Longitudinal proteomic profiling of the inflammatory response in dengue patients.

BACKGROUND: The immunopathogenesis of dengue virus (DENV) infection remains incompletely understood. To increase our understanding of inflammatory response in non-severe dengue, we assessed longitudinal changes in the inflammatory proteome in patients with an acute DENV infection. METHODS: Using a multiplex proximity extension assay (PEA), we measured relative levels of 368 inflammatory markers in plasma samples from hospitalized patients with non-severe DENV infection in the acute (n = 43) and convalescence (n = 35) phase of the infection and samples of healthy controls (n = 10). RESULTS: We identified 203 upregulated and 39 downregulated proteins in acute versus convalescent plasma samples. The upregulated proteins had a strong representation of interferon (IFN) and IFN-inducible effector proteins, cytokines (e.g. IL-10, IL-33) and cytokine receptors, chemokines, pro-apoptotic proteins (e.g. granzymes) and endothelial markers. A number of differentially expressed proteins (DEPs) have not been reported in previous studies. Functional network analysis highlighted a central role for IFNγ, IL-10, IL-33 and chemokines. We identified different novel associations between inflammatory proteins and circulating concentrations of the endothelial glycocalyx disruption surrogate marker syndecan-1. Conclusion: This unbiased proteome analysis provides a comprehensive insight in the inflammatory response in DENV infection and its association with glycocalyx disruption.

Identifying platelet-derived factors as amplifiers of B. burgdorferi-induced cytokine production.

Previous studies have shown that monocytes can be 'trained' or tolerized by certain stimuli to respond stronger or weaker to a secondary stimulation. Rewiring of glucose metabolism was found to be important in inducing this phenotype. As we previously found that Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme borreliosis (LB), alters glucose metabolism in monocytes, we hypothesized that this may also induce long-term changes in innate immune responses. We found that exposure to B. burgdorferi decreased cytokine production in response to the TLR4-ligand lipopolysaccharide (LPS). In addition, B. burgdorferi exposure decreased baseline levels of glycolysis, as assessed by lactate production. Using GWAS analysis, we identified a gene, microfibril-associated protein 3-like (MFAP3L) as a factor influencing lactate production after B. burgdorferi exposure. Validation experiments proved that MFAP3L affects lactate- and cytokine production following B. burgdorferi stimulation. This is mediated by functions of MFAP3L, which includes activating ERK2 and through activation of platelet degranulation. Moreover, we showed that platelets and platelet-derived factors play important roles in B. burgdorferi-induced cytokine production. Certain platelet-derived factors, such chemokine C-X-C motif ligand 7 (CXCL7) and (C-C motif) ligand 5 (CCL5), were elevated in the circulation of LB patients in comparison to healthy individuals.

Extrapulmonary Manifestations COVID-19.

After being declared as a pandemic on March 11, 2020 by the World Health Organization, COVID-19 has affected 497 million people worldwide as of 9 April 2022. COVID-19 is a disease with a plethora of clinical manifestations, which extends to those beyond pulmonary signs and symptoms. Studies that report on the clinical presentation of COVID-19 rarely report specifically on cases with only extrapulmonary manifestations of COVID-19. Extrapulmonary clinical presentations of COVID-19 without pulmonary signs and symptoms is rare, and in such cases, COVID-19 is rarely suspected.We herewith describe four patients with extrapulmonary manifestations of COVID-19, with positive SARS-COV-2 PCR when the test was performed for initial patient screening. The first patient is a 44-year-old female who developed painful ulcer with burning sensation at the lateral side of the tongue along with low grade fever. This symptom appeared after the initial complaints of coughing and nasal congestion subsided.  The second patient is a 37-year-old male, who complained of red eyes  with itchiness and increased tear production for 3 days before seeing an ophthalmologist. The third patient is a 44-year-old female who developed burning sensation and soreness on her throat upon swallowing with fever and chills. These symptoms appear consecutively without any respiratory complaint. The fourth patient is a previously healthy, 30-year-old female, with a normal weight and BMI, and without any comorbidity, cardiovascular risk and neither personal nor family history of cardiovascular disease. In these 4 patients, COVID-19 stomatitis, conjunctivitis, pharyngitis and COVID-19-associated atrial fibrillation was subsequently diagnosed, respectively.In the pandemic stage of COVID-19, COVID-19 screening has often been routinely performed due to the high risk of transmission. However, the decrease in the number of COVID-19 cases may prompt physicians to perform SARS-COV-2 testing based on clinical suspicion. It is imperative to consider the likelihood of COVID-19 and perform SARS-COV-2 PCR in patients with extrapulmonary complaints that have persisting complaints despite treatment.

Parenteral and Oral Anticoagulant Treatment for Hospitalized and Post-Discharge COVID-19 Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: The use of anticoagulants has been endorsed by different hematological societies as coagulation abnormalities are key features of COVID-19 patients. This systematic review and meta-analysis aims to provide the most recent update on available evidence on the clinical benefits and risk of oral and parenteral anticoagulants, as well agents with anticoagulant properties, in hospitalized and post-discharge COVID-19 patients. METHODS: This systematic review synthesizes data on the outcome of anticoagulation in hospitalized and post-discharge COVID-19 patients. Dichotomous variables from individual studies were pooled by risk ratio (RR) and their 95% confidence interval (95% CI) using the random-effects model. Meta-analyses were performed when feasible. RESULTS: We included 32 studies from 2.815 unique citations, including 7 randomized clinical trials. A total of 33.494 patients were included. Outcomes measured include mortality and survival rates, the requirement for ICU care and mechanical ventilation. A pooled meta-analysis favors anticoagulant compared to no anticoagulant with reduced mortality in hospitalized patients (RR 0,55; 95%CI 0,43-0,66; p<0,001). Higher dose of anticoagulant also showed treatment benefit compared to standard prophylactic dose in selected populations (RR 0,68; 95%CI 0,40-0,96; p<0,001). Regular, pre-hospital anticoagulation prior to hospitalization yielded mixed result. There are currently no data on the benefit of anticoagulation on post-discharge COVID-19 patients. CONCLUSION: Determination of the presence of thrombosis in COVID-19 is important, as therapeutic dosage of anticoagulants, rather than prophylatic dose, would be indicated in such clinical situation. Anticoagulants were found to decrease the mortality of hospitalized COVID-19. The results from this study are important in the tailored treatment of COVID-19 patients. Further studies on the need for oral anticoagulation for outpatients or post-discharge is warranted. This study has been registered in PROSPERO database (CRD42020201418).

Effect of oseltamivir phosphate versus placebo on platelet recovery and plasma leakage in adults with dengue and thrombocytopenia; a phase 2, multicenter, double-blind, randomized trial.

BACKGROUND: Thrombocytopenia, bleeding and plasma leakage are major complications of dengue. Activation of endogenous sialidases with desialylation of platelets and endothelial cells may underlie these complications. We aimed to assess the effects of the neuraminidase inhibitor oseltamivir on platelet recovery and plasma leakage in dengue. METHODS: We performed a phase 2, double-blind, multicenter, randomized trial in adult dengue patients with thrombocytopenia (<70,000/µl) and a duration of illness ≤ 6 days. Oseltamivir phosphate 75mg BID or placebo were given for a maximum of five days. Primary outcomes were the time to platelet recovery (≥ 100,000/µl) or discharge from hospital and the course of measures of plasma leakage. RESULTS: A total of 70 patients were enrolled; the primary outcome could be assessed in 64 patients (31 oseltamivir; 33 placebo). Time to platelet count ≥100,000/µl (n = 55) or discharge (n = 9) were similar in the oseltamivir and placebo group (3.0 days [95% confidence interval, 2.7 to 3.3] vs. 2.9 days [2.5 to 3.3], P = 0.055). The kinetics of platelet count and parameters of plasma leakage (gall bladder thickness, hematocrit, plasma albumin, syndecan-1) were also similar between the groups. DISCUSSION: In this trial, adjunctive therapy with oseltamivir phosphate had no effect on platelet recovery or plasma leakage parameters. TRIAL REGISTRATION: ISRCTN35227717.

Untargeted Plasma Metabolomics and Gut Microbiome Profiling Provide Novel Insights into the Regulation of Platelet Reactivity in Healthy Individuals.

BACKGROUND: Considerable variation exists in platelet reactivity to stimulation among healthy individuals. Various metabolites and metabolic pathways influence platelet reactivity, but a comprehensive overview of these associations is missing. The gut microbiome has a strong influence on the plasma metabolome. Here, we investigated the association of platelet reactivity with results of untargeted plasma metabolomics and gut microbiome profiling. METHODS: We used data from a cohort of 534 healthy adult Dutch volunteers (the 500 Functional Genomics study). Platelet activation and reactivity were measured by the expression of the alpha-granule protein P-selectin and the binding of fibrinogen to the activated integrin αIIbß3, both in unstimulated blood and after ex vivo stimulation with platelet agonists. Plasma metabolome was measured using an untargeted metabolic profiling approach by quadrupole time-of-flight mass spectrometry. Gut microbiome data were measured by shotgun metagenomic sequencing from stool samples. RESULTS: Untargeted metabolomics yielded 1,979 metabolites, of which 422 were identified to play a role in a human metabolic pathway. Overall, 92/422 (21.8%) metabolites were significantly associated with at least one readout of platelet reactivity. The majority of associations involved lipids, especially members of eicosanoids, including prostaglandins and leukotrienes. Dietary-derived polyphenols were also found to inhibit platelet reactivity. Validation of metabolic pathways with functional microbial profiles revealed two overlapping metabolic pathways ("alanine, aspartate, and glutamate metabolism" and "arginine biosynthesis") that were associated with platelet reactivity. CONCLUSION: This comprehensive overview is an resource for understanding the regulation of platelet reactivity by the plasma metabolome and the possible contribution of the gut microbiota.

Vaccine-Associated Disease Enhancement (VADE): Considerations in Postvaccination COVID-19.

INTRODUCTION: The COVID-19 pandemic has entered a new phase with the roll-out of several vaccines worldwide at an accelerated phase. The occurrence of a more severe presentation of COVID-19 after vaccination may affect policymakers' decision-making and vaccine uptake by the public. Vaccine-associated disease enhancement (VADE) is the modified presentation of infections in individuals after having received a prior vaccination. Currently, little is known about the potential of vaccine-associated disease enhancement (VADE) following COVID-19 immunization. Case Illustration. We herewith report two patients admitted with confirmed COVID-19 pneumonia with a history of CoronaVac vaccination. The first patient with a relatively milder course of the disease had received two doses of CoronaVac, whereas the second patient with a more progressive course of the disease received only one dose before developing symptoms and being admitted to the hospital. Our observations suggest that vaccination could act in boosting the inflammatory process and reveal the previously asymptomatic COVID-19 illness. Theoretically, vaccines could induce VADE, where only suboptimal, nonprotective titers of neutralizing antibodies were produced or proinflammatory T-helper type 2 response was induced. Secondly, enhanced respiratory disease (ERD) could manifest, where pulmonary symptoms are more severe due to peribronchial monocytic and eosinophilic infiltration. Understanding VADE is important for the decision-making by the public, clinicians, and policymakers and is warranted for successful vaccination uptake. CONCLUSION: We report two cases of patients developing COVID-19 shortly after CoronaVac vaccination in which VADE is likely. We recommend that current vaccination strategies consider the measurement of neutralizing antibody titer as a guide in ensuring the safest strategy for mass immunization. Studies are needed to investigate the true incidence of VADE on vaccinated individuals as well as on how to differentiate between VADE and severe manifestations of COVID-19 that are unrelated to vaccination.

Black Fungus Complicated with COVID-19 in a Man with Underlying Non-Hodgkin's Lymphoma.

COVID-19 is a disease reported to suppress cellular immunity. This may lead to the development of opportunistic infections, among others black fungus, or mucormycosis. On the other hand, pre-existing defect in immunity may render patients susceptible to both mucormycosis and COVID-19. Mucormycosis is a relatively rare fungal infection with rapid progression unless diagnosed promptly and treated adequately, and urgent surgical and medical intervention is lifesaving. The manifestation of mucormycosis largely depends on the presence of exposure to the pathogen and the existing risk factor of the host. As black fungus is locally invasive, the majority of cases will involve tissue damage with local destruction and contiguous spread to nearby structure. We here with present a case of black fungus complicated with COVID-19 in a man with underlying non-Hodgkin's lymphoma.

Hospital-acquired Skin and Skin-structure Infection in COVID-19 Infected Patient with Prolonged Hospitalization.

Acute bacterial skin and skin-structure infections (ABSSSI) is defined in 2013 by the US Food and Drug Administration as a bacterial cellulitis/erysipelas, major skin abscesses, and wound infections. The Infectious Diseases Society of America (IDSA) in 2014 classifies skin and soft-tissue infection (SSTI) as either non-purulent (which includes cellulitis, erysipelas, and necrotizing infection) or purulent (including furuncle, carbuncle, and abscess). Among hospitalized patients with SSTI, healthcare-associated infections account for 73.5% of all cases. Notably, skin and skin-structure infections caused by Pseudomonas aeruginosa, a common hospital pathogen, was reported to cause higher total cost and longer hospital length of stay compared to non-P. aeruginosa cases, despite causing only approximately 5.7% of all healthcare-associated SSTIs. Infection with P. aeruginosa should always be considered in non-healing skin infections in patients with prolonged hospitalization and antibiotic exposure. Tissue culture, preferably taken by surgical debridement, should be promptly performed; and when hospital-infection is suspected, appropriate antibiotics should be started along with removal of all devitalized tissue and to promote skin and soft tissue healing. Expedited discharge should be considered when possible, with adequate antibiotic treatment and follow up for definitive wound treatment.

Increased Plasma Heparanase Activity and Endothelial Glycocalyx Degradation in Dengue Patients Is Associated With Plasma Leakage.

Background: Endothelial hyper-permeability with plasma leakage and thrombocytopenia are predominant features of severe dengue virus infection. It is well established that heparanase, the endothelial glycocalyx degrading enzyme, plays a major role in various diseases with vascular leakage. It is yet to be elucidated whether heparanase activity plays a major role in dengue-associated plasma leakage. Moreover, the major source of heparanase secretion and activation in dengue remains elusive. Since a relatively high amount of heparanase is stored in platelets, we postulate that heparanase released by activated platelets contributes to the increased plasma heparanase activity during dengue virus infection. Methods: Heparanase activity (plasma and urine), and heparan sulfate and syndecan-1 (plasma levels) were measured in dengue patients with thrombocytopenia in acute phase (n=30), during course of disease (n=10) and in convalescent phase (n=25). Associations with clinical parameters and plasma leakage markers were explored. Platelets from healthy donors were stimulated with dengue non-structural protein-1, DENV2 virus and thrombin to evaluate heparanase release and activity ex vivo. Results: Heparanase activity was elevated in acute dengue and normalized during convalescence. Similarly, glycocalyx components, such as heparan sulfate and syndecan-1, were increased in acute dengue and restored during convalescence. Increased heparanase activity correlated with the endothelial dysfunction markers heparan sulfate and syndecan-1, as well as clinical markers of plasma leakage such as ascites, hematocrit concentration and gall-bladder wall thickening. Notably, platelet number inversely correlated with heparanase activity. Ex vivo incubation of platelets with thrombin and live DENV2 virus, but not dengue virus-2-derived non-structural protein 1 induced heparanase release from platelets. Conclusion: Taken together, our findings suggest that the increase of heparanase activity in dengue patients is associated with endothelial glycocalyx degradation and plasma leakage. Furthermore, thrombin or DENV2 activated platelets may be considered as a potential source of heparanase.

Platelet Integrin αIIbß3 Activation is Associated with 25-Hydroxyvitamin D Concentrations in Healthy Adults.

BACKGROUND: Cardiovascular events are associated with low circulating vitamin D concentrations, although the underlying mechanisms are poorly understood. This study investigated associations between 25-hydroxyvitamin D concentrations, platelet function, and single-nucleotide polymorphisms (SNPs) in genes influencing vitamin D biology in the 500 Functional Genomics (500FG) cohort. METHODS: In this observational study, platelet activation and function were measured by flow cytometry by binding of fibrinogen to the activated fibrinogen receptor integrin αIIbß3 and expression of P-selectin, markers of platelet aggregation and degranulation, respectively. These parameters were correlated to serum 25-hydroxyvitamin D and genotyping was performed to investigate SNPs in genes important for vitamin D biology. RESULTS: Circulating 25-hydroxyvitamin D concentrations correlated inversely with baseline platelet binding of fibrinogen to integrin αIIbß3 (Pearson's r= -0.172, p = 0.002) and platelet responses to platelet agonist cross-linked collagen-related peptide (CRP-XL) (Pearson's r= -0.196,p = 0.002). This effect was due to circulating vitamin D levels ≤50nmol/L, since no differences in platelet fibrinogen binding were observed between subjects with normal 25-hydroxyvitamin D concentrations (>75nmol/L) and a 25-hydroxyvitamin D insufficiency (50-75 nmol/L). No correlations between 25-hydroxyvitamin D concentrations and platelet P-selectin expression were found. Several SNPs in the GC region of the vitamin D binding proteingene were associated with platelet responses to CRP-XL. CONCLUSION: Low circulating vitamin D concentrations are associated with increased platelet fibrinogen binding to integrin αIIbß3 in unstimulated samples and after stimulation with CRP-XL. These findings may contribute to the increased incidence of cardiovascular events in vitamin D deficient adults and its seasonal variation. Further studies are needed to investigate causality.

Desialylation of platelets induced by Von Willebrand Factor is a novel mechanism of platelet clearance in dengue.

Thrombocytopenia and platelet dysfunction are commonly observed in patients with dengue virus (DENV) infection and may contribute to complications such as bleeding and plasma leakage. The etiology of dengue-associated thrombocytopenia is multifactorial and includes increased platelet clearance. The binding of the coagulation protein von Willebrand factor (VWF) to the platelet membrane and removal of sialic acid (desialylation) are two well-known mechanisms of platelet clearance, but whether these conditions also contribute to thrombocytopenia in dengue infection is unknown. In two observational cohort studies in Bandung and Jepara, Indonesia, we show that adult patients with dengue not only had higher plasma concentrations of plasma VWF antigen and active VWF, but that circulating platelets had also bound more VWF to their membrane. The amount of platelet-VWF binding correlated well with platelet count. Furthermore, sialic acid levels in dengue patients were significantly reduced as assessed by the binding of Sambucus nigra lectin (SNA) and Maackia amurensis lectin II (MAL-II) to platelets. Sialic acid on the platelet membrane is neuraminidase-labile, but dengue virus has no known neuraminidase activity. Indeed, no detectable activity of neuraminidase was present in plasma of dengue patients and no desialylation was found of plasma transferrin. Platelet sialylation was also not altered by in vitro exposure of platelets to DENV nonstructural protein 1 or cultured DENV. In contrast, induction of binding of VWF to glycoprotein 1b on platelets using the VWF-activating protein ristocetin resulted in the removal of platelet sialic acid by translocation of platelet neuraminidase to the platelet surface. The neuraminidase inhibitor oseltamivir reduced VWF-induced platelet desialylation. Our data demonstrate that excessive binding of VWF to platelets in dengue results in neuraminidase-mediated platelet desialylation and platelet clearance. Oseltamivir might be a novel treatment option for severe thrombocytopenia in dengue infection.

The influence of hypoxia on platelet function and plasmatic coagulation during systemic inflammation in humans in vivo.

Systemic inflammation and hypoxia frequently occur simultaneously in critically ill patients, and are both associated with platelet activation and coagulopathy. However, human in vivo data on the effects of hypoxia on platelet function and plasmatic coagulation under systemic inflammatory conditions are lacking. In the present study, 20 healthy male volunteers were randomized to either 3.5 h of hypoxia (peripheral saturation 80-85%) or normoxia (room air), and systemic inflammation was elicited by intravenous administration of 2 ng/kg endotoxin. Various parameters of platelet function and plasmatic coagulation were determined serially. Endotoxemia resulted in increased circulating platelet-monocyte complexes and enhanced platelet reactivity, effects which were attenuated by hypoxia. Furthermore, endotoxin administration resulted in decreased plasma levels of platelet factor-4 levels and increased concentrations of von Willebrand factor. These endotoxemia-induced effects were not influenced by hypoxia. Neither endotoxemia nor hypoxia affected thrombin generation. In conclusion, our data reveal that hypoxia attenuates the endotoxemia-induced increases in platelet-monocyte formation and platelet reactivity, while leaving parameters of plasmatic coagulation unaffected.

The Inter-Relationship of Platelets with Interleukin-1ß-Mediated Inflammation in Humans.

BACKGROUND: Inflammation and coagulation are key processes in cardiovascular diseases (CVDs). The Canakinumab Anti-inflammatory Thrombosis Outcome Study trial affirmed the importance of inflammation in CVD by showing that inhibition of the interleukin (IL)-1ß pathway prevents recurrent CVD. A bi-directional relationship exists between inflammation and coagulation, but the precise interaction of platelets and IL-1ß-mediated inflammation is incompletely understood. We aimed to determine the inter-relationship between platelets and inflammation-and especially IL-1ß-in a cohort of healthy volunteers. METHODS: We used data from the 500-Human Functional Genomics cohort, which consists of approximately 500 Caucasian, healthy individuals. We determined associations of plasma levels of IL-1ß and other inflammatory proteins with platelet number and reactivity, the association of platelet reactivity with ex vivo cytokine production as well as the impact of genetic variations through a genome-wide association study (GWAS). RESULTS: Platelets were associated with IL-1ß on different levels. First, platelet number was positively associated with plasma IL-1ß concentrations (p = 8.9 × 10-9) and inversely with concentrations of α-1-anti-trypsin (p = 1.04 × 10-18), which is a known antagonist of IL-1ß. Second, platelet degranulation capacity, as determined by agonist-induced P-selectin expression, was associated with ex vivo IL-1ß and IL-6 production. Third, several platelet single-nucleotide polymorphisms (SNPs) were associated with cytokine production and there was a significant platelet SNP enrichment in specific biological important pathways. Finally, platelet SNPs were enriched among SNPs earlier identified in GWAS studies in blood-related diseases and immune-mediated diseases. CONCLUSION: This comprehensive assessment of factors associated with platelet number and reactivity reinforces the important inter-relationship of platelets and IL-1ß-mediated inflammation.

Thrombocytopenia and Platelet Dysfunction in Acute Tropical Infectious Diseases.

Thrombocytopenia is a well-known manifestation of acute tropical infectious diseases. The role of platelets in infections has received much attention recently because of their emerging activities in modulation of inflammatory responses, host defense, and vascular integrity. However, while many studies have addressed thrombocytopenia in tropical infections, abnormalities in platelet function have been largely overlooked. This is an important research gap, as platelet dysfunction may contribute to the bleeding tendency that characterizes some tropical infections. The development of novel platelet function assays that can be used in thrombocytopenic conditions (e.g., flow cytometry assays) has contributed to important new insights in recent years. In this review, the importance of platelets in tropical infections is discussed with special emphasis on the underlying mechanisms and consequences of thrombocytopenia and platelet dysfunction in these infections. Special attention is paid to malaria, a disease characterized by microvascular obstruction in which bleeding is rare, and to infections in which bleeding is common, such as dengue, other viral hemorrhagic fevers, and the bacterial infection leptospirosis. Given the importance of platelet function abnormalities in these infections, the development of affordable assays for monitoring of platelet function in low-resource countries, as well as pharmacologic interventions to prevent or reverse platelet function abnormalities, might improve clinical care and the prognosis of these infections.

The effects of signal transducer and activator of transcription three mutations on human platelets.

Involvement of signal transducer and activator of transcription 3 (STAT3) in inflammation is well known. Recently, a role for STAT3 in platelet activation and platelet production has been suggested. Platelets exhibit important immune functions and engagement of STAT3 in platelet physiology may link inflammation and hemostasis. This study investigated the effects of STAT3 loss-of-function mutations and single nucleotide polymorphisms (SNPs) in STAT3 on glycoprotein VI (GPVI)-mediated platelet activation and platelet numbers in humans. Two cohorts were studied. The first cohort concerned patients with STAT3 loss-of-function mutations. Platelet numbers were investigated in eight patients and GPVI-mediated platelet activation was functionally tested in four patients. Additional experiments were performed to investigate underlying mechanisms. The second cohort concerned 334 healthy volunteers and investigated the consequences of SNPs in STAT3 on GPVI-mediated platelet activation and platelet numbers. Platelet activation was lower in STAT3 loss-of-function patients at baseline and after stimulation of the GPVI receptor, reflected by decreased P-selectin expression. This was independent of gene transcription. Blockade of the adenosine di-phosphate (ADP) pathway resulted in a further decrease of P-selectin expression, particularly in STAT3 loss-of-function patients. In contrast, the SNPs in STAT3 did not influence GPVI-mediated platelet activation. Also, platelet numbers were not affected by STAT3 loss-of-function mutations, nor was there an association with the SNPs. In conclusion, STAT3 signaling does not seem to play a major role in thrombopoiesis. We confirm that STAT3 is involved in GPVI-mediated platelet activation in humans, independent of gene transcription. GPVI-mediated platelet activation is highly dependent on secondary ADP release. Our findings suggest that STAT3 modulation may affect inflammation, hemostasis, and their interaction.

Platelet dysfunction contributes to bleeding complications in patients with probable leptospirosis.

BACKGROUND: Severe leptospirosis is frequently complicated by a hemorrhagic diathesis, of which the pathogenesis is still largely unknown. Thrombocytopenia is common, but often not to the degree that spontaneous bleeding is expected. We hypothesized that the hemorrhagic complications are not only related to thrombocytopenia, but also to platelet dysfunction, and that increased binding of von Willebrand factor (VWF) to platelets is involved in both platelet dysfunction and increased platelet clearance. METHODOLOGY/PRINCIPAL FINDINGS: A prospective study was carried out in Semarang, Indonesia, enrolling 33 hospitalized patients with probable leptospirosis, of whom 15 developed clinical bleeding, and 25 healthy controls. Platelet activation and reactivity were determined using flow cytometry by measuring the expression of P-selectin and activation of the αIIbß3 integrin by the binding of fibrinogen in unstimulated samples and after ex vivo stimulation by the platelet agonists adenosine-diphosphate (ADP) and thrombin-receptor activating peptide (TRAP). Platelet-VWF binding, before and after VWF stimulation by ristocetin, as well as plasma levels of VWF, active VWF, the VWF-inactivating enzyme ADAMTS13, thrombin-antithrombin complexes (TAT) and P-selectin were also measured. Bleeding complications were graded using the WHO bleeding scale. Our study revealed that platelet activation, with a secondary platelet dysfunction, is a feature of patients with probable leptospirosis, especially in those with bleeding manifestations. There was a significant inverse correlation of bleeding score with TRAP-stimulated P-selectin and platelet-fibrinogen binding (R = -0.72, P = 0.003 and R = -0.66, P = 0.01, respectively) but not with platelet count. Patients with bleeding also had a significantly higher platelet-VWF binding. Platelet counts were inversely correlated with platelet-VWF binding (R = -0.74; P = 0.0009. There were no correlations between platelet-VWF binding and the degree of platelet dysfunction, suggesting that increased platelet-VWF binding does not directly interfere with the platelet αIIbß3 signaling pathway in patients with probable leptospirosis. CONCLUSION/SIGNIFICANCE: Platelet dysfunction is common in probable leptospirosis patients with manifest bleeding. Increased VWF-platelet binding may contribute to the activation and clearance of platelets.

Phage-Derived Protein Induces Increased Platelet Activation and Is Associated with Mortality in Patients with Invasive Pneumococcal Disease.

To improve our understanding about the severity of invasive pneumococcal disease (IPD), we investigated the association between the genotype of Streptococcus pneumoniae and disease outcomes for 349 bacteremic patients. A pneumococcal genome-wide association study (GWAS) demonstrated a strong correlation between 30-day mortality and the presence of the phage-derived gene pblB, encoding a platelet-binding protein whose effects on platelet activation were previously unknown. Platelets are increasingly recognized as key players of the innate immune system, and in sepsis, excessive platelet activation contributes to microvascular obstruction, tissue hypoperfusion, and finally multiorgan failure, leading to mortality. Our in vitro studies revealed that pblB expression was induced by fluoroquinolones but not by the beta-lactam antibiotic penicillin G. Subsequently, we determined pblB induction and platelet activation by incubating whole blood with the wild type or a pblB knockout mutant in the presence or absence of antibiotics commonly administered to our patient cohort. pblB-dependent enhancement of platelet activation, as measured by increased expression of the α-granule protein P-selectin, the binding of fibrinogen to the activated αIIbß3 receptor, and the formation of platelet-monocyte complex occurred irrespective of antibiotic exposure. In conclusion, the presence of pblB on the pneumococcal chromosome potentially leads to increased mortality in patients with an invasive S. pneumoniae infection, which may be explained by enhanced platelet activation. This study highlights the clinical utility of a bacterial GWAS, followed by functional characterization, to identify bacterial factors involved in disease severity. IMPORTANCE: The exact mechanisms causing mortality in invasive pneumococcal disease (IPD) patients are not completely understood. We examined 349 patients with IPD and found in a bacterial genome-wide association study (GWAS) that the presence of the phage-derived gene pblB was associated with mortality in the first 30 days after hospitalization. Although pblB has been extensively studied in Streptococcus mitis, its consequence for the interaction between platelets and Streptococcus pneumoniae is largely unknown. Platelets are important in immunity and inflammation, and excessive platelet activation contributes to microvascular obstruction and multiorgan failure, leading to mortality. We therefore developed this study to assess whether the expression of pblB might increase the risk of death for IPD patients through its effect on enhanced platelet activation. This study also shows the value of integrating extensive bacterial genomics and clinical data in predicting and understanding pathogen virulence, which in turn will help to improve prognosis and therapy.

Higher platelet reactivity and platelet-monocyte complex formation in Gram-positive sepsis compared to Gram-negative sepsis.

Platelets may play a role in the high risk for vascular complications in Gram-positive sepsis. We compared the platelet reactivity of 15 patients with Gram-positive sepsis, 17 with Gram-negative sepsis and 20 healthy controls using a whole blood flow cytometry-based assay. Patients with Gram-positive sepsis had the highest median fluorescence intensity (MFI) of the platelet membrane expression of P-selectin upon stimulation with high dose adenosine diphosphate (ADP; P = 0.002 vs. Gram-negative and P = 0.005 vs. control groups) and cross-linked collagen-related peptide (CRP-XL; P = 0.02 vs. Gram-negative and P = 0.0001 vs. control groups). The Gram-positive group also demonstrated significantly higher ADP-induced fibrinogen binding (P = 0.001), as wll as platelet-monocyte complex formation (P = 0.02), compared to the Gram-negative group and had the highest plasma levels of platelet factor 4, ß-thromboglobulin and soluble P-selectin. In contrast, thrombin-antithrombin complex and C-reactive protein levels were comparable in both patient groups. In conclusion, common Gram-positive pathogens induce platelet hyperreactivity, which may contribute to a higher risk for vascular complications.

Human recombinant alkaline phosphatase inhibits ex vivo platelet activation in humans.

Sepsis-associated acute kidney injury (AKI) is associated with high morbidity and mortality. Excessive platelet activation contributes to AKI through the formation of microthrombi and amplification of systemic inflammation. Two phase II trials demonstrated that bovine-intestinal alkaline phosphatase (AP) improved renal function in critically ill patients with sepsis-associated AKI. In this study, we characterised the platelet-inhibiting effects of a human recombinant AP. Whole blood and platelet-rich plasma (PRP) of healthy volunteers (n=6) was pre-treated ex vivo with recAP, whereafter platelet reactivity to ADP, collagen-related peptide (CRP-XL) and Pam3CSK4 was determined by flow cytometry. RecAP (40 U/ml) reduced the platelet reactivity to ADP (inhibition with a median of 47 %, interquartile range 43-49 %; p<0.001) and tended to reduce platelet reactivity to CRP-XL (9 %, 2-25 %; p=0.08) in whole blood. The platelet-inhibiting effects of recAP were more pronounced in PRP both for ADP- (64 %, 54-68 %; p=0.002) and CRP-XL-stimulated samples (60 %, 46-71 %; p=0.002). RecAP rapidly converted ADP into adenosine, whereas antagonism of the A2A adenosine receptor partially reversed the platelet inhibitory effects of recAP. Platelets of septic shock patients (n=5) showed a 31% (22-34%; p=0.03) more pronounced reactivity compared to healthy volunteers, and this was completely reversed by recAP treatment. In conclusion, we demonstrate that recAP inhibits ex vivo human platelet activation through dephosphorylation of ADP and formation of adenosine as its turnover product. RecAP is able to reverse the platelet hyperreactivity present in septic shock patients. These effects may contribute to the beneficial effects of recAP as a new therapeutic candidate for sepsis-associated AKI.