Platelet Subpopulations Research Articles

Hemin-induced platelet activation is regulated by the ACKR3 chemokine surface receptor and has implications for passivation of vulnerable atherosclerotic plaques.

In vulnerable atherosclerotic plaques, intraplaque hemorrhages (IPH) result in hemolysis of red blood cells and release of hemoglobin and free hemin. Hemin activates platelets and leads to thrombosis. Agonism of the inhibitory platelet receptor ACKR3 inhibits hemin-dependent platelet activation and thrombus formation. To characterize the effect of hemin and ACKR3 agonism on isolated human platelets, multi-color flow cytometry and classical experimental setup such as light transmission aggregometry and a flow chamber assay were used. Hemin induces platelet aggregation and ex vivo platelet-dependent thrombus formation on immobilized collagen under a low shear rate of 500 s-1, indicating that free hemin is a strong activator of platelet-dependent thrombosis. Recently, we described that ACKR3 is a prominent inhibitory receptor of platelet activation. Specific ACKR3 agonists but not conventional antiplatelet compounds such as COX-1 inhibitor (indometacin), ADP-receptor blocker (cangrelor), or PAR1 inhibitor (ML161) inhibit both hemin-dependent aggregation and thrombus formation. To further characterize the effect of hemin on platelet subpopulations, we established a multi-color flow cytometry assay. We found that hemin induces procoagulant (CD42bpos/PAC-1neg/AnnexinVpos), aggregatory (CD42bpos/PAC-1pos/AnnexinVneg), and inflammatory (CD42bpos/CXCR4pos/ACKR3pos/AnnexinVpos) platelet subpopulations. Treatment with ACKR3 agonists significantly decreased the formation of procoagulant and ACKR3pos platelets in response to hemin. We conclude that hemin is a strong activator for the formation of procoagulant platelets and thrombus formation which is dependent on the function of ACKR3. Activation of ACKR3 using specific agonists may offer a therapeutic strategy to regulate the vulnerability of atherosclerotic plaques in areas of IPH.

Modulating Immune Responses: The Double-Edged Sword of Platelet CD40L.

The CD40-CD40L receptor ligand pair plays a fundamental role in the modulation of the innate as well as the adaptive immune response, regulating monocyte, T and B cell activation, and antibody isotype switching. Although the expression and function of the CD40-CD40L dyad is mainly attributed to the classical immune cells, the majority of CD40L is expressed by activated platelets, either in a membrane-bound form or shed as soluble molecules in the circulation. Platelet-derived CD40L is involved in the communication with different immune cell subpopulations and regulates their functions effectively. Thus, platelet CD40L contributes to the containment and clearance of bacterial and viral infections, and additionally guides leukocytes to sites of infection. However, platelet CD40L promotes inflammatory cellular responses also in a pathophysiological context. For example, in HIV infections, platelet CD40L is supportive of neuronal inflammation, damage, and finally HIV-related dementia. In sepsis, platelet CD40L can induce extensive endothelial and epithelial damage resulting in barrier dysfunction of the gut, whereby the translocation of microbiota into the circulation further aggravates the uncontrolled systemic inflammation. Nevertheless, a distinct platelet subpopulation expressing CD40L under septic conditions can attenuate systemic inflammation and reduce mortality in mice. This review focuses on recent findings in the field of platelet CD40L biology and its physiological and pathophysiological implications, and thereby highlights platelets as vital immune cells that are essential for a proper immune surveillance. In this context, platelet CD40L proves to be an interesting target for various inflammatory diseases. However, either an agonism or a blockade of CD40L needs to be well balanced since both the approaches can cause severe adverse events, ranging from hyperinflammation to immune deficiency. Thus, an interference in CD40L activities should be likely done in a context-dependent and timely restricted manner.

Elevated levels of pro-thrombotic eNOS-negative platelets in COVID-19 patients

BackgroundPlatelet-rich microvascular thrombi are common in severe COVID-19. Endogenous nitric oxide (NO)-signaling limits thrombus formation and previously we identified platelet subpopulations with a differential ability to produce NO based on the presence or absence of endothelial nitric oxide synthase (eNOS). eNOS expression is counter-regulated by cytokines, and COVID-19-associated immune/inflammatory responses may affect the transcriptome profile of megakaryocytes and their platelet progeny. ObjectivesWe investigated whether the percentage of eNOS-negative to eNOS-positive platelets increases in COVID-19 patients and whether this change may be due to the actions of pro-inflammatory cytokines on megakaryocytes. MethodsPlatelets were isolated from hospitalized COVID-19 patients and COVID-19-negative controls. Platelet eNOS was measured by flow cytometry and plasma inflammatory cytokines by ELISA. Megakaryocytes from eNOS-GFP transgenic mice and the Meg-01 cell line were characterized to identify an appropriate model to study eNOS-based platelet subpopulation formation in response to inflammatory cytokines. ResultsCOVID-19 patients demonstrated a significant increase in eNOS-negative and a concomitant decrease in eNOS-positive platelets compared to controls, and this change was associated with disease severity as assessed by ICU admission. A higher eNOS-negative to –positive platelet percentage was associated with enhanced platelet activation as measured by surface CD62P. Accordingly, COVID-19 patients demonstrated higher TNF-α, IL-6, and IL-1β plasma concentrations than controls. Inflammatory cytokines associated with COVID-19 promoted eNOS-negative Meg-01 formation and enhanced subsequent eNOS-negative platelet-like particle formation. ConclusionsCOVID-19 patients have a higher percentage of eNOS-negative to –positive platelets, likely as a result of inflammatory response reducing megakaryocyte eNOS expression, which predisposes to thrombosis.

HLA class I expression on human platelets is highly variable and correlates with distinct allele group frequencies.

Human leukocyte antigen (HLA) class I molecules are expressed on platelets and can represent a source of alloimmunization in recipients of platelet transfusions. HLA mismatch between donors and recipients may be associated with the induction of anti-HLA antibodies, which can culminate in refractoriness to platelet transfusions. In the present study we analyzed HLA allele group frequencies and HLA expression levels on human platelets from blood donors. Platelet-rich plasma was collected from 139 donors to monitor platelet HLA class I expression by flow cytometry. DNA from donors with high and low platelet HLA expression was used in the genotype studies. Frequencies of large and normal-sized platelet subpopulations were determined and HLA class I expression was studied. Mean platelet volume (MPV) and platelet large-cell ratio (P-LCR) were analyzed in both groups of donors. The analysis showed variable platelet HLA class I expression with significant differences among donors. HLA class I allele group frequencies in donors with high and low platelet HLA expression showed distinctive genotypic features strictly related to expression level. The main allele groups found in samples with high platelet HLA class I expression were HLA-A*02, -A*68, -B*15, -B*49, and -C*03. Platelet HLA class I expression did not change over time or during freezing-thawing cycles. The analysis of platelet subpopulations showed a statistically significant higher expression of HLA class I molecules on large platelets than on normal-sized platelets. Moreover, donors with high HLA class I expression showed a higher frequency of large platelets (p<0.0001). The analysis of P-LCR in both groups of donors showed a statistically significant difference (p<0.05) within high HLA-expressing donors. Our data suggest an allele-dependent expression of HLA class I molecules on human platelets with distinct HLA allele group frequencies and different platelet subpopulation frequencies among blood donors.

High-Dimensional Single-Cell Mass Cytometry Demonstrates Differential Platelet Functional Phenotypes in Infants With Congenital Heart Disease.

Congenital heart disease (CHD) is a group of complex heart defects associated with hematologic abnormalities, including increased risk of thrombotic and bleeding events. Past studies have observed evidence of platelet hyperreactivity, while other studies showed decreased platelet activation in patients with CHD. The goal of this study was to develop a mass spectrometry approach to characterize single platelets in infants with CHD and identify potential etiology for such discrepant results. We enrolled 19 infants with CHD along with 21 non-CHD controls at Yale New Haven Children's Heart Center. A single-cell high-dimensional mass cytometry method was developed to quantitatively interrogate platelet surface markers in whole blood. Additionally, plasma cytokine analysis was performed through a multiplexed panel of 52 vascular and inflammatory markers to assess for platelet releasates. We found that infants with CHD had significant differences in platelet activation and functional markers by mass cytometry compared with non-CHD controls. Based on cell surface markers, we classified the platelets into 8 subpopulations (P0 to P7). Distinct subpopulations of platelets (P1, P4, and P5) exhibiting decreased aggregatory phenotype but altered secretory phenotypes were also identified and found to be more abundant in the blood of infants with CHD. Electron microscopy identified increased proportion of hypogranular platelets in CHD. Moreover, cytokine analysis demonstrated an overall increase in plasma cytokines and biomarkers in CHD, including IL (interleukin)-6, IL-8, IL-27, RANTES, and VWF (von Willebrand factor), which are expressed in platelet granules and can be released upon activation. We developed a robust mass cytometry approach to identify platelet phenotypic heterogeneity. Infants with CHD had alterations in distinct subpopulations of platelets with overall reduced aggregatory phenotype and secretory dysfunction. These findings suggest that platelets in infants with CHD may be exhausted due to persistent stimulation and may explain both bleeding and thrombotic vascular complications associated with CHD.

Multiparameter phenotyping of platelets and characterization of the effects of agonists using machine learning

BackgroundPlatelet function is driven by the expression of specialized surface markers. The concept of distinct circulating subpopulations of platelets has emerged in recent years, but their exact nature remains debatable. ObjectivesTo design a spectral flow cytometry–based phenotyping workflow to provide a more comprehensive characterization, at a global and individual level, of surface markers in resting and activated healthy platelets, and to apply this workflow to investigate how responses differ according to platelet age. MethodsA 14-marker flow cytometry panel was developed and applied to vehicle- or agonist-stimulated platelet-rich plasma and whole blood samples obtained from healthy volunteers, or to platelets sorted according to SYTO-13 (Thermo Fisher Scientific) staining intensity as an indicator of platelet age. Data were analyzed using both user-led and independent approaches incorporating novel machine learning–based algorithms. ResultsThe assay detected differences in marker expression in healthy platelets, at rest and on agonist activation, in both platelet-rich plasma and whole blood samples, that are consistent with the literature. Machine learning identified stimulated populations of platelets with high accuracy (>80%). Similarly, machine learning differentiation between young and old platelet populations achieved 76% accuracy, primarily weighted by forward scatter, cluster of differentiation (CD) 41, side scatter, glycoprotein VI, CD61, and CD42b expression patterns. ConclusionOur approach provides a powerful phenotypic assay coupled with robust bioinformatic and machine learning workflows for deep analysis of platelet subpopulations. Cleavable receptors, glycoprotein VI and CD42b, contribute to defining shared and unique subpopulations. This adoptable, low-volume approach will be valuable in deep characterization of platelets in disease.

Dissecting the roles of dynamin and clathrin in platelet pinocytosis

Platelets endocytose many molecules from their environment. However, this process of pinocytosis in platelets is poorly understood. Key endocytic regulators such as dynamin, clathrin, CDC42 and Arf6 are expressed in platelets but their roles in pinocytosis is not known. Stimulated platelets form two subpopulations of pro-aggregatory and procoagulant platelets. The effect of stimulation on pinocytosis is also poorly understood. In this study, washed human platelets were treated with a range of endocytosis inhibitors and stimulated using different activators. The rate of pinocytosis was assessed using pHrodo green, a pH-sensitive 10 kDa dextran. In unstimulated platelets, pHrodo fluorescence increased over time and accumulated as intracellular puncta indicating constituently active pinocytosis. Stimulated platelets (both pro-aggregatory and procoagulant) had an elevated pinocytosis rate compared to unstimulated platelets. Dynamin inhibition blocked pinocytosis in unstimulated, pro-aggregatory and procoagulant platelets indicating that most platelet pinocytosis is dynamin dependent. Although pinocytosis was clathrin-independent in unstimulated and procoagulant populations, clathrin partially contributed to pinocytosis in pro-aggregatory platelets.

Deciphering Abnormal Platelet Subpopulations in COVID-19, Sepsis and Systemic Lupus Erythematosus through Machine Learning and Single-Cell Transcriptomics.

This study focuses on understanding the transcriptional heterogeneity of activated platelets and its impact on diseases such as sepsis, COVID-19, and systemic lupus erythematosus (SLE). Recognizing the limited knowledge in this area, our research aims to dissect the complex transcriptional profiles of activated platelets to aid in developing targeted therapies for abnormal and pathogenic platelet subtypes. We analyzed single-cell transcriptional profiles from 47,977 platelets derived from 413 samples of patients with these diseases, utilizing Deep Neural Network (DNN) and eXtreme Gradient Boosting (XGB) to distinguish transcriptomic signatures predictive of fatal or survival outcomes. Our approach included source data annotations and platelet markers, along with SingleR and Seurat for comprehensive profiling. Additionally, we employed Uniform Manifold Approximation and Projection (UMAP) for effective dimensionality reduction and visualization, aiding in the identification of various platelet subtypes and their relation to disease severity and patient outcomes. Our results highlighted distinct platelet subpopulations that correlate with disease severity, revealing that changes in platelet transcription patterns can intensify endotheliopathy, increasing the risk of coagulation in fatal cases. Moreover, these changes may impact lymphocyte function, indicating a more extensive role for platelets in inflammatory and immune responses. This study identifies crucial biomarkers of platelet heterogeneity in serious health conditions, paving the way for innovative therapeutic approaches targeting platelet activation, which could improve patient outcomes in diseases characterized by altered platelet function.

Binding of coagulation factor IXa to procoagulant platelets revisited: Low affinity and interactions with other factors

Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, Kd at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3–5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.

The activity of platelet enzymes and subpopulation composition of monocytes in schizophrenia

IntroductionThe studies on various groups of patients with schizophrenia revealed impairments in immune system, glutamatergic, and antioxidant systems contributing substantially in schizophrenia pathogenesis.Objectives To search for links between the activities of platelet enzymes involved in glutamate and glutathione metabolism and monocytes’ subpopulation compositions in patients with schizophrenia and to identify possible correlations of the biomarkers with clinical data. Research objectives: determination of subpopulation ratio of monocytes; measurement of the activity levels of glutamate dehydrogenase (GDH), phosphate-activated glutaminase (PAG), glutathione reductase (GR) and glutathione S-transferase (GST) in blood platelets; search for correlations between these parameters and the scores by psychometric scales.Methods The study included 36 women aged 16-45 years with acute schizophrenia hospitalized in the Mental Health Research Centre with their current condition assessed as depressive-delusional. The control group consisted of 17 women 18-45 years old without somatic or mental pathology. GDH, PAG, GR and GST activities were measured by spectrophotometric methods, and numbers of monocyte subpopulations - “classical”, “intermediate”, “non-classical” - by flow cytometry. The Hamilton Depression Rating Scale (HAMD-17) was used to assess depression severity. The data was processed using the Statistica 8.0 software.Results The detected changes in monocyte subpopulations’ composition towards the increase in the proportion of cells having a pro-inflammatory phenotype (CD14++CD16+ “intermediate”) indicated the activation of inflammatory reactions. Also, the activities of platelet enzymes of glutathione metabolism (GR and GST) were significantly decreased (p&lt;0.05). Moreover, GDH and GST activities significantly correlated with the scores by HAMD-17 (r=0.40, p=0.022 and r=0.45, p=0.030, respectively). The results indicate the presence of pathological inflammatory process, the decrease in activities of glutathione antioxidant metabolism enzymes and a link to glutamate metabolism involvement (GDH) in the studied patient group.Conclusions The identified redistribution in the monocyte subpopulations’ composition and decrease in the activity of enzymes involved in glutamate metabolism and antioxidant system indicate the involvement of the immune, glutamate and antioxidant systems in the pathogenesis of schizophrenia and may reflect a functional interaction between these systems.Disclosure of InterestNone Declared

CGMP modulates hemin-mediated platelet death

Background and aimsHemolysis is a known risk factor for thrombosis resulting in critical limb ischemia and microcirculatory disturbance and organ failure. Intravasal hemolysis may lead to life-threatening complications due to uncontrolled thrombo-inflammation. Until now, conventional antithrombotic therapies failed to control development and progression of these thrombotic events. Thus, the pathophysiology of these thrombotic events needs to be investigated to unravel underlying pathways and thereby identify targets for novel treatment strategies. MethodsHere we used classical experimental set-ups as well as high-end flow cytometry, metabolomics and lipidomic analysis to in-depth analyze the effects of hemin on platelet physiology and morphology. ResultsHemin does strongly and swiftly induce platelet activation and this process is modulated by the sGC-cGMP-cGKI signaling axis. cGMP modulation also reduced the pro-aggregatory potential of plasma derived from patients with hemolysis. Furthermore, hemin-induced platelet death evokes distinct platelet subpopulations. Typical cell death markers, such as ROS, were induced by hemin-stimulation and the platelet lipidome was specifically altered by high hemin concentration. Specifically, arachidonic acid derivates, such as PGE2, TXB2 or 12-HHT, were significantly increased. Balancing the cGMP levels by modulation of the sGC-cGMP-cGKI axis diminished the ferroptotic effect of hemin. ConclusionWe found that cGMP modulates hemin-induced platelet activation and thrombus formation in vitro and cGMP effects hemin-mediated platelet death and changes in the platelet lipidome. Thus, it is tempting to speculate that modulating platelet cGMP levels may be a novel strategy to control thrombosis and critical limb ischemia in patients with hemolytic crisis.

Platelet Subpopulations and Distinct Platelet Function Defects after Hematopoietic Stem Cell Transplantation in Mice and Humans

Allogeneic hematopoietic stem cell transplantation (HSCT) is often accompanied by bleeding complications due to delayed platelet engraftment. Despite having prolonged thrombocytopenia, patients can also develop thrombotic events. The mechanisms leading to HSCT-related hemorrhage and thrombosis, and especially platelet activity in these patients are yet unknown. Our study aimed to decipher post HSCT megakaryocyte (MK) engraftment and platelet function in a murine model of bone marrow transplantation (BMT) and in patients undergoing HSCT. C57BL/6 mice were lethally irradiated with 10 Gy total body irradiation prior to BMT. Radiation exposure led to massive BM remodeling, including extracellular matrix degradation, vasodilation and aberrant pericyte morphology, accompanied by extensive ectopic platelet release into the BM cavity, which might contribute to thrombocytopenia. We used Actb dsRed reporter mice as BM donors allowing us to track all donor-derived (dsRed +) cells after BMT. Femurs were removed after 14 days and subjected to confocal laser immunofluorescence microscopy using lineage-specific markers. While we found an overall homogenous engraftment pattern for red blood cells and most leukocyte sub-lineages, the engraftment pattern for MKs and Ly6G + cells was characterized by long triangular clusters at the bone cortex. We manipulated BM engraftment by transplanting different numbers of graft cells. Within 2 weeks after BMT with 6 x 10 6 BM cells per mouse, a complete chimerism was achieved for both MKs in BM and platelets in peripheral blood. These newly formed platelets had an increased platelet size, while the surface expression levels (CD41/CD42/GPVI) were decreased by half, implying a massive reduction in receptor density. Flow cytometry analyses of agonist-stimulated platelets revealed a severely impaired granule release and integrin activation that was confirmed by aggregometry. When only 1 x 10 6 BM cells per mouse were transplanted, we found an extended mixed MK chimerism after BMT. During the first 28 days, we detected both dsRed - (recipient-derived) and dsRed + (donor-derived) MKs in the recipient BM and, concomitantly, dsRed - and dsRed + platelets in peripheral blood. Unexpectedly, we observed opposing agonist-induced platelet reactivity with hyporeactive dsRed - platelets, while dsRed + platelets were hyperreactive, indicating that 2 BM MK subpopulations generate 2 functionally distinct platelet subpopulations. Intriguingly, we found an upregulation of annexin V binding on dsRed -, but not on dsRed + resting platelets, implying that the myeloablative conditions preceding HSCT result in increased phosphatidylserine (PS) exposure, a key sign of pro-coagulant platelets. In vitro thrombus formation was assessed under flow using a whole blood collagen-coated microfluidic device. While surface coverage was mildly increased, thrombus volume remained comparable to controls. dsRed + platelets exhibited a distinct incorporation pattern by preferentially attaching to the edges of growing thrombi, indicating that dsRed - and dsRed + platelets may differ in thrombus initiation and growth. While platelet count and other blood cell parameters are routinely monitored in HSCT patients, the functional capacity of graft-derived platelets has not yet been systematically assessed. We thus performed extended platelet function analyses in 13 adult patients (aged 24-73 years) undergoing allogeneic HSCT for malignant leukemic diseases (mostly acute myeloid leukemia) and detected a massive platelet hyporeactivity for up to 100 days observation time after HSCT. Integrin activation was severely impaired upon stimulation of PAR-1 or GPVI. In contrast to mice, α granule release, monitored by P-selectin neo-exposure, was only slightly reduced compared to healthy controls, suggesting a decoupling of integrin activation and P-selectin exposure. In line with our findings in mice, we detected a pronounced upregulation in the proportion of pro-coagulant PS-positive platelets with a reduced mitochondrial membrane potential. Our data demonstrate that a mixed chimerism after HSCT results in two co-existing platelet subpopulations with opposing response profiles. This unexpected finding provides the first experimental evidence why bleedings and thromboembolic events can both occur after HSCT and hints toward pro-coagulant platelets as a potential therapeutic target.

PB0601 Identification of a Novel Growth Factor-Enriched Platelet Subpopulation: Potential Implications for COVID-19

PB0601 Identification of a Novel Growth Factor-Enriched Platelet Subpopulation: Potential Implications for COVID-19

PB0904 A Novel Quantitative Flow Cytometry Approach in Determining Platelet Subpopulations and Quantifying Cell Surface Receptors

PB0904 A Novel Quantitative Flow Cytometry Approach in Determining Platelet Subpopulations and Quantifying Cell Surface Receptors

PB1368 Platelet Subpopulations in Decompensated Cirrhotic Children

PB1368 Platelet Subpopulations in Decompensated Cirrhotic Children

A thrombus is formed by a gradient of platelet activation and procoagulant endothelium

BackgroundThe contribution of platelets in thrombosis within microcirculation has been extensively documented in the literature. We previously showed, in vivo, that platelet activation revealed by intracellular calcium mobilization was a crucial step in the growth of thrombi following laser-induced injury, a model of thromboinflammation. ObjectiveOur goal was to investigate the extent of platelet activation and the spatial distribution of platelets throughout a growing thrombus. MethodsWe employed a multimodal, correlative microscopy approach and computational biology to study the state of platelets on a growing thrombus obtained after a laser injury. ResultsWe observed a reversible intracellular platelet calcium mobilization that correlates with the time a platelet resides during thrombus growth. Our bioinformatics analysis displayed the following 3 distinct platelet subpopulations resident within a thrombus: (1) resting, (2) partially activated, and (3) “fully” activated platelets. The spatial distribution of the platelet subpopulations in the thrombus creates a double gradient in both the transversal and longitudinal axis, with the maximal percentage of fully activated platelets close to the site of injury. However, these activated platelets did not express negative phospholipids. The injured endothelium was identified to play a vital role in activating the blood coagulation cascade in this model of thrombosis. ConclusionFollowing a laser-induced injury, thrombi are formed by a gradient of activated platelets from the injury site to the periphery of the thrombus. These different activation states of platelets throughout the thrombi regulate the biomechanics of the thrombus. The injured endothelium, rather than platelets, was identified to play a key role in the activation of the blood coagulation cascade in this model of thromboinflammation.

The phenotype of cryopreserved platelets influences the formation of platelet-leukocyte aggregates in an in vitro model

Cryopreservation significantly alters the phenotype of platelets; generating distinct subpopulations, which may influence the formation of platelet leukocyte aggregates (PLA). PLAs are immunomodulatory and have been associated with transfusion-associated adverse events. As such, the aim of this study was to examine the effect of cryopreservation on the ability of platelets to form PLAs, using a monocyte-like cell line (THP-1). Platelets were tested pre-freeze, post-thaw and following stimulation with TRAP-6 or A23187, both alone and following co-culture with THP-1 cells for 1 and 24 hours (n = 6). Platelet subpopulations and platelet-THP-1 cell aggregates were analyzed using multi-color imaging flow cytometry using Apotracker Green (ApoT), CD42b, CD62P, CD61, and CD45. Cryopreservation resulted in the generation of activated (ApoT-/CD42b+/CD62P+), procoagulant (ApoT+/CD42b+/CD62P+) and a novel (ApoT+/CD42b+/CD62P-) platelet subpopulation. Co-incubation of cryopreserved platelets with THP-1 cells increased PLA formation compared to pre-freeze but not TRAP-6 or A23187 stimulated platelets. P-selectin on the surface membrane was correlated with increased PLA formation. Our findings demonstrate that cryopreservation increases the interaction between platelets and THP-1 cells, largely due to an increase in procoagulant platelets. Further investigation is required to determine the immunological consequences of this interaction.

Ontogenesis of functional platelet subpopulations from preterm and term neonates to adulthood: The PLINIUS study

AbstractErythrocytes undergo a well-defined switch from fetal to postnatal circulation, which is mainly reflected by the stage-specific expression of hemoglobin chains. Perinatal alterations in thrombopoiesis are poorly understood. We assessed the ontogenesis of platelet phenotype and function from early prematurity to adulthood. We recruited 64 subjects comprising 7 extremely preterm (27-31 weeks gestational age), 25 moderately preterm (32-36 weeks), 10 term neonates, 8 infants (<2 years), 5 children (2-13 years), and 9 adults (>13 years). Blood was withdrawn at up to 3 different time points in neonates (t1: 0-2, t2: 3-7, and t3: 8-14 days after birth). We found that the expression levels of the major surface receptors for fibrinogen, collagen, vWF, fibronectin, and laminin were reduced but correlated with decreased platelet size, indicating a normal surface density. Although CD62P and CD63 surface exposure upon stimulation with TRAP-6, ADP, or U46619 was unaltered or only slightly reduced in neonates, GPIIb/IIIa inside-out and outside-in activation was blunted but showed a continuous increase until adulthood, correlating with the expression of the GPIIb/IIIa regulating tetraspanin CD151. Platelet subpopulation analysis using automated clustering revealed that neonates presented with a CD63+/PAC-1– pattern, followed by a continuous increase in CD63+/PAC-1+ platelets until adulthood. Our findings revealed that the number of platelet-monocyte and platelet-neutrophil aggregates, but not platelet-lymphocyte aggregates, is increased in neonates and that neonatal aggregate formation depends in part on CD62P activation. Our PLatelets In Neonatal Infants Study (PLINIUS) provides several lines of evidence that the platelet phenotype and function evolve continuously from neonates to adulthood.

Identification of High Platelet Reactivity Despite ADP P2Y12 Inhibitor Treatment: Two Populations in the Vasodilator-Stimulated Phosphoprotein Assay and Variable PFA-P2Y Shapes of Curve

Introduction Response to ADP P2Y12 receptor inhibition by clopidogrel can be evaluated by various techniques. Here, we compared a functional rapid point-of-care technique (PFA-P2Y) with the degree of biochemical inhibition assessed by the VASP/P2Y12 assay. Methods Platelet response to clopidogrel was investigated in 173 patients undergoing elective intracerebral stenting (derivation cohort n = 117; validation cohort n = 56). High platelet reactivity (HPR) was defined as PFA-P2Y occlusion time &lt;106 seconds or VASP/P2Y12 platelet reactivity index (PRI) &gt;50%. Results In the derivation cohort, receiver operator characteristics analysis for the ability of PFA-P2Y to detect biochemical HPR showed high specificity (98.4%) but poor sensitivity (20.0%) and a very low area under the curve (0.59). The VASP/P2Y12 assay revealed two coexisting platelet populations with different levels of vasodilator-stimulated phosphoprotein (VASP) phosphorylation: a fraction of highly phosphorylated, inhibited platelets and another of poorly phosphorylated, reactive platelets. Analysis of the PFA-P2Y curve shape revealed different types, categorized by time of occlusion (&lt;106 seconds, 106 to 300 seconds, &gt;300 seconds), and pattern (regular, irregular, and atypical). Noteworthy, curves with late occlusion and permeable curves with an irregular or atypical pattern correlated with VASP-PRI &gt;50% and smaller sizes of the inhibited platelet subpopulation. Considering the PFA-P2Y shape of the curve for the detection of HPR improved sensitivity (72.7%) and preserved specificity (91.9%), with a rather high AUC (0.823). The validation cohort confirmed the VASP/P2Y12 assay data and the usefulness of considering the PFA-P2Y curve shape. Conclusion In patients treated with acetylsalicylic acid and clopidogrel for 7-10 days, the VASP/P2Y12 assay reveals two coexisting subpopulations of differentially inhibited platelets, whose relative sizes predict global PRI and distinct PFA-P2Y curve patterns, indicating incomplete clopidogrel efficacy. The detailed analysis of both VASP/P2Y12 and PFA-P2Y is necessary for optimal detection of HPR.

Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry

Cryopreservation of platelets, at − 80 °C with 5–6% DMSO, results in externalisation of phosphatidylserine and the formation of extracellular vesicles (EVs), which may mediate their procoagulant function. The phenotypic features of procoagulant platelets overlap with other platelet subpopulations. The aim of this study was to define the phenotype of in vitro generated platelet subpopulations, and subsequently identify the subpopulations present in cryopreserved components. Fresh platelet components (n = 6 in each group) were either unstimulated as a source of resting platelets; or stimulated with thrombin and collagen to generate a mixture of aggregatory and procoagulant platelets; calcium ionophore (A23187) to generate procoagulant platelets; or ABT-737 to generate apoptotic platelets. Platelet components (n = 6) were cryopreserved with DMSO, thawed and resuspended in a unit of thawed plasma. Multi-colour panels of fluorescent antibodies and dyes were used to identify the features of subpopulations by imaging flow cytometry. A combination of annexin-V (AnnV), CD42b, and either PAC1 or CD62P was able to distinguish the four subpopulations. Cryopreserved platelets contained procoagulant platelets (AnnV+/PAC1−/CD42b+/CD62P+) and a novel population (AnnV+/PAC1−/CD42b+/CD62P−) that did not align with the phenotype of aggregatory (AnnV−/PAC1+/CD42b+/CD62P+) or apoptotic (AnnV+/PAC1−/CD42b−/CD62P−) subpopulations. These data suggests that the enhanced haemostatic potential of cryopreserved platelets may be due to the cryo-induced development of procoagulant platelets, and that additional subpopulations may exist.