Platelet Functional Responses Research Articles

Pim Kinase Inhibition Disrupts CXCR4 Signalling in Megakaryocytes and Platelets by Reducing Receptor Availability at the Surface.

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.

Difluorinated thromboxane A2 reveals crosstalk between platelet activatory and inhibitory pathways by targeting both the TP and IP receptors.

Thromboxane A2 (TXA2) is a prostanoid produced during platelet activaton, important in enhancing platelet reactivity by activation of TP receptors. However, due to the short half-life, studying TXA2 signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA2 analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function. Platelet functional and signalling responses were studied using aggregometry, Ca2+ mobilisation experiments and immunoblotting and compared with an analogue of the TXA2 precursor prostaglandin H2, U46619. Gαq/Gαs receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system. BRET studies revealed that F-TXA2 and F2-TXA2 promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F2-TXA2 caused reversible aggregation in platelets, whereas F-TXA2 and U46619 induced sustained aggregation. Blocking the IP receptor switched F2-TXA2-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F2-TXA2-mediated IP receptor activation. F2-TXA2 rapidly and potently stimulated platelet TP receptor-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed. F-TXA2 is a close analogue to TXA2 used as a selective tool for TP receptor platelet activation. In contrast, F2-TXA2 acts on both TP and IP receptors differently over time, resulting in an initial wave of TP receptor-mediated platelet aggregation followed by IP receptor-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation.

Sex Dependent Occlusive Cardiovascular Disease Effects of Short-Term Thirdhand Smoke Exposure.

Thirdhand smoke (THS) is associated with many public health and disease concerns, such as respiratory illness, cancer, lipidemia, and cardiovascular disease (CVD). We have previously shown a moderate to long-term exposure to THS increased risk of thrombosis. However, whether short-term exposure to THS would produce any effects in causing disease remains to be discovered. Therefore, this study investigated the impact of one-month THS exposure on platelet function and cytokine response in sex-dependent. Secondhand smoke or clean air (CA) exposed upholstery materials for 1 week were kept in cages housed with 5-6 mice, and the procedure was repeated for 4 weeks. These THS-exposed mice were evaluated for thrombogenesis and platelet function assays. In addition, the cytokines expressions were evaluated from pooled serum (n=5). Compared to the CA group, the THS exposure significantly shortened tail bleeding time and carotid artery thrombus formation. Moreover, the female mice appeared more sensitive to THS exposure than males. Furthermore, platelet aggregation, dense granule secretion, and P-selectin activation markers were significantly elevated due to THS exposure. In addition, the high throughput screening showed at least 30 cytokines differentially modulated by THS in females relative to 26 in male mice. Collectively, these results demonstrate that one month of THS exposure represents a high health risk, in part, by triggering a prothrombotic phenotype that appears to be more significant in females, who are at a much higher risk for occlusive CVD. Additionally, changes in cytokine levels mediate some of the THS-induced occlusive effects. This study revealed that THS exposure in one month is detrimental to the cardiovascular health of both sexes; however, females could be more aggressively affected than males. In addition, interleukins and chemokines could be critical factors for initiating prothrombotic activity due to THS exposure.

Comparison of platelet proteomic profiles between children and adults reveals origins of functional differences.

Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors. Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data. We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content. Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content. Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed.

Role of heat shock protein 47 in platelet glycoprotein VI dimerization and signaling

BackgroundHeat shock protein 47 (HSP47) is an intracellular chaperone protein with an indispensable role in collagen biosynthesis in collagen-secreting cells. This chaperone has also been shown to be released and present on the surface of platelets. The inhibition of HSP47 in human platelets or its ablation in mouse platelets reduces platelet function in response to collagen and the glycoprotein (GP) VI collagen receptor agonist CRP-XL. ObjectivesIn this study, we sought, through experiments, to explore cellular distribution, trafficking, and influence on GPVI interactions to understand how HSP47 modulates collagen receptor signaling. MethodsHSP47-deficient mouse platelets and SMIH- treated human platelets were used to study the role of HSP47 in collagen mediated responses and signaling. ResultsUsing subcellular fractionation analysis and immunofluorescence microscopy, HSP47 was found to be localized to the platelet-dense tubular system. Following platelet stimulation, HSP47 mobilization to the cell surface was shown to be dependent on actin polymerization, a feature common to other dense tubular system resident platelet proteins that are released to the cell surface during activation. In this location, HSP47 was found to contribute to platelet adhesion to collagen or CRP-XL but not to GFOGER peptide (an integrin α2β1-binding sequence within collagens), indicating selective effects of HSP47 on GPVI function. Dimerization of GPVI on the platelet surface increases its affinity for collagen. GPVI dimerization was reduced following HSP47 inhibition, as was collagen and CRP-XL-mediated signaling. ConclusionThe present study identifies a role for cell surface-localized HSP47 in modulating platelet responses to collagen through dimerization of GPVI, thereby enhancing platelet signaling and activation.

Association of Antiretroviral Therapy with Platelet Function and Systemic Inflammatory Response in People Living with HIV: A Cross-Sectional Study.

People living with HIV (PLWHIV) present an increased risk of adverse cardiovascular events. We aimed to assess whether antiretroviral therapy (ART) pharmacologically enhances platelet reactivity and platelet activation intensity, and explore the potential association with underlying inflammatory status. This was a cross-sectional cohort study carried out among PLWHIV on diverse ART regimens. Platelet reactivity and activation intensity were assessed using the bedside point-of-care VerifyNow assay, in P2Y12 reaction units (PRU), measurements of monocyte-platelet complexes, and P-selectin and GPIIb/IIIa expression increase, following activation with ADP, respectively. Levels of major inflammatory markers and whole blood parameters were also evaluated. In total, 71 PLWHIV, 59 on ART and 22 healthy controls, were included in this study. PRU values were significantly elevated in PLWHIV compared to controls [Mean; 257.85 vs. 196.67, p < 0.0001], but no significant differences were noted between ART-naïve or ART-experienced PLWHIV, or between TAF/TDF and ABC based regimens, similar to systemic inflammatory response. However, within-group analysis showed that PRUs were significantly higher in ABC/PI vs ABC/INSTI or TAF/TDF + PI patients, in line with levels of IL-2. PRU values did not correlate strongly with CD4 counts, viral load, or cytokine values. P-selectin and GPIIb/IIIa expression increased following ADP activation and were significantly more prominent in PLWHIV (p < 0.005). Platelet reactivity and platelet activation intensity were shown to be increased in PLWHIV, but they did not appear to be related to ART initiation, similar to the underlying systemic inflammatory response.

Alterations in platelet proteome signature and impaired platelet integrin αIIbβ3 activation in patients with COVID-19

BackgroundPatients with COVID-19 are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. ObjectivesTo characterize the mechanism of altered platelet function in COVID-19 patients. MethodsThe platelet proteome, platelet functional responses, and platelet-neutrophil aggregates were compared between patients hospitalized with COVID-19 and healthy control subjects using tandem mass tag proteomic analysis, Western blotting, and flow cytometry. ResultsCOVID-19 patients showed a different profile of platelet protein expression (858 altered of the 5773 quantified). Levels of COVID-19 plasma markers were enhanced in the platelets of COVID-19 patients. Gene ontology pathway analysis demonstrated that the levels of granule secretory proteins were raised, whereas those of platelet activation proteins, such as the thrombopoietin receptor and protein kinase Cα, were lowered. Basally, platelets of COVID-19 patients showed enhanced phosphatidylserine exposure, with unaltered integrin αIIbβ3 activation and P-selectin expression. Agonist–stimulated integrin αIIbβ3 activation and phosphatidylserine exposure, but not P-selectin expression, were decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This association was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. ConclusionsOverall, our data suggest the presence of 2 platelet populations in patients with COVID-19: one of circulating platelets with an altered proteome and reduced functional responses and another of P-selectin-expressing neutrophil–associated platelets. Platelet–driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.

Characterisation of Platelet Sensitivity in Severe COVID-19

Background: Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterised by a hyperinflammatory and prothrombotic state. In the most severe cases, patients can develop lung complications such as pneumonitis and acute respiratory distress syndrome, frequently complicated by venous and arterial thrombosis. Recent studies suggest that platelets play a role as immune mediators, and may therefore contribute to the development of hyperinflammation and thrombosis in severe COVID-19 patients. The expression and signalling of Glycoprotein VI on platelets are dependent on its association and cross-linking with the dimeric Fc receptor γ-chain, which, upon platelet activation, leads to a signalling cascade involving the Spleen Tyrosine Kinase (SYK). Fostamatinib, a potent SYK inhibitor, has been approved for treatment of Immune Thrombocytopenia (ITP), and has been implicated in abrogating thrombosis by mitigating SYK signalling in platelets. We have recently shown that SYK inhibition can abrogate the thrombosis caused by cross-linking of COVID-19 antibodies. In this study, we assessed platelet reactivity using the Platelet Phenomic Analysis (PPAnalysis), which identifies platelet sensitivity and functional response as independent measures of platelet reactivity by flow cytometry. We hypothesised that platelets in patients with severe COVID-19 are hypersensitive to platelet agonists, in particular to the collagen-related peptide agonist, which involves the SYK pathway of platelet activation. Methods: Patients with COVID-19 pneumonia, recruited to the Multi-Arm Trial of Inflammatory Signal inhibitors for COVID-19 (MATIS) clinical trial - an interventional study assessing the impact of Fostamatinib (FOS) or Ruxolitinib (RUX) against Standard of Care (SOC) to prevent escalation of COVID-19 from moderate (WHO grades 3 and 4) to severe (WHO grade 5) - were included in this study, REC reference 20/HRA/2618. Samples were collected in citrated vacutainers, taken at day 0, before starting study medication or SOC, and at days 7, 14 and 28, where possible. Samples were processed immediately to obtain platelet-rich plasma (PRP). PRP was labelled with fluorescein isothiocyanate (FITC)-conjugated anti-fibrinogen and PE-Cy5-conjugated P-Selectin antibodies, and stimulated with increasing concentrations of the platelet agonists, adenosine diphosphate (ADP; 0.03-30 µM), collagen-related peptide (CRP; 0.003-3 µg/mL), and thrombin receptor activator peptide 6 (TRAP-6; 0.05-15 µM). Changes in platelet sensitivity, measured as LogEC50, were analysed using R. Results: 40 of 182 patients recruited into MATIS were included. A total of 50 COVID-19 samples (24 at day 0, 14 at day 7, 2 at day 14, and 10 at day 28) and 59 healthy control samples were processed using PPAnalysis. The 24 samples taken at day 0 showed extensive variability in platelet sensitivities between the responses to 3 agonists, with outliers both above and below the 95% centile of controls: 0-4% (P-Selectin response) and 13-42% (fibrinogen response) of patients displayed higher platelet sensitivity (below the 95% centile for controls), and 4-42% (P-Selectin response) and 8-16% (fibrinogen response) had lower sensitivity (above the 95% centile for controls), (Figure 1). Of the 9 patients with samples also taken at day 7, no patients showed an increase in platelet sensitivity from the P-Selectin response to all agonists. 5 patients showed reduced sensitivity. 2 patients in this cohort required non-invasive ventilation (NIV). Conversely, fibrinogen response to all agonists showed increased sensitivity at day 7 in 6 patients, with 1 patient requiring NIV, and reduced sensitivity was observed in 2 patients, both requiring NIV (Figure 2). Conclusions: Patients with severe COVID-19 display a large variability in platelet function, with either higher or lower platelet sensitivity to the agonists at day 0 when compared to controls. This implicates a role for aberrant platelet reactivity in COVID-19 and its associated prothrombotic state, and could contribute to the morbidity and mortality of patients. Interpretation of day 7 results at present is limited due to small numbers. However, it does show utility of the PPAnalysis, as a useful tool in the stratification of different patient cohorts, and in studying the associations between platelet function and disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Plasma Exchange for Immune-Related Adverse Events Due to Immune Checkpoint Inhibitor Therapy: Implications for Clinical Care and Clinical Trial Design

Therapeutic plasma exchange (TPE) is increasingly utilized for Grade 3-4 immunotherapy adverse events (irAEs) to accelerate elimination of drug, autoantibodies, and cytokines. Immune checkpoint inhibitors (ICI) are IgG1 or IgG4 constructs with long half-lives (t1/2) and volume of distributions (Vd) favorable for clearance by apheresis. As drug levels are not readily available, the number of sessions needed to eliminate ICIs is estimated based on the interval from last drug dose, drug Vd, and drug t1/2. Increased use of TPE is anticipated as ICIs are now given despite preexisting autoimmunity, prior organ transplantation, and/or prior irAEs. TPE protocols are well established for the management of diseases that can also present as irAEs [e.g. thrombotic thrombocytopenic purpura (TTP), chronic inflammatory demyelinating polyneuropathy (CIDP), myasthenia gravis (MG), hemophagocytic lymphohistiocytosis (HLH)] and for drug removal (e.g. natalizumab). While TPE is included in irAE management guidelines as second line therapy or later (Brahmer JR, et al, J Immunother Cancer, 2021 PMID: 34172516), no randomized controlled trial (RCT) data are available. TPE can reduce the level of clinical biomarkers for irAE monitoring: creatinine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT),fibrinogen, platelet count, ferritin. We treated 4 adults (median age 66 years, range 65-77 years) with TPE for steroid-refractory irAEs [myositis (2), myocarditis (1)] and, as part of initial therapy, in combination with dexamethasone and anakinra, for ICI-associated HLH. ICI constructs were IgG1 anti-PD-L1 (envafolimab, durvalumab) and IgG4 anti-PD-1 (nivolumab, cemiplimab). Data collection was IRB approved and with patient written consent. ICI indications were sarcoma, thymic carcinoma, cutaneous squamous cell carcinoma (SCC), and lung SCC. Only one patient (HLH irAE) had preexisting autoimmunity (thyroiditis). Median time from last ICI dose to first irAE symptoms or signs was 16 days (range 5-29 days). Three of 4 patients developed irAE after the first ICI dose. One patient (myocarditis) required full systemic anticoagulation (heparin, then enoxaparin) for atrial fibrillation and deep venous thrombosis, and dual antiplatelet therapy (DAPT) for a new coronary stent. The two myositis patients received prophylactic enoxaparin. All TPE sessions were one plasma volume exchange through central venous access, utilized Anticoagulant Citrate Dextrose Solution A (ACDA), and scheduled daily or every other day. Number of sessions ranged from 2-4. Except for the patient with HLH, TPE was initiated with 5% albumin replacement. Fibrinogen levels and estimated bleeding risk guided the need for fresh frozen plasma (FFP) replacement. Session intervals, exchange fluid, transfusion, biomarkers, changes in fibrinogen and platelet count, anticoagulation, and functional response are detailed in Table 1. Three of 4 patients, who had normal baseline fibrinogen and platelet counts, experienced clinically significant hypofibrinogenemia resulting in discontinuation of chemical DVT prophylaxis and/or cryoprecipitate transfusion, but did not have clinically significant thrombocytopenia. The fourth patient (HLH) required ongoing transfusion support due to pre-TPE hypofibrinogenemia and thrombocytopenia. TPE confounded interpretation of biomarkers in all. All irAEs demonstrated prompt clinical improvement, however, due to rapid corticosteroid tapers, 2 patients (myositis, HLH) relapsed within 1 month from last TPE. Both responded to additional immunosuppression. One patient (myositis) did not relapse. Grade 4 bleeding occurred in the patient receiving enoxaparin and DAPT and led to discontinuation of TPE after 2 sessions. Our experience supports the following when considering TPE for irAEs: 1) exclusion of patients with high bleeding risk, or use of 100% FFP replacement and every other day TPE; 2) meticulous non-biomarker irAE assessment during and after TPE; 3) RCT design includes irAE relapse timing and rates and long term follow-up to assess cumulative immunosuppression and tumor outcome; 4) serial drug levels and correlative studies (e.g. cytokines, autoantibodies) to better understand TPE mechanisms, persistence of irAEs, and long term ICI anti-tumor benefit. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterisation of Platelet Sensitivity in Immune Thrombocytopenia Identifies Distinct Subgroups with Either Higher or Lower Platelet Sensitivity

Background: Immune Thrombocytopenia (ITP) is an autoimmune bleeding disorder characterised by a low platelet count (≤100 × 109/L). Anti-platelet autoantibodies, present in approximately 60% of ITP patients, are known to target platelet glycoproteins (GP) Ib-IX and IIb/IIIa (integrin αIIbβ3) on the platelet surface, contributing to platelet clearance and impaired platelet production. Current therapeutic strategies, such as corticosteroids, intravenous immunoglobulins (IVIg), rituximab, and thrombopoietin receptor agonists (TPO-RAs) aim to increase platelet number. More recently, inhibitors of the Spleen Tyrosine Kinase (SYK) and Bruton's Tyrosine Kinase (BTK) are either recently licensed (Fostamatinib) or in clinical trial (Rilzabrutinib) for the treatment of ITP. However, diagnosis and treatment remain a challenge, as it can be difficult to predict treatment response and disease prognosis. One feature complicating the management of ITP is the wide heterogeneity of the disease. This extends also to haemostatic risks. While many patients have minimal symptoms, some patients have increased bleeding tendencies. Paradoxically, others suffer from thrombosis, even at low platelet counts. Hence, there is a need to further characterise patient variability within ITP and elucidate personalised therapeutic strategies for patients at varying stages of disease. The Platelet Phenomic Analysis (PPAnalysis) identifies platelet sensitivity and functional response as independent measures of platelet reactivity by flow cytometry. The assay measures fibrinogen binding as a marker of αIIbβ3 activation and the exposure of P-selectin as a marker of α-granule secretion. We hypothesised that this assay could be used to categorise patients into different subgroups: those who are more at risk of bleeding or more at risk of thrombosis. Methods: Patients and controls were recruited from the ITP Centre at Imperial College Healthcare NHS Trust and consented to the Multi Centre Research Ethics Committee in Wales MREC Wales reference 07/MRE09/54. Venous blood samples collected in sodium citrate vacutainers were processed immediately to obtain platelet-rich plasma (PRP). PRP was labelled with fluorescein isothiocyanate (FITC)-conjugated anti-fibrinogen and PE-Cy5-conjugated P-Selectin antibodies, and stimulated with increasing concentrations of the platelet agonists, adenosine diphosphate (ADP; 0.03-30 µM), collagen-related peptide (CRP; 0.003-3 µg/mL), and thrombin receptor activator peptide 6 (TRAP-6; 0.05-15 µM). Changes in platelet sensitivity, measured as LogEC50, were analysed using R. Results: 126 patients (providing 262 samples at multiple time points) and 59 healthy control samples were included in the study: 5 patients had refractory disease, 7 were in remission, and 12 had vaccine-associated ITP. Patients with ITP had more variable platelet sensitivities than controls, with outliers both above and below the 95% centile of controls: 11-19% of ITP patients exhibited higher platelet sensitivity (below the 95% centile for controls) and 2-10% had lower sensitivity (above the 95% centile for controls), (Figure 1). The variability in platelet sensitivity did not correlate with platelet count (Figure 2). We also found no correlations between hypo- or hypersensitive platelets in ITP post COVID-19 vaccine. Patients with either higher or lower sensitivities were more likely to vary over time, suggesting a more volatile group of patients. Conclusions: Here we show that cohorts of patients with ITP display either higher platelet sensitivity or lower platelet sensitivity compared to controls, irrespective of their platelet count. This signifies a role for aberrant platelet reactivity in the pathogenesis of ITP and may contribute to the heterogeneity of the disease. We also highlight the usefulness of PPAnalysis in distinguishing different subgroups of patients, demonstrating an assessment of platelet function, regardless of platelet count, that could ultimately allow for more personalised treatment for patients. Larger cohorts of patients and assessment of responses to treatment could help to further define this disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Antibody Targeting the Second Extracellular Loop of the Serotonin 5-HT2A Receptor Inhibits Platelet Function

Serotonin (5-hydroxytriptamine or 5-HT) is known to be a weak platelet agonist, and is involved in thrombus formation. While 5-HT cannot induce platelet aggregation on its own, when secreted from the alpha granules, it binds to its G-protein Coupled Receptor (GPCR; i.e., 5HT2AR), thereby acting to amplify platelet functional responses (e.g., aggregation). Thus, 5HT2AR-mediated responses are more involved in the secondary amplification of platelet aggregation in the growing thrombus. Therefore, even though 5-HT can be seen as a weak inducer of platelet activation, it is an important amplifier of aggregation triggered by agonists such as ADP, collagen, and epinephrine, thereby enhancing thrombogenesis. The 5HT2AR/5HT2A signaling pathway is of clinical interest to the scientific and medical communities as it has been implicated in the genesis of several forms of cardiovascular disorders. However, efforts to develop antagonists for 5HT2AR as therapeutic agents in cardiovascular diseases have thus far failed due to these reagents having deleterious side-effects, and/or to lack of selectivity, amongst other reasons. In light of research efforts that identified that the 5HT2AR ligand binding domain resides in the second extracellular loop (EL2; amino acids P209-N233), we developed an antibody, i.e., referred to as 5HT2ARAb, against the EL2 region, and characterized its pharmacological activity in the context of platelets. Thus, we utilized platelets from healthy human donors, as well as C57BL/6J mice (10–12 weeks old) to analyze the inhibitory effects of the 5HT2ARAb on platelet activation in vitro, ex vivo, and on thrombogenesis in vivo as well as on 5HT2AR ligand binding. Our results indicate that the 5HT2ARAb inhibits 5-HT-enhanced platelet activation in vitro and ex vivo, but has no apparent effects on that which is agonist-induced. The 5HT2ARAb was also found to prolong the thrombus occlusion time, and it did so without modulating the tail bleeding time, in mice unlike the P2Y12 antagonist clopidogrel and the 5HT2AR antagonist ketanserin. Moreover, it was found that the 5HT2ARAb does so by directly antagonizing the platelet 5HT2AR. Our findings document that the custom-made 5HT2ARAb exhibits platelet function blocking activity and protects against thrombogenesis without impairing normal hemostasis.

Platelets and tyrosine kinase inhibitors: clinical features, mechanisms of action, and effects on physiology.

Tyrosine kinase inhibitors (TKIs) have emerged as a promising class of target-directed, small molecule inhibitors used to treat hematologic malignancies, inflammatory diseases, and autoimmune disorders. Recently, TKIs have also gained interest as potential antiplatelet-directed therapeutics that could be leveraged to reduce pathologic thrombus formation and atherothrombotic complications, while minimally affecting platelet hemostatic function. This review provides a mechanistic overview and summarizes the known effects of tyrosine kinase inhibitors on platelet signaling and function, detailing prominent platelet signaling pathways downstream of the glycoprotein VI (GPVI) receptor, integrin αIIbβ3, and G protein-coupled receptors (GPCRs). This review focuses on mechanistic as well as clinically relevant and emerging TKIs targeting major families of tyrosine kinases including but not limited to Bruton's tyrosine kinase (BTK), spleen tyrosine kinase (Syk), Src family kinases (SFKs), Janus kinases (JAK), and signal transducers and activators of transcription (STAT) and evaluates their effects on platelet aggregation and adhesion, granule secretion, receptor expression and activation, and protein phosphorylation events. In summation, this review highlights current advances and knowledge on the effects of select TKIs on platelet biology and furthers insight on signaling pathways that may represent novel druggable targets coupled to specific platelet functional responses.

Analysis of change in esophageal varices and clinical characteristics in hepatitis B virus-related cirrhosis after antiviral therapy

Objective: To clarify the effect and related factors of antiviral therapy on the change of esophageal varices in patients with hepatitis B virus-related cirrhosis. Methods: Fifty-two cases with hepatitis B virus-related cirrhosis who underwent endoscopy before and after antiviral therapy were selected from prospective cohorts. Patients were divided into three groups: no, mild, and moderate-severe based on the degree of esophageal varices. The changes in the severity of esophageal varices in each group were compared after antiviral therapy. Clinical characteristics (platelet, liver and kidney function, liver stiffness, and virological response) of patients with different regressions were analyzed. Measurement data were analyzed by independent sample t-test, one-way ANOVA, Mann-Whitney U test and Kruskal-Wallis H test, and Chi-Square test was used for count data. Results: All patients received entecavir-based antiviral therapy. The median treatment time was 3.1 (2.5-4.4) years. The proportion of patients without esophageal varices increased from 30.8% to 51.9%, the proportion of mild esophageal varices decreased from 40.4% to 30.8%, and the proportion of patients with moderate-to-severe esophageal varices decreased from 28.8% to 17.3% (χ2=14.067, P=0.001). A total of 40.4% of patients had esophageal varices regression, and 13.5% had esophageal varices progression. The progression rate was significantly higher in patients with moderate-severe esophageal varices than patients with mild and no esophageal varices (χ2=28.126, P<0.001), and 60.0% of patients with moderate-severe esophageal varices still remained in moderate-severe state after antiviral treatment. Baseline platelet count and 5-year mean change rates were significantly lower in patients with progressive moderate-to-severe esophageal varices than in those without progression (+3.3% vs. +34.1%, Z=7.00, P=0.027). Conclusion: After effective antiviral treatment, 40.4% of patients with hepatitis B virus-related cirrhosis combined with esophageal varices has obtained esophageal varices regression, but those with moderate to severe esophageal varices still have a considerable risk of progression while receiving mono antiviral treatment only. Thrombocytopenia and without significant improving are the clinical signs of progression risk after receiving antiviral treatment.

Human and mouse PAR4 are functionally distinct receptors: Studies in novel humanized mice

BackgroundHuman and mouse platelets both express protease‐activated receptor (PAR) 4 but sequence alignment reveals differences in several functional domains. These differences may result in functional disparities between the receptors which make it difficult to translate PAR4 studies using mice to human platelet physiology. ObjectivesTo generate transgenic mice that express human, but not mouse, PAR4 and directly compare human and mouse PAR4 function in the same platelet environment. MethodsTransgenic mice were made using a genomic clone of the F2RL3 gene (encoding PAR4) and backcrossed with Par4 KO mice. For certain experiments, mice were bred with GRK6 KO mice. Tail bleeding time and platelet function in response to PAR4‐activating peptide were assessed. ResultsHuman F2RL3 was successfully integrated into the mouse genome, transgenic mice were crossed to the mPar4 KO background (PAR4 tg/KO), and PAR4 was functionally expressed on platelets. Compared to WT, PAR4 tg/KO mice exhibited shortened tail bleeding time and their platelets were more responsive to PAR4‐AP as assessed by α‐granule release and integrin activation. The opposite was observed with thrombin. Knocking out GRK6 had no effect on human PAR4‐expressing platelets, unlike mouse Par4‐expressing platelets. PAR4 tg/KO platelets exhibited greater Ca2+ area under the curve and more robust extracellular vesicle release than WT stimulated with PAR4‐AP. ConclusionThese data suggest that (1) human PAR4‐ and mouse Par4‐mediated signaling are different and (2) the feedback regulation mechanisms of human and mouse PAR4 are different. These functional differences are important to consider when interpreting PAR4 studies done with mice.

Ex vivo anticoagulants affect human blood platelet biomechanics with implications for high-throughput functional mechanophenotyping

Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.

Longitudinal multiparametric characterization of platelet dysfunction in COVID-19: Effects of disease severity, anticoagulation therapy and inflammatory status

IntroductionDefects of platelet functional responses in COVID-19 were reported, but their origin and pathophysiological significance are unclear. The objective of this study was to characterize the thrombocytopathy in COVID-19. Materials and methodsAnalysis of platelet functional responses to activation by flow cytometry and aggregometry in 46 patients with confirmed COVID-19 of different severity (non-ICU, ICU, and ECMO) over the course of hospitalization alongside with plasma coagulation, inflammatory markers (CRP, fibrinogen, NETosis assays in smears) was performed. Results and conclusionsAll patients had increased baseline percentage of procoagulant platelets (healthy: 0.9 ± 0.5%; COVID-19: 1.7 ± 0.6%). Patients had decreased agonist-induced platelet GPIb shedding (1.8 ± 0.7 vs 1.25 ± 0.4), P-Selectin exposure (1.51 ± 0.21 vs 1.1 ± 0.3) and aggregation. The values of these parameters among the non-ICU and ICU cohorts differed modestly, while the ECMO cohort differed significantly. Only ECMO patients had pronounced thrombocytopenia. While inflammatory markers improved over time, the observed platelet functional responses changed only moderately. SARS-CoV-2 RNA was found in 8% of blood samples and it did not correlate with platelet counts or responses. All patients had increased NETosis that moderately correlated with platelet dysfunction. High cumulative dosages of LMWH (average > 12,000 IU/day over 5 days) resulted in an improvement in platelet parameters. The observed pattern of platelet refractoriness was reproduced by in vitro pre-treatment of washed platelets with subnanomolar thrombin or perfusion of blood through a collagen-covered flow chamber.We conclude that platelet dysfunction in COVID-19 is consistent with the intravascular-coagulation-induced refractoriness rather than with an inflammation-induced mechanism or a direct activation by the virus.

Increased Platelet S100A8/S100A9 Associated with Vasculitis in Granulomatosis with Polyangiitis (GPA)

Granulomatosis with polyangiitis (GPA), formerly known as Wegener's Granulomatosis, is characterized by vasculitis that predominantly affects small- and medium-sized blood vessels in the sinuses, lungs, and kidneys. In addition to vascular inflammation, GPA is also characterized by an increased risk of thrombosis. The role of platelets in GPA pathogenesis remains incompletely understood. We aimed to better understand the changes in platelet gene expression and function in patients with GPA. Forty-two patients diagnosed with GPA (n=9 with active GPA and n=33 with GPA in remission) and 25 healthy, age-, gender-, and race-matched donors were enrolled. Patients with GPA showed typical disease manifestations, with an average Birmingham Vasculitis Activity Score of 1.6 (Mean±SD 1.6±3.5). One sixth of GPA patients (7/42) had a history of thrombosis. When stimulated with thrombin receptor activating peptide (TRAP, 50nM), platelets from patients with GPA showed significantly increased expression of P-selectin as compared to healthy controls (P-selectin+% Mean±SEM: Healthy 15.50±1.84 vs GPA 25.71±16.05, P<0.05). This suggests increased platelet activation in GPA, consistent with previous findings of increased platelet aggregation in vitro in GPA. In addition, released chemokines sCD40L and platelet-derived growth factor (PDGF) by activated platelets were increased in patients with GPA when we measured the cytokines in the platelet poor plasma using the Miliplex human cytokine Assay [sCD40L (ng/mL) Mean±SEM: Healthy 63.05±6.63 vs GPA 100.40±11.86, P<0.05, PDGF-AA (pg/mL) Mean±SEM: Healthy 137.50±46.52 vs GPA 357.30±79.65, P=0.052]. Next, we performed RNA-sequencing on platelets from GPA patients (n=8, 3 with active GPA disease and 5 in remission) and, for comparison, 4 healthy donors. We identified 75 genes that were significantly differentially expressed between GPA patients and healthy donors. The top 30 genes are listed in Figure 1A. S100A8 and S100A9 were the top two significantly differentially expressed transcripts in patients with GPA (Fig. 1B). These two genes encode proteins that form a heterodimer S100A8/S100A9 (commonly known as calprotectin) known to be increased in the plasma of GPA patients and associated with disease activity. Interestingly, platelets have not been identified as the cellular source of plasma calprotectin in GPA previously. Significantly increased RNA and protein expression of S100A8 and S100A9 in GPA patients was independently validated by qRT-PCR and immunoblot, respectively. The mRNA expression of S100A8 and S100A9 in platelets were significantly correlated with p-ANCA and anti-MPO antibodies, indicating platelet S100A8/S100A9 promotes neutrophil activation and inflammation (Mann-Whitney nonparametric test, P<0.05). As previously reported, plasma levels of calprotectin were also increased in GPA patients. To further evaluate if plateletS100A8/S100A9 mediates endothelial inflammation and vasculitis, we co-cultured platelets activated with thrombin (which increases S100A8/S100A9 secretion) with endothelial cells in the presence or absence of an anti-S100A8/S100A9 blocking antibody. Activated platelets triggered endothelial cell inflammation (e.g., increased expression of ICAM-1) that was significantly reduced when S100A8/S100A9 was blocked. In summary, the platelet transcriptome is altered in patients with GPA, with S100A8 and S100A9 being the top upregulated genes. Platelet functional responses are enhanced in patients with GPA, and our data suggests that increased plasma calprotectin levels in GPA patients may be platelet derived. Platelets and platelet S100A8/S100A9 appear to mediate vascular inflammation and thrombosis in GPA. [Display omitted] DisclosuresRondina: Novartis: Research Funding; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Transcriptomics: Patents & Royalties.

A new trauma frontier: Exploratory pilot study of platelet transcriptomics in trauma patients.

The earliest measurable changes to postinjury platelet biology may be in the platelet transcriptome, as platelets are known to carry messenger ribonucleic acids (RNAs), and there is evidence in other inflammatory and infectious disease states of differential and alternative platelet RNA splicing in response to changing physiology. Thus, the aim of this exploratory pilot study was to examine the platelet transcriptome and platelet RNA splicing signatures in trauma patients compared with healthy donors. Preresuscitation platelets purified from trauma patients (n = 9) and healthy donors (n = 5) were assayed using deep RNA sequencing. Differential gene expression analysis, weighted gene coexpression network analysis, and differential alternative splicing analyses were performed. In parallel samples, platelet function was measured with platelet aggregometry, and clot formation was measured with thromboelastography. Differential gene expression analysis identified 49 platelet RNAs to have differing abundance between trauma patients and healthy donors. Weighted gene coexpression network analysis identified coexpressed platelet RNAs that correlated with platelet aggregation. Differential alternative splicing analyses revealed 1,188 splicing events across 462 platelet RNAs that were highly statistically significant (false discovery rate <0.001) in trauma patients compared with healthy donors. Unsupervised principal component analysis of these platelet RNA splicing signatures segregated trauma patients in two main clusters separate from healthy controls. Our findings provide evidence of finetuning of the platelet transcriptome through differential alternative splicing of platelet RNA in trauma patients and that this finetuning may have relevance to downstream platelet signaling. Additional investigations of the trauma platelet transcriptome should be pursued to improve our understanding of the platelet functional responses to trauma on a molecular level.

α-Ketoglutarate Inhibits Thrombosis and Inflammation by Prolyl Hydroxylase-2 Mediated Inactivation of Phospho-Akt.

BackgroundPhospho-Akt1 (pAkt1) undergoes prolyl hydroxylation at Pro125 and Pro313 by the prolyl hydroxylase-2 (PHD2) in a reaction decarboxylating α-ketoglutarate (αKG). We investigated whether the αKG supplementation could inhibit Akt-mediated activation of platelets and monocytes, in vitro as well as in vivo, by augmenting PHD2 activity.MethodsWe treated platelets or monocytes isolated from healthy individuals with αKG in presence of agonists in vitro and assessed the signalling molecules including pAkt1. We supplemented mice with dietary αKG and estimated the functional responses of platelets and monocytes ex vivo. Further, we investigated the impact of dietary αKG on inflammation and thrombosis in lungs of mice either treated with thrombosis-inducing agent carrageenan or infected with SARS-CoV-2.FindingsOctyl αKG supplementation to platelets promoted PHD2 activity through elevated intracellular αKG to succinate ratio, and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline and enhanced binding of PHD2 to pAkt in αKG-treated platelets. Contrastingly, inhibitors of PHD2 significantly increased pAkt1 in platelets. Octyl-αKG followed similar mechanism in monocytes to inhibit cytokine secretion in vitro. Our data also describe a suppressed pAkt1 and reduced activation of platelets and leukocytes ex vivo from mice supplemented with dietary αKG, unaccompanied by alteration in their number. Dietary αKG significantly reduced clot formation and leukocyte accumulation in various organs including lungs of mice treated with thrombosis-inducing agent carrageenan. Importantly, in SARS-CoV-2 infected hamsters, we observed a significant rescue effect of dietary αKG on inflamed lungs with significantly reduced leukocyte accumulation, clot formation and viral load alongside down-modulation of pAkt in the lung of the infected animals.InterpretationOur study suggests that dietary αKG supplementation prevents Akt-driven maladies such as thrombosis and inflammation and rescues pathology of COVID19-infected lungs.FundingStudy was funded by the Department of Biotechnology (DBT), Govt. of India (grants: BT/PR22881 and BT/PR22985); and the Science and Engineering Research Board, Govt. of India (CRG/000092).

The Predominant Role of Arrestin3 in General GPCR Desensitization in Platelets.

Arrestins in concert with GPCR kinases (GRKs) function in G protein-coupled receptor (GPCR) desensitization in various cells. Therefore, we characterized the functional differences of arrestin3 versus arrestin2 in the regulation of GPCR signaling and its desensitization in platelets using mice lacking arrestin3 and arrestin2. In contrast to arrestin2, platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in arrestin3-deficient platelets compared to wild-type (WT) platelets, while non-GPCR agonist CRP-induced platelet aggregation and secretion were not affected. Surprisingly, in contrast to GRK6, platelet aggregation induced by the co-stimulation of serotonin and epinephrine was significantly potentiated in arrestin3-deficient platelets, suggesting the central role of arrestin3 in general GPCR desensitization in platelets. In addition, the second challenge of ADP and AYPGKF restored platelet aggregation in arrestin3-deficient platelets but failed to do so in WT and arrestin2-deficient platelets, confirming that arrestin3 contributes to GPCR desensitization. Furthermore, ADP- and AYPGKF-induced Akt and ERK phosphorylation were significantly increased in arrestin3-deficient platelets. Finally, we found that arrestin3 is critical for thrombus formation in vivo. In conclusion, arrestin3, not arrestin2, plays a central role in the regulation of platelet functional responses and thrombus formation through general GPCR desensitization in platelets.