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. Multiple inducible (C3b, CD63, CD107a, CD154, and TLT-1) and constitutive (CD29, CD31, CD32, CD36, CD42a, CD61, and GPVI) markers are also measurable, and we demonstrate the use of automatic gating for platelet analysis. The panel is widely applicable to research and clinical settings and can be readily modified, should users wish to tailor the panel to more specific needs.

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. Studies are needed to evaluate the value of these new tests for diagnosis and monitoring of therapy in patients with platelet disorders.

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. Here, we describe the analysis of platelets by full spectrum flow cytometry and discuss a range of controls and methods for interpreting results. © 2023 Wiley Periodicals LLC. Basic Protocol: Platelet phenotyping by full spectrum flow cytometry Support Protocol 1: Spectral unmixing Support Protocol 2: Data preprocessing.

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. Among these substances, prothrombotic agonists, vasodilators, and bloodborne pathogens modulate platelet phenotypes via distinct signaling cascades. The ability of platelets to respond to (patho)physiologic signals is incompletely understood but likely depends on their repertoire of surface receptors, which may partition them into discrete subsets with specialized functions and divergent abilities. The single-cell resolution of flow and mass cytometry is ideal for immunophenotyping and allows the identification of platelet subsets in remarkable detail. In this report, we describe the surface markers and gating strategies needed for the comprehensive characterization of platelets.

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. Unlike traditional platelet tests, immunophenotypic analysis of platelets by flow cytometry allows the analysis of platelet function in samples with very low platelet counts as often encountered in clinical situations. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Immunophenotyping of platelet surface receptors Alternate Protocol: Fix-first method for immunophenotyping of platelet surface receptors Basic Protocol 2: Determination of platelet activation using P-selectin expression and/or PAC1 binding Basic Protocol 3: Determination of procoagulant platelets using annexin V binding or antibodies specific for coagulation factor V/Va or X/Xa Support Protocol: Preparation of isolated platelets.

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. This powerful new approach provides a vastly more detailed picture of platelet immunophenotypes than conventional flow cytometry and enables investigation of the roles of platelet subsets in health and disease. © 2021 Wiley Periodicals LLC. Basic Protocol: Platelet immunophenotyping by high-dimensional mass cytometry Support Protocol: Data preprocessing.

Multidimensional flow cytometry reveals novel platelet subpopulations in response to prostacyclin.

BACKGROUND: Robust platelet activation leads to the generation of subpopulations characterized by differential expression of phosphatidylserine (PS). Prostacyclin (PGI2 ) modulates many aspects of platelet function, but its influence on platelet subpopulations is unknown. OBJECTIVES AND METHODS: We used fluorescent flow cytometry coupled to multidimensional fast Fourier transform-accelerated interpolation-based t-stochastic neighborhood embedding analysis to examine the influence of PGI2 on platelet subpopulations. RESULTS: Platelet activation (SFLLRN/CRP-XL) in whole blood revealed three platelet subpopulations with unique combinations of fibrinogen (fb) binding and PS exposure. These subsets, PSlo /fbhi (68%), PShi /fblo (23%), and PShi /fbhi (8%), all expressed CD62P and partially shed CD42b. PGI2 significantly reduced fibrinogen binding and prevented the majority of PS exposure, but did not significantly reduce CD62P, CD154, or CD63 leading to the generation of four novel subpopulations, CD62Phi /PSlo /fblo (64%), CD62Phi /PSlo /fbhi (22%), CD62Phi /PShi /fblo (3%), and CD62Plo /PSlo /fblo (12%). Mechanistically this was linked to PGI2 -mediated inhibition of mitochondrial depolarization upstream of PS exposure. Combining phosphoflow with surface staining, we showed that PGI2 -treated platelets were characterized by both elevated vasodilator-stimulated phosphoprotein phosphorylation and CD62P. The resistance to cyclic AMP signaling was also observed for CD154 and CD63 expression. Consistent with the functional role of CD62P, exposure of blood to PGI2 failed to prevent SFLLRN/CRP-XL-induced platelet-monocyte aggregation despite reducing markers of hemostatic function. CONCLUSION: The combination of multicolor flow cytometry assays with unbiased computational tools has identified novel platelet subpopulations that suggest differential regulation of platelet functions by PGI2 . Development of this approach with increased surface and intracellular markers will allow the identification of rare platelet subtypes and novel biomarkers.

Thrombospondin-1 promotes hemostasis through modulation of cAMP signaling in blood platelets.

Thrombospondin-1 (TSP-1) is released by platelets upon activation and can increase platelet activation, but its role in hemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of hemostasis that promotes platelet activation by modulating inhibitory cyclic adenosine monophosphate (cAMP) signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of hemostasis and thrombosis showed that TSP-1-deficient mice had prolonged bleeding, defective thrombosis, and increased sensitivity to the prostacyclin mimetic iloprost. Adoptive transfer of wild-type (WT) but not TSP-1-/- platelets ameliorated the thrombotic phenotype, suggesting a key role for platelet-derived TSP-1. In functional assays, TSP-1-deficient platelets showed an increased sensitivity to cAMP signaling, inhibition of platelet aggregation, and arrest under flow by prostacyclin (PGI2). Plasma swap experiments showed that plasma TSP-1 did not correct PGI2 hypersensitivity in TSP-1-/- platelets. By contrast, incubation of TSP-1-/- platelets with releasates from WT platelets or purified TSP-1, but not releasates from TSP-1-/- platelets, reduced the inhibitory effects of PGI2. Activation of WT platelets resulted in diminished cAMP accumulation and downstream signaling, which was associated with increased activity of the cAMP hydrolyzing enzyme phosphodiesterase 3A (PDE3A). PDE3A activity and cAMP accumulation were unaffected in platelets from TSP-1-/- mice. Platelets deficient in CD36, a TSP-1 receptor, showed increased sensitivity to PGI2/cAMP signaling and diminished PDE3A activity, which was unaffected by platelet-derived or purified TSP-1. This scenario suggests that the release of TSP-1 regulates hemostasis in vivo through modulation of platelet cAMP signaling at sites of vascular injury.

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. We recently reported a method to identify platelet subsets by mass cytometry using a panel of 14 metal-tagged antibodies directed at platelet surface markers. Here, we describe the technical considerations and best practices for platelet sample preparation, processing, and analysis by mass cytometry. Specifically, we show that anticoagulant choice alters platelet phenotype and function and that antibody cocktail storage and sample processing are critical for reproducibility. Additionally, we optimize sample density and instrument setup for maximal platelet transmission. Lastly, we demonstrate the importance of panel design and compensation and the use of clustering and dimension reduction to map platelet heterogeneity across resting and stimulated samples.

Platelet Flow Cytometry: Instrument Setup, Controls, and Panel Performance.

Platelet flow cytometry is widely used in cardiovascular medicine as the platelet surface is rich in clinical biomarkers. Surface profiling is critical in disease management, but current assays can abet clinical errors as they are suboptimal and prone to bias. Accordingly, the technical and analytical advances that can be used to create high quality assays with minimal error and maximal sensitivity were reviewed. Specifically, the best practices for instrument setup, quality control, panel design, titration, gating, and compensation were described. Adherence to these practices will enhance the validity and reliability of platelet flow cytometry in clinical/research settings. © 2019 International Clinical Cytometry Society.

Atherogenic lipid stress induces platelet hyperactivity through CD36-mediated hyposensitivity to prostacyclin: the role of phosphodiesterase 3A.

Prostacyclin (PGI2) controls platelet activation and thrombosis through a cyclic adenosine monophosphate (cAMP) signaling cascade. However, in patients with cardiovascular diseases this protective mechanism fails for reasons that are unclear. Using both pharmacological and genetic approaches we describe a mechanism by which oxidized low density lipoproteins (oxLDL) associated with dyslipidemia promote platelet activation through impaired PGI2 sensitivity and diminished cAMP signaling. In functional assays using human platelets, oxLDL modulated the inhibitory effects of PGI2, but not a phosphodiesterase (PDE)-insensitive cAMP analog, on platelet aggregation, granule secretion and in vitro thrombosis. Examination of the mechanism revealed that oxLDL promoted the hydrolysis of cAMP through the phosphorylation and activation of PDE3A, leading to diminished cAMP signaling. PDE3A activation by oxLDL required Src family kinases, Syk and protein kinase C. The effects of oxLDL on platelet function and cAMP signaling were blocked by pharmacological inhibition of CD36, mimicked by CD36-specific oxidized phospholipids and ablated in CD36-/- murine platelets. The injection of oxLDL into wild-type mice strongly promoted FeCl3-induced carotid thrombosis in vivo, which was prevented by pharmacological inhibition of PDE3A. Furthermore, blood from dyslipidemic mice was associated with increased oxidative lipid stress, reduced platelet sensitivity to PGI2 ex vivo and diminished PKA signaling. In contrast, platelet sensitivity to a PDE-resistant cAMP analog remained normal. Genetic deletion of CD36 protected dyslipidemic animals from PGI2 hyposensitivity and restored PKA signaling. These data suggest that CD36 can translate atherogenic lipid stress into platelet hyperactivity through modulation of inhibitory cAMP signaling.

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. Recent advances in multiplex flow cytometry (barcoding) allow the rapid acquisition of highly batched samples with standard laboratory equipment. However, many assays have not been standardized, and success is largely dependent on protocol/reagent selection. Accordingly, we review the technical steps that are key to success with an emphasis on fixation, permeabilization, staining, controls, and data visualization.

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

High-Throughput Signaling Profiling in Blood Platelets by Multiplexed Phosphoflow Cytometry.

Multiplexed phosphoflow cytometry is a novel method that provides rapid and quantitative readouts on intracellular phosphoprotein signaling. In this approach, flow cytometry is combined with fluorescent cell barcoding (FCB) to facilitate high-throughput analyses of signaling events. After stimulation, fixed and permeabilized platelets are labeled with distinct dye intensities to create unique fluorescent signatures for individual samples. These uniquely labeled samples can be combined for simultaneous antibody staining and acquisition. During software analysis, multiplexed samples can be differentiated by their distinct fluorescence intensities and analyzed as if they had been acquired individually. Multiplexing eliminates intersample variation, increases statistical robustness, and allows 4-96 samples to be processed with no appreciable increase in antibody consumption or runtime. The method can be performed on washed platelets, platelet-rich plasma (PRP), and whole blood. Its inherent versatility can fulfil wide-ranging experimental requirements from simple dose titrations to complex pharmacologic screens.

Corrigendum: The Effect of a Simulated Commercial Flight Environment With Hypoxia and Low Humidity on Clotting, Platelet, and Endothelial Function in Participants With Type 2 Diabetes - A Cross-Over Study.

[This corrects the article on p. 26 in vol. 9, PMID: 29487564.].

The Effect of a Simulated Commercial Flight Environment with Hypoxia and Low Humidity on Clotting, Platelet, and Endothelial Function in Participants with Type 2 Diabetes - A Cross-over Study.

Aims: To determine if clotting, platelet, and endothelial function were affected by simulated short-haul commercial air flight conditions (SF) in participants with type 2 diabetes (T2DM) compared to controls. Methods: 10 participants with T2DM (7 females, 3 males) and 10 controls (3 females, 7 males) completed the study. Participants were randomized to either spend 2 h in an environmental chamber at sea level conditions (temperature: 23°C, oxygen concentration 21%, humidity 45%), or subject to a simulated 2-h simulated flight (SF: temperature: 23°C, oxygen concentration 15%, humidity 15%), and crossed over 7 days later. Main outcome measures: clot formation and clot lysis parameters, functional platelet activation markers, and endothelial function measured by reactive hyperemia index (RHI) by EndoPAT and serum microparticles. Results: Comparing baseline with SF conditions, clot maximal absorption was increased in controls (0.375 ± 0.05 vs. 0.39 ± 0.05, p < 0.05) and participants with T2DM (0.378 ± 0.089 vs. 0.397 ± 0.089, p < 0.01), while increased basal platelet activation for both fibrinogen binding and P-selectin expression (p < 0.05) was seen in participants with T2DM. Parameters of clot formation and clot lysis, stimulated platelet function (stimulated platelet response to ADP and sensitivity to prostacyclin), and endothelial function were unchanged. Conclusion: While SF resulted in the potential of denser clot formation with enhanced basal platelet activation in T2DM, the dynamic clotting, platelet, and endothelial markers were not affected, suggesting that short-haul commercial flying adds no additional hazard for venous thromboembolism for participants with T2DM compared to controls.