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Endometriosis is a chronic inflammatory condition characterized by the presence of endometrium‐like tissue outside the uterus, primarily affecting pelvic organs and tissues. In this study, we explored platelet activation in endometriosis. We utilized the STRING database to analyze the functional interactions among proteins previously identified in small extracellular vesicles (EVs) isolated from the peritoneal fluid of endometriosis patients and controls. The bioinformatic analysis indicated enriched signaling pathways related to platelet activation, hemostasis, and neutrophil degranulation. Double immunohistochemistry analysis for CD61 and MPO revealed a significant presence of neutrophils and platelets in close contact infiltrating endometriotic lesions, suggesting potential cell–cell interactions. Subsequently, we isolated small EVs from the peritoneal fluid of women diagnosed with endometriosis and from women without endometriosis who underwent surgery for non‐inflammatory benign diseases. We performed single‐particle phenotyping analysis based on platelet biomarkers GPIIb/IIIa and PF4 using nanoflow cytometry, as well as single‐particle morphological and nanomechanical characterization through atomic force microscopy. The study demonstrated that patients with endometriosis had a notably higher proportion of particles testing positive for platelet biomarkers compared to the total number of EVs. This finding implies a potential role for platelets in the pathogenesis of endometriosis. Further research is necessary to delve into the mechanisms underlying this phenomenon and its implications for disease progression. [ABSTRACT FROM AUTHOR]

Background/Aim: SARS-CoV-2 infection presents different severity levels that suggest the influence of genetic factors on the clinical outcome of the disease. In cases of severe COVID-19, the presence of elevated coagulation markers, increased platelet activation and aggregation and the risk of thrombotic complications are described. Given the participation of these cells in several serious viral infections and their negative role when associated with a prothrombotic response, it is important to understand the mechanistic role of SARS-CoV-2 in platelet physiology. This study evaluated the hyperexpression of platelet-activating factor receptor (PTAFR) and platelet factor 4 (PF4) in unvaccinated and hospitalized patients with COVID-19. Patients and Methods: The study included 43 COVID-19 patients stratified according to WHO guidelines. Subsequently, the expression of the PTAFR and PF4 genes were evaluated using the real-time quantitative PCR and their possible correlation with the severity of the disease and clinical variables including hospitalization, outcome, sex, age and laboratory parameters (platelet count, INR and D-dimer). Results: The analysis demonstrated a significant (p<0.05) hyperexpression of these genes COVID-19 patients (n=43) compared to healthy controls. Expression of these genes in patients was not statistically significant (p>0.05) different between patients stratified according to clinical variables. Conclusion: The expression of PTAFR and PF4 suggests an important molecular pathway in the pathophysiology of the disease and may be valuable platelet biomarkers to indicate increased risk in patients with COVID-19 who require hospital care, contributing to personalized intervention strategies and improving their clinical management. [ABSTRACT FROM AUTHOR]

Summary No risk factors have been identified for vaccine‐induced immune thrombotic thrombocytopenia (VITT) so far. The aim of this study was to identify human leucocyte antigen (HLA) alleles potentially associated with VITT susceptibility. Specific HLA class II alleles were detected with significantly higher frequency in VITT patients compared with Italian controls: DPB1*17:01, DQA1*05:01, and DRB1*11:04. In silico analysis revealed increased affinity of DRB1*11:04 for a platelet factor 4 (PF4)‐derived peptide, ITSLEVIKA, that contains two amino acids present in the specific binding site of anti‐PF4 antibodies from VITT patients. Our findings show for the first time a genetic predisposition to developing anti‐PF4 antibodies in response to Ad‐vector vaccines. [ABSTRACT FROM AUTHOR]

Angiogenesis is essential for many tumours to grow and metastasise, including breast tumours. Captopril, an Angiotensin- Converting Enzyme inhibitor is known to have anti-angiogenic activity. Recently, novel derivatives of captopril that include thiosemicarbazide moiety have shown enhanced ACE inhibition activity compared to captopril. This study aimed to assess the anti-angiogenic activity of one of these derivatives designated as (8) in the Estrogen receptor-positive MCF-7, and the Estrogen-Progesterone receptor-negative AMJ13 breast cancer cells. The study included a microarray screening for 24 angiogenic factors, and genes were confirmed by RT-qPCR. Results demonstrated a stronger anti-angiogenic effect in the MCF-7 cells compared to those on AMJ13. In MCF7, the derivative caused a significant decrease in the pro-angiogenic bFGF mRNA, VEGF-A mRNA expression, thrombopoietin protein level, PECAM -1 (CD31) mRNA, G-CSF protein, and a significant increase in the anti-angiogenic factors MIG mRNA level, IL-13 protein level, PF-4 both protein and mRNA level. The derivative also significantly decreased TIMP-1 mRNA and IFN-γ protein levels, whereas in AMJ13, a significant increase in MIG protein expression (but not mRNA), a significant decrease in IL-β protein expression, as well as thrombopoietin and PECAM-1 mRNA were documented. This work has shown the thiosemicarbazide derivative of captopril (8) as a potential anti-angiogenic agent targeting multiple factors in the angiogenesis of breast cancer cells. This study has demonstrated derivative (8) as a very promising molecule to be further investigated in other modes of angiogenesis and types of cancer. [ABSTRACT FROM AUTHOR]

Inhibiting ferroptosis represents a promising strategy for managing neuronal injury caused by intracerebral hemorrhage (ICH). Platelet factor 4 (PF4), a chemokine with diverse biological functions, has an unclear role in ICH and its impact on neuronal ferroptosis. To investigate this, a hemin-induced injury model was established in PC12 cells in vitro, and an ICH model was created in vivo using IV collagenase injection. Hemin-treated PC12 cells were co-cultured with recombinant mouse PF4 (Rm-PF4) protein to examine the effects of PF4 on ferroptosis. Additionally, Rm-PF4 was administered intraperitoneally to ICH mice, and its influence on neurological dysfunction, brain edema, and neuronal ferroptosis was evaluated. Western blot analysis was employed to assess PF4 levels, CXCR3/phosphatidylinositol 3-kinase (PI3K)/AKT/nuclear factor erythroid-2-related factor 2 (Nrf2) pathway activation, and ferroptosis-related protein expression. PF4 levels were found to be reduced in both perihematomal brain tissues of ICH mice and hemin-treated PC12 cells. Treatment with Rm-PF4 decreased ferrous ion, malondialdehyde (MDA), and reactive oxygen species (ROS) levels, effectively inhibiting ferroptosis in PC12 cells. Furthermore, Rm-PF4 administration alleviated neurological dysfunction, neuronal damage, and brain edema while suppressing neuronal ferroptosis in ICH mice. Mechanistically, Rm-PF4 activated the CXCR3/PI3K/AKT/Nrf2 pathway, and this protective effect was diminished by a CXCR3 antagonist in both ICH mice and hemin-treated PC12 cells. In conclusion, PF4 mitigates ICH-induced neuronal ferroptosis in mouse models and PC12 cells by activating the CXCR3/PI3K/AKT/Nrf2 pathway.

Ferroptosis plays a crucial role in the secondary pathophysiological damage to brain tissue surrounding hematomas after intracerebral hemorrhage (ICH). While platelet factor 4 (PF4) is known to promote regeneration following peripheral nerve injury, its role in brain tissue repair after cerebral hemorrhage remains unclear. In this study, Hemin-induced PC12 cells were treated with various inhibitors and assessed for viability, oxidative stress, and ferroptosis using a combination of assays, including CCK-8 (Cell Counting Kit-8), EdU (5-Ethynyl-2’-deoxyuridine), flow cytometry, and immunofluorescence. ICH cells were also treated with recombinant PF4 (Rm-PF4) and a CXCR3 antagonist (AMG487) to investigate the mechanism by which Rm-PF4 influences Hemin-induced PC12 cell injury and inflammation. Subsequently, ICH mouse models were established via collagenase injection. Neurological function in these mice was evaluated using the Cylinder and Corner tests. Histopathological damage to brain tissue was analyzed through HE, TUNEL, and Nissl staining, as well as immunohistochemistry, to further explore the role of Rm-PF4 in controlling neuroinflammation in vivo. Results showed that Rm-PF4 inhibited Hemin-mediated ferroptosis-induced PC12 cell damage and inflammation by activating the CXCR3/AKT1/SLC7A11 signaling pathway. Blocking the CXCR3/AKT1/SLC7A11 pathway partially reversed PF4's protective effects on Hemin-induced PC12 cells.In ICH mice, pro-inflammatory marker CD16 (3rd day) and anti-inflammatory marker Arg1 (7th day) were significantly decreased and increased, respectively (p

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. [ABSTRACT FROM AUTHOR]

Recently, it is discovered PF4 is a cognitive enhancer that improved the cognitive abilities of younger mice and gave older animals their middle-aged acuity back. PF4 works by reducing inflammation during the aging process. As we all known, aging is undoubtedly the main risk factor of neurodegenerative diseases. Furthermore, inflammation has been extensively investigated and attracted even more interest. Therefore, the aim of the proposal is to highlight the worth of PF4 in inflammaging of neurodegenerative diseases, which might provide a potential therapeutic strategy.

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. [ABSTRACT FROM AUTHOR]

Background & Aims: The nuclear receptor coactivator 5 (NCOA5) is a putative type 2 diabetes susceptibility gene. NCOA5 haploinsufficiency results in the spontaneous development of nonalcoholic fatty liver disease (NAFLD), insulin resistance, and hepatocellular carcinoma (HCC) in male mice; however, the cell-specific effect of NCOA5 haploinsufficiency in various types of cells, including macrophages, on the development of NAFLD and HCC remains unknown. Methods: Control and myeloid-lineage–specific Ncoa5 deletion (Ncoa5ΔM/+) mice fed a normal diet were examined for the development of NAFLD, nonalcoholic steatohepatitis (NASH), and HCC. Altered genes and signaling pathways in the intrahepatic macrophages of Ncoa5ΔM/+ male mice were analyzed and compared with those of obese human individuals. The role of platelet factor 4 (PF4) in macrophages and the underlying mechanism by which PF4 affects NAFLD/NASH were explored in vitro and in vivo. PF4 expression in HCC patient specimens and prognosis was examined. Results: Myeloid-lineage–specific Ncoa5 deletion sufficiently causes spontaneous NASH and HCC development in male mice fed a normal diet. PF4 overexpression in Ncoa5ΔM/+ intrahepatic macrophages is identified as a potent mediator to trigger lipid accumulation in hepatocytes by inducing lipogenesis-promoting gene expression. The transcriptome of intrahepatic macrophages from Ncoa5ΔM/+ male mice resembles that of obese human individuals. High PF4 expression correlated with poor prognosis of HCC patients and increased infiltrations of M2 macrophages, regulatory T cells, and myeloid-derived suppressor cells in HCCs. Conclusions: Our findings reveal a novel mechanism for the onset of NAFLD/NASH and HCC initiated by NCOA5-deficient macrophages, suggesting the NCOA5-PF4 axis in macrophages as a potential target for developing preventive and therapeutic interventions against NAFLD/NASH and HCC.

Intracerebral haemorrhage (ICH) is a lethal cerebrovascular disorder with a high mortality and morbidity. Although it is a major public health problem, there is no effective treatment for ICH. After ICH, the primary and secondary mechanisms are mentioned when discussing brain injury. The transcription factor, nuclear factor-kappa B (NF-kB), is an important regulator of inflammatory responses. The role of platelet factor 4 (PF4) in ICH is unclear. To study the effect of PF4 on inflammatory response of rats in ICH, a rat model of striatum ICH was established by injecting autologous blood from the autogenous femoral artery into the right striatum of rats. Forty-eight hours after ICH, the expression of PF4, NF-kB (P-P65) and inflammatory changes in rats were determined with WB and ELISA. Heme was used to induce PC12 cell damage, simulate the ICH model in vitro, and detect PF4, P-P65 and striatal inflammatory changes. Short hairpin RNA (shRNA-PF4) was used to knock-down the expression of PF4 in PC12 cells to detect changes in inflammatory factors. The results showed that 48 hours after surgery, the behavioural score of cerebral haemorrhage was the lowest. The expression of PF4 and P-P65 in the striatum of the ICH group was significantly higher compared with the sham surgery group. The expression of interleukin (IL)-6 and IL-1b in the ICH group was also greatly improved. After inhibiting NF-kB expression, PF4 expression was decreased. In short, ICH enhances the expression of PF4, which induces an inflammatory response in rats with cerebral haemorrhage through the NF-kB signalling pathway. Reducing the expression of PF4 can attenuate the inflammatory response.

Background: The inovirus Pf4 is a lysogenic bacteriophage of Pseudomonas aeruginosa (Pa). People with Cystic Fibrosis (pwCF) experience chronic airway infection with Pa and a significant proportion have high numbers of Pf4 in their airway secretions. Given the known severe damage in the airways of Pa-infected pwCF, we hypothesized a high Pf4 burden can affect airway healing and inflammatory responses. In the airway, basal epithelial cells (BCs) are a multipotent stem cell population critical to epithelium homeostasis and repair. We sought to investigate the transcriptional responses of BCs under conditions that emulate infection with Pa and exposure to high Pf4 burden. Methods: Primary BCs isolated from pwCF and wild-type (WT) donors were cultured in vitro and exposed to Pf4 or bacterial Lipopolysaccharide (LPS) followed by transcriptomic and functional assays. Results: We found that BCs internalized Pf4 and this elicits a strong antiviral response as well as neutrophil chemokine production. Further, we found that BCs that take up Pf4 demonstrate defective migration and proliferation. Conclusions: Our findings are highly suggestive of Pf4 playing a role in the pathogenicity of Pa in the airways. These findings provide additional evidence for the ability of inoviruses to interact with mammalian cells and disrupt cell function. [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]

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. [ABSTRACT FROM AUTHOR]

Intracerebral haemorrhage (ICH) is a lethal cerebrovascular disorder with a high mortality and morbidity. Although it is a major public health problem, there is no effective treatment for ICH. After ICH, the primary and secondary mechanisms are mentioned when discussing brain injury. The transcription factor, nuclear factor-kappa B (NF-κB), is an important regulator of inflammatory responses. The role of platelet factor 4 (PF4) in ICH is unclear. To study the effect of PF4 on inflammatory response of rats in ICH, a rat model of striatum ICH was established by injecting autologous blood from the autogenous femoral artery into the right striatum of rats. Forty-eight hours after ICH, the expression of PF4, NF-κB (P-P65) and inflammatory changes in rats were determined with WB and ELISA. Heme was used to induce PC12 cell damage, simulate the ICH model in vitro, and detect PF4, P-P65 and striatal inflammatory changes. Short hairpin RNA (shRNA-PF4) was used to knock-down the expression of PF4 in PC12 cells to detect changes in inflammatory factors. The results showed that 48 hours after surgery, the behavioural score of cerebral haemorrhage was the lowest. The expression of PF4 and P-P65 in the striatum of the ICH group was significantly higher compared with the sham surgery group. The expression of inter-leukin (IL)-6 and IL-1β in the ICH group was also greatly improved. After inhibiting NF-κB expression, PF4 expression was decreased. In short, ICH enhances the expression of PF4, which induces an inflammatory response in rats with cerebral haemorrhage through the NF-κB signalling pathway. Reducing the expression of PF4 can attenuate the inflammatory response. [ABSTRACT FROM AUTHOR]

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.

Pseudomonas aeruginosa is a model quorum sensing (QS) pathogen with three interconnected QS circuits that control the production of virulence factors and antibiotic tolerant biofilms. The pqs QS system of P. aeruginosa is responsible for the biosynthesis of diverse 2-alkyl-4-quinolones (AQs), of which 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) function as QS signal molecules. Transcriptomic analyses revealed that HHQ and PQS influenced the expression of multiple genes via PqsR-dependent and -independent pathways whereas 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) had no effect on P. aeruginosa transcriptome. HQNO is a cytochrome bc1 inhibitor that causes P. aeruginosa programmed cell death and autolysis. However, P. aeruginosa pqsL mutants unable to synthesize HQNO undergo autolysis when grown as colony biofilms. The mechanism by which such autolysis occurs is not understood. Through the generation and phenotypic characterization of multiple P. aeruginosa PAO1 mutants producing altered levels of AQs in different combinations, we demonstrate that mutation of pqsL results in the accumulation of HHQ which in turn leads to Pf4 prophage activation and consequently autolysis. Notably, the effect of HHQ on Pf4 activation is not mediated via its cognate receptor PqsR. These data indicate that the synthesis of HQNO in PAO1 limits HHQ-induced autolysis mediated by Pf4 in colony biofilms. A similar phenomenon is shown to occur in P. aeruginosa cystic fibrosis (CF) isolates, in which the autolytic phenotype can be abrogated by ectopic expression of pqsL. [ABSTRACT FROM AUTHOR]

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a highly prothrombotic disorder caused by anti-PF4 antibodies that activate platelets and neutrophils, leading to thrombosis. Heparin-induced thrombocytopenia (HIT) is a related anti-PF4 mediated disorder, with similar pathophysiology and clinical manifestations but different triggers (i.e., heparin vs adenoviral vector vaccine). Clinically, both HIT and VITT typically present with thrombocytopenia and thrombosis, although the risk of thrombosis is significantly higher in VITT, and the thromboses occur in unusual anatomical sites (e.g., cerebral venous sinus thrombosis and hepatic vein thrombosis). The diagnostic accuracy of available laboratory testing differs between HIT and VITT; for VITT, ELISAs have better specificity compared to HIT and platelet activation assays require the addition of PF4. Treatment of VITT and HIT is anticoagulation non-heparin anticoagulants; however, heparin may be considered for VITT if no other option is available. [ABSTRACT FROM AUTHOR]

Abstract Background Platelets play an important role in the progression of atherosclerosis and cardiovascular events. The inhibition of platelet function is a main strategy to reduce risk of cardiovascular events. Some studies have shown that tomato extracts inhibit platelet function, but the molecular mechanisms remain unclear. Fruitflow is a water-solute tomato extract and the main ingredients including flavonoids, adenosine, chlorogenic acid, phytosterols, naringenin, and carotenoids. The present study investigated the effects of fruitflow on adenosine diphosphate (ADP)- and collagen- stimulated platelet aggregation, platelet adhesion, and levels of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (PGF1α), and platelet factor 4 (PF4) and explored the underlying molecular mechanisms. Methods Platelet-rich plasma (PRP) was used for measurement of platelet aggregation, TXB2, 6-keto- PGF1α, and PF4 levels. Platelet aggregation was analyzed using a Chrono-Log aggregometer. TXB2, 6-keto- PGF1α, and PF4 levels were determined using enzyme-linked immunosorbent assay kits. Immunoblotting was used to detect protein expression and phosphorylation on washed platelets. Platelet adhesion and spreading were determined by immunofluorescence. Results Fruitflow (1, 3, 10 and 100 μg/ml) dose-dependently inhibited platelet aggregation that was induced by ADP and collagen. Fruitflow (100 μg/ml) treatment completely suppressed ADP- and collagen-stimulated platelet aggregation. Fruitflow (100 μg/ml) significantly decreased TXB2 and 6-keto-PGF1α generation and PF4 release in ADP- and collagen-stimulated platelets. Treatment with fruitflow effectively blocked collagen-induced platelet spreading. To determine the potential molecule mechanism of action of fruitflow, we investigated the protein expression and phosphorylation of several signaling molecules in collagen-activated platelets. Fruitflow dose-dependently suppressed Akt, Glycogen synthase kinase-3β (GSK-3β), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) and p38 MAPK phosphorylation that was induced by collagen. Conclusion Fruitflow inhibited platelet aggregation and reduced TXB2, 6-keto-PGF1α, and PF4 levels in ADP- and collagen-stimulated platelets. The mechanism of action of fruitflow may be associated with the suppression of Akt/GSK3β, Syk/PLCγ2, and p38 MAPK phosphorylation in collagen-activated platelets. Fruitflow is a natural product derived from tomato and can be used as a health food for decreasing platelet activity.

Severe COVID-19 is characterized by a prothrombotic state associated with thrombocytopenia, with microvascular thrombosis being almost invariably present in the lung and other organs at postmortem examination. We evaluated the presence of antibodies to platelet factor 4 (PF4)–polyanion complexes using a clinically validated immunoassay in 100 hospitalized patients with COVID-19 with moderate or severe disease (World Health Organization score, 4 to 10), 25 patients with acute COVID-19 visiting the emergency department, and 65 convalescent individuals. Anti-PF4 antibodies were detected in 95 of 100 hospitalized patients with COVID-19 (95.0%) irrespective of prior heparin treatment, with a mean optical density value of 0.871 ± 0.405 SD (range, 0.177 to 2.706). In contrast, patients hospitalized for severe acute respiratory disease unrelated to COVID-19 had markedly lower levels of the antibodies. In a high proportion of patients with COVID-19, levels of all three immunoglobulin (Ig) isotypes tested (IgG, IgM, and IgA) were simultaneously elevated. Antibody levels were higher in male than in female patients and higher in African Americans and Hispanics than in White patients. Anti-PF4 antibody levels were correlated with the maximum disease severity score and with significant reductions in circulating platelet counts during hospitalization. In individuals convalescent from COVID-19, the antibody levels returned to near-normal values. Sera from patients with COVID-19 induced higher levels of platelet activation than did sera from healthy blood donors, but the results were not correlated with the levels of anti-PF4 antibodies. These results demonstrate that the vast majority of patients with severe COVID-19 develop anti-PF4 antibodies, which may play a role in the clinical complications of COVID-19. [ABSTRACT FROM AUTHOR]

Platelet factor 4 (PF4) has been shown to regulate several viral infections. Our previous study demonstrated that PF4 inhibits the entry of enterovirus A 71 (EV71) and coxsackievirus A16 (CA16), which cause hand, foot, and mouth disease (HFMD). In this study, we report that PF4 also inhibits the circulating HFMD pathogen coxsackievirus A6 (CA6) and the re-emerging enterovirus D68 (EVD68). A 15-amino acid peptide, C15, at the C-terminus of PF4 confers anti-viral activity against multiple enteroviruses (EVs) besides CA6 and EVD68, including EV71 and CA16. Mechanistic studies revealed that wild-type C15 with a net-positive charge (+3), but not its mutants C15M and C15A (both -1), specifically binds to the VP3 capsid protein of CA6 and EVD68, thereby disrupting their attachment to the host cell surface. In addition, VP3 of EVs contains a conserved domain (residues 155-170) crucial for binding to C15. An aspartic acid residue at position 156 imparts a net-negative charge to this domain, which, when substituted with a neutrally charged amino acid, reduces the binding affinity of VP3 for C15. Additionally, C15 protects neonatal mice from lethal challenge upon a CA6 infection. These results suggest that C15 is a promising broad-spectrum anti-viral candidate against multiple EVs., Importance: EVs, which pose a significant public health threat, can be classified into 15 species, with EV-A, -B, -C, and -D infecting humans and causing a wide range of diseases, from mild illnesses, such as HFMD, to more severe conditions, such as acute flaccid paralysis. The emergence of new and alternative strains highlights the urgent need for broad-spectrum anti-viral agents. In this study, we identified that the C15 of PF4 exhibits potent anti-viral activity against multiple EVs by binding to their surface and blocking their entry into host cells. Furthermore, C15 provides significant protection in vivo. These findings highlight the potential of C15 as a broad-spectrum anti-viral candidate. Our study opens a new avenue for developing treatments to combat the diverse and evolving threats posed by EVs.

Background: Recurrent Miscarriage (RM) is a chronic and heterogeneous autoimmune disease. Platelet factor 4 (PF4) has been found to be involved in the pathogenesis of RM., Objective: We aimed to explore the role and mechanism of PF4 on trophoblasts in RM in vitro., Methods: In this study, the expression of PF4 and PF4 receptor CXC chemokine receptor 3 (CXCR3) in the placentas of patients with RM were analyzed by RT-qPCR and western blotting. Serum PF4 level was tested by ELISA. Following PF4 silencing and SOCS3 overexpression in HTR-8/SVneo cells, cell proliferation was detected by CCK-8, colony formation, and EDU assays. Wound healing and transwell assays separately evaluated cell migration and invasion. Immunofluorescence assay determined E-cadherin expression. Tube formation assay was used to measure the angiogenesis. Western blotting examined the expression of metastasis, epithelial-mesenchymal transition (EMT) and suppressor of cytokine signaling 3 (SOCS3)/signal transducer and activator of transcription 3 (STAT3) signaling-associated proteins., Results: The results revealed that PF4 displayed increased expression in placental villus tissues of RM patients. Serum PF4 level was also elevated in RM patients. PF4 silencing promoted the proliferation, migration, invasion, EMT, and angiogenesis of HTR-8/SVneo cells. Additionally, PF4 knockdown downregulated SOCS3 expression to activate STAT3 signaling. SOCS3 overexpression countervailed the effects of PF4 deficiency on HTR-8/SVneo cells., Conclusion: In summary, PF4 participated in the proliferation, migration, invasion, EMT and angiogenesis of trophoblast cells by modulating the SOCS3/STAT3 signaling pathway, indicating that targeting the PF4/SOCS3/STAT3 pathway could be a novel therapy for RM., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Over 60% of End Stage Renal Disease (ESRD) patients are relying on hemodialysis (HD) to survive, and the arteriovenous fistula (AVF) is the preferred vascular access method for HD. However approximately half of all newly created AVF fail to mature and cannot be used without a salvage procedure. We have recently demonstrated an association between AVF maturation failure and post-operative fibrosis, while our RNA-seq study also revealed that veins that ultimately failed during AVF maturation had elevated levels of platelet factor 4 (PF4/CXCL4). However, a link between these two findings was yet to be established., Methods: In this study, we investigated potential mechanisms between PF4 levels and fibrotic remodeling in veins. We compared the local expression of PF4 and fibrosis marker integrin β 6 (ITGB6) in veins that successfully underwent maturation with that in veins that ultimately failed to mature. We also measured the changes of expression level of α-smooth muscle actin (αSMA/ ACTA2 ) and collagen (Col1/ COL1A1 ) in venous fibroblasts upon various treatments, such as PF4 pharmacological treatment, alteration of PF4 expression, and blocking of PF4 receptors., Results: We found that PF4 is expressed in veins and co-localizes with αSMA. In venous fibroblasts, PF4 stimulates expression of αSMA and Col1 via different pathways. The former requires integrins α v β 5 and α 5 β 1 , while chemokine receptor CXCR3 is needed for the latter. Interestingly, we also discovered that the expression of PF4 is associated with that of ITGB6, the β subunit of integrin α v β 6 . This integrin is critical for the activation of the major fibrosis factor TGFβ, and overexpression of PF4 promotes activation of the TGFβ pathway., Conclusions: These results indicate that upregulation of PF4 may cause venous fibrosis both directly by stimulating fibroblast differentiation and expression of extracellular matrix (ECM) molecules and indirectly by facilitating the activation of the TGFβ pathway., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Simple Summary: Platelets play a critical role in coagulation and fibrinolysis processes, but recent literature also indicates their central involvement in immune response, cancer progression and metastasis. During platelet activation, small extracellular vesicles (EVs) are released. The ascites are a fluid developing in the peritoneum of ovarian cancer patients in an advanced stage. This study analysed the expression of platelet markers GPIIb/IIIa and PF4 in small-EVs populations isolated from the ascitic fluid of patients with advanced ovarian cancer. The percentage of platelet-derived small-EVs was positively correlated with platelet distribution width to platelet count in sera (PDW/PLT), a surrogate indicator of platelet activation. Overall, we presented a method that can be helpful in future studies to determine the correlation between the extent of platelet activation in ascites and disease status. In ovarian cancer, ascites represent the microenvironment in which the platelets extravasate to play their role in the disease progression. We aimed to develop an assay to measure ascites' platelet activation. We enriched small extracellular vesicles (EVs) (40–200 nm) from ascites of high-grade epithelial ovarian cancer patients (n = 12) using precipitation with polyethylene glycol, and we conducted single-particle phenotyping analysis by nano-flow cytometry after labelling and ultra-centrifugation. Atomic force microscopy single-particle nanomechanical analysis showed heterogeneous distributions in the size of the precipitated particles and their mechanical stiffness. Samples were fluorescently labelled with antibodies specific to the platelet markers GPIIb/IIIa and PF4, showing 2.6 to 18.16% of all particles stained positive for the biomarkers and, simultaneously, the EV membrane labelling. Single-particle phenotyping analysis allowed us to quantify the total number of non-EV particles, the number of small-EVs and the number of platelet-derived small-EVs, providing a platelet activation assessment independent of the ascites volume. The percentage of platelet-derived small-EVs was positively correlated with platelet distribution width to platelet count in sera (PDW/PLT). Overall, we presented a high-throughput method that can be helpful in future studies to determine the correlation between the extent of platelet activation in ascites and disease status. [ABSTRACT FROM AUTHOR]

Abstract Background This is a review article on heparin-induced thrombocytopenia, an adverse effect of heparin therapy, and vaccine-induced immune thrombotic thrombocytopenia, occurring in some patients administered certain coronavirus vaccines. Main body/text Immune-mediated thrombocytopenia occurs when specific antibodies bind to platelet factor 4 /heparin complexes. Platelet factor 4 is a naturally occurring chemokine, and under certain conditions, may complex with negatively charged molecules and polyanions, including heparin. The antibody-platelet factor 4/heparin complex may lead to platelet activation, accompanied by other cascading reactions, resulting in cerebral sinus thrombosis, deep vein thrombosis, lower limb arterial thrombosis, myocardial infarction, pulmonary embolism, skin necrosis, and thrombotic stroke. If untreated, heparin-induced thrombocytopenia can be life threatening. In parallel, rare incidents of spontaneous vaccine-induced immune thrombotic thrombocytopenia can also occur in some patients administered certain coronavirus vaccines. The role of platelet factor 4 in vaccine-induced thrombosis with thrombocytopenia syndrome further reinforces the importance the platelet factor 4/polyanion immune complexes and the complications that this might pose to susceptible individuals. These findings demonstrate, how auxiliary factors can complicate heparin therapy and drug development. An increasing interest in biomanufacturing heparins from non-animal sources has driven a growing interest in understanding the biology of immune-mediated heparin-induced thrombocytopenia, and therefore, the development of safe and effective biosynthetic heparins. Short conclusion In conclusion, these findings further reinforce the importance of the binding of platelet factor 4 with known and unknown polyanions, and the complications that these might pose to susceptible patients. In parallel, these findings also demonstrate how auxiliary factors can complicate the heparin drug development.

Platelet factor 4 (PF4) or the CXC chemokine CXCL4 is the most abundant protein within the a-granules of platelets. Previous studies found that PF4 regulates infections of several viruses, including HIV-1, H1N1, hepatitis C virus (HCV), and dengue virus. Here, we show that PF4 is an inhibitor of enterovirus A71 (EV71) and coxsackievirus A16 (CA16) infections. The secreted form of PF4 from transfected cells or soluble purified PF4 from Escherichia coli, even lacking signal peptide affected secretion, obviously inhibited the propagation of EV71 and CA16. Mechanistically, we demonstrated that PF4 blocked the entry of the virus into the host cells by interactions with VP3 proteins of EV71/CA16 and the interaction with SCARB2 receptor-mediated EV71 and CA16 endocytosis. As expected, the incubation of anti-PF4 antibody with PF4 blocked PF4 inhibition on EV71 and CA16 infections further supported the above conclusion. Importantly, pretreatment of EV71 viruses with PF4 significantly protected the neonatal mice from EV71 lethal challenge and promoted the survival rate of infected mice. PF4 derived from natural platelets by EV71/CA16 activation also presented strong inhibition on EV71 and CA16. In summary, our study identified a new host factor against EV71 and CA16 infections, providing a novel strategy for EV71 and CA16 treatment. IMPORTANCE The virus's life cycle starts with binding to cell surface receptors, resulting in receptor-mediated endocytosis. Targeting the entry of the virus into target cells is an effective strategy to develop a novel drug. EV71 and CA16 are the major pathogens that cause hand, foot, and mouth disease (HFMD) outbreaks worldwide since 2008. However, the treatment of EV71 and CA16 infections is mainly symptomatic because there is no approved drug. Therefore, the underlying pathogenesis of EV71/CA16 and the interaction between host-EV71/CA16 need to be further investigated to develop an inhibitor. Here, we identified PF4 as a potent entry inhibitor of EV71 and CA16 via binding to VP3 proteins of EV71 and CA16 or binding to receptor SCARB2. In the EV71 infection model, PF4 protected mice from EV71 lethal challenge and promoted the survival rate of EV71-infected mice. Our study suggests that PF4 represents a potential candidate host factor for anti-EV71 and CA16 infections. [ABSTRACT FROM AUTHOR]

Experimental evidence has emerged that local platelet activation contributes to inflammation and infarct formation in acute ischemic stroke (AIS) which awaits confirmation in human studies. We conducted a prospective observational study on 258 consecutive patients undergoing mechanical thrombectomy (MT) due to large-vessel-occlusion stroke of the anterior circulation (08/2018–05/2020). Intraprocedural microcatheter aspiration of 1 ml of local (occlusion condition) and systemic arterial blood samples (self-control) was performed according to a prespecified protocol. The samples were analyzed for differential leukocyte counts, platelet counts, and plasma levels of the platelet-derived neutrophil-activating chemokine C-X-C-motif ligand (CXCL) 4 (PF-4), the neutrophil attractant CXCL7 (NAP-2), and myeloperoxidase (MPO). The clinical-biological relevance of these variables was corroborated by specific associations with molecular-cellular, structural-radiological, hemodynamic, and clinical-functional parameters. Seventy consecutive patients fulfilling all predefined criteria entered analysis. Mean local CXCL4 (+ 39%: 571 vs 410 ng/ml, P =.0095) and CXCL7 (+ 9%: 693 vs 636 ng/ml, P =.013) concentrations were higher compared with self-controls. Local platelet counts were lower (− 10%: 347,582 vs 383,284/µl, P =.0052), whereas neutrophil counts were elevated (+ 10%: 6022 vs 5485/µl, P = 0.0027). Correlation analyses revealed associations between local platelet and neutrophil counts (r = 0.27, P =.034), and between CXCL7 and MPO (r = 0.24, P =.048). Local CXCL4 was associated with the angiographic degree of reperfusion following recanalization (r = − 0.2523, P =.0479). Functional outcome at discharge correlated with local MPO concentrations (r = 0.3832, P =.0014) and platelet counts (r = 0.288, P =.0181). This study provides human evidence of cerebral platelet activation and platelet-neutrophil interactions during AIS and points to the relevance of per-ischemic thrombo-inflammatory mechanisms to impaired reperfusion and worse functional outcome following recanalization. [ABSTRACT FROM AUTHOR]

ObjectivesSerial kidney biopsy for repeat evaluation and monitoring of lupus nephritis (LN) in childhood-onset Systemic Lupus Erythematosus (cSLE) remains challenging, thus non-invasive biomarkers are needed. Here, we evaluate the performance of ten urine protein markers of diverse nature including cytokines, chemokines, and adhesion molecules in distinguishing disease activity in cSLE.MethodsEighty-four pediatric patients meeting ≥4 ACR criteria for SLE were prospectively enrolled for urine assay of 10 protein markers normalized to urine creatinine, namely ALCAM, cystatin-C, hemopexin, KIM-1, MCP-1, NGAL, PF-4, Timp-1, TWEAK, and VCAM-1 by ELISA. Samples from active renal (LN) and active non-renal SLE patients were obtained prior to onset/escalation of immunosuppression. SLE disease activity was evaluated using SLEDAI-2000. 59 patients had clinically-active SLE (SLEDAI score ≥4 or having a flare), of whom 29 patients (34.5%) were classified as active renal, and 30 patients (35.7%) were active non-renal. Twenty-five healthy subjects were recruited as controls.ResultsUrine concentrations of ALCAM, KIM-1, PF4 and VCAM-1 were significantly increased in active LN patients versus active non-renal SLE, inactive SLE and healthy controls. Five urine proteins differed significantly between 2 (hemopexin, NGAL, MCP1) or 3 (Cystatin-C, TWEAK) groups only, with the highest levels detected in active LN patients. Urine ALCAM, VCAM-1, PF4 and hemopexin correlated best with total SLEDAI as well as renal-SLEDAI scores (p < 0.05). Urine ALCAM, VCAM-1 and hemopexin outperformed conventional laboratory measures (anti-dsDNA, complement C3 and C4) in identifying concurrent SLE disease activity among patients (AUCs 0.75, 0.81, 0.81 respectively), while urine ALCAM, VCAM-1 and PF4 were the best discriminators of renal disease activity in cSLE (AUCs 0.83, 0.88, 0.78 respectively), surpassing conventional biomarkers, including proteinuria. Unsupervised Bayesian network analysis based on conditional probabilities re-affirmed urine ALCAM as being most predictive of active LN in cSLE patients.ConclusionUrinary ALCAM, PF4, and VCAM-1 are potential biomarkers for predicting kidney disease activity in cSLE and hold potential as surrogate markers of nephritis flares in these patients.

Background: Platelets play an important role in the progression of atherosclerosis and cardiovascular events. The inhibition of platelet function is a main strategy to reduce risk of cardiovascular events. Some studies have shown that tomato extracts inhibit platelet function, but the molecular mechanisms remain unclear. Fruitflow is a water-solute tomato extract and the main ingredients including flavonoids, adenosine, chlorogenic acid, phytosterols, naringenin, and carotenoids. The present study investigated the effects of fruitflow on adenosine diphosphate (ADP)- and collagen- stimulated platelet aggregation, platelet adhesion, and levels of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (PGF1α), and platelet factor 4 (PF4) and explored the underlying molecular mechanisms. Methods: Platelet-rich plasma (PRP) was used for measurement of platelet aggregation, TXB2, 6-keto- PGF1α, and PF4 levels. Platelet aggregation was analyzed using a Chrono-Log aggregometer. TXB2, 6-keto- PGF1α, and PF4 levels were determined using enzyme-linked immunosorbent assay kits. Immunoblotting was used to detect protein expression and phosphorylation on washed platelets. Platelet adhesion and spreading were determined by immunofluorescence. Results: Fruitflow (1, 3, 10 and 100 μg/ml) dose-dependently inhibited platelet aggregation that was induced by ADP and collagen. Fruitflow (100 μg/ml) treatment completely suppressed ADP- and collagen-stimulated platelet aggregation. Fruitflow (100 μg/ml) significantly decreased TXB2 and 6-keto-PGF1α generation and PF4 release in ADP- and collagen-stimulated platelets. Treatment with fruitflow effectively blocked collagen-induced platelet spreading. To determine the potential molecule mechanism of action of fruitflow, we investigated the protein expression and phosphorylation of several signaling molecules in collagen-activated platelets. Fruitflow dose-dependently suppressed Akt, Glycogen synthase kinase-3β (GSK-3β), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) and p38 MAPK phosphorylation that was induced by collagen. Conclusion: Fruitflow inhibited platelet aggregation and reduced TXB2, 6-keto-PGF1α, and PF4 levels in ADP- and collagen-stimulated platelets. The mechanism of action of fruitflow may be associated with the suppression of Akt/GSK3β, Syk/PLCγ2, and p38 MAPK phosphorylation in collagen-activated platelets. Fruitflow is a natural product derived from tomato and can be used as a health food for decreasing platelet activity. [ABSTRACT FROM AUTHOR]

Introduction: Patients with gastrointestinal cancer are at high risk of developing thrombosis and postoperative infection. Antico-agulation therapy for such patients is provided by low molecular weight heparin (LMWH) and elastic stockings. The latter, however, is linked to immunoregulatory activities and immunosuppression in vivo and in vitro.Aim: Therefore, the present study aimed to examine the link between LMWH and infection in patients with gastrointestinal cancer.Materials and methods: The study is a retrospective report of 51 patients operated on at the Second Department of Surgery at Metaxa Cancer Hospital. The sample was divided into groups based on the presence or absence of diabetes and preoperative anticoagu-lation therapy. Afterwards, the data were statistically analysed. Results: The results of the study show a statistically significant correlation between LMWH and infection. Moreover, the risk of infec-tion increases by 13.3% for each day of heparin intake. The theory of this correlation is explained in detail. Conclusions: The findings of the present study raise an essential question about postoperative management of cancer patients. How-ever, the study sample size is rather small so further studies with larger sample size are required to give greater credence to results.

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in a variety of settings. Many P. aeruginosa isolates are infected by filamentous Pf bacteriophage integrated into the bacterial chromosome as a prophage. Pf virions can be produced without lysing P. aeruginosa. However, cell lysis can occur during superinfection, which occurs when Pf virions successfully infect a host lysogenized by a Pf prophage. Temperate phages typically encode superinfection exclusion mechanisms to prevent host lysis by virions of the same or similar species. In this study, we sought to elucidate the superinfection exclusion mechanism of Pf phage. Initially, we observed that P. aeruginosa that survive Pf superinfection are transiently resistant to Pf-induced plaquing and are deficient in twitching motility, which is mediated by type IV pili (T4P). Pf utilize T4P as a cell surface receptor, suggesting that T4P are suppressed in bacteria that survive superinfection. We tested the hypothesis that a Pf-encoded protein suppresses T4P to mediate superinfection exclusion by expressing Pf proteins in P. aeruginosa and measuring plaquing and twitching motility. We found that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), promoted resistance to Pf infection and suppressed twitching motility by binding the T4P protein PilC. Because T4P play key roles in biofilm formation and virulence, the ability of Pf phage to modulate T4P via PfsE has implications in the ability of P. aeruginosa to persist at sites of infection. IMPORTANCE Pf bacteriophage (phage) are filamentous viruses that infect Pseudomonas aeruginosa and enhance its virulence potential. Pf virions can lyse and kill P. aeruginosa through superinfection, which occurs when an already infected cell is infected by the same or similar phage. Here, we show that a small, highly conserved Pf phage protein (PA0721, PfsE) provides resistance to superinfection by phages that use the type IV pilus as a cell surface receptor. PfsE does this by inhibiting assembly of the type IV pilus via an interaction with PilC. As the type IV pilus plays important roles in virulence, the ability of Pf phage to modulate its assembly has implications for P. aeruginosa pathogenesis.

Recent reports of rare ChAdOx1-S vaccine-related venous thrombosis led to the suspension of its usage in several countries. Vaccine-induced thrombotic thrombocytopenia (VITT) is characterized by thrombocytopenia and thrombosis in association with anti-platelet factor 4 (PF4) antibodies. Herein, we propose five potential anionic substances of the ChAdOx1-S vaccine that can combine with PF4 and trigger VITT, including (1) the proteins on the surface of adenovirus, e.g., negative charged glycoprotein, (2) the adjuvant components of the vaccine, e.g., Tween 80, (3) the DNA of adenovirus, (4) the S protein antigen expressed by the vaccine, and (5) the negatively charged impurity proteins expressed by the vaccine, e.g., adenovirus skeleton proteins. After analysis of each case, we consider the most possible trigger to be the negatively charged impurity proteins expressed by the vaccine. Then, we display the possible extravascular route and intravascular route of the formation of PF4 autoantibodies triggered by the negatively charged impurity proteins, which is accordant with the clinical situation. Accordingly, the susceptible individuals of VITT after ChAdOx1-S vaccination may be people who express negatively charged impurity proteins and reach a certain high titer.

Recent reports of rare ChAdOx1-S vaccine-related venous thrombosis led to the suspension of its usage in several countries. Vaccine-induced thrombotic thrombocytopenia (VITT) is characterized by thrombocytopenia and thrombosis in association with anti-platelet factor 4 (PF4) antibodies. Herein, we propose five potential anionic substances of the ChAdOx1-S vaccine that can combine with PF4 and trigger VITT, including (1) the proteins on the surface of adenovirus, e.g., negative charged glycoprotein, (2) the adjuvant components of the vaccine, e.g., Tween 80, (3) the DNA of adenovirus, (4) the S protein antigen expressed by the vaccine, and (5) the negatively charged impurity proteins expressed by the vaccine, e.g., adenovirus skeleton proteins. After analysis of each case, we consider the most possible trigger to be the negatively charged impurity proteins expressed by the vaccine. Then, we display the possible extravascular route and intravascular route of the formation of PF4 autoantibodies triggered by the negatively charged impurity proteins, which is accordant with the clinical situation. Accordingly, the susceptible individuals of VITT after ChAdOx1-S vaccination may be people who express negatively charged impurity proteins and reach a certain high titer. [ABSTRACT FROM AUTHOR]

The chemokine CXCL4/platelet factor 4 (PF4) gene, a key player in myelofibrosis, was knocked out by CRISPR/Cas9 in induced pluripotent stem cells (iPS cells) of a polycythemia vera (PV) patient with JAK2 V617F mutation. Two CXCL4KO iPS cell lines with and without JAK2 V617F mutation (UKAi002-B-1 and UKAi002-A-1, respectively) were generated. CXCL4KO iPS cells showed deletion of exon 1 and complete loss of CXCL4 protein. Pluripotency of iPS cells was confirmed by expression of pluripotency markers and trilineage differentiation. CXCL4KO iPS cells are expected to provide a valuable tool for investigating the role of CXCL4 in human diseases.

Background: This is a review article on heparin-induced thrombocytopenia, an adverse effect of heparin therapy, and vaccine-induced immune thrombotic thrombocytopenia, occurring in some patients administered certain coronavirus vaccines. Main body/text: Immune-mediated thrombocytopenia occurs when specific antibodies bind to platelet factor 4 /heparin complexes. Platelet factor 4 is a naturally occurring chemokine, and under certain conditions, may complex with negatively charged molecules and polyanions, including heparin. The antibody-platelet factor 4/heparin complex may lead to platelet activation, accompanied by other cascading reactions, resulting in cerebral sinus thrombosis, deep vein thrombosis, lower limb arterial thrombosis, myocardial infarction, pulmonary embolism, skin necrosis, and thrombotic stroke. If untreated, heparin-induced thrombocytopenia can be life threatening. In parallel, rare incidents of spontaneous vaccine-induced immune thrombotic thrombocytopenia can also occur in some patients administered certain coronavirus vaccines. The role of platelet factor 4 in vaccine-induced thrombosis with thrombocytopenia syndrome further reinforces the importance the platelet factor 4/polyanion immune complexes and the complications that this might pose to susceptible individuals. These findings demonstrate, how auxiliary factors can complicate heparin therapy and drug development. An increasing interest in biomanufacturing heparins from non-animal sources has driven a growing interest in understanding the biology of immune-mediated heparin-induced thrombocytopenia, and therefore, the development of safe and effective biosynthetic heparins. Short conclusion: In conclusion, these findings further reinforce the importance of the binding of platelet factor 4 with known and unknown polyanions, and the complications that these might pose to susceptible patients. In parallel, these findings also demonstrate how auxiliary factors can complicate the heparin drug development. [ABSTRACT FROM AUTHOR]

Platelets are anucleated blood cells derived from bone marrow megakaryocytes and play a crucial role in hemostasis and thrombosis. Platelets contain specialized storage organelles, called alpha-granules, contents of which are rich in cytokines such as C-X-C Motif Chemokine Ligand (CXCL) 1/4/7, (C-C motif) ligand (CCL) 5/3, CXCL8 (also called as interleukin 8, IL-8), and transforming growth factor β (TGF-β). Activation of platelets lead to degranulation and release of contents into the plasma. Platelet activation is a common event in many viral infections including human immunodeficiency virus (HIV), H1N1 influenza, Hepatitis C virus (HCV), Ebola virus (EBV), and Dengue virus (DENV). The cytokines CXCL8, CCL5 (also known as Regulated on Activation, Normal T Expressed and Secreted, RANTES), tumor necrosis factor α (TNF-α), CXCL1/5 and CCL3 released, promote development of a pro-inflammatory state along with the recruitment of other immune cells to the site of infection. Platelets also interact with Monocytes and Neutrophils and facilitate their activation to release different cytokines which further enhances inflammation. Upon activation, platelets also secrete factors such as CXCL4 (also known as platelet factor, PF4), CCL5 and fibrinopeptides which are critical regulators of replication and propagation of several viruses in the host. Studies suggest that CXCL4 can both inhibit as well as enhance HIV1 infection. Data from our lab show that CXCL4 inhibits interferon (IFN) pathway and promotes DENV replication in monocytes in vitro and in patients significantly. Inhibition of CXCL4 mediated signaling results in increased IFN production and suppressed DENV and JEV replication in monocytes. In this review, we discuss the role of platelets in viral disease progression with a focus on dengue infection.

Malignant pleural effusion (MPE) is defined as the presence of tumor cells in pleural fluid and it is a fatal complication of advanced lung adenocarcinoma (LAC). To understand the immune response to the tumor in MPE, we compared the concentration of immunomodulatory factors in MPE of LAC and pleural effusion of heart failure (HF) patients by ELISA, and the proliferation and cytotoxic phenotype of T cells stimulated in the presence of LAC and HF pleural fluids by cytometry. Platelet factor 4 (PF4), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β) and P-selectin levels were higher in LAC than in HF pleural fluids. However, plasmatic PF4 and P-selectin levels were similar in LAC and HF. VEGF positively correlated with TGF-β and sPD-L1 in LAC but not in HF pleural fluids. LAC pleural fluids also inhibited T lymphocyte proliferation and cytotoxicity and reduced IL-17 production. PF4 levels inversely correlated with T cell function. The high content of PF4 in MPE was associated with poor prognosis. Our findings suggest that an impaired response of T lymphocytes induced by PF4 provides a significant advantage for tumor progression. [ABSTRACT FROM AUTHOR]

Platelets are anucleated blood cells derived from bone marrow megakaryocytes and play a crucial role in hemostasis and thrombosis. Platelets contain specialized storage organelles, called alpha-granules, contents of which are rich in cytokines such as C-X-C Motif Chemokine Ligand (CXCL) 1/4/7, (C-C motif) ligand (CCL) 5/3, CXCL8 (also called as interleukin 8, IL-8), and transforming growth factor β (TGF-β). Activation of platelets lead to degranulation and release of contents into the plasma. Platelet activation is a common event in many viral infections including human immunodeficiency virus (HIV), H1N1 influenza, Hepatitis C virus (HCV), Ebola virus (EBV), and Dengue virus (DENV). The cytokines CXCL8, CCL5 (also known as Regulated on Activation, Normal T Expressed and Secreted, RANTES), tumor necrosis factor α (TNF-α), CXCL1/5 and CCL3 released, promote development of a pro-inflammatory state along with the recruitment of other immune cells to the site of infection. Platelets also interact with Monocytes and Neutrophils and facilitate their activation to release different cytokines which further enhances inflammation. Upon activation, platelets also secrete factors such as CXCL4 (also known as platelet factor, PF4), CCL5 and fibrinopeptides which are critical regulators of replication and propagation of several viruses in the host. Studies suggest that CXCL4 can both inhibit as well as enhance HIV1 infection. Data from our lab show that CXCL4 inhibits interferon (IFN) pathway and promotes DENV replication in monocytes in vitro and in patients significantly. Inhibition of CXCL4 mediated signaling results in increased IFN production and suppressed DENV and JEV replication in monocytes. In this review, we discuss the role of platelets in viral disease progression with a focus on dengue infection. [ABSTRACT FROM AUTHOR]

Antibodies against platelet factor 4 (PF4), a protein released from alpha-granules of activated platelets, may cause a number of pathophysiological conditions. The most commonly known is heparin-induced thrombocytopenia (HIT), which develops in a small proportion of people treated with the anticoagulant drug heparin. Notably, PF4 binds with high affinity to heparin, and in HIT, complexes of PF4/H may, in a small proportion of susceptible patients, trigger the development of anti-PF4 antibodies and subsequent platelet activation and aggregation, ultimately leading to the development of pathological thrombosis at sites of vessel occlusion. Of more modern interest, antibodies against PF4 may also arise in patients with COVID-19 (Coronavirus Disease 2019) or in patients who have been vaccinated against COVID-19, especially in recipients of adenovirus-based vaccines. For this latter group of patients, the terms VITT (vaccine-induced [immune] thrombotic thrombocytopenia) and TTS (thrombotic thrombocytopenia syndrome) have been coined. Another category associated with this pathophysiology comprises those in whom a precipitating event is not clear; this category is referred to as ‘spontaneous HIT-like syndrome’. Despite its name, it arises as an HIT-mimicking disorder but without antecedent heparin exposure. In this narrative review, we describe the development of antibodies against PF4, and associated pathophysiology, in such conditions.

Platelet factor 4 (PF4) is a pleiotropic inflammatory chemokine, which has been implicated in various inflammatory disorders including liver fibrosis. However, its role in acute liver diseases has not yet been elucidated. Here we describe an unexpected, anti-inflammatory role of PF4. Serum concentrations of PF4 were measured in patients and mice with acute liver diseases. Acute liver injury in mice was induced either by carbon tetrachloride or by D-galactosamine hydrochloride and lipopolysaccharide. Serum levels of PF4 were decreased in patients and mice with acute liver diseases. PF4-/- mice displayed increased liver damage in both models compared to control which was associated with increased apoptosis of hepatocytes and an enhanced pro-inflammatory response of liver macrophages. In this experimental setting, PF4-/- mice were unable to generate activated Protein C (APC), a protein with anti-inflammatory activities on monocytes/macrophages. In vitro, PF4 limited the activation of liver resident macrophages. Hence, the systemic application of PF4 led to a strong amelioration of experimental liver injury. Along with reduced liver injury, PF4 improved the severity of the pro-inflammatory response of liver macrophages and induced increased levels of APC. PF4 has a yet unidentified direct anti-inflammatory effect in two models of acute liver injury. Thus, attenuation of acute liver injury by systemic administration of PF4 might offer a novel therapeutic approach for acute liver diseases.

Background: COVID-19 vaccines have been widely used to control the SARS-CoV-2 pandemic. In individuals receiving replication-incompetent, adenovirus vector-based COVID-19 vaccines (eg, ChAdOx1 nCoV-19 [AstraZeneca] or Ad26.COV2.S [Johnson & Johnson/Janssen] vaccines), a very rare but serious adverse reaction has been reported and described as vaccine-induced immune thrombotic thrombocytopenia (VITT). The exact mechanism of VITT following Ad26.COV2.S vaccination is under investigation. Antibodies directed against human platelet factor 4 (PF4) are considered critical in the pathogenesis of VITT, suggesting similarities with heparin-induced thrombocytopenia. It has been postulated that components of these vaccines mimic the role of heparin by binding to PF4, triggering production of these anti-PF4 antibodies., Objectives: This study aimed to investigate the potential interaction between human PF4 and Ad26.COV2.S vaccine using several biophysical techniques., Methods: Direct interaction of PF4 with Ad26.COV2.S vaccine was investigated using dynamic light scattering, biolayer interferometry, and surface plasmon resonance. For both biosensing methods, the Ad26.COV2.S vaccine was immobilized to the sensor surface and PF4 was used as analyte., Results: No direct interactions between PF4 and Ad26.COV2.S vaccine could be detected using dynamic light scattering and biolayer interferometry. Surface plasmon resonance technology was shown to be unsuitable to investigate these types of interactions., Conclusion: Our findings make it very unlikely that direct binding of PF4 to Ad26.COV2.S vaccine or components thereof is driving the onset of VITT, although the occurrence of such interactions after immunization (potentially facilitated by unknown plasma or cellular factors) cannot be excluded. Further research is warranted to improve the understanding of the full mechanism of this adverse reaction., Competing Interests: Declaration of competing interests R.B. is a co-inventor on related vaccine patents. M.v.d.N.K., H.I., J.M.M., N.v.D., M.P., E.K., D.v.M., J.C., and R.B. are employees of Janssen Vaccines & Prevention B.V. M.v.d.N.K., N.v.D., D.v.M., J.V., J.C., and R.B. hold stock of Johnson & Johnson. C.P.-P., J.C.d.S.F., and J.V. are former employees of Janssen Vaccines & Prevention B.V., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA.

Platelet factor 4 (PF4) is a pleiotropic inflammatory chemokine, which has been implicated in various inflammatory disorders including liver fibrosis. However, its role in acute liver diseases has not yet been elucidated. Here we describe an unexpected, anti-inflammatory role of PF4. Serum concentrations of PF4 were measured in patients and mice with acute liver diseases. Acute liver injury in mice was induced either by carbon tetrachloride or by D-galactosamine hydrochloride and lipopolysaccharide. Serum levels of PF4 were decreased in patients and mice with acute liver diseases. PF4-/- mice displayed increased liver damage in both models compared to control which was associated with increased apoptosis of hepatocytes and an enhanced pro-inflammatory response of liver macrophages. In this experimental setting, PF4-/- mice were unable to generate activated Protein C (APC), a protein with anti-inflammatory activities on monocytes/macrophages. In vitro , PF4 limited the activation of liver resident macrophages. Hence, the systemic application of PF4 led to a strong amelioration of experimental liver injury. Along with reduced liver injury, PF4 improved the severity of the pro-inflammatory response of liver macrophages and induced increased levels of APC. PF4 has a yet unidentified direct anti-inflammatory effect in two models of acute liver injury. Thus, attenuation of acute liver injury by systemic administration of PF4 might offer a novel therapeutic approach for acute liver diseases. [ABSTRACT FROM AUTHOR]

Platelet factor 4 (PF4, CXCL4) is a small chemokine protein released by activated platelets. Although a major physiological function of PF4 is to promote blood coagulation, this cytokine is involved in innate and adaptive immunity in events when platelets are activated in response to infections. Coronavirus disease 2019 (COVID-19) patients have abnormal coagulation activities, and severe patients develop higher D-dimer levels. D-dimers are small protein products present in the blood after blood clots are degraded by fibrinolysis. To prevent clotting, heparin is often clinically used in COVID-19 patients. Some clinical procedures for the management of COVID-19 patients may include extracorporeal membrane oxygenation (ECMO) and renal replacement therapy (CRRT), which also require the use of heparin. Anti-PF4 antibodies are frequently detected in severe patients and heparin-induced thrombocytopenia (HIT) can also be observed. PF4 and its role in HIT as well as in pathologies seen in COVID-19 patients define a potential therapeutic option of using blocking antibodies in the treatment of COVID-19.

Background The PIFA PLUSS PF4 Rapid Assay (PIFA) is a rapid screening test used for the diagnosis of heparin-induced thrombocytopenia (HIT). Objective To determine the usefulness of this assay as a screening method in our institution. Methods A total of 159 specimens from patients with suspected HIT were included in our study. We simultaneously performed PIFA assay and confirmatory polyspecific enzyme-linked immunosorbent assay (ELISA). We subjected most of the specimens with false-negative results to serotonin release assay (SRA). Results The initial sensitivity and specificity of the PIFA assay were calculated as 27.3% and 71.5%, respectively. A total of 12 of 16 false-negative results were further tested using the SRA method. The revised sensitivity and specificity were 50.0% and 73.5%, respectively. Conclusions Despite its appealing feature of yielding rapid results, the PIFA assay is inadequate as a sole screening test for HIT because of its high probability of missing many true cases of HIT. [ABSTRACT FROM AUTHOR]

Introduction: This study focused on PF4 effects on caspase-3,-6, -7, -8 and -9 which regulate the apopotosis process in breast cancer. Materials and Methods: Breast tumours were induced in forty 21-day-old female Sprague Dawley rats (SDRs) using MNU until tumour size reached 14.5 mm (SD: 0.5 mm). The rats were then divided into two groups: Group 1 (control injected with 0.9% saline; n = 20), and Group 2 (platelet factor 4 (PF4); n = 20). PF4 was administered through focal intralesional injection at 20 μg/lesion dose. Following 5-day treatment, the SDRs were sacrificed. Subsequently, representative sections from the tumour were obtained for haematoxylin and eosin (H&E) staining. The expressions of caspase-3, -6, -7, -8 and -9 were evaluated using immunohistochemistry (IHC) staining. Results: The majority of breast tumour specimens were of aggressive types [ncontrol = 13 (65%); nPF4 = 12 (60%)]. Invasive ductal carcinoma not otherwise specified (IDC-NOS) was the most commonly observed breast tumour histology for control and PF4 groups (n = 8 (40%) in respective groups). PF4-treated group exhibited significant differences in the caspase-3, -6 and -8 expression levels compared to the control group (all p < 0.001). There were no significant differences in caspase-7 (p = 0.347) and caspase-9 (p = 0.373) expression levels between both groups. Conclusion: This study found that PF4 acts via the caspase-mediated extrinsic apoptosis pathway without the involvement of the intrinsic pathway. [ABSTRACT FROM AUTHOR]

For the last two decades, heparins have been widely used as anticoagulants. Besides numerous advantages, up to 5% patients with heparin administration suffer from a major adverse drug effect known as heparin-induced thrombocytopenia (HIT). This typical HIT can result in deep vein thrombosis, pulmonary embolism, occlusion of a limb artery, acute myocardial infarct, stroke, and a systemic reaction or skin necrosis. The basis of HIT may lead to clinical insights. Recent studies using single-molecule force spectroscopy (SMFS)-based atomic force microscopy revealed detailed binding mechanisms of the interactions between platelet factor 4 (PF4) and heparins of different lengths in typical HIT. Especially, SMFS results allowed identifying a new mechanism of the autoimmune HIT caused by a subset of human-derived antibodies in patients without heparin exposure. The findings proved that not only heparin but also a subset of antibodies induce thrombocytopenia. In this review, the role of SMFS in unraveling a major adverse drug effect and insights into molecular mechanisms inducing thrombocytopenia by both heparins and antibodies will be discussed. [ABSTRACT FROM AUTHOR]