Role of deubiquitinase USP47 in cardiac function alleviation and anti-inflammatory immunity after myocardial infarction by regulating NLRP3 inflammasome-mediated pyroptotic signal pathways.

Myocardial infarction (MI) is an event of heart attack due to the formation of plaques in the interior walls of the arteries. This study is conducted to explore the role of ubiquitin-specific peptidase 47 (USP47) in cardiac function and inflammatory immunity. MI mouse models were established, followed by an appraisal of cardiac functions, infarct size, pathological changes, and USP47 and NLRP3 levels. MI cell models were established in HL-1 cells using anoxia. Levels of cardiac function-associated proteins, USP7, interferon regulatory factor 1 (IRF1), platelet factor-4 (CXCL4), pyroptotic factors, and neutrophil extracellular traps (NETs) were determined. The bindings of IRF1 to USP47 and the CXCL4 promoter and the ubiquitination of IRF1 were analyzed. USP47 was upregulated in myocardial tissues of MI mice. USP47 inhibition alleviated cardiac functions, and decreased infarct size, pro-inflammatory cytokines, NETs, NLRP3, and pyroptosis. The ubiquitination and expression levels of IRF1 were increased by silencing USP47, and IRF1 bound to the CXCL4 promoter to promote CXCL4. Overexpression of IRF1 or CXCL4 in vitro and injection of Nigericin in vivo reversed the effect of silencing USP47 on alleviating pyroptosis and cardiac functions. Collectively, USP47 stabilized IRF1 and promoted CXCL4, further promoting pyroptosis, impairing cardiac functions, and aggravating immune inflammation through NLRP3 pathways.

CXCL4:NLRP3-mediated pyroptosis product that regulates cardiac fibrosis.

Severe myocarditis is often accompanied by cardiac fibrosis, but the underlying mechanism has not been fully elucidated. NOD-like receptor protein 3 (NLRP3) inflammation is involved in the development of myocarditis and is closely related to the form of cell death. Inhibiting pyroptosis mediated by NLRP3 inflammasome can reduce cardiac fibrosis, although its exact mechanism remains unknown. In this study, we induced Viral myocarditis (VMC) via infection of CVB3 to explore the relationship between pyroptosis and fibrosis. Our results showed that intraperitoneal injection of an NLRP3 inhibitor MCC950 or use of NLRP3-/- mice inhibited cardiac pyroptosis mediated by NLRP3 inflammasome in VMC. CXCL4 is a chemokine that has been reported to have pro-inflammatory and pro-fibrotic functions. In VMC, we further found that pyroptosis of Mouse myocardial fibroblasts (MCF) promoted the secretion of CXCL4 by activating Wnt/ß-Catenin signaling. Subsequently, the transcriptome sequencing data showed that CXCL4 could promote cardiac fibrosis by activating PI3K/AKT pathway. In summary, infection of CVB3 induced host oxidative stress to further activate the NLRP3 inflammasome and ultimately lead to heart pyroptosis, in which MCF secreted CXCL4 by activating Wnt/ß-Catenin signaling and CXCL4 participated in cardiac fibrosis by activating PI3K/AKT pathway. Therefore, our findings revealed the role of CXCL4 in VMC and unveiled its underlying mechanism. CXCL4 appears to be a potential target for the treatment of VMC.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Platelet Factor 4 and Longevity of Patients with Essential Thromobocythemia: An Example of Antagonistic Pathogenic Pleiotropy.

This article presents the concept of Antagonistic Pathogenic Pleiotropy, in which an abnormality that causes a specific pathology can simultaneously reduce other morbidities through unrelated mechanisms, resulting in the pathology causing less morbidity or mortality than expected. The concept is illustrated by the case of essential thrombocythemia (ET). Patients with ET have substantially elevated platelets and are therefore expected to have increased thrombotic events leading to reduced life expectancy. However, patients with ET do not have reduced life expectancy. A possible explanation is that elevated platelets produce higher levels of platelet factor 4 (PF4), which has been found to reduce age-associated decline in immune and cognitive function in mice and has been suggested as a treatment for age-associated illness. The benefit of elevated PF4 is hypothesized to balance the increased morbidity from hematological causes. Searches for other indications where a well-defined pathology is not associated with concomitant reduction in overall mortality may be a route to identifying factors that could protect against, prevent, or treat chronic disease.

In platelet single donor apheresis, platelet factor 4 levels correlated with donor's age and decreased during storage.

The human population is ageing worldwide. The World Health Organization estimated that the world's population of people aged 60 years and older will increase to at least 30%, coinciding with a growing frequency of cognitive and cardiovascular disease. Recently, in preclinical studies platelet Factor 4 (PF4) was presented as a pro-cognitive factor. This molecule is released by platelets in the circulation and could be present in blood products destined for transfusion. We wondered if PF4 levels are correlated to the age of the blood donor or to the storage time of platelet concentrates (PCs) intended for transfusion? We observed higher levels of PF4 in PCs from elderly donors compared to younger donors, while PC storage time did not determine PF4 levels expression.

Molecular architecture and platelet-activating properties of small immune complexes assembled on heparin and platelet factor 4.

Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin leading to a reduction in circulating platelets with an increased risk of thrombosis. It is precipitated by polymerized immune complexes consisting of pathogenic antibodies that recognize a small chemokine platelet factor 4 (PF4) bound to heparin. Characterization of these immune complexes is extremely challenging due to the enormous structural heterogeneity of such macromolecular assemblies and their constituents. Native mass spectrometry demonstrates that up to three PF4 tetramers can be assembled on a heparin chain, consistent with the molecular modeling studies showing facile polyanion wrapping along the polycationic belt on the PF4 surface. Although these assemblies can accommodate a maximum of only two antibodies, the resulting immune complexes are capable of platelet activation despite their modest size. Taken together, these studies provide further insight into molecular mechanisms of HIT and other immune disorders where anti-PF4 antibodies play a central role.

Platelet factor 4 promotes deep venous thrombosis by regulating the formation of neutrophil extracellular traps.

The presence of neutrophil extracellular traps (NETs) in thrombotic diseases has been extensively studied. The exact mechanism of NET formation in deep venous thrombosis (DVT) has not been largely studied. This study is aimed to explore the role of NETs and their interaction with platelet factor 4 (PF4) in DVT. In plasma samples from 51 healthy volunteers and 52 DVT patients, NET markers and PF4 were measured using enzyme-linked immunosorbent assays (ELISA). NET generation in blood samples from healthy subjects and DVT patients was analyzed by confocal microscopy and flow cytometry. The plasma levels of NETs were significantly elevated in DVT patients, and neutrophils from patients showed a stronger ability to generate NETs after treatment. PF4 was upregulated in plasma samples from DVT patients and mediated NET formation. NETs enhanced procoagulant (PCA) via tissue factor and activating platelets to induce procoagulant activity. In addition, we established an inferior vena cava ligation (IVC) model to examine the role of NETs in thrombogenicity in DVT. In conclusion, NET formation was mediated by PF4 and enhance the procoagulant activity in DVT.

The Binding of the SARS-CoV-2 Spike Protein to Platelet Factor 4: A Proposed Mechanism for the Generation of Pathogenic Antibodies.

Pathogenic platelet factor 4 (PF4) antibodies contributed to the abnormal coagulation profiles in COVID-19 and vaccinated patients. However, the mechanism of what triggers the body to produce these antibodies has not yet been clarified. Similar patterns and many comparable features between the COVID-19 virus and heparin-induced thrombocytopenia (HIT) have been reported. Previously, we identified a new mechanism of autoimmunity in HIT in which PF4-antibodies self-clustered PF4 and exposed binding epitopes for other pathogenic PF4/eparin antibodies. Here, we first proved that the SARS-CoV-2 spike protein (SP) also binds to PF4. The binding was evidenced by the increase in mass and optical intensity as observed through quartz crystal microbalance and immunosorbent assay, while the switching of the surface zeta potential caused by protein interactions and binding affinity of PF4-SP were evaluated by dynamic light scattering and isothermal spectral shift analysis. Based on our results, we proposed a mechanism for the generation of PF4 antibodies in COVID-19 patients. We further validated the changes in zeta potential and interaction affinity between PF4 and SP and found that their binding mechanism differs from ACE2-SP binding. Importantly, the PF4/SP complexes facilitate the binding of anti-PF4/Heparin antibodies. Our findings offer a fresh perspective on PF4 engagement with the SARS-CoV-2 SP, illuminating the role of PF4/SP complexes in severe thrombotic events.

A career in solving clinical-pathological conundrums: Heyde syndrome, anti-platelet factor 4 disorders, and microvascular limb ischemic necrosis.

Hematology is a clinical specialty with strong roots in the laboratory; accordingly, the lab can help solve perplexing clinical problems. This review highlights clinical-pathological conundrums addressed during my 35-year hematology career at McMaster University. Heyde syndrome is the association between aortic stenosis and bleeding gastrointestinal (GI) angiodysplasia where the bleeding is usually cured by aortic valve replacement; the chance reading of a neonatal study showing reversible deficiency of high-molecular-weight (HMW) multimers of von Willebrand factor (vWF) following surgical correction of congenital heart disease provided the key insight that a subtle deficiency of HMW multimers of vWF explains Heyde syndrome. The unusual immunobiology of heparin-induced thrombocytopenia (HIT)-a highly prothrombotic, antibody-mediated, anti-platelet factor 4 (PF4) disorder featuring rapid appearance and then disappearance (seroreversion) of the pathological heparin-dependent platelet-activating antibodies-permitted identification of key clinical features that informed development of a scoring system (4Ts) to aid in HIT diagnosis. Atypical clinical presentations of HIT prompted identification of heparin-independent anti-PF4 antibodies, now recognized as the explanation for vaccine-induced immune thrombotic thrombocytopenia (VITT), as well as VITT-like disorders triggered by adenovirus infection. Another unusual feature of HIT is its strong association with limb ischemia, including limb necrosis secondary to deep-vein/microvascular thrombosis (venous limb gangrene). The remarkable observation that supratherapeutic warfarin anticoagulation predisposes to HIT- and cancer-associated venous limb gangrene provided insight into disturbed procoagulant/anticoagulant balance; these concepts are relevant to microvascular thrombosis in critical illness (symmetrical peripheral gangrene), including a pathophysiological role for proximate "shock liver" (impaired hepatic synthesis of natural anticoagulants).

Characterization of complexes of PF4 and heparins by size-exclusion chromatography coupled with multi-angle light scattering detector.

Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy. Antibodies binding to complexes of platelet factor 4 (PF4) and heparin is the trigger of HIT. A method using size exclusion chromatography with multi-angle laser light scattering detector (SEC-MALS) was developed in this work. The soluble ultra-large complex (ULC) was separated from the small complex (SC) and their molecular weights (MWs) were firstly measured. The complexes of PF4 and three heparins with different MW, including unfractionated heparin (UFH), dalteparin (Daltep) and enoxaparin (Eno) were characterized using this method. The contents and the sizes of ULC increased gradually when heparins were added to PF4 to certain amounts. While, they reduced after more heparins were added. It is the first time to measure the MWs of the biggest ULC of PF4-heparins as millions of Dalton. at the proper ratios of PF4 to heparin (PHR). Meanwhile, those mixtures at those certain PHRs induced the higher expression of CD83 and CD14 markers on dendritic cells (DCs) suggesting that they had stronger immunogenicity and is critical for HIT.

Platelet factor 4(PF4) and its multiple roles in diseases.

Platelet factor 4 (PF4) combines with heparin to form an antigen that could produce IgG antibodies and participate in heparin-induced thrombocytopenia (HIT). PF4 has attracted wide attention due to its role in novel coronavirus vaccine-19 (COVID-9)-induced immune thrombotic thrombocytopenia (VITT) and cognitive impairments. The electrostatic interaction between PF4 and negatively charged molecules is vital in the progression of VITT, which is similar to HIT. Emerging evidence suggests its multiple roles in hematopoietic and angiogenic inhibition, platelet coagulation interference, host inflammatory response promotion, vascular inhibition, and antitumor properties. The emerging pharmacological effects of PF4 may help deepen the exploration of its mechanism, thus accelerating the development of targeted therapies. However, due to its pleiotropic properties, the development of drugs targeting PF4 is at an early stage and faces many challenges. Herein, we discussed the characteristics and biological functions of PF4, summarized PF4-mediated signaling pathways, and discussed its multiple roles in diseases to inform novel approaches for successful clinical translational research.

Thrombotic anti-PF4 immune disorders: HIT, VITT, and beyond.

Antibodies against the chemokine platelet factor 4 (PF4) occur often, but only those that activate platelets induce severe prothrombotic disorders with associated thrombocytopenia. Heparin-induced thrombocytopenia (HIT) is the prototypic anti-PF4 disorder, mediated by strong activation of platelets through their FcγIIa (immunoglobulin G [IgG]) receptors (FcγRIIa). Concomitant pancellular activation (monocytes, neutrophils, endothelium) triggers thromboinflammation with a high risk for venous and arterial thrombosis. The classic concept of HIT is that anti-PF4/heparin IgG, recognizing antigen sites on (cationic) PF4 that form in the presence of (anionic) heparin, constitute the heparin-dependent antibodies that cause HIT. Accordingly, HIT is managed by anticoagulation with a nonheparin anticoagulant. In 2021, adenovirus vector COVID-19 vaccines triggered the rare adverse effect "vaccine-induced immune thrombotic thrombocytopenia" (VITT), also caused by anti-PF4 IgG. VITT is a predominantly heparin-independent platelet-activating disorder that requires both therapeutic-dose anticoagulation and inhibition of FcγRIIa-mediated platelet activation by high-dose intravenous immunoglobulin (IVIG). HIT and VITT antibodies bind to different epitopes on PF4; new immunoassays can differentiate between these distinct HIT-like and VITT-like antibodies. These studies indicate that (1) severe, atypical presentations of HIT ("autoimmune HIT") are associated with both HIT-like (heparin-dependent) and VITT-like (heparin-independent) anti-PF4 antibodies; (2) in some patients with severe acute (and sometimes chronic, recurrent) thrombosis, VITT-like antibodies can be identified independent of proximate heparin exposure or vaccination. We propose to classify anti-PF4 antibodies as type 1 (nonpathogenic, non- platelet activating), type 2 (heparin dependent, platelet activating), and type 3 (heparin independent, platelet activating). A key concept is that type 3 antibodies (autoimmune HIT, VITT) require anticoagulation plus an adjunct treatment, namely high-dose IVIG, to deescalate the severe anti-PF4 IgG-mediated hypercoagulability state.

Platelet factor 4 improves survival in a murine model of antibiotic-susceptible and methicillin-resistant Staphylococcus aureus peritonitis.

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria.

Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure.

ABSTRACT: Platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. Diagnostic and treatment considerations differ somewhat between HIT and VITT. We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or adenovirus-based vaccination who tested strongly positive by PF4/polyanion enzyme-immunoassays and negative/weakly positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (ie, VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL AcuStar HIT-IgG(PF4-H)) as well as a novel chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera included an exploratory anti-PF4 antibody-positive but HIPA-negative/weak cohort obtained before 2020 (n = 188). We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n = 3; cerebral venous sinus thrombosis, n = 4), thrombocytopenia (median platelet count nadir, 49 × 109/L), and hypercoagulability (greatly elevated D-dimer levels). VITT-like serological features included strong reactivity by PIPA (aggregation <10 minutes in 9/9 sera) and positive testing in the novel anti-PF4 chemiluminescence assay (3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay). Our exploratory cohort identified 13 additional patient sera obtained before 2020 with VITT-like anti-PF4 antibodies. Platelet-activating VITT-like anti-PF4 antibodies should be considered in patients with thrombocytopenia, thrombosis, and very high D-dimer levels, even without a proximate exposure to heparin or adenovirus vector vaccines.

Platelet factor 4 (PF4) induces cluster of differentiation 40 (CD40) expression in human aortic endothelial cells (HAECs) through the SIRT1/NF-κB/p65 signaling pathway.

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.

TLR9 ligand sequestration by chemokine CXCL4 negatively affects central B cell tolerance.

Central B cell tolerance is believed to be regulated by B cell receptor signaling induced by the recognition of self-antigens in immature B cells. Using humanized mice with defective MyD88, TLR7, or TLR9 expression, we demonstrate that TLR9/MYD88 are required for central B cell tolerance and the removal of developing autoreactive clones. We also show that CXCL4, a chemokine involved in systemic sclerosis (SSc), abrogates TLR9 function in B cells by sequestering TLR9 ligands away from the endosomal compartments where this receptor resides. The in vivo production of CXCL4 thereby impedes both TLR9 responses in B cells and the establishment of central B cell tolerance. We conclude that TLR9 plays an essential early tolerogenic function required for the establishment of central B cell tolerance and that correcting defective TLR9 function in B cells from SSc patients may represent a novel therapeutic strategy to restore B cell tolerance.

Platelet factors attenuate inflammation and rescue cognition in ageing.

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 immunology of PF4 polyanion interactions.

PURPOSE OF REVIEW: Platelet factor 4 (PF4, CXCL4), the most abundant α-granule platelet-specific chemokine, forms tetramers with an equatorial ring of high positive charge that bind to a wide range of polyanions, after which it changes conformation to expose antigenic epitopes. Antibodies directed against PF4 not only help to clear infection but can also lead to the development of thrombotic disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombocytopenia and thrombosis (VITT). This review will outline the different mechanisms through which PF4 engagement with polyanions combats infection but also contributes to the pathogenesis of inflammatory and thrombotic disease states. RECENT FINDINGS: Recent work has shown that PF4 binding to microbial polyanions may improve outcomes in infection by enhancing leukocyte-bacterial binding, tethering pathogens to neutrophil extracellular traps (NETs), decreasing the thrombotic potential of NET DNA, and modulating viral infectivity. However, PF4 binding to nucleic acids may enhance their recognition by innate immune receptors, leading to autoinflammation. Lastly, while HIT is induced by platelet activating antibodies that bind to PF4/polyanion complexes, VITT, which occurs in a small subset of patients treated with COVID-19 adenovirus vector vaccines, is characterized by prothrombotic antibodies that bind to PF4 alone. SUMMARY: Investigating the complex interplay of PF4 and polyanions may provide insights relevant to the treatment of infectious disease while also improving our understanding of the pathogenesis of thrombotic disorders driven by anti-PF4/polyanion and anti-PF4 antibodies.

Platelet factor 4 induces bone loss by inhibiting the integrin α5-FAK-ERK pathway.

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.

Novel Knowledge about Molecular Mechanisms of Heparin-Induced Thrombocytopenia Type II and Treatment Targets.

Heparin-induced thrombocytopenia type II (HIT II), as stated in the literature, occurs in about 3% of all patients and in 0.1-5% of surgical patients. Thrombosis develops in 20-64% of patients with HIT. The mortality rate in HIT II has not decreased using non-heparin treatment with anticoagulants such as argatroban and lepirudin. An improved understanding of the pathophysiology of HIT may help identify targeted therapies to prevent thrombosis without subjecting patients to the risk of intense anticoagulation. The review will summarize the current knowledge about the pathogenesis of HIT II, potential new therapeutic targets related to it, and new treatments being developed. HIT II pathogenesis involves multi-step immune-mediated pathways dependent on the ratio of PF4/heparin and platelet, monocyte, neutrophil, and endothelium activation. For years, only platelets were known to take part in HIT II development. A few years ago, specific receptors and signal-induced pathways in monocytes, neutrophils and endothelium were revealed. It had been shown that the cells that had become active realised different newly formed compounds (platelet-released TF, TNFα, NAP2, CXCL-7, ENA-78, platelet-derived microparticles; monocytes-TF-MPs; neutrophils-NETs), leading to additional cell activation and consequently thrombin generation, resulting in thrombosis. Knowledge about FcγIIa receptors on platelets, monocytes, neutrophils and FcγIIIa on endothelium, chemokine (CXCR-2), and PSGL-1 receptors on neutrophils could allow for the development of a new non-anticoagulant treatment for HIT II. IgG degradation, Syk kinase and NETosis inhibition are in the field of developing new treatment possibilities too. Accordingly, IdeS and DNases-related pathways should be investigated for better understanding of HIT pathogenesis and the possibilities of being the HIT II treatment targets.