Flow Cytometry and Platelets.

Clinical assessment of platelet activation by flow cytometry is useful in the characterization and diagnosis of platelet-specific disorders and as a measure of risk for thrombosis or bleeding. Platelets circulate in a resting, "unactivated" state, but when activated they undergo alterations in surface glycoprotein function and/or expression level, exposure of granule membrane proteins, and exposure of procoagulant phospholipids. Flow cytometry provides the means to detect these changes and, unlike other platelet tests, is appropriate for measuring platelet function in samples from patients with low platelet counts.

Dysregulation of platelet serotonin, 14-3-3, and GPIX in sudden infant death syndrome.

Sudden infant death syndrome (SIDS) is the leading cause of post-neonatal infant mortality, but the underlying cause(s) are unclear. A subset of SIDS infants has abnormalities in the neurotransmitter, serotonin (5-hydroxytryptamine [5-HT]) and the adaptor molecule, 14-3-3 pathways in regions of the brain involved in gasping, response to hypoxia, and arousal. To evaluate our hypothesis that SIDS is, at least in part, a multi-organ dysregulation of 5-HT, we examined whether blood platelets, which have 5-HT and 14-3-3 signaling pathways similar to brain neurons, are abnormal in SIDS.

OMIP-097: High-parameter phenotyping of human platelets by spectral flow cytometry.

Using spectral flow cytometry, we developed a 16-color panel for analysis of platelet phenotype and function in human whole blood. The panel contains markers of clinical relevance and follows an optimized protocol for the high-parameter phenotyping of (phosphatidylserine positive) procoagulant platelets. Inclusion of established markers, such as CD62P and PAC-1, allows the subsetting of classic (proinflammatory and proaggregatory) phenotypes, while addition of novel markers, such as TLR9, allows the resolution of platelets with nonclassic functions.

Clinical Cytometry for Platelets and Platelet Disorders.

Clinical flow cytometry tests for inherited and acquired platelet disorders are useful diagnostic tools but are not widely available. Flow cytometric methods are available to detect inherited glycoprotein deficiencies, granule release (secretion defects), drug-induced thrombocytopenias, presence of antiplatelet antibodies, and pharmacodynamic inhibition by antiplatelet agents. New tests take advantage of advanced multicolor cytometers and allow identification of novel platelet subsets by high-dimensional immunophenotyping.

Platelet Phenotyping by Full Spectrum Flow Cytometry.

Platelets play key roles in hemostasis, immunity, and inflammation, and tests of platelet phenotype and function are useful in studies of disease biology and pathology. Full spectrum flow cytometry offers distinct advantages over standard tests and enables the sensitive and simultaneous detection of many biomarkers. A typical assay provides a wealth of information on platelet biology and allows the assessment of in vivo activation and in vitro reactivity, as well as the discovery of novel phenotypes.

Ticagrelor vs placebo for the reduction of vaso-occlusive crises in pediatric sickle cell disease: the HESTIA3 study.

The phase 3 HESTIA3 study assessed the efficacy and safety of the reversible P2Y12 inhibitor ticagrelor vs placebo in preventing vaso-occlusive crises in pediatric patients with sickle cell disease (SCD). Patients aged 2 to 17 years were randomly assigned 1:1 to receive weight-based doses of ticagrelor or matching placebo. The primary end point was the rate of vaso-occlusive crises, a composite of painful crises and/or acute chest syndrome (ACS).

Expert opinion on the use of platelet secretion assay for the diagnosis of inherited platelet function disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

Assessment of platelet secretion is crucial for diagnosing suspected inherited platelet function disorders (IPFD). A previous survey of the SSC on Platelet Physiology of the ISTH and a comprehensive review highlighted that most of the platelet secretion assays (PSAs) lack standardization and validation. The aim of this study was to provide expert consensus guidance on the use of PSAs for IPFD diagnosis.

Comprehensive phenotyping of human platelets by single-cell cytometry.

Platelets are small anucleate blood cells that contribute to hemostasis, immunity, and inflammation. Circulating platelets are heterogeneous in size, age, receptor expression, and reactivity. They inherit many features from megakaryocytes and are further modified on exposure to bioactive substances in the bloodstream.

Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia.

During gram-negative septicemia, interactions between platelets and neutrophils initiate a detrimental feedback loop that sustains neutrophil extracellular trap (NET) induction, disseminated intravascular coagulation, and inflammation. Understanding intracellular pathways that control platelet-neutrophil interactions is essential for identifying new therapeutic targets. Here, we found that thrombin signaling induced activation of the transcription factor NFAT in platelets.

Sex-specific platelet activation through protease-activated receptor-1 in patients undergoing cardiac catheterization.

Background And Aims: Protease-activated receptor (PAR)-1-mediated platelet activation may vary according to sex and clinical situation. In order to investigate sex-specific platelet activation through PAR-1, we assessed platelet response to thrombin receptor-activating peptide (TRAP) in 562 patients undergoing cardiac catheterization without (Group 1A) and with (Group 1B) acute coronary syndrome (ACS). Subsequently, we sought to confirm our findings in 287 patients undergoing elective (Group 2A) or acute (Group 2B) percutaneous coronary intervention.

Consensus recommendations on flow cytometry for the assessment of inherited and acquired disorders of platelet number and function: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

Flow cytometry is increasingly used in the study of platelets in inherited and acquired disorders of platelet number and function. However, wide variation exists in specific reagents, methods, and equipment used, making interpretation and comparison of results difficult. The goal of the present study was to provide expert consensus guidance on the use of flow cytometry for the evaluation of platelet disorders.

Platelet Activation and Reactivity in a Large Cohort of Patients with Gaucher Disease.

Objectives: Patients with Gaucher disease (GD) are at increased risk of bleeding and have varying degrees of thrombocytopenia, making the analysis of platelet function difficult. This study aimed to provide a clinically relevant quantitative assessment of platelet function and determine its relationship with bleeding and GD-related data.

Methods: Unstimulated and stimulated platelet function was measured by whole blood flow cytometry of platelet surface-activated αIIbβ3 integrin (detected with monoclonal antibody PAC1), P-selectin (CD62P), and lysosomal-associated membrane protein (LAMP3/CD63) in 149 GD patients.

Immunophenotypic Analysis of Platelets by Flow Cytometry.

Platelets are small but very abundant blood cells that play a key role in hemostasis, contributing to thrombus formation at sites of injury. The ability of platelets to perform this function, as well as functions in immunity and inflammation, is dependent on the presence of cell surface glycoproteins and changes in their quantity and conformation after platelet stimulation. In this article, we describe the characterization of platelet surface markers and platelet function using platelet-specific fluorescent probes and flow cytometry.

Platelet Immunophenotyping by High-Dimensional Mass Cytometry.

Platelets are small blood cells that contribute to hemostasis, immunity, and inflammation. Characterization of platelet surface markers allows for differentiation of activated platelets from resting platelets, diagnosis of platelet disorders, and investigation of platelet biology and pathology. In this article, we describe the use of mass cytometry or "CyTOF" (mass spectroscopy detection of metal-tagged antibodies on individual cells) to measure a large number of markers on each platelet and to identify platelet subsets based on the shared expression of multiple markers.

Activation of platelet-rich plasma by pulse electric fields: Voltage, pulse width and calcium concentration can be used to control and tune the release of growth factors, serotonin and hemoglobin.

Activated platelet-rich plasma (PRP) has been used in the clinical settings of wound healing and regenerative medicine, with activation typically induced by the addition of bovine thrombin. To eliminate issues with availability, cost and potential side effects associated with bovine thrombin, ex vivo PRP activation using pulse electric fields (PEF) has been proposed and demonstrated. The present study characterizes the effect of PEF voltage and pulse width, in combination with a range of calcium concentrations, on clot formation, growth factor release, and serotonin (5-HT) release from dense granules.

Platelet mass cytometry: Optimization of sample, reagent, and analysis parameters.

Platelets mediate key biological processes, including hemostasis, immunity, and inflammation. Although platelets are often treated as a homogeneous cell population, they are known to be heterogeneous in size, age, surface receptor expression, and response to agonist stimulation, raising the possibility that distinct platelet subsets perform specialized functions and that such subsets may be altered in disease settings. Attempts to identify platelet subsets by flow cytometry have had limited success due in part to limits on the number of probes that can be used at the same time and due to the challenges of compensating for probes that have large spectral overlap.

Decreased platelet surface phosphatidylserine predicts increased bleeding in patients with severe factor VIII deficiency.

Background: Bleeding phenotypes among individuals with severe factor VIII (FVIII) deficiency are variable, even with routine prophylactic FVIII administration. Activated platelets, in addition to their role in primary hemostasis, play a major role in coagulation by providing a phospholipid surface to which coagulation factors bind.

Objectives: The aim of this study was to determine whether platelet function is associated with past and/or future bleeding in patients with severe FVIII deficiency on prophylaxis.

Platelet surface GPIbα, activated GPIIb-IIIa, and -selectin levels in adult veno-arterial extracorporeal membrane oxygenation patients.

Our objective was to characterize platelet surface glycoprotein (GP)Ibα, activated GPIIb-IIIa, and -selectin levels during and after extracorporeal membrane oxygenation (ECMO). We performed a single center cohort study of 10 adult patients on ECMO for cardiogenic shock. Patients had blood samples drawn on ECMO day 1 or 2, day 3, day 5, and 48-72 hours after ECMO decannulation.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2.

Biomarkers of platelet activation and cardiovascular risk in the DAPT trial.

Prolonged use of dual antiplatelet therapy (DAPT) post-percutaneous coronary intervention (PCI) has been shown to reduce the risk of major adverse cardiovascular events (MACE), but with increased bleeding. It remains unknown whether biomarkers of platelet activation may be useful for identifying patients at increased risk of MACE. The DAPT study was a randomized trial of 12 versus 30 months of DAPT in patients who underwent PCI.

Novel Antiplatelet Agents in Cardiovascular Disease.

Antiplatelet therapy reduces atherothrombotic risk and has therefore become a cornerstone in the treatment of cardiovascular disease. Aspirin, adenosine diphosphate P2Y receptor antagonists, glycoprotein IIb/IIIa inhibitors, and the thrombin receptor blocker vorapaxar are effective antiplatelet agents but significantly increase the risk of bleeding. Moreover, atherothrombotic events still impair the prognosis of many patients with cardiovascular disease despite established antiplatelet therapy.

An exploratory, randomised, placebo-controlled, 14 day trial of the soluble guanylate cyclase stimulator praliciguat in participants with type 2 diabetes and hypertension.

Aims/hypothesis: Praliciguat (IW-1973), a soluble guanylate cyclase stimulator, amplifies nitric oxide signalling. This exploratory trial investigated the safety, tolerability, pharmacokinetic profile and pharmacodynamic effects of praliciguat in individuals with type 2 diabetes and hypertension.

Methods: This Phase IIA, double-blind, placebo-controlled trial investigated praliciguat in 26 participants with type 2 diabetes and hypertension on stable glucose- and BP-lowering therapies.

Phosphoflow cytometry and barcoding in blood platelets: Technical and analytical considerations.

Platelet function is regulated by finely tuned phosphoprotein signals. Subtle aberrations in signaling can cause platelet hyperactivity and severe cardiovascular events. Mapping phosphorylation profiles in health and disease could accelerate antiplatelet discovery and enhance cardiovascular management, but traditional assays are ill-suited to clinical application as they are laborious and low throughput.