lungs and intestines, thereby exerting anti-inflammatory and regulatory effects on gut microbiota. Conclusion ZTOI can protect from lung inflammation via PF4 and regulate gut microbiota disorder in RSV-infected young mice by sesquiterpenes, which provides reference for its clinical application in RSV-induced lung diseases.

Platelet hyperreactivity and hyperlipidaemia contribute significantly to atherosclerosis. Thus, it is desirable to review the platelet–hyperlipidaemia interplay and its impact on atherogenesis. Native low‐density lipoprotein (nLDL) and oxidized LDL (oxLDL) are the key proatherosclerotic components of hyperlipidaemia. nLDL binds to the platelet‐specific LDL receptor (LDLR) ApoE‐R2′, whereas oxLDL binds to the platelet‐expressed scavenger receptor CD36, lectin‐type oxidized LDLR 1 and scavenger receptor class A 1. Ligation of nLDL/oxLDL induces mild platelet activation and may prime platelets for other platelet agonists. Platelets, in turn, can modulate lipoprotein metabolisms. Platelets contribute to LDL oxidation by enhancing the production of reactive oxygen species and LDLR degradation via proprotein convertase subtilisin/kexin type 9 release. Platelet‐released platelet factor 4 and transforming growth factor β modulate LDL uptake and foam cell formation. Thus, platelet dysfunction and hyperlipidaemia work in concert to aggravate atherogenesis. Hypolipidemic drugs modulate platelet function, whereas antiplatelet drugs influence lipid metabolism. The research prospects of the platelet–hyperlipidaemia interplay in atherosclerosis are also discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Identifying therapeutics to delay, and potentially reverse, age-related cognitive decline is critical in light of the increased incidence of dementia-related disorders forecasted in the growing older population1. Here we show that platelet factors transfer the benefits of young blood to the ageing brain. Systemic exposure of aged male mice to a fraction of blood plasma from young mice containing platelets decreased neuroinflammation in the hippocampus at the transcriptional and cellular level and ameliorated hippocampal-dependent cognitive impairments. Circulating levels of the platelet-derived chemokine platelet factor 4 (PF4) (also known as CXCL4) were elevated in blood plasma preparations of young mice and humans relative to older individuals. Systemic administration of exogenous PF4 attenuated age-related hippocampal neuroinflammation, elicited synaptic-plasticity-related molecular changes and improved cognition in aged mice. We implicate decreased levels of circulating pro-ageing immune factors and restoration of the ageing peripheral immune system in the beneficial effects of systemic PF4 on the aged brain. Mechanistically, we identified CXCR3 as a chemokine receptor that, in part, mediates the cellular, molecular and cognitive benefits of systemic PF4 on the aged brain. Together, our data identify platelet-derived factors as potential therapeutic targets to abate inflammation and rescue cognition in old age.

The beneficial effects of physical activity on brain ageing are well recognised, with exerkines, factors that are secreted into the circulation in response to exercise, emerging as likely mediators of this response. However, the source and identity of these exerkines remain unclear. Here we provide evidence that an anti-geronic exerkine is secreted by platelets. We show that platelets are activated by exercise and are required for the exercise-induced increase in hippocampal precursor cell proliferation in aged mice. We also demonstrate that increasing the systemic levels of the platelet-derived exerkine CXCL4/platelet factor 4 (PF4) ameliorates age-related regenerative and cognitive impairments in a hippocampal neurogenesis-dependent manner. Together these findings highlight the role of platelets in mediating the rejuvenating effects of exercise during physiological brain ageing.

Background: Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse response to heparin therapy, characterized by decreased platelet count and increased risk of thrombosis. HIT, without the tell-tale sign of thrombocytopenia, has rarely been described. Key Clinical Question: Can HIT be diagnosed in the presence of thrombocytosis? What clinical clues and diagnostic tools facilitate accurate diagnosis in such cases? Clinical Approach: We report a case of HIT with thrombocytosis in a young male who initially presented after traumatic knee dislocation. HIT was diagnosed clinically through the discovery of a white thrombus during a vascular surgery procedure and corroborated by a positive latex immunoturbidimetric immunoassay (HemosIL HIT-Ab (platelet-factor 4(PF4)-heparin)), a rapid immunoassay. Conclusion: With its high sensitivity, specificity, and rapid results, the latex immunoturbidimetric immunoassay is a valuable diagnostic tool, even among patients with a seemingly low pretest probability. This case underscores the guidance imparted by Dr Andreas Greinacher: “[HIT] must be considered if thrombosis occurs or progresses despite effective heparinization even in the absence of thrombocytopenia.” Access to rapid and effective laboratory testing reduces the probability of diagnostic error.

Anti-PF4 disorders are a group of platelet-consumptive disorders characterized by platelet-activating antibodies against platelet factor 4 (PF4), thrombocytopenia and an increased risk of thrombosis. PF4 is a chemokine released by platelet alpha granules upon activation, which can form immune complexes with negatively charged substances, such as heparin, cartilage components, nucleic acids, and viral and bacterial agents. Antibodies formed in response to PF4-polyanion complexes may display platelet-activating properties and cause pancellular activation, leading to the marked prothrombotic state of anti-PF4 disorders. In recent years, the landscape of anti-PF4 disorders has evolved to include classic heparin-induced thrombocytopenia (cHIT), autoimmune HIT (aHIT), spontaneous HIT (SpHIT), vaccine-induced immune thrombotic thrombocytopenia (VITT), and the newly recognized spontaneous VITT (SpVITT). These disorders have garnered increased attention due to their association with severe clinical outcomes. Recent discoveries have expanded the understanding of these conditions, highlighting the role of various triggers, such as upper respiratory tract infections and monoclonal gammopathy of undetermined significance, in their development. Compared to cHIT, the less common anti-PF4 disorders VITT, aHIT, SpHIT and SpVITT generally appear more severe, with aggressive disease courses, more severe thrombocytopenia and a higher frequency of bleeding, thrombosis at unusual sites, involvement of the central nervous system and of multiple vascular beds. Clinical suspicion and knowledge of the less well-known triggers of anti-PF4 disorders are pivotal to ordering the appropriate laboratory tests and initiating the necessary treatments. Herein, we will review cHIT, aHIT, SpHIT and VITT, focusing on their clinical presentation and therapeutic management. [ABSTRACT FROM AUTHOR]

Les thrombopénies et thromboses induites par les anticorps dirigés contre le facteur plaquettaire 4 (FP4) constituent un groupe hétérogène de syndromes clinico-biologiques rares mais sévères, comprenant les thrombopénies induites par l'héparine (TIH) classiques, et plus rarement auto-immunes, les thrombopénies et thromboses induites par les vaccins contre la Covid-19 (VITT pour vaccine-induced immune thrombotic thrombocytopenia), et, récemment, les syndromes VITT-like. Ces pathologies induisent toutes une hypercoagulabilité majeure, en lien avec une activation des cellules sanguines par les anticorps anti-FP4, conduisant à des thromboses potentiellement sévères, qu'il convient de traiter en urgence. La gravité de la thrombopénie et des thromboses, ainsi que leur localisation varient selon la pathologie, les TIH auto-immunes et les VITT étant associées à des formes particulièrement agressives, notamment des thromboses veineuses cérébrales. Le diagnostic des thrombopénies et thromboses induites par les anticorps anti-FP4 s'appuie sur un faisceau d'arguments, tout d'abord cliniques, puis biologiques, reposant sur la mise en évidence des anticorps anti-FP4 par des tests immunologiques et, le plus souvent, confirmation de leur pathogénicité à l'aide de tests d'activation plaquettaire. Les performances de ces différents outils étant très variables selon la pathologie, le biologiste possède un rôle majeur dans le choix des tests utilisés, et leur interprétation, pour assurer un diagnostic correct et une prise en charge adaptée du patient. Thrombocytopenia and thromboses induced by antibodies to platelet factor 4 (PF4) constitute a heterogeneous group of rare but severe clinical and biological syndromes, including classical heparin-induced thrombocytopenia (HIT), and more rarely autoimmune HIT, vaccine-induced immune thrombotic thrombocy-topenia (VITT), and, recently, VITT-like syndromes. These pathologies all induce major hypercoagulability, related to blood cell activation by anti-PF4 antibodies, leading to potentially severe thromboses, which require urgent treatment. The severity of thrombocytopenia and thromboses, and their location, vary with the disease; autoimmune HIT and VITT are associated with particularly aggressive forms, including cerebral venous thrombosis. The diagnosis of thrombocytope-nia and thromboses induced by anti-PF4 antibodies relies on a combination of clinical and biological factors, including the detection of anti-PF4 antibodies by immunological assays and, in most cases, confirmation of their pathogenicity by platelet activation tests. As the performance of these tools varies greatly depending on the pathology, the biologist plays a major role in the choice of the assays, and their interpretation, to ensure correct diagnosis and appropriate management of the patient. [ABSTRACT FROM AUTHOR]

The prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation. Here we demonstrate significantly increased concentrations of von Willebrand factor (VWF), platelet factor 4 (PF4), serum amyloid A (SAA), α-2 antiplasmin (α-2AP), endothelial-leukocyte adhesion molecule 1 (E-selectin), and platelet endothelial cell adhesion molecule (PECAM-1) in the soluble part of the blood. It was noteworthy that the mean level of α-2 antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls. This is alarming if we take into consideration that a significant amount of the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules). We conclude that presence of microclotting, together with relatively high levels of six biomarkers known to be key drivers of endothelial and clotting pathology, points to thrombotic endothelialitis as a key pathological process in Long COVID. [ABSTRACT FROM AUTHOR]

Not available.

Anti-platelet factor 4 (PF4) disorders are a group of platelet-consumptive disorders characterized by platelet-activating antibodies against PF4, thrombocytopenia and an increased risk of thrombosis. PF4 is a chemokine released by platelet alpha granules upon activation, which can form immune complexes with negatively charged substances, such as heparin, cartilage components, nucleic acids, and viral and bacterial agents. Antibodies formed in response to PF4-polyanion complexes may display platelet-activating properties and cause pan-cellular activation, leading to the marked prothrombotic state of anti-PF4 disorders. In recent years, the landscape of anti-PF4 disorders has evolved to include classic heparin-induced thrombocytopenia (cHIT), autoimmune HIT (aHIT), spontaneous HIT (SpHIT), vaccine-induced immune thrombotic thrombocytopenia (VITT), and the newly recognized spontaneous VITT (SpVITT). These disorders have garnered increased attention due to their association with severe clinical outcomes. Recent discoveries have expanded the understanding of these conditions, highlighting the role of various triggers, such as upper respiratory tract infections and monoclonal gammopathy of undetermined significance, in their development. Compared to cHIT, the less common anti-PF4 disorders VITT, aHIT, SpHIT and SpVITT generally appear more severe, with aggressive disease courses, more severe thrombocytopenia and a higher frequency of bleeding, thrombosis at unusual sites, involvement of the central nervous system and of multiple vascular beds. Clinical suspicion and knowledge of the less well-known triggers of anti-PF4 disorders are pivotal to ordering the appropriate laboratory tests and initiating the necessary treatments. Herein, we will review cHIT, aHIT, SpHIT and VITT, focusing on their clinical presentation and therapeutic management.

Abstract Background The effect of platelet factor 4 (PF4) on bone marrow mesenchymal stem cells (BMMSCs) and osteoporosis is poorly understood. Therefore, this study aimed to evaluate the effects of PF4‐triggered bone destruction in mice and determine the underlying mechanism. Methods First, in vitro cell proliferation and cell cycle of BMMSCs were assessed using a CCK8 assay and flow cytometry, respectively. Osteogenic differentiation was confirmed using staining and quantification of alkaline phosphatase and Alizarin Red S. Next, an osteoporotic mouse model was established by performing bilateral ovariectomy (OVX). Furthermore, the PF4 concentrations were obtained using enzyme‐linked immunosorbent assay. The bone microarchitecture of the femur was evaluated using microCT and histological analyses. Finally, the key regulators of osteogenesis and pathways were investigated using quantitative real‐time polymerase chain reaction and Western blotting. Results Human PF4 widely and moderately decreased the cell proliferation and osteogenic differentiation ability of BMMSCs. Furthermore, the levels of PF4 in the serum and bone marrow were generally increased, whereas bone microarchitecture deteriorated due to OVX. Moreover, in vivo mouse PF4 supplementation triggered bone deterioration of the femur. In addition, several key regulators of osteogenesis were downregulated, and the integrin α5‐focal adhesion kinase‐extracellular signal‐regulated kinase (ITGA5‐FAK‐ERK) pathway was inhibited due to PF4 supplementation. Conclusions PF4 may be attributed to OVX‐induced bone loss triggered by the suppression of bone formation in vivo and alleviate BMMSC osteogenic differentiation by inhibiting the ITGA5‐FAK‐ERK pathway.

Heparin-induced thrombocytopenia (HIT) occurs with the development of IgG antibodies that bind complexes of heparin and platelet factor 4 (PF4), which activate platelets and result in a profoundly prothrombotic condition. In rare instances, this syndrome develops in the absence of proximate heparin administration, referred to as spontaneous HIT, for which less than three dozen cases have been reported. Spontaneous HIT is considered a subtype of "autoimmune HIT" (aHIT), characterized by platelet activation in the serotonin release assay (SRA) without the addition of exogenous heparin. Here, we report spontaneous HIT as the presenting feature in a patient with 2019 coronavirus disease infection (COVID-19).A 66-year-old male presented with progressive leg pain and was found to have a platelet count of 39 × 109/L and multiple lower extremity arterial thromboses requiring fasciotomy and thrombectomy. He had no recent hospitalization, heparin exposure, vaccinations, or known thrombophilia. He had a strongly positive IgG-specific enzyme-linked immunosorbent assay for heparin-PF4 antibodies, and the SRA was strongly positive both with and without the addition of heparin. He was treated successfully with bivalirudin, intravenous immunoglobulin, and apixaban.

Introduction: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet severe adverse complication first identified during the global vaccination effort against SARS-CoV-2 infection, predominantly observed following administration of the ChAdOx1-S (Oxford-AstraZeneca) and Ad26.CoV2.S (Johnson & Johnson/Janssen) adenoviral vector-based vaccines. Unlike other anti-platelet factor 4 (PF4) antibody-mediated disorders, such as heparin-induced thrombocytopenia (HIT), VITT arises with the development of platelet-activating anti-PF4 antibodies 4–42 days post-vaccination, typically featuring thrombocytopenia and thrombosis at unusual sites. Aim: To explore the unique properties, pathogenic mechanisms, and long-term persistence of VITT antibodies in patients, in comparison with other anti-PF4 antibody-mediated disorders. Discussion: This review highlights the complexity of VITT as it differs in antibody behavior and clinical presentation from other anti-PF4-mediated disorders, including the high incidence rate of cerebral venous sinus thrombosis (CVST) and the persistence of anti-PF4 antibodies, necessitating a re-evaluation of long-term patient care strategies. The nature of VITT antibodies and the underlying mechanisms triggering their production remain largely unknown. Conclusion: The rise in awareness and subsequent prompt recognition of VITT is paramount in reducing mortality. As vaccination campaigns continue, understanding the role of adenoviral vector-based vaccines in VITT antibody production is crucial, not only for its immediate clinical implications, but also for developing safer vaccines in the future. [ABSTRACT FROM AUTHOR]

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. 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. 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. [ABSTRACT FROM AUTHOR]

This article provides guidelines for the diagnosis and management of heparin-induced thrombocytopenia (HIT), a condition characterized by low platelet count caused by an immune response to heparin. It discusses different types of HIT, the incidence of the condition, and the presentation and assessment of HIT. The article also provides recommendations for platelet monitoring and laboratory diagnosis of HIT, as well as management strategies including the use of alternative anticoagulants and therapies such as intravenous immunoglobulin and plasma exchange. The recommendations are evidence-based and aim to assist healthcare professionals in managing HIT. [Extracted from the article]

Objectives Heparin-induced thrombocytopenia (HIT) is a rare but life-threatening condition that requires rapid diagnosis for proper management. Laboratory testing should only be performed on patients with intermediate- or high-risk pretest probability. The platelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA) is the screening test that should be confirmed by higher specificity testing such as the heparin-induced platelet aggregation (HIPA) or the serotonin release assay (SRA). This study aims to evaluate the performance of the HIPA in comparison to the SRA, establish cutoffs of the PF4 ELISA to predict positivity for HIPA/SRA, and study the mortality rate between patients with suspected HIT confirmed as HIT positive vs negative. Methods All positive PF4 ELISA cases with confirmatory HIPA and SRA testing were included. As the SRA was considered the gold standard, the HIPA performance was evaluated in comparison to SRA before and after the implementation of a new standardized interpretation guide in 2022. The mortality of these cases was also documented by chart reviews. Results In total, 232 cases with positive or indeterminate anti-PF4 IgG ELISA had confirmatory testing with HIPA and SRA. The sensitivity of the HIPA improved from 55.4% in 2018 to 2021 to 73.8% in 2022. The specificity remained similarly high in 2018 to 2021 vs 2022 (94.9% vs 87.5%). The negative predictive value (NPV) improved in 2022. The PF4 optical density cutoff to predict the positivity of SRA was 0.85 vs 1.47 to predict the positivity of HIPA but decreased to 0.83 when combining HIPA and/or SRA. There was no significant difference in mortality between patients with suspected HIT confirmed positive vs negative. Conclusions Although the HIPA has a lower sensitivity than the SRA, the new standardized interpretation guide improved its sensitivity and NPV in 2022. Future improvements are needed to use the HIPA as a stand-alone confirmatory test with the goal to shorten hospital length of stay and expedite proper anticoagulation management. [ABSTRACT FROM AUTHOR]

Objective The aim of this study was to determine an optimal cutoff value for the newly available HemosIL-AcuStar-HIT-IgG assay (AcuStar) for the diagnosis of heparin-induced thrombocytopenia (HIT). Method We evaluated the performance of AcuStar using serotonin release assay (SRA) as the gold standard and incorporated 4T score calculation in a cohort of suspected HIT cases. Statistical analysis was performed to determine optimal cutoff value for the diagnosis of HIT. Result A diagnosis of HIT can be excluded with a platelet factor 4 (PF4) value of <0.4 U/mL by AcuStar and 4T score in the low-risk category (≤3). All other cases will require confirmation with a functional test. Conclusion Our study resulted in the implementation of a diagnostic algorithm for laboratory diagnosis of HIT, which incorporates pretest calculation of 4T score and AcuStar as a screening test, with reflex confirmation by SRA. This new algorithm resulted in extended hours of test availability and a more rapid turnaround time in reporting PF4 results. [ABSTRACT FROM AUTHOR]

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare autoimmune condition associated with recombinant adenovirus (rAV)-based COVID-19 vaccines. It is thought to arise from autoantibodies targeting platelet factor 4 (aPF4), triggered by vaccine-induced inflammation and the formation of neo-antigenic complexes between PF4 and the rAV vector. To investigate the specific induction of aPF4 by rAV-based vaccines, we examined sera from rAV vaccine recipients (AZD1222, AD26.COV2.S) and messenger RNA (mRNA) based (mRNA-1273, BNT162b2) COVID-19 vaccine recipients. We compared the antibody fold change (FC) for aPF4 and for antiphospholipid antibodies (aPL) of rAV to mRNA vaccine recipients. We combined two biobanks of Dutch healthcare workers and matched rAV-vaccinated individuals to mRNA-vaccinated controls, based on age, sex and prior history of COVID-19 (AZD1222: 37, Ad26.COV2.S: 35, mRNA-1273: 47, BNT162b2: 26). We found no significant differences in aPF4 FCs after the first (0.99 vs. 1.08, mean difference (MD) = −0.11 (95% CI −0.23 to 0.057)) and second doses of AZD1222 (0.99 vs. 1.10, MD = −0.11 (95% CI −0.31 to 0.10)) and after a single dose of Ad26.COV2.S compared to mRNA-based vaccines (1.01 vs. 0.99, MD = 0.026 (95% CI −0.13 to 0.18)). The mean FCs for the aPL in rAV-based vaccine recipients were similar to those in mRNA-based vaccines. No correlation was observed between post-vaccination aPF4 levels and vaccine type (mean aPF difference −0.070 (95% CI −0.14 to 0.002) mRNA vs. rAV). In summary, our study indicates that rAV and mRNA-based COVID-19 vaccines do not substantially elevate aPF4 levels in healthy individuals. [ABSTRACT FROM AUTHOR]

Background & Aims: Platelet factor 4 (PF4) is considered as a chemokine mainly strode in the granules of platelets. Its important role in heparin-induced thrombocytopenia (HIT) was the basis of many investigations about this chemokine. High affinity to heparin was used to extract of PF4 from platelet concentrates (PCs). Despite many advantages of recombinant PF4 (rPF4), some researchers prefer to purify rich proteins of platelets from outdated PCs mainly because of cost effectiveness. The main aim of this study was introducing a home-made method to purify PF4 from PCs in blood banks. Materials & Methods: In this experimental study, we presented a customized procedure based on immunoaffinity chromatography to isolate PF4 that may be useful for laboratory access of PCs from blood bank centers. Briefly, platelet lysate (PL) was extracted from PCs by freeze/thaw cycles and then treated with anti-PF4 antibody to elute PF4 extract in the final product. Data were analyzed by SPSS software (version 26). Student t-test was used to compare the results. A probability of < 0.05 was accepted as significant statistically. Results: Our experiments showed that immunoaffinity chromatography might be considered as an alternative source to provide PF4 particularly in view of cost effectiveness. Conclusion: PF4 is one of the most bulk growth factors stored in platelets. It has the versatile applications both in diagnosis of HIT and in the study of platelet biology. A home-made protocol presented in this investigation can be helpful to obtain PF4 from blood bank in a reasonable scale.

Therapeutic plasma exchange (TPE) has been proposed to remove heparin-induced thrombocytopenia (HIT) antibodies before planned thoracic surgery in patients with acute HIT and to allow brief re-exposure to heparin during surgery. In patients on extracorporeal membrane oxygenation (ECMO), simultaneous administration of TPE and alternative nonheparin anticoagulant therapies is challenging.

Autoimmune thrombocytopenia (aHIT) is a severe subtype of heparin-induced thrombocytopenia (HIT) with atypical clinical features caused by highly pathological IgG antibodies ("aHIT antibodies") that activate platelets even in the absence of heparin. The clinical features of aHIT include: the onset or worsening of thrombocytopenia despite stopping heparin ("delayed-onset HIT"), thrombocytopenia persistence despite stopping heparin ("persisting" or "refractory HIT"), or triggered by small amounts of heparin (heparin "flush" HIT), most cases of fondaparinux-induced HIT, and patients with unusually severe HIT (e.g., multi-site or microvascular thrombosis, overt disseminated intravascular coagulation [DIC]). Special treatment approaches are required. For example, unlike classic HIT, heparin cessation does not result in de-escalation of antibody-induced hemostasis activation, and thus high-dose intravenous immunoglobulin (IVIG) may be indicated to interrupt aHIT-induced platelet activation; therapeutic plasma exchange may be required if high-dose IVIG is ineffective. Also, aHIT patients are at risk for treatment failure with (activated partial thromboplastin time [APTT]-adjusted) direct thrombin inhibitor (DTI) therapy (argatroban, bivalirudin), either because of APTT confounding (where aHIT-associated DIC and resulting APTT prolongation lead to systematic underdosing/interruption of DTI therapy) or because DTI inhibits thrombin-induced protein C activation. Most HIT laboratories do not test for aHIT antibodies, contributing to aHIT under-recognition. [ABSTRACT FROM AUTHOR]

An effective worldwide vaccination campaign started and is still being carried out in the face of the coronavirus disease 2019 (COVID-19) pandemic. While vaccines are great tools to confront the pandemic, predominantly adenoviral vector-based vaccines can cause a rare severe adverse effect, termed vaccine-induced immune thrombocytopenia and thrombosis (VITT), in about 1 in 100,000 vaccinated individuals. VITT is diagnosed 5–30 days post-vaccination and clinically characterized by thrombocytopenia, strongly elevated D-dimer levels, platelet-activating anti-platelet factor 4 (PF4) antibodies and thrombosis, especially at atypical sites such as the cerebral venous sinus and/or splanchnic veins. There are striking similarities between heparin-induced thrombocytopenia (HIT) and VITT. Both are caused by anti-PF4 antibodies, causing platelet and leukocyte activation which results in massive thrombo-inflammation. However, it is still to be determined why PF4 becomes immunogenic in VITT and which constituent of the vaccine triggers the immune response. As VITT-like syndromes are increasingly reported in patients shortly after viral infections, direct virus-PF4 interactions might be most relevant. Here we summarize the current information and hypotheses on the pathogenesis of VITT and address in vivo models, especially murine models for further studies on VITT. [ABSTRACT FROM AUTHOR]

Heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT) are highly prothrombotic (thrombosis frequency ≥50%). Both are caused by platelet-activating anti-platelet factor 4 (PF4) antibodies, forming PF4/IgG-containing immune complexes that engage platelet FcγIIa receptors, producing strong platelet activation. In HIT, heparin crosslinks several PF4 molecules, whereas in VITT, anti-PF4 antibodies alone crosslink PF4. Sufficient levels of circulating anti-PF4 antibodies are needed to create the pathogenic immune complexes on platelet surfaces; this explains why certain serum (plasma)-based assays are highly sensitive for detecting HIT/VITT antibodies. Accordingly, HIT and VITT are "clinical-pathological" disorders, that is, positive testing for such antibodies—together with a compatible clinical picture—is integral for diagnosis. Heparin (low concentrations) enhances HIT antibody-induced platelet activation, but platelet activation by VITT sera is usually inhibited by heparin. For both HIT and VITT, high sensitivity (>99% and >95%, respectively) characterizes PF4-dependent enzyme immunoassays (EIAs) and PF4-enhanced platelet activation assays; in contrast, certain rapid immunoassays have high sensitivity for HIT (>90-97%) but poor sensitivity (<25%) for VITT. HIT and VITT antibodies are directed at distinct sites on PF4: solid-phase EIAs and platelet activation assays are indifferent to these distinct antigen targets, but rapid immunoassays are not. We discuss a conceptual model where PF4 is viewed as a "globe," with the heparin-binding site the "equator"; in this model, HIT antibodies are primarily directed at antigen site(s) at the north and south "poles" of PF4 (formed when PF4 binds to heparin), whereas VITT antibodies recognize sites on the equator. [ABSTRACT FROM AUTHOR]

PF4 is a pro-atherosclerotic molecule. Endothelial CD40, upon binding to its ligand CD40L, induces endothelial cell (EC) activation, which is a vital pathophysiological process in the initiation and progression of atherosclerosis. However, the relationship between PF4 and endothelial CD40 remains elusive. This study aims to investigate whether and how PF4 affects endothelial CD40 expression using primary HAECs. PF4 treatment down-regulated sirtuin 1 (SIRT1) expression but upregulated the expression of acetylated NF-κB p65 (Ac-p65) and CD40 in HAECs in a concentration- and time-dependent manner. Pretreatment with SIRT1 agonist (SRT1720 or RSV) or SIRT1-overexpressing lentivirus attenuated PF4-induced Ac-p65 and CD40 expression in HAECs, whereas preincubation with SIRT1 antagonist (NAM or EX527) or SIRT1 shRNA had the opposite effect. To investigate whether NF-κB/p65 signaling pathway modulates CD40 expression in PF4-treated HAECs, PDTC, a NF-κB inhibitor, and p65-shRNA were introduced. PDTC or p65-shRNA treatment down-regulated Ac-p65 expression in HAECs. PDTC or p65-shRNA preincubation suppressed CD40 expression in HAECs after PF4 treatment. To better determine whether SIRT1 regulates CD40 expression in PF4-treated HAECs via the NF-κB/p65 signaling pathway, p65-knockdown HAECs were preincubated with SIRT1 agonists before PF4 treatment. SIRT1 agonist preincubation further decreased CD40 expression in p65-knockdown HAECs treated with PF4. Moreover, PF4 treatment promoted p65 nuclear translocation in HAECs. The results of dual luciferase assay demonstrated that four NF-κB binding sites in the promoter of human CD40 gene were activated in PF4-treated HAECs. In conclusion, our findings suggest that PF4 treatment facilitates CD40 expression in HAECs through the SIRT1/NF-κB/p65 pathway. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) contains variably repeating disaccharide units organized into high‐ and low‐sulfated domains. This rich structural diversity enables HS to interact with many proteins and regulate key signaling pathways. Efforts to understand structure‐function relationships and harness the therapeutic potential of HS are hindered by the inability to synthesize an extensive library of well‐defined HS structures. We herein report a rational and expedient approach to access a library of 27 oligosaccharides from natural aminoglycosides as HS mimetics in 7–12 steps. This strategy significantly reduces the number of steps as compared to the traditional synthesis of HS oligosaccharides from monosaccharide building blocks. Combined with computational insight, we identify a new class of four trisaccharide compounds derived from the aminoglycoside tobramycin that mimic natural HS and have a strong binding to heparanase but a low affinity for off‐target platelet factor‐4 protein. [ABSTRACT FROM AUTHOR]

Aside from their key protective roles in hemostasis and innate immunity, platelets are now recognized as having multifaceted, adverse roles in the pathogenesis, progression and outcome of many types of human malignancy. The most consistent and compelling evidence in this context has been derived from the notable association of elevated circulating platelet counts with the onset and prognosis of various human malignancies, particularly lung cancer, which represents the primary focus of the current review. Key topics include an overview of the association of lung cancer with the circulating platelet count, as well as the mechanisms of platelet-mediated, pro-tumorigenic immunosuppression, particularly the role of transforming growth factor beta 1. These issues are followed by a discussion regarding the pro-tumorigenic role of platelet-derived microparticles (PMPs), the most abundant type of microparticles (MPs) in human blood. In this context, the presence of increased levels of PMPs in the blood of lung cancer patients has been associated with tumor growth, invasion, angiogenesis and metastasis, which correlate with disease progression and decreased survival times. The final section of the review addresses, firstly, the role of cancer-related platelet activation and thrombosis in the pathogenesis of secondary cardiovascular disorders and the associated mortality, particularly in lung cancer, which is second only to disease progression; secondly, the review addresses the potential role of antiplatelet agents in the adjunctive therapy of cancer. [ABSTRACT FROM AUTHOR]

Heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT) are rare, iatrogenic immune-mediated conditions with high rates of thrombosis-related morbidity and mortality. HIT is a long-recognized reaction to the administration of the common parenterally administered anticoagulant heparin (or its derivatives), while VITT is a new, distinct syndrome occurring in response to adenovirus-based vaccines against coronavirus disease 2019 and potentially other types of vaccines. A feature of both HIT and VITT is paradoxical thrombosis despite a characteristic low platelet count, mediated by the presence of platelet-activating antibodies to platelet factor 4. Several additional factors have also been suggested to contribute to clot formation in HIT and/or VITT, including monocytes, tissue factor, microparticles, endothelium, the formation of neutrophil extracellular traps, complement, procoagulant platelets, and vaccine components. In this review, we discuss the literature to date regarding mechanisms contributing to thrombosis in both HIT and VITT and explore the pathophysiological similarities and differences between the two conditions. [ABSTRACT FROM AUTHOR]

Platelet factor 4 (PF4, synonym: CXCL4) is an evolutionary old chemokine with proposed roles in hemostasis and antimicrobial defense. In addition, PF4 has attracted considerable attention as a crucial mediator of one of the most prothrombotic adverse drug effects affecting blood cells, heparin‐induced thrombocytopenia (HIT). Interest in PF4 substantially increased in 2021 when it was identified as the target antigen in the life‐threatening adverse effect, vaccine‐induced immune thrombotic thrombocytopenia (VITT). We address the concept that a major biological function of PF4—a strongly cationic chemokine—is to bind to negatively‐charged prokaryotic microorganisms, resulting in structural changes in PF4 that trigger a danger signal recognized by the adaptive immune system. Application of biophysical tools has provided substantial insights into the molecular mechanisms by which PF4 becomes immunogenic, providing insights into a new mechanism of autoimmunity. Binding of autoantibodies with high affinity induces conformational change(s) in the endogenous protein, which are then recognized as foreign antigen, as exemplified by the prothrombotic disorders, autoimmune HIT and VITT. The final part of our review summarizes current assays for HIT and VITT, explaining how structural aspects of anti‐PF4 pathobiology relate to assay design and performance characteristics. Currently, functional (platelet activation) assays using washed platelets detect HIT antibodies when heparin is added, and VITT antibodies when PF4 is added. Solid‐phase PF4‐dependent immunoassays using microtiter plates are sensitive for both HIT and VITT antibodies, while rapid immunoassays, in which the PF4/heparin antigen is coated on beads, are sensitive and specific for HIT, but not for VITT antibodies. [ABSTRACT FROM AUTHOR]

In response to the ongoing COVID-19 pandemic, pharmaceutical companies have been able to rapidly develop and distribute effective vaccines. Despite some common, minor, local, and systemic adverse effects following vaccination, there is also, in rare cases, a potential for the development of anti-platelet factor 4 antibodies, resulting in platelet activation and aggregation leading to potentially life-threatening thrombosis. Vaccine-induced immune thrombocytopaenia (VITT) is a rare immune response following administration of adenoviral vector vaccines against severe acute respiratory coronavirus 2 (SARS-CoV-2) such as ChAdOx1 nCoV-19 AstraZeneca – Oxford and Ad26.COV2. S Johnson & Johnson. Since the discovery of the syndrome, the mortality rate has decreased by 90%. Therefore, in this paper, data including epidemiology and pathophysiology of the syndrome, followed by the diagnosis criteria and management options, were collected. The authors believe that spreading knowledge further among primary care physicians and other healthcare professionals will lead to better VITT treatment outcomes.

Abstract Background Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation. Methods Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician. Results Our long COVID cohort’s symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots. Conclusion Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare autoimmune condition associated with recombinant adenovirus (rAV)-based COVID-19 vaccines. It is thought to arise from autoantibodies targeting platelet factor 4 (aPF4), triggered by vaccine-induced inflammation and the formation of neo-antigenic complexes between PF4 and the rAV vector. To investigate the specific induction of aPF4 by rAV-based vaccines, we examined sera from rAV vaccine recipients (AZD1222, AD26.COV2.S) and messenger RNA (mRNA) based (mRNA-1273, BNT162b2) COVID-19 vaccine recipients. We compared the antibody fold change (FC) for aPF4 and for antiphospholipid antibodies (aPL) of rAV to mRNA vaccine recipients. We combined two biobanks of Dutch healthcare workers and matched rAV-vaccinated individuals to mRNA-vaccinated controls, based on age, sex and prior history of COVID-19 (AZD1222: 37, Ad26.COV2.S: 35, mRNA-1273: 47, BNT162b2: 26). We found no significant differences in aPF4 FCs after the first (0.99 vs. 1.08, mean difference (MD) = −0.11 (95% CI −0.23 to 0.057)) and second doses of AZD1222 (0.99 vs. 1.10, MD = −0.11 (95% CI −0.31 to 0.10)) and after a single dose of Ad26.COV2.S compared to mRNA-based vaccines (1.01 vs. 0.99, MD = 0.026 (95% CI −0.13 to 0.18)). The mean FCs for the aPL in rAV-based vaccine recipients were similar to those in mRNA-based vaccines. No correlation was observed between post-vaccination aPF4 levels and vaccine type (mean aPF difference −0.070 (95% CI −0.14 to 0.002) mRNA vs. rAV). In summary, our study indicates that rAV and mRNA-based COVID-19 vaccines do not substantially elevate aPF4 levels in healthy individuals.

In ultrarare cases, patients vaccinated with DNA adenovirus vector vaccine against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), develop a vaccine-induced immune thrombotic thrombocytopenia (VITT), with a high incidence of fatal cases. The causative agent is the development of platelet factor 4 (PF4)-dependent antibodies that resemble heparin-induced thrombocytopenia (HIT) complication, although many differences can be noticed in clinical presentation, antibody reactivity, involved epitopes on the PF4 protein, and pathological mechanisms. From the literature review, and the experience of HIT and testing a few plasmas from patients with VITT, this review analyzes the possible mechanisms, which show the strong immunoglobulin G (IgG) antibody reactivity to PF4 alone, in the absence of heparin, and to a lesser extend to stoichiometric complexes of PF4 and heparin (H-PF4). In addition, much lower heparin concentrations are required for inhibiting antibody binding to PF4. These concentrations are much lower than those required for disrupting the stoichiometric H-PF4 complexes. This confirms that IgG antibodies responsible for HIT bind preferentially to PF4, to epitopes that are readily masked by low concentrations of heparin. These antibodies are at a much higher concentration than the current ones observed for HIT, keeping a strong reactivity even for plasma dilutions as high as 1/500 to 1/5,000, whilst the current dilution for testing heparin-dependent antibodies in HIT is 1/100. Although VITT anti-PF4 antibodies can be detected with the current anti-H-PF4 enzyme-linked immunosorbent assays (ELISAs) designed for HIT, some assays have low sensitivity or are unreactive, like lateral immunofiltration methods or chemiluminescent automated assays. The preferred method should concern the use of capture assays using PF4 coated solid surfaces. This report proposes that the immune response is only targeted to the binding domain of PF4 with the hexons present on the adenovirus vector, through an epitope spreading mechanism, without any exposure of neo-epitopes on PF4 protein.

Abstract Background Neutrophil extracellular traps (NETs) contribute to the creation of a coagulation state in various diseases. Currently, it is not clear whether NETs are present in the thrombi and plasma of patients with cerebral venous sinus thrombosis (CVST). This study aimed to investigate the presence of NETs in thrombi and blood samples from CVST patients and the procoagulant activity (PCA) of NETs during the progression of CVST. Results Thrombi obtained from CVST patients undergoing thrombectomy were examined by immunochemistry using neutrophil elastase (NE), CD66b and citrullinated histone H3(citH3). The presence of NET markers in samples from 37 CVST patients and 32 healthy people was evaluated by ELISA. NET-producing neutrophils and neutrophil-platelet (PLT) aggregates were examined in samples obtained from CVST patients and healthy people by flow cytometry. The TAT complex in plasma sample from each group was detected by ELISA to evaluate the procoagulant activity of NETs in CVST patients. Neutrophils from healthy subjects were treated with PLT-rich plasma in the presence of anti-PF4 antibodies or an autophagy inhibitor and analyzed by flow cytometry and confocal microscopy. After treatment with NETs, the expression of von Willebrand factor (VWF), tissue factor (TF) and CD31 in human brain microvascular endothelial cells (HBMECs) was measured by confocal microscopy and western blotting. Our results showed that NETs were abundant in the plasma and thrombi from CVST patients. Platelet factor 4 (PF4) from CVST PLTs induced NET generation through autophagy. NETs could induce PCA by modulating TF and phosphatidylserine (PS) in CVST. NETs also disrupted the endothelial barrier and transformed ECs into a procoagulant phenotype to exacerbate thrombogenicity. Conclusions NET generation was mediated by PF4 from PLTs through autophagy and contribute to thrombosis in CVST patients.