ABSTRACT
The control of malaria, a disease caused by Plasmodium parasites that kills over half a million people every year, is threatened by the continual emergence and spread of drug resistance. Therefore, new molecules with different mechanisms of action are needed in the antimalarial drug development pipeline. Peptides developed from host defense molecules are gaining traction as anti-infectives due to theood of inducing drug resistance. Human platelet factor 4 (PF4) has intrinsic activity against P. falciparum, and a macrocyclic helix-loop-helix peptide derived from its active domain recapitulates this activity. In this study, we used a stepwise approach to optimize first-generation PF4-derived internalization peptides (PDIPs) by producing analogues with substitutions to charged and hydrophobic amino acid residues or with modifications to terminal residues including backbone cyclization. We evaluated the in vitro activity of PDIP analogues against P. falciparum compared to their overall helical structure, resistance to breakdown by serum proteases, selective binding to negatively charged membranes, and hemolytic activity. Next, we combined antiplasmodial potency-enhancing substitutions that retained favorable membrane and cell-selective properties onto the most stable scaffold to produce a backbone cyclic PDIP analogue with four-fold improved activity against P. falciparum compared to first-generation peptides. These studies demonstrate the ability to modify PDIP to select for and combine desirable properties and further validate the suitability of this unique peptide scaffold for developing a new molecule class that is distinct from existing antimalarial drugs.
Subject(s)
Antimalarials , Peptides , Plasmodium falciparum , Platelet Factor 4 , Plasmodium falciparum/drug effects , Antimalarials/pharmacology , Antimalarials/chemistry , Humans , Platelet Factor 4/chemistry , Platelet Factor 4/pharmacology , Peptides/pharmacology , Peptides/chemistry , Structure-Activity RelationshipABSTRACT
Background: In February 2021, a few cases of unusual, severe thrombotic events associated with thrombocytopenia reported after vaccination with ChAdOx1 nCoV-19 (Vaxzevria) or with Johnson & Johnson's Janssen vaccine raise concern about safety. The vaccine-induced thrombotic thrombocytopenia (VITT) has been related to the presence of platelet-activating antibodies directed against platelet Factor 4. Objectives: We investigated VITT subject genetic background by a high-throughput whole exome sequencing (WES) approach in order to investigate VITT genetic predisposition. Methods: Six consecutive patients (females of Caucasian origin with a mean age of 64 years) were referred to the Atherothrombotic Diseases Center (Department of Experimental and Clinical Medicine, Azienda Ospedaliero-Universitaria Careggi, Florence) with a diagnosis of definite VITT underwent WES analysis. WES analysis was performed on the Illumina NextSeq500 platform. Results:WES analysis revealed a total of 140,563 genetic variants. Due to VITT's rare occurrence, we focused attention on rare variants. The global analysis of all high-quality rare variants did not reveal a significant enrichment of mutated genes in biological/functional pathways common to patients analyzed. Afterwards, we focused on rare variants in genes associated with blood coagulation and fibrinolysis, platelet activation and aggregation, integrin-mediated signaling pathway, and inflammation with particular attention to those involved in vascular damage, as well as autoimmune thrombocytopenia. According to ACMG criteria, 47/194 (24.2%) rare variants were classified as uncertain significance variants (VUS), whereas the remaining were likely benign/benign. Conclusion: WES analysis identifies rare variants possibly favoring the prothrombotic state triggered by the exposure to the vaccine. Functional studies and/or extensions to a larger number of patients might allow a more comprehensive definition of these molecular pathways.
Subject(s)
Exome Sequencing , Humans , Middle Aged , Female , Aged , Thrombocytopenia/genetics , Thrombocytopenia/chemically induced , ChAdOx1 nCoV-19/adverse effects , Thrombosis/genetics , Genetic Predisposition to Disease , Platelet Factor 4/genetics , Male , Vaccination/adverse effectsABSTRACT
As a versatile element for maintaining homeostasis, the chemokine system has been reported to be implicated in the pathogenesis of immune thrombocytopenia (ITP). However, research pertaining to chemokine receptors and related ligands in adult ITP is still limited. The states of several typical chemokine receptors and cognate ligands in the circulation were comparatively assessed through various methodologies. Multiple variable analyses of correlation matrixes were conducted to characterize the correlation signatures of various chemokine receptors or candidate ligands with platelet counts. Our data illustrated a significant decrease in relative CXCR3 expression and elevated plasma levels of CXCL4, 9-11, 13, and CCL3 chemokines in ITP patients with varied platelet counts. Flow cytometry assays revealed eminently diminished CXCR3 levels on T and B lymphocytes and increased CXCR5 on cytotoxic T cell (Tc) subsets in ITP patients with certain platelet counts. Meanwhile, circulating CX3CR1 levels were markedly higher on T cells with a concomitant increase in plasma CX3CL1 level in ITP patients, highlighting the importance of aberrant alterations of the CX3CR1-CX3CL1 axis in ITP pathogenesis. Spearman's correlation analyses revealed a strong positive association of peripheral CXCL4 mRNA level, and negative correlations of plasma CXCL4 concentration and certain chemokine receptors with platelet counts, which might serve as a potential biomarker of platelet destruction in ITP development. Overall, these results indicate that the differential expression patterns and distinct activation states of peripheral chemokine network, and the subsequent expansion of circulating CXCR5+ Tc cells and CX3CR1+ T cells, may be a hallmark during ITP progression, which ultimately contributes to thrombocytopenia in ITP patients.
Subject(s)
CX3C Chemokine Receptor 1 , Purpura, Thrombocytopenic, Idiopathic , Receptors, CXCR3 , Receptors, CXCR5 , Humans , Receptors, CXCR3/metabolism , Purpura, Thrombocytopenic, Idiopathic/blood , Purpura, Thrombocytopenic, Idiopathic/immunology , CX3C Chemokine Receptor 1/metabolism , Male , Receptors, CXCR5/metabolism , Female , Adult , Middle Aged , Platelet Count , Platelet Factor 4/blood , Platelet Factor 4/metabolism , Aged , B-Lymphocytes/immunology , B-Lymphocytes/metabolismABSTRACT
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.
Subject(s)
Inflammasomes , Mice, Inbred C57BL , Myocardial Infarction , NLR Family, Pyrin Domain-Containing 3 Protein , Signal Transduction , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Myocardial Infarction/immunology , Myocardial Infarction/metabolism , Mice , Inflammasomes/metabolism , Male , Pyroptosis , Interferon Regulatory Factor-1/metabolism , Interferon Regulatory Factor-1/genetics , Disease Models, Animal , Cell Line , Extracellular Traps/metabolism , Extracellular Traps/immunology , Ubiquitin-Specific Proteases/metabolism , Ubiquitin-Specific Proteases/genetics , Platelet Factor 4/metabolism , Platelet Factor 4/genetics , Ubiquitination , HumansABSTRACT
BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a complication of adenoviral-based vaccine against SARS-CoV-2 due to prothrombotic immunoglobulin (Ig) G antibodies to platelet factor 4 (PF4) and may be difficult to distinguish from heparin-induced thrombocytopenia (HIT) in patients treated with heparin. OBJECTIVES: We assessed the usefulness of competitive anti-PF4 enzyme immunoassays (EIAs) in this context. METHODS: The ability of F(ab')2 fragments of 1E12, 1C12, and 2E1, 3 monoclonal anti-PF4 antibodies, to inhibit the binding of human VITT or HIT antibodies to PF4 was evaluated using EIAs. Alanine-scanning mutagenesis was performed to define the amino acids involved in the interactions between the monoclonal antibodies and PF4. RESULTS: A strong inhibition of VITT IgG binding to PF4 was measured with 1E12 (median inhibition, 93%; n = 8), whereas it had no effect on the binding of HIT antibodies (median, 6%; n = 8). In contrast, 1C12 and 2E1 inhibited VITT (median, 74% and 76%, respectively) and HIT antibodies (median, 68% and 53%, respectively) binding to PF4. When a competitive anti-PF4 EIA was performed with 1E12 for 19 additional VITT samples, it strongly inhibited IgG binding to PF4, except for 1 patient, who had actually developed HIT according to the clinical history. Epitope mapping showed that 1E12 interacts with 5 key amino acids on PF4, of which 4 are also required for the binding of human VITT antibodies, thus explaining the competitive inhibition. CONCLUSION: A simple competitive anti-PF4 EIA with 1E12 could help confirm VITT diagnosis and distinguish it from HIT in patients when both diagnoses are possible.
Subject(s)
Antibodies, Monoclonal , COVID-19 Vaccines , Heparin , Platelet Factor 4 , Humans , Platelet Factor 4/immunology , Heparin/adverse effects , Heparin/immunology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/adverse effects , COVID-19 Vaccines/adverse effects , COVID-19 Vaccines/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Predictive Value of Tests , Anticoagulants/adverse effects , Anticoagulants/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19/diagnosis , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/adverse effects , Protein Binding , Purpura, Thrombocytopenic, Idiopathic/immunology , Purpura, Thrombocytopenic, Idiopathic/diagnosis , Purpura, Thrombocytopenic, Idiopathic/chemically induced , SARS-CoV-2/immunology , Binding, Competitive , Purpura, Thrombotic Thrombocytopenic/immunology , Purpura, Thrombotic Thrombocytopenic/diagnosis , Purpura, Thrombotic Thrombocytopenic/blood , Purpura, Thrombotic Thrombocytopenic/chemically inducedSubject(s)
Autoantibodies , Platelet Factor 4 , Purpura, Thrombocytopenic, Idiopathic , Humans , Adenoviridae , Antibodies, Viral/blood , Autoantibodies/blood , Autoantibodies/immunology , Platelet Factor 4/immunology , Purpura, Thrombocytopenic, Idiopathic/etiology , Purpura, Thrombocytopenic, Idiopathic/immunology , ChAdOx1 nCoV-19/adverse effects , ChAdOx1 nCoV-19/immunology , Ad26COVS1/adverse effects , Ad26COVS1/immunology , Adenovirus Infections, Human/complications , Adenovirus Infections, Human/immunology , Adenovirus Infections, Human/virologySubject(s)
Intracranial Hemorrhages , Platelet Factor 4 , Sinus Thrombosis, Intracranial , Thrombocytopenia , Humans , Sinus Thrombosis, Intracranial/diagnostic imaging , Sinus Thrombosis, Intracranial/complications , Thrombocytopenia/complications , Thrombocytopenia/diagnosis , Intracranial Hemorrhages/diagnostic imaging , Intracranial Hemorrhages/etiology , Intracranial Hemorrhages/diagnosis , Platelet Factor 4/immunology , Female , Autoantibodies/blood , Male , AdultABSTRACT
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1-IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.
Subject(s)
Platelet Factor 4 , Humans , Platelet Factor 4/immunology , Platelet Factor 4/chemistry , COVID-19 Vaccines/adverse effects , COVID-19 Vaccines/immunology , COVID-19 Vaccines/chemistry , Autoantibodies/immunology , Autoantibodies/blood , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/chemistry , Amino Acid Sequence , Purpura, Thrombotic Thrombocytopenic/chemically induced , Purpura, Thrombotic Thrombocytopenic/immunologyABSTRACT
BACKGROUNDFeatures of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM.METHODSPlasma from children with uncomplicated malaria (UM) (n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-platelet factor 4/polyanion (anti-PF4/P), anti-phospholipid, anti-phosphatidylserine, anti-myeloperoxidase, anti-proteinase 3, anti-dsDNA, anti-ß-2-glycoprotein I, and anti-cardiolipin. Plasma samples from individuals with nonmalarial coma (NMC) (n = 49) and healthy controls (HCs) (n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses.RESULTSMedian anti-PF4/P and anti-PS IgG levels were elevated in individuals with malaria infection relative to levels in HCs (P < 0.001) and patients with NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in children with CM (median = 0.27, IQR: 0.19-0.41) compared with those with UM (median = 0.19, IQR: 0.14-0.22, P < 0.0001). Anti-PS IgG levels did not differ between patients with UM and those with CM (P = 0.39). When patients with CM were stratified by malaria retinopathy (Ret) status, the levels of anti-PF4/P IgG correlated negatively with the peripheral platelet count in patients with Ret+ CM (Spearman's rho [Rs] = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02-275, P = 0.048). Plasma from patients with CM induced greater platelet activation in an ex vivo assay relative to plasma from patients with UM (P = 0.02), and the observed platelet activation was associated with anti-PF4/P IgG levels (Rs= 0.293, P = 0.035).CONCLUSIONSThrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.
Subject(s)
Autoantibodies , Malaria, Cerebral , Platelet Factor 4 , Humans , Malaria, Cerebral/immunology , Malaria, Cerebral/blood , Autoantibodies/blood , Autoantibodies/immunology , Female , Male , Platelet Factor 4/immunology , Platelet Factor 4/blood , Child , Child, Preschool , Infant , Polyelectrolytes , Thrombosis/immunology , Thrombosis/bloodABSTRACT
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.
Subject(s)
COVID-19 Vaccines , Platelet Activation , Platelet Factor 4 , Receptors, IgG , Thrombocytopenia , Thrombosis , Humans , Netherlands , Platelet Factor 4/immunology , Female , Male , Middle Aged , Thrombocytopenia/chemically induced , Thrombocytopenia/diagnosis , Thrombocytopenia/blood , Thrombosis/blood , Thrombosis/immunology , Thrombosis/diagnosis , Thrombosis/etiology , COVID-19 Vaccines/adverse effects , COVID-19 Vaccines/immunology , Platelet Activation/immunology , Adult , Aged , COVID-19 , Heparin/adverse effects , Blood Platelets/immunology , Blood Platelets/metabolism , Immunoglobulin G/bloodABSTRACT
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.
Subject(s)
Blood Platelets , Cyclooxygenase 1 , Disease Models, Animal , Integrases , Mice, Inbred C57BL , Mice, Knockout , Platelet Aggregation , Platelet Factor 4 , Receptors, LDL , Animals , Blood Platelets/metabolism , Blood Platelets/drug effects , Blood Platelets/enzymology , Cyclooxygenase 1/metabolism , Cyclooxygenase 1/genetics , Cyclooxygenase 1/deficiency , Platelet Aggregation/drug effects , Platelet Factor 4/genetics , Platelet Factor 4/metabolism , Integrases/genetics , Receptors, LDL/genetics , Receptors, LDL/deficiency , Male , Mice , Atherosclerosis/genetics , Atherosclerosis/pathology , Atherosclerosis/enzymology , Atherosclerosis/prevention & control , Atherosclerosis/blood , Hyperlipidemias/blood , Hyperlipidemias/genetics , Hyperlipidemias/enzymology , Phenotype , Membrane Proteins , Platelet Glycoprotein GPIb-IX ComplexABSTRACT
ABSTRACT: New analytical techniques can assess hundreds of proteins simultaneously with high sensitivity, facilitating the observation of their complex interplay and role in disease mechanisms. We hypothesized that proteomic profiling targeting proteins involved in thrombus formation, inflammation, and the immune response would identify potentially new biomarkers for heparin-induced thrombocytopenia (HIT). Four existing panels of the Olink proximity extension assay covering 356 proteins involved in thrombus formation, inflammation, and immune response were applied to randomly selected patients with suspected HIT (confirmed HIT, n = 32; HIT ruled out, n = 38; and positive heparin/platelet factor 4 [H/PF4] antibodies, n = 28). The relative difference in protein concentration was analyzed using a linear regression model adjusted for sex and age. To confirm the test results, soluble P-selectin was determined using enzyme-linked immunosorbent assay (ELISA) in above mentioned patients and an additional second data set (n = 49). HIT was defined as a positive heparin-induced platelet activation assay (washed platelet assay). Among 98 patients of the primary data set, the median 4Ts score was 5 in patients with HIT, 4 in patients with positive H/PF4 antibodies, and 3 in patients without HIT. The median optical density of a polyspecific H/PF4 ELISA were 3.0, 0.9, and 0.3. Soluble P-selectin remained statistically significant after multiple test adjustments. The area under the receiver operating characteristic curve was 0.81 for Olink and 0.8 for ELISA. Future studies shall assess the diagnostic and prognostic value of soluble P-selectin in the management of HIT.
Subject(s)
Biomarkers , Heparin , Proteomics , Thrombocytopenia , Humans , Heparin/adverse effects , Female , Proteomics/methods , Male , Thrombocytopenia/chemically induced , Thrombocytopenia/diagnosis , Thrombocytopenia/blood , Middle Aged , Aged , P-Selectin/blood , Platelet Factor 4 , Adult , Platelet ActivationABSTRACT
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.
Subject(s)
Fibrosis , Myocarditis , NLR Family, Pyrin Domain-Containing 3 Protein , Platelet Factor 4 , Pyroptosis , Animals , Humans , Male , Mice , Fibroblasts/metabolism , Furans/pharmacology , Indenes , Inflammasomes/metabolism , Mice, Inbred C57BL , Mice, Knockout , Myocarditis/metabolism , Myocardium/pathology , Myocardium/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Platelet Factor 4/metabolism , Signal Transduction , Sulfonamides/pharmacology , Sulfones/pharmacologyABSTRACT
Platelet-viral interactions are evolving as a new concern. Coagulation disorder is a major consequence of the COVID-19 infection. In chronic hepatitis virus infections, defect in coagulation factors, thrombocytopenia and platelet function abnormalities are common. A SARS-CoV-2 infection on top of chronic viral hepatitis infection can be common in areas where viral hepatitis is endemic. Here, we investigate the platelet ultrastructural changes and estimate the serum platelet factor-4 (PF-4), ferritin, CRP, and D-dimer in COVID-19 patients (n = 60), COVID-19 patients with associated chronic viral hepatitis (n = 20), and healthy subjects (n = 20). Ultrastructural changes were demonstrated in all test groups, denoting platelet activation. In chronic viral hepatitis patients, Platelet ultrastrustural apoptotic changes were also seen. Significantly high levels of PF-4 were confirmed in moderate and severe COVID-19 patients (P.value <0.001), with a cut off value of 17 ng/ml for predicting disease severity. A positive correlation of PF-4 with the level of serum ferritin, CRP, and D-dimer (p value < 0.001) was noted, while negatively correlated with platelet count and platelet granule count (p value < 0.001). In our study, chronic viral hepatitis patients presented mild COVID-19 signs, and their PF-4 level was comparable with the subgroup of mild COVID-19 infection. The platelet's critical role in COVID-19 coagulopathy and chronic viral hepatitis is evidenced by the ultrastructural changes and the high levels of PF4. Moreover, a dual viral infection poses a substantial burden on the platelets, necessitating close monitoring of the patient's coagulation profile.
Subject(s)
Blood Platelets , COVID-19 , Humans , COVID-19/complications , COVID-19/blood , COVID-19/pathology , Blood Platelets/ultrastructure , Male , Female , Middle Aged , Adult , SARS-CoV-2 , Fibrin Fibrinogen Degradation Products/analysis , Platelet Count , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/blood , Hepatitis C, Chronic/pathology , Aged , Platelet Factor 4/blood , Platelet ActivationABSTRACT
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.
Subject(s)
Longevity , Platelet Factor 4 , Thrombocythemia, Essential , Humans , Thrombocythemia, Essential/pathology , Thrombocythemia, Essential/genetics , Thrombocythemia, Essential/blood , Platelet Factor 4/metabolism , Animals , Blood Platelets/metabolismABSTRACT
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).
Subject(s)
Platelet Factor 4 , Humans , Platelet Factor 4/immunology , Platelet Factor 4/metabolism , Necrosis , Ischemia/etiology , Ischemia/pathology , Ischemia/metabolism , Heparin/adverse effects , Aortic Valve Stenosis , Thrombocytopenia/etiology , Thrombocytopenia/pathology , Autoantibodies/immunologyABSTRACT
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.
Subject(s)
Extracellular Traps , Platelet Factor 4 , Venous Thrombosis , Adult , Animals , Female , Humans , Male , Mice , Middle Aged , Blood Platelets/metabolism , Extracellular Traps/metabolism , Neutrophils/metabolism , Platelet Factor 4/blood , Platelet Factor 4/metabolism , Venous Thrombosis/blood , Venous Thrombosis/pathologyABSTRACT
INTRODUCTION: Fcγ-receptors (FcγR) are membrane receptors expressed on a variety of immune cells, specialized in recognition of the Fc part of immunoglobulin G (IgG) antibodies. FcγRIIA-dependent platelet activation in platelet factor 4 (PF4) antibody-related disorders have gained major attention, when these antibodies were identified as the cause of the adverse vaccination event termed vaccine-induced immune thrombocytopenia and thrombosis (VITT) during the COVID-19 vaccination campaign. With the recognition of anti-PF4 antibodies as cause for severe spontaneous and sometimes recurrent thromboses independent of vaccination, their clinical relevance extended far beyond heparin-induced thrombocytopenia (HIT) and VITT. AREAS COVERED: Patients developing these disorders show life-threatening thromboses, and the outcome is highly dependent on effective treatment. This narrative literature review summarizes treatment options for HIT and VITT that are currently available for clinical application and provides the perspective toward new developments. EXPERT OPINION: Nearly all these novel approaches are based on in vitro, preclinical observations, or case reports with only limited implementation in clinical practice. The therapeutic potential of these approaches still needs to be proven in larger cohort studies to ensure treatment efficacy and long-term patient safety.
Subject(s)
COVID-19 Vaccines , Heparin , Receptors, IgG , Thrombocytopenia , Thrombosis , Humans , Anticoagulants/adverse effects , COVID-19/complications , COVID-19/prevention & control , COVID-19/immunology , COVID-19 Vaccines/adverse effects , COVID-19 Vaccines/immunology , Heparin/adverse effects , Platelet Activation/drug effects , Platelet Factor 4/immunology , Purpura, Thrombocytopenic, Idiopathic/chemically induced , Purpura, Thrombocytopenic, Idiopathic/immunology , Purpura, Thrombocytopenic, Idiopathic/drug therapy , Receptors, IgG/metabolism , Receptors, IgG/immunology , Thrombocytopenia/chemically induced , Thrombocytopenia/immunology , Thromboinflammation/drug therapy , Thrombosis/drug therapy , Thrombosis/immunologyABSTRACT
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.
Subject(s)
Platelet Factor 4 , Plateletpheresis , Aged , Humans , Middle Aged , Platelet Factor 4/metabolism , Blood Platelets/metabolism , Platelet Transfusion , Blood Donors , Blood PreservationABSTRACT
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.