Platelet-neutrophil Interactions Research Articles

Platelet hyperresponsiveness and increased platelet-neutrophil aggregates in dogs with myxomatous mitral valve disease and pulmonary hypertension.

Pulmonary hypertension (PH) in dogs with myxomatous mitral valve disease (MMVD) is caused by increased pulmonary venous pressure. Thrombosis, vascular remodeling, and vasoconstriction mediated by platelets could exacerbate PH. Dogs with PH will exhibit a hypercoagulable state, characterized by increased platelet activation, platelet-leukocyte, and platelet-neutrophil aggregate formation. Eleven dogs (≥3.5 kg) diagnosed with MMVD and PH and 10 dogs with MMVD lacking PH. Prospective cohort ex vivo study. All dogs underwent echocardiographic examination, CBC, 3-view thoracic radiographs, and heartworm antigen testing. Severity of PH and MMVD were assessed by echocardiography. Viscoelastic monitoring of coagulation was assessed using thromboelastography (TEG). Platelet activation and platelet-leukocyte/platelet-neutrophil interactions were assessed using flow cytometry. Plasma serotonin concentrations were measured by ELISA. Unstimulated platelets from dogs with MMVD and PH expressed more surface P-selectin than MMVD controls (P = .03). Platelets from dogs with MMVD and PH had persistent activation in response to agonists. The number of platelet-leukocyte aggregates was higher in dogs with MMVD and PH compared with MMVD controls (P = .01). Ex vivo stimulation of whole blood resulted in higher numbers of platelet-neutrophil aggregates in dogs with MMVD and PH (P = .01). Assessment of hypercoagulability based on TEG or plasma serotonin concentrations did not differ between groups. Platelet hyperresponsiveness and increased platelet-neutrophil interaction occur in dogs with MMVD and PH, suggesting that platelets play a role of in the pathogenesis of PH. Clinical benefits of antiplatelet drugs in dogs with MMVD and PH require further investigation.

Neutrophil Extracellular DNA Traps in Response to Infection or Inflammation, and the Roles of Platelet Interactions.

Neutrophils present the host's first line of defense against bacterial infections. These immune effector cells are mobilized rapidly to destroy invading pathogens by (a) reactive oxygen species (ROS)-mediated oxidative bursts and (b) via phagocytosis. In addition, their antimicrobial service is capped via a distinct cell death mechanism, by the release of their own decondensed nuclear DNA, supplemented with a variety of embedded proteins and enzymes. The extracellular DNA meshwork ensnares the pathogenic bacteria and neutralizes them. Such neutrophil extracellular DNA traps (NETs) have the potential to trigger a hemostatic response to pathogenic infections. The web-like chromatin serves as a prothrombotic scaffold for platelet adhesion and activation. What is less obvious is that platelets can also be involved during the initial release of NETs, forming heterotypic interactions with neutrophils and facilitating their responses to pathogens. Together, the platelet and neutrophil responses can effectively localize an infection until it is cleared. However, not all microbial infections are easily cleared. Certain pathogenic organisms may trigger dysregulated platelet-neutrophil interactions, with a potential to subsequently propagate thromboinflammatory processes. These may also include the release of some NETs. Therefore, in order to make rational intervention easier, further elucidation of platelet, neutrophil, and pathogen interactions is still needed.

PAD4-Induced NETosis Via Cathepsin G-Mediated Platelet-Neutrophil Interaction in ChAdOx1 Vaccine-Induced Thrombosis.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare syndrome characterized by platelet anti-PF4 (platelet-activating antiplatelet factor 4)-related thrombosis. Platelet-neutrophil interaction has been suggested to play a role, but the underlying mechanism has not been fully elucidated. The study included 10 patients with VITT after ChAdOx1 nCoV-19 (Oxford-AstraZeneca) vaccine administration, 10 patients with ischemic stroke (IS), 10 patients with acute deep vein thrombosis, and 10 control subjects in whom blood levels of neutrophil extracellular traps (NETs), soluble TF (tissue factor), and thrombin generation were examined. Furthermore, we performed in vitro studies comparing the effect of serum from patients and controls on NETs formation. Finally, immunohistochemistry was performed in cerebral thrombi retrieved from a patients with VITT and 3 patients with IS. Compared with patients with IS, patients with deep vein thrombosis, controls, and patients with VITT had significantly higher blood values of CitH3 (citrullinated histone H3), soluble TF, D-dimer, and prothrombin fragment 1+2 (P<0.0001). Blood CitH3 significantly correlated with blood soluble TF (Spearman rank correlation coefficient=0.7295; P=0.0206) and prothrombin fragment 1+2 (Spearman rank correlation coefficient=0.6809; P<0.0350) in patients with VITT. Platelet-neutrophil mixture added with VITT plasma resulted in higher NETs formation, soluble TF and thrombin generation, and platelet-dependent thrombus growth under laminar flow compared with IS and deep vein thrombosis plasma; these effects were blunted by PAD4 (protein arginine deiminase 4) and cathepsin G inhibitors, anti-FcγRIIa, and high doses of heparin. Immunohistochemistry analysis showed a more marked expression of PAD4 along with more diffuse neutrophil infiltration and NETs formation as well as TF and cathepsin expression in VITT thrombus compared with thrombi from patients with IS. Patients with VITT display enhanced thrombogenesis by PAD4-mediated NETs formation via cathepsin G-mediated platelet/neutrophil interaction.

Platelet-neutrophil interaction in COVID-19 and vaccine-induced thrombotic thrombocytopenia.

Coronavirus disease 2019 (COVID-19) is known to commonly induce a thrombotic diathesis, particularly in severely affected individuals. So far, this COVID-19-associated coagulopathy (CAC) has been partially explained by hyperactivated platelets as well as by the prothrombotic effects of neutrophil extracellular traps (NETs) released from neutrophils. However, precise insight into the bidirectional relationship between platelets and neutrophils in the pathophysiology of CAC still lags behind. Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare autoimmune disorder caused by auto-antibody formation in response to immunization with adenoviral vector vaccines. VITT is associated with life-threatening thromboembolic events and thus, high fatality rates. Our concept of the thrombophilia observed in VITT is relatively new, hence a better understanding could help in the management of such patients with the potential to also prevent VITT. In this review we aim to summarize the current knowledge on platelet-neutrophil interplay in COVID-19 and VITT.

Platelet function suggests cardioembolic aetiology in cryptogenic stroke

Platelet-monocyte (PMA) and platelet-neutrophil aggregations (PNA) play critical roles in the evolution of acute ischemic stroke (AIS). The present study investigates the mechanistic basis of platelet responsiveness in cryptogenic stroke compared with cardioembolic stroke. Platelet from 16 subjects, each from cryptogenic and cardioembolic stroke groups and 18 age-matched healthy controls were subjected to different investigations. Compared to healthy controls, platelet-monocyte and platelet-neutrophil interactions were significantly elevated in cryptogenic (2.7 and 2.1 times) and cardioembolic stroke (3.9 and 2.4 times). P-selectin expression on platelet surface was 1.89 and 2.59 times higher in cryptogenic and cardioembolic strokes, respectively, compared to healthy control. Cell population with [Ca2+i] in either stroke group was significantly outnumbered (by 83% and 72%, respectively, in cryptogenic and cardioembolic stroke) in comparison to healthy controls. Noteworthy, TEG experiment revealed that the cryptogenic stroke exhibited significant decline in Reaction Time (R) and amplitude of 20 mm (K) (by 32% and 33%, respectively) while thrombin burst (α-angle) was augmented by 12%, which reflected substantial boost in thrombus formation in cryptogenic stroke. Although TEG analysis reveals a state of hypercoagulability in patients with cryptogenic stroke. However, platelets from both stroke subtypes switch to a ‘hyperactive’ phenotype.

Abstract 140: A Common, Racially Diverse Protease Activated Receptor 4 Functional Variant Impacts Ischemic Stroke Outcomes: Evidence For A Pharmacogenetic Effect

Black individuals have a higher incidence of ischemic stroke (IS) than White individuals. The platelet receptor PAR4 harbors a functional dimorphism: rs773902 A/G (encoding Thr120/Ala120, respectively), and the A allele is associated with greater ex vivo platelet aggregation. The A allele frequency is 0.6 in Black individuals and 0.2 in White subjects. However, it is unknown whether the A allele contributes to racial differences in IS. In a prospective cohort, the REGARDS study, a Black subject-only analysis of 487 IS cases and 7,133 controls was performed, examining if the A allele was a risk for incident IS and worse IS outcome. We used Cox proportional hazards models to calculate hazard ratios (HR) with 95% confidence intervals (CI) of incident IS for the rs773902 A allele. Models were adjusted for age, sex, the first 5 genetic principal components, smoking, hypertension and diabetes. To directly test the effect of this PAR4 variant on IS, we generated the first humanized PAR4 (hPAR4) mouse strains expressing either hPAR4 Thr120 or hPAR4 Ala120. In REGARDS, the A allele was a risk factor for incident IS (HR 1.24; CI 1.04-1.53, p=0.046) and worse IS outcome (HR 2.04; CI 1.10-3.79, p=0.025). In a murine stroke model, hPAR4 Thr120 mice had worse stroke outcomes compared to hPAR4 Ala120 mice (60% increase, p=0.001). This phenotype was mediated, in part, by enhanced platelet activation (p=0.002), platelet-neutrophil interactions (p=0.014), and neutrophil extracellular traps (p=0.001). Direct inhibition of PAR4 with BMS-986120 was equally effective in improving stroke outcomes in hPAR4 Ala120 and Thr120 mice. In contrast, ticagrelor treatment improved stroke outcomes in hPAR4 Ala120 mice (50% decrease, p=0.007), but was ineffective in hPAR4 Thr120 mice, even when combined with aspirin. Importantly, blocking platelet-neutrophil interactions downstream of PAR4 with a murine analog of the FDA-approved P-selectin inhibitor crizanlizumab improved stroke outcomes in hPAR4 Thr120 mice (55% decrease, p=0.004). These results support a causal role for PAR4 Thr120 in IS that may explain part of the racial disparity in stroke. Our findings further suggest that different anti-platelet therapies may be needed for patients expressing PAR4 Thr120.

Short-term exposure of female BALB/cJ mice to e-cigarette aerosol promotes neutrophil recruitment and enhances neutrophil-platelet aggregation in pulmonary microvasculature

ABSTRACT Despite the perception that e-cigarettes are safer than conventional cigarettes, numerous findings demonstrated that e-cigarette aerosol (EC) exposure induced compromised immune functionality, vascular changes even after acute exposure, and lung injury. Notably, altered neutrophil functionality and platelet hemodynamics have been observed post-EC exposure. It was hypothesized that EC exposure initiates an inflammatory response resulting in altered neutrophil behavior and increased neutrophil-platelet interaction in the pulmonary microvasculature. Neutrophil and platelet responses were examined up to 48 hrs following whole-body, short-term EC exposure without flavorants or nicotine in a murine model, which most closely modeled secondhand exposure. This study is the first to investigate the impact of EC exposure through lung intravital imaging. Compared to room air-exposed mice, EC-exposed mice displayed significantly increased 1.7‒1.9-fold number of neutrophils in the pulmonary microvasculature associated with no marked change in neutrophils within whole blood or bronchoalveolar lavage fluid (BALF). Neutrophil-platelet interactions were also significantly elevated 1.9‒2.5-fold in exposed mice. Plasma concentration of myeloperoxidase was markedly reduced 1.5-fold 48 hr following exposure cessation, suggesting suppressed neutrophil antimicrobial activity. Cytokine expression exhibited changes indicating vascular damage. Effects persisted for 48 hr post-EC exposure. Data demonstrated that EC exposure repeated for 3 consecutive days in 2.5 hr intervals in the absence of flavorants or nicotine resulted in modified pulmonary vasculature hemodynamics, altered immune functionality, and a pro-inflammatory state in female BALB/cJ mice.

Platelets mediate acute hepatic microcirculatory injury in a protease-activated-receptor-4-dependent manner after extended liver resection

BackgroundSmall-for-size syndrome (SFSS) is a major complication following extended liver resection. The role of platelets in the early development of SFSS remains to be cleared. We aimed to investigate the impact of platelets and PAR-4, a receptor for platelet activation, on the acute phase microcirculatory injury after liver resection by in vivo microscopy analyzing the changes in leukocyte recruitment, platelet-neutrophil interaction, and microthrombosis-induced perfusion failure. MethodsSixty-percent partial hepatectomy (PH) models using C57BL/6 mice receiving platelet depletion with anti-GPIbα, PAR-4 blockade with tcY-NH2, or vehicle treatment with saline were used. Sham-operated animals served as controls. Epifluorescence microscopic analysis was performed 2 h after PH to quantify the leukocyte recruitment and microcirculatory changes. Sinusoidal neutrophil recruitment, platelet-neutrophil interaction, and microthrombosis were evaluated using two-photon microscopy. ICAM-1 expression and liver liver injury were assessed in tissue/blood samples. ResultsThe increments of leukocyte recruitment in post-sinusoidal venules and sinusoidal perfusion failure, the upregulation of ICAM-1 expression, and the deterioration of liver function 2 h after 60% PH were alleviated in the absence of platelets or by PAR-4 blockade. Intensified platelet-neutrophil interaction and microthrombosis in sinusoids were observed 2 h after 60% PH, which significantly attenuated after PAR-4 blockade. ConclusionPlatelets play a critical role in acute liver injury after extended liver resection within 2 h. The deactivation of platelets via PAR-4 blockade ameliorated liver function deterioration by suppressing early leukocyte recruitment, platelet-neutrophil interaction, and microthrombosis in hepatic sinusoids.

IRGM/Irgm1 deficiency inhibits neutrophil-platelet interactions and thrombosis in experimental atherosclerosis and arterial injury

BackgroundNeutrophil extracellular traps (NETs) closely link inflammation and thrombosis. The immune-related GTPase family M protein (IRGM) and its ortholog of mouse IRGM1 are positively correlated with plaque rupture during atherosclerosis process. However, whether and how IRGM/IRGM1 affects NETs formation and atherosclerotic thrombosis remains unknown, which will further promote the development of antithrombotic treatment tools. MethodsThe thrombi images, platelet activation makers and NETs makers were detected in the serum of STEMI patients and controls. To futher investigate IRGM/IRGM1 affects NETs formation and atherothrombosis in vivo, ApoE-/-Irgm1+/- and ApoE-/- mice received diets rich in fat and 2.5% FeCl3 was then used to induce experimental arterial thrombosis in an atherosclerosis background. In vitro, PMA and thrombin were used to stimulate neutrophils and platelets, respectively, and the expression of IRGM/IRGM1 were modified. To reveal the molecular mechanisms, MAPK-cPLA2 signals inhibitors were used. ResultsSerum IRGM was positively correlated with PF4 and neutrophil elastase. Subsequently, Irgm1 deficient mice have a longer occlusion time and lower growth rate. In vitro, as expected, IRGM/Irgm1 deficiency inhibits platelet activation and platelet-neutrophil interaction. More importantly, IRGM promoted NETs production through activating MAPK-cPLA2 signals in PMA stimulated neuropils, whereas inhibiting the production of NETs eliminated the difference in platelet activation and thrombosis caused by IRGM/Irgm1 modification in vivo and vitro. Similarly, inhibition of platelet activation also eliminated the influence of IRGM/Irgm1 modification on NETs production. ConclusionsOverall, our data indicate that IRGM/Irgm1 deficiency in neuropils inhibits the intense interaction between neutrophils and platelets, and ultimately inhibits thrombosis.

Role of S100A8/A9 in Platelet–Neutrophil Complex Formation during Acute Inflammation

Acute respiratory distress syndrome (ARDS) due to pulmonary infections is associated with high morbidity and mortality. Upon inflammation, the alarmin S100A8/A9 is released and stimulates neutrophil recruitment mainly via binding to Toll-like receptor 4 (TLR4). TLR4 is also expressed on platelets, which modulate the immune response through direct interaction with leukocytes. In a murine model of Klebsiella pneumoniae-induced pulmonary inflammation, global S100A9 deficiency resulted in diminished neutrophil recruitment into the lung alveoli and neutrophil accumulation in the intravascular space, indicating an impaired neutrophil migration. A lack of TLR4 on platelets resulted in reduced neutrophil counts in the whole lung, emphasising the impact of TLR4-mediated platelet activity on neutrophil behaviour. Flow cytometry-based analysis indicated elevated numbers of platelet-neutrophil complexes in the blood of S100A9-/- mice. Intravital microscopy of the murine cremaster muscle confirmed these findings and further indicated a significant increase in neutrophil-platelet complex formation in S100A9-/- mice, which was reversed by administration of the S100A8/A9 tetramer. An in vitro bilayer assay simulated the murine alveolar capillary barrier during inflammation and validated significant differences in transmigration behaviour between wild-type and S100A9-/- neutrophils. This study demonstrates the role of S100A8/A9 during platelet-neutrophil interactions and neutrophil recruitment during pulmonary inflammation.

Hypercoagulability and Inflammatory Markers in a Case of Congenital Thrombotic Thrombocytopenic Purpura Complicated by Fetal Demise

Congenital thrombotic thrombocytopenic purpura (cTTP) is a rare disorder caused by an inherited genetic deficiency of ADAMTS13 and affects less than one per million individuals. Patients who are diagnosed with TTP during pregnancy are at increased risk of maternal and fetal complications including fetal demise. We present a case of a 32-year-old G3P0 (gravida 3, para 0) who presented at 20 weeks gestation with a new diagnosis of congenital TTP (cTTP) and fetal demise. We describe the pathophysiology of pregnancy complications in a patient with cTTP using platelet procoagulant membrane dynamics analysis and quantitative proteomic studies, compared to four pregnant patients with gestational hypertension, four pregnant patients with preeclampsia, and four healthy pregnant controls. The cTTP patient had increased P-selectin, tissue factor expression, annexin-V binding on platelets and neutrophils, and localized thrombin generation, suggestive of hypercoagulability. Among 15 proteins that were upregulated, S100A8 and S100A9 were distinctly overexpressed. There is platelet-neutrophil activation and interaction, platelet hypercoagulability, and proinflammation in our case of cTTP with fetal demise.

The Common Racially Diverse Human PAR4 Variant Thr120 Causes Worse Outcomes in a Mouse Stroke Model Via Platelet-Mediated NET Formation: Prevention By P-Selectin Blockade

Background: Human platelets express two thrombin-activated G-protein coupled receptors, PAR1 and PAR4. Because PAR1 blockade is contraindicated in treating stroke, PAR4 has been considered as a potential anti-platelet target. Human PAR4 has a common, racially divergent Ala120Thr variant; the Thr120 allele frequency is ~0.7 in blacks and ~0.2 in whites. The Thr120 variant induces platelet hyperreactivity, and a previous GWAS study suggested the PAR4 Thr120 allele conferred an increased risk of ischemic stroke. Mouse platelets express Par4 and not Par1. Because human PAR4 (hPAR4) differs substantially from mouse Par4, in vivo studies of hPAR4 and the Ala120Thr variant have not been possible. Aims: The goals of the current study were to (1) express and characterize hPAR4 in vivo, (2) determine if the PAR4 Thr120 variant results in platelet hyperactivity and worse outcomes in a murine stroke model, and (3) to test for a pharmacogenetic interaction between the hPAR4 Ala120Thr variants and FDA-approved anti-thrombotic agents for stroke outcome. Methods: Humanized PAR4 Ala120 (hPAR4Ala) and PAR4 Thr120 (hPAR4Thr) mouse lines were generated using CRISPR/Cas9 to replace mouse F2rl3 with either human variant of F2RL3 under control of mouse regulatory elements. Using these mice and a transient middle cerebral artery occlusion (tMCAO) model of stroke, we assessed stroke volume and neurologic outcomes, as well as in vivo and ex vivo measures of platelet and neutrophil function. Results: hPAR4 mRNA and protein were expressed at similar levels in hPAR4Ala and hPAR4Thr lines. Compared to hPAR4Ala platelets, hPAR4Thr platelets stimulated with the PAR4 activation peptide AYPGKF, showed a higher dose-dependent activation of aIIbb3 and P-selectin expression (p<0.05). To directly assess the effect of the hPAR4 variant on stroke outcomes, hPAR4Ala or hPAR4Thr mice underwent a tMCAO model of stroke. Compared to hPAR4Ala mice, hPAR4Thr mice had larger infarct volume (p<0.05), more circulating platelet-neutrophil aggregates (PNAs) (p<0.05), and more neutrophil extracellular traps (NETs) in the brain (p<0.05). Since platelets activate neutrophils to induce NETs that regulate ischemic stroke, we tested if purified platelets and neutrophils from each mouse line directly altered NET formation. We observed that compared to AYPGKF-activated platelets from hPAR4Ala mice, platelets from hPAR4Thr mice produced more NETs in vitro (p<0.05). Similar results were observed in human platelets expressing either one or two PAR4 Thr120 alleles compared to platelets expressing two PAR4 Ala120 alleles (p<0.05). Both hPAR4Ala and hPAR4Thr mice were significantly protected in the tMCAO model after administration of the specific PAR4 blocker BMS-986120, indicating PAR4 is directly involved in brain injury. We then examined if expression of the PAR4 Thr120 variant altered therapeutic benefit during ischemic stroke using clinically-approved treatments. The P2Y12 blocker ticagrelor indirectly inhibits PAR4 activation and is used in brain ischemic conditions. Single agent ticagrelor administration by gavage reduced ex vivo ADP-induced aggregation in both mouse lines (indicating an effective therapeutic dose) and reduced infarct volume, PNAs, and NET formation in hPAR4Ala mice but not in hPAR4Thr mice (p<0.05). Moreover, dual anti-platelet therapy (combined aspirin and ticagrelor) also failed to protect hPAR4Thr mice from ischemic stroke brain injury. Since more PNAs and NETs were produced after stroke in hPAR4Thr mice (see above), we tested if inhibiting platelet-neutrophil interactions with anti-P-selectin therapy was beneficial. In hPAR4Thr mice, P-selectin blockade reduced PNAs and NET formation and improved stroke outcomes (p<0.05). Conclusion: We have generated humanized PAR4 mouse strains that directly demonstrate the role of human PAR4 in stroke. This new model will allow for novel insights into PAR4 biology in both platelets and other tissues, and provides a system to test other antithrombotic therapies in disease settings where the hyperactive PAR4 Thr120 variant induces worse outcomes. Importantly, these findings suggest blockade of platelet-neutrophil interactions may be an effective stroke treatment, especially for subjects with the hyperactive PAR4 Thr120 variant.

Analysis of the Platelet Proteome Reveals Insights into the Pro-Inflammatory and Pro-Thrombotic State Associated with the Philadelphia Chromosome-Negative Myeloproliferative Neoplasms

Introduction: Hypercoagulability, thrombosis and microvascular dysfunction are hallmarks of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPN). Elevated peripheral blood cell counts appear to contribute to thrombotic risk but the precise underlying mechanisms remain to be fully elucidated. The interplay between haemostatic and pro-inflammatory pathway activity ('thrombo-inflammation') has emerged in recent years as a source of hypercoagulability in MPN. Platelets are recognised as being mediators of thrombo-inflammation in other diseases and may also be effectors of pro-inflammatory/pro-coagulant activity in MPN. We hypothesized that the platelet proteome is altered in MPN and that its characterisation would reveal insights into the pathophysiology of the disease and the associated thrombotic risk. Aim: To determine if differences exist in the pattern of platelet protein expression in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) in contrast to healthy donors. Methods: 62 patients (ET, n=38; PV, n=24) and 9 healthy volunteers were recruited at Papa Giovanni XXIII Hospital, Bergamo, Italy and the Mater Misericordiae University hospital, Dublin, Ireland. Platelets were isolated from platelet rich plasma, washed in Krebs Ringer buffer and resuspended at 1x 109 platelets/mL in phosphate buffered saline. Whole platelets were lysed in RIPA buffer and differential proteomic signatures established using label-free quantification (LFQ) mass spectrometry (MS), where platelet lysate proteins were double digested using the commercially available PreOmics kit and analysed in a Bruker TimsTOF mass spectrometer connected to a EvoSep liquid chromatography system. Identified peptides were searched against a human FASTA using MaxQuant. For statistical analysis, proteins identified in a minimum of 70% of samples in at least one group were included. Differences in protein expression were determined using an unpaired t-test with a false discovery rate of 5% and a minimal fold change of 0.1 within the Perseus software; p- values below 0.05 were considered significant. Results: 2,180 proteins from platelet lysates were quantified across all patient and control samples. In MPN platelet lysates, 49 proteins were found to be differentially expressed in comparison to controls (p< 0.05) (Figure 1), including increased expression of markers of platelet activity such as vesicle-associated membrane protein 7 (VAMP-7; regulator of granule exocytosis and actin cytoskeleton activity) and CD109 (a GPI-linked glycoprotein expressed by activated platelets). Bioinformatical analysis revealed a cohort of mitochondrial, cytoskeletal & ribosomal proteins as well as potential effectors of thrombopoiesis and thrombo-inflammation which were significantly upregulated in the MPN cohort. Strikingly, protein disulfide-isomerase (PDI, a member of the thioredoxin superfamily of redox proteins) was increased in MPN lysates. PDI has been shown to contribute to thrombosis, platelet activation and platelet-neutrophil interactions in vivo; plasma levels of PDI have been shown to be increased in MPN and are associated with thrombosis risk. Conclusion: The platelet proteome is altered in MPN and is suggestive of an activated platelet phenotype with over-expression of proteins with known pro-coagulant activity. To our knowledge the over expression of PDI in MPN platelets has not previously been described. The identification of differentially expressed proteins, such as PDI, provides mechanistic insights into the pathophysiology underlying the substantial burden of arterial and venous thrombosis in MPN and may also assist in the identification of novel therapeutic targets. Additional proteomic analysis of platelet releasate and plasma extracellular vesicles is currently ongoing and may provide additional pathophysiological insights. Figure 1: Volcano plot of MPN versus control platelet lysate proteomes (x-axis, t-test difference between the mean log2 of the LFQ values; y-axis, the negative log transformed p-value) representing the proteins significantly altered in MPN. Black hyperbolic curves show the threshold for statistical significance. 36 proteins were found to be increased in MPN platelet lysates (including CD109, PDIA4 and VAMP-7) in comparison to control lysates (red) while 13 proteins were decreased in MPN platelet lysates (blue). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune Checkpoint Inhibitors (ICI) Promote Neutrophil-Platelet Aggregate and NET Formation in Tumor-Bearing Mice

Introduction: Immune checkpoint inhibitor (ICI) therapy has revolutionized cancer treatment. Despite remarkable clinical benefit, there are numerous immune-related adverse events (irAEs) associated with ICI therapy. Cancer-associated thrombosis (CAT) is a common complication of cancer, and ICI-associated thrombosis (ICI-CAT) is an underappreciated irAE. There is no data available concerning the mechanisms underlying ICI-CAT. To address this issue, we have established a murine model of ICI-CAT in which tumor-bearing mice treated with ICI develop larger thrombi than those treated with control IgG. We have observed that these ICI-treated mice display greater numbers of neutrophils in thrombi, and increased levels of circulating neutrophil extracellular traps (NETs). Methods: ICI (anti-PD-1 and anti-CTLA-4) was infused into Balb/c mice bearing subcutaneous CT26 colorectal tumors. Neutrophils (CD11b+ Ly6G+) as a percentage of total WBC was assessed using an automated hematology analyzer. Neutrophil-platelet doublets and aggregates were determined by flow cytometry analysis in using antibodies for CD41 (GPIIb) and CD62P (P-selectin). Circulating neutrophils were isolated from tumor-bearing mice treated with IgG and ICI 48h after IVC occlusion using an EasySep mouse neutrophil enrichment kit (STEMCELL technologies). For NET staining, isolated neutrophils (5x105 cells/well) were seeded on poly-L lysine coated coverslips, and treated with or without phorbol myristate acetate (PMA, 100 nM) for 4 h in 37°C. Neutrophils were then blocked using 5% FBS and stained, without permeabilization, using rabbit monoclonal anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, Abcam). Extracellular CitH3 was detected using anti-rabbit Alexa Fluor 488 secondary antibody. Results: Thrombi in ICI-treated mice were larger and contained greater numbers of neutrophils than thrombi from IgG-treated mice. Complete blood counts demonstrated that neutrophil numbers increased 3-fold in tumor bearing (38.2%) vs non-tumor bearing mice (12.8%) (Data not shown). However, neutrophils from ICI-treated tumor-bearing mice demonstrated significantly more spontaneous NET formation than those isolated from mice treated with control IgG (66.7% vs 18.2%). Smaller differences were also noted in response to PMA (Fig. A). Since binding of platelets to neutrophils is one cause of NET release, we also determined the frequency of neutrophil-platelet aggregates circulating in tumor-bearing mice treated with ICI or control IgG. Increased levels of circulating neutrophil-platelet aggregate in ICI-treated mice were present in a doublet CD41+Ly6G+ population (44.8% vs 22.3%); similar results were noted when platelets were stained with the activation marker CD62 (P-selectin) (8.5% vs 1%) (Fig. B, Left). The frequency of larger neutrophil-platelet aggregates followed a similar trend (ICI vs IgG; 75.8% vs 30% for CD41; 48.1% vs 7.7% for CD62P) (Fig. B, Right). Conclusions: Circulating neutrophils from ICI-treated, tumor bearing mice demonstrate increased spontaneous NET release. Moreover, elevated levels of circulating platelet-neutrophil doublets and aggregates are present in ICI-treated mice compared to those treated with control IgG. These results suggest a potential mechanism of ICI-induced thrombosis, and that platelet-neutrophil interactions may stimulate NET release in ICI-treated mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Platelet-Neutrophil Interaction and Thromboinflammation in Diabetes: Considerations for Novel Therapeutic Approaches.

Thromboinflammation has become a topic of key interest in cardiovascular disease and the prevention of diabetes complications because of the interplay between thrombosis and inflammation in diabetes. Specifically, the significant risk of vascular thrombotic disease in diabetes highlights the need for new and better therapeutic targets to help manage and prevent vascular thrombo-occlusive disease in this condition. Similarly, the prominent role of inflammation in diabetes has sparked interest in anti-inflammatory agents to better prevent and control vascular disease. Investigations on the effects of anticoagulation and antiplatelet interventions in patients with diabetes and cardiovascular disease show a potential role for these agents in decreasing morbidity and mortality. Neutrophils and platelets are key players in inflammation and wound-healing response, respectively. The interaction between neutrophils and platelets is thought to be an important driver of thromboinflammation. Therefore, this review describes the mechanisms involved in platelet-neutrophil interactions that contribute to the development or exacerbation of thromboinflammation in the context of diabetes and its associated comorbidities. The effects observed by the antithrombotic/antidiabetic treatments and physical activity/dietary interventions on attenuating thromboinflammation are discussed. These data suggest that mechanisms involved in platelet-neutrophil interaction, platelet activation/aggregation, and the recruitment of neutrophils have a promising potential to become therapeutic targets to decrease thromboinflammation in patients with diabetes.

PLATELET- NEUTROPHIL COMPLEXES – DEFINITION, MECHANISMS AND IMPLICATIONS (REVIEW)

Neutrophils are innate immune cells that protect the host from infection by eliminating the invadingpathogens. In recent years, neutrophils and platelets have been shown to be active not only in inflammatoryprocesses but also in some pathological conditions, including the development of neoplastic diseases andthrombosis. The interaction between circulating platelets and neutrophils is characterised by the intensive exchangeof intracellular signals. Overexpression of some of these molecules has been reported, with increased leukocyte andplatelet activation as well as increased adhesion of blood cells to the vascular endothelium. Platelet-neutrophilinteractions are increased in thrombo-inflammatory diseases and are sensitive biomarkers for platelet activation andtargets for the development of new therapies. The crosstalk between platelets and innate immune cells promotesthrombosis, inflammation, and tissue damage.A key step in the formation of platelet-neutrophil complexes (PNCs) is the process of interaction between theadhesion molecule P-selectin (CD62P) on the platelet membrane and the PSGL-1 ligand on neutrophils. Theformation of PNCs modulates a wide range of pathological conditions, including both innate and acquired immunesystems. PNCs are involved in the pathogenesis of inflammatory diseases, malignancies and thrombotic processes.The formation of PNCs is associated with the initiation of a number of processes at the cellular and molecular levels.Malignant diseases are risk factors for the occurrence of arterial, venous and microthrombosis, which are most oftenassociated with hypercoagulable processes that form platelet aggregates. By modulating the immune system throughdirect and indirect interactions with leukocytes, platelets regulate several aspects of tumor-related pathology.Thisreview aims at summarizing the role of platelet-neutrophil interactions in the development and progression of cancerand puts its focus on cancer-related alterations of platelet and leukocyte functions and their impact on cancerpathology.The analysis and evaluation of the levels of the circulating platelet- neutrophil complexes in blood in neoplasticdiseases can be used as potential predictors of tumor progression, metastasis and the occurrence of thrombosis.

Stachydrine exhibits a novel antiplatelet property and ameliorates platelet-mediated thrombo-inflammation.

Platelets are versatile anucleate cells involved in thrombosis as well as inflammation. Stachydrine (STA), a major bioactive compound extracted from Motherwort, has multiple pharmacological properties. Nevertheless, the significance of STA in platelet regulation and whether STA could ameliorate platelet-mediated thrombo-inflammation still remain elusive. Human platelets were used to assess the regulatory effects of STA on platelet activation and interactions with neutrophils in vitro. FeCl3 injury-induced carotid/mesenteric thrombosis and collagen/epinephrine-induced pulmonary thromboembolism model were used to explore whether STA could regulate thrombosis in vivo. Furthermore, a cecal ligation and puncture-induced sepsis model was employed to investigate the role of STA in thrombo-inflammatory diseases. STA markedly suppressed platelet activation represented by aggregation, secretion, αIIbβ3-mediated signaling events and calcium mobilization, etc. by inhibiting agonists-induced activation signaling and potentiating cGMP-dependent inhibitory signaling. Mice receiving STA-treated platelets were less susceptible to thrombosis in vivo. In addition, decreased platelet-neutrophil interactions including platelet-neutrophil aggregates and neutrophil extracellular traps, and alleviative sepsis-induced multiorgan damage were observed due to STA-mediated platelet inhibition. This study suggested the potential therapeutic role of STA in thrombotic and thrombo-inflammatory disorders.

Live imaging of platelets and neutrophils during antibody-mediated neurovascular thrombosis

AbstractImmune complexes form in systemic disorders such as rheumatological, autoimmune, and allergic diseases or in response to infections or medications. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adenoviral vector vaccines have been associated with rare yet serious thrombotic complications in the brain due to the formation of immune complexes that activate platelets. There are currently no data visualizing the interplay of platelets with leukocytes and the brain vasculature endothelium in response to immune complexes. This is in part due to the absence of FcγRIIA in mice, a receptor for immune complexes implicated in these thrombotic incidents. Here, we describe and illustrate events at the cellular level that take place in the brain vasculature in response to systemic administration of surrogate immune complexes. We used Ly6gCre+/−::Rosa26-TdT+/−::CD41-YFP+/− mice expressing the FcγRIIA transgene and fluorescence in neutrophils and platelets. Using real-time videomicroscopy to capture high-velocity events in conjunction with unbiased computer-assisted analyses, we provide images and quantifications of the cellular responses downstream of FcγRIIA stimulation. We observed transient and stable platelet–neutrophil interactions, platelets forming thrombi, and neutrophil adhesion to blood vessel walls. This imaging approach in a quadruple transgenic animal model can be used for the study of the pathogenic roles of immune complexes in disease.

Thrombosis and Inflammation-A Dynamic Interplay and the Role of Glycosaminoglycans and Activated Protein C.

Hemostasis, thrombosis, and inflammation are tightly interconnected processes which may give rise to thrombo-inflammation, involved in infectious and non-infectious acute and chronic diseases, including cardiovascular diseases (CVD). Traditionally, due to its hemostatic role, blood coagulation is isolated from the inflammation, and its critical contribution in the progressing CVD is underrated, until the full occlusion of a critical vessel occurs. Underlying vascular injury exposes extracellular matrix to deposit platelets and inflammatory cells. Platelets being key effector cells, bridge all the three key processes (hemostasis, thrombosis, and inflammation) associated with thrombo-inflammation. Under physiological conditions, platelets remain in an inert state despite the proximity to the endothelium and other cells which are decorated with glycosaminoglycan (GAG)-rich glycocalyx (GAGs). A pathological insult to the endothelium results in an imbalanced blood coagulation system hallmarked by increased thrombin generation due to losses of anticoagulant and cytoprotective mechanisms, i.e., the endothelial GAGs enhancing antithrombin, tissue factor pathway-inhibitor (TFPI) and thrombomodulin-protein C system. Moreover, the loss of GAGs promotes the release of mediators, such as von Willebrand factor (VWF), platelet factor 4 (PF4), and P-selectin, both locally on vascular surfaces and to circulation, further enhancing the adhesion of platelets to the affected sites. Platelet-neutrophil interaction and formation of neutrophil extracellular traps foster thrombo-inflammatory mechanisms exacerbating the cardiovascular disease course. Therefore, therapies which not only target the clotting mechanisms but simultaneously or independently convey potent cytoprotective effects hemming the inflammatory mechanisms are expected to provide clinical benefits. In this regard, we review the cytoprotective protease activated protein C (aPC) and its strong anti-inflammatory effects thereby preventing the ensuing thrombotic complications in CVD. Furthermore, restoring GAG-like vasculo-protection, such as providing heparin-proteoglycan mimetics to improve regulation of platelet and coagulation activity and to suppress of endothelial perturbance and leukocyte-derived pro-inflammatory cytokines, may provide a path to alleviate thrombo-inflammatory disorders in the future. The vascular tissue-modeled heparin proteoglycan mimic, antiplatelet and anticoagulant compound (APAC), dual antiplatelet and anticoagulant, is an injury-targeting and locally acting arterial antithrombotic which downplays collagen- and thrombin-induced and complement-induced activation and protects from organ injury.

Potential Role of Neutrophil-Platelet Interaction in Increased Susceptibility to Infection of Patients with Down Syndrome.

Recurrent infection in Down syndrome (DS) has been previously documented; the potential role of platelets and neutrophil-platelet interaction has not been addressed in previous studies. Using flow cytometry, we evaluated CD40 and CD18 expression as activation markers for neutrophils and CD62p as an activation marker for platelets, before and after lipopolysaccharide (LPS) stimulation, in 34 patients with DS and 39 control patients. Markers were evaluated as percentage of positivity, mean fluorescent intensity (MFI), and activation index (MFI after stimulation/MFI before stimulation). Patients showed a significantly lower CD40 MFI (P = .019) after LPS stimulation, a lower CD62p percentage before and after LPS stimulation (P = .013 and P = .029), and a higher CD62p MFI (P = .011) after LPS stimulation. Patients showed a lower activation index for CD40 and CD18 (P ≤ .001) but not for CD62p (P = .338). Dysfunctional efficiency in neutrophils and in the neutrophil-platelet interaction could not be correlated to infection. A consensus on a scoring system for infection is needed for an objective evaluation of correlation to infection.