Background: Despite the increasing number of treatments for immune thrombocytopenia (ITP), there are no established laboratory predictors of treatment response. Immature platelet fraction (IPF) and immature platelet count (IPC) are clinical laboratory measurements reflecting bone marrow thrombopoietic activity. The association between IPF and IPC and response to treatments, including thrombopoietin receptor agonists, has not been previously studied in children. Objectives: This study evaluated the relationship among IPF, IPC, and treatment response in children with ITP. Methods: This observational cohort study included 4 pediatric medical centers. Inclusion criteria were a diagnosis of ITP, ITP medication treatment, and an available clinical IPF measurement. Laboratory and clinical data were collected via medical record review. Results: A total of 195 patients were included, with a median age at diagnosis of 7.5 years (range, 0.1-20.7). IPF was inversely associated with platelet count, whereas IPC was positively associated with platelet count. IPC at diagnosis and pretreatment was higher in overall responders than in nonresponders (P = .03 and P = .001, respectively). There were higher pretreatment platelet counts in overall responders than in nonresponders (P < .001), but no differences in IPF at diagnosis or pretreatment between the treatment groups. Pretreatment IPC and platelet count were higher in corticosteroid responders than in nonresponders (P = .046 and P = .005, respectively), and in intravenous immunoglobulin responders than in nonresponders (P = .04 and P = .003, respectively). However, pretreatment IPF, IPC, and platelet counts did not differ between thrombopoietin receptor agonist responders and nonresponders. Conclusion: Higher pretreatment platelet count and IPC correlate with overall treatment response, but the association varies by individual treatment. Biomarkers of treatment response are needed to inform individualized management.

ABSTRACT:Blood platelets are crucial in hemostasis, thrombosis, and thromboinflammation. Current evidence highlights the considerable heterogeneity within individuals in platelet structure, age, and activation properties. This heterogeneity has major implications for the diverse functions of platelets in physiology and pathophysiology, extending to therapeutic targeting in hemostasis and cancer. In this review, we provide a general concept of heterogeneity or diversity of platelet populations, with emphasis on the diagnostic and advanced methodologies to assess and study differences between platelets. We describe conventional and novel approaches to address clinical and in research questions addressing platelet heterogeneity and discuss strengths and limitations of the available techniques.

Millions of platelet units are needed each year to manage thrombocytopenia and other conditions linked to excessive bleeding. These life-saving treatments still depend entirely on donated platelets, despite the numerous shortcomings associated with them, such as limited shelf life, supply shortages, unpredictable functionality, potential for infection, as well as immune-incompatibility issues. These challenges could be overcome with universal donor platelets generated from human induced pluripotent stem cell (hiPSC)-derived megakaryocytes (MKs). We recently developed expandable hiPSC-derived megakaryocytic cell lines (imMKCLs) as a potentially unlimited source for platelet production. imMKCL-derived platelets are functional and have already been tested in patients. In this study, we demonstrate through single-cell time-course imaging that imMKCL maturation is heterogeneous and asynchronous, with only a few imMKCLs generating platelets at any given time under static culture conditions. Using a chemical screen, we identify microtubule (MT) destabilizing agents, including vincristine (VCR), as promising hits, with a larger proportion of VCR-exposed imMKCLs developing proplatelet extensions and more platelets being produced per imMKCL. VCR use reduces the MT content of imMKCLs and results in the production of platelets with a diminished peripheral MT ring structure. Nevertheless, these platelets are functional, as evidenced by their normal response to agonists, their ability to attach to and spread on fibrinogen-coated surfaces, and their capacity to restore hemostasis in vivo. Interestingly, we also observed a negative correlation between the MT content of imMKCLs and platelet yields when we compared imMKCLs differentiated under static conditions (MThigh, low yield) to our turbulence-optimized VerMES™ bioreactor (MTlow, high yield). Taken together, our findings highlight the importance of MT dynamics in megakaryocyte biology, provide a possible explanation for the still poorly understood link between vinca alkaloid in vivo use and thrombocytosis, and bring us closer to realizing the clinical potential of affordable, off-the-shelf hiPSC-derived platelets.

Background:We have reported that trivalent and tetravalent nanobodies against glycoprotein (GP)VI, C-type lectin-like receptor (CLEC)-2, and platelet endothelial aggregation receptor (PEAR)1 stimulate powerful aggregation and adenosine triphosphate secretion in human platelets. Objectives:This study aimed to evaluate changes in platelet surface GPs elicited by activation of GPVI, CLEC-2, and PEAR1 using trivalent and tetravalent nanobodies. Methods:The effect of the crosslinked nanobodies on P-selectin was measured in whole blood and washed platelets with and without secondary mediator inhibitors using classical flow cytometry and on 16 platelet surface GPs in whole blood using multispectral flow cytometry. Results:Trivalent nanobodies to GPVI and CLEC-2 stimulated modest (<60% of collagen-related peptide) expression of P-selectin in whole blood (10-fold dilution) and washed platelets (2 × 107 mL), whereas tetravalent nanobodies induced a response approaching that of collagen-related peptide. Stimulation of P-selectin expression was partially reduced by inhibitors of adenosine diphosphate (ADP) and thromboxane A2, indicating secondary platelet activation despite the low platelet concentration. By multispectral flow cytometry, tetravalent nanobodies to GPVI and CLEC-2 stimulated similar maximal fibrinogen binding and platelet surface α-granule (TLT-1 and CD154) and δ-granule (CD63) markers, but lower levels of the lysosomal marker CD107a. The tetravalent PEAR1 nanobody showed partial agonist activity in some donors but full activity in others. Conclusion:Tetravalent nanobodies to GPVI and CLEC-2 stimulate powerful activation of platelets at low nanomolar concentrations in flow cytometry. In contrast, trivalent nanobodies are partial agonists. The defined stoichiometry of the nanobodies will aid development of standardized platelet flow cytometry assays.

Blood cell aggregates are clinically useful biomarkers in a number of medical disorders. This protocol provides accurate and quantitative analysis of cell aggregates using a small volume of whole blood and imaging flow cytometry. We describe steps for sample collection, staining, and measurement. We then detail gating procedures and analysis of cell morphology. Sample preparation artifacts, activation, and morphological changes of cells are mitigated by omitting erythrocyte lysis and leukocyte isolation while maintaining high-throughput accurate imaging of leukocytes and platelets.

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. We also studied platelet surface glycoprotein IX (GPIX), a cell adhesion receptor which is physically linked to 14-3-3. In infants dying of SIDS compared to infants dying of known causes, we found significantly higher intra-platelet 5-HT and 14-3-3 and lower platelet surface GPIX. Serum and plasma 5-HT were also elevated in SIDS compared to controls. The presence in SIDS of both platelet and brainstem 5-HT and 14-3-3 abnormalities suggests a global dysregulation of these pathways and the potential for platelets to be used as a model system to study 5-HT and 14-3-3 interactions in SIDS. Platelet and serum biomarkers may aid in the forensic determination of SIDS and have the potential to be predictive of SIDS risk in living infants.

Platelets are the smallest blood cells, numbering 150 to 350 x 10(9)/L in healthy individuals. The ability of activated platelets to adhere to an injured vessel wall and form aggregates was first described in the 19th century. Besides their long-established roles in thrombosis and hemostasis, platelets are increasingly recognized as pivotal players in numerous other pathophysiological processes including inflammation and atherogenesis, antimicrobial host defense, and tumor growth and metastasis. Consequently, profound knowledge of platelet structure and function is becoming more important in research and in many fields of modern medicine. This review provides an overview of platelet physiology focusing particularly on the structure, granules, surface glycoproteins, and activation pathways of platelets.

Neonates admitted to neonatal intensive care units are often thrombocytopenic and receive platelet transfusions to prevent or treat bleeding. However, a large recent randomized trial found higher mortality and morbidity among preterm neonates receiving more platelet transfusions. The mechanisms underlying these findings are unknown but are thought to be related to functional differences between the transfused (adult) and recipient's (neonatal) platelets. We recently demonstrated distinct procoagulant, proinflammatory, and proaggregatory platelet subpopulations in adults by 16-color spectral flow cytometry and hypothesized that neonatal and adult platelets would exhibit differential abundance of these subpopulations. The goal of this study was to characterize the platelet subpopulations present in cord vs adult blood in response to agonist stimulation. All studies were IRB approved. Cord blood (CB) was collected from healthy full-term infants born by elective cesarean section. Adult blood was from healthy volunteers free of antiplatelet medications. Anticoagulated CB and adult blood +/- platelet agonist (varied concentrations of adenosine diphosphate (ADP), thrombin receptor activator peptide (TRAP), cross-linked collagen related peptide (CRP), or rhodocytin) was stained with a panel of 15 platelet surface markers (PAC-1, CD61, CD42a, CD29, CD31, CD32, CD62P, CD63, CD107a, CD154, annexin V, TLR9, TLT-1, CD36 and GPVI) and analyzed as described (Spurgeon & Frelinger, Cytometry A 2023). Platelet subsets were identified using FAUST. CB (n=13, 7 male) and adult blood (n=13, 4 male) did not differ significantly with respect to platelet count (206 ± 6.4 vs 256 ± 48, CB vs. adult), immature platelet fraction (3.0 ± 1.3 vs. 2.2 ± 1.4), or mean platelet volume (9.5 ± 0.7 vs. 8.8 ± 0.7). At baseline, of the 15 measured platelet surface markers, only CD42a (GPIX) and CD32 (FcgRIIa) differed, even after correction for platelet size, on CB vs adult platelets (each was 1.7-fold higher on CB platelets). Consistent with prior reports, CB platelets showed significantly lower platelet surface PAC-1 and CD62P with low TRAP. In addition, TLR9 was reduced in CB vs adult platelets with CRP and CRP+TRAP. However, other activation markers (TLT-1, CD63, CD107a and CD154) did not significantly differ between CB and adult platelets activated with other agonists. This led us to investigate lower concentrations of ADP, TRAP and CRP using dose-response curves. Increasing concentrations of ADP, TRAP, and CRP led to dose-dependent increases in 7 activation markers (PAC-1, CD62P, CD63, CD107a, CD154, TLT-1 and TLR9) in CB and adult platelets. The EC50s for TRAP- and CRP-induced increase of these markers were significantly higher for CB vs adult platelets (TRAP 3-fold, range 2.7-3.2; CRP 9.2-fold, range 5.3-12.6) while EC50s for ADP were similar in CB vs adult platelets. These results suggest that the developmental differences in platelet activation are highly agonist- and signaling pathway-dependent, with the GPVI-mediated pathway being the most hyporeactive in neonatal compared to adult platelets. In contrast to the higher TRAP and CRP EC50s in CB vs adults for all markers, the maximal expression levels for 5 of the 7 surface markers (CD63, CD107a, CD154, TLT-1 and TLR9) were similar in CB vs adult platelets, while maximal PAC-1 and CD62P were significantly lower in CB vs adult platelets across all agonists. High-dimensional (FAUST) analysis identified 12 platelet subsets based on the expression patterns of 6 markers (annexin V, PAC-1, CD62P, CD107a, CD63 and TLT-1). These platelet subsets were functionally categorized as resting, proinflammatory, proaggregatory, procoagulant, and combinations thereof. In baseline and agonist-stimulated samples, significant developmental differences in the abundance of subsets were observed. Notably, proinflammatory (FAUST07: Annexin V- PAC-1- CD62P+ CD107a+ CD63+ TLT-1+) platelets were more abundant in CB than adult blood across agonists, while proaggregatory/proinflammatory platelets (FAUST10: Annexin V- PAC-1+ CD62P+ CD107a+ CD63+ TLT-1+) were more abundant in adults. In sum, high-parameter spectral flow cytometry provided new insights into neonatal platelet function and identified developmental differences in the abundance of platelet subpopulations. How these differences impact the neonatal responses to platelet transfusions remains to be investigated.

In summary, whole blood flow cytometry is a powerful laboratory technique for assessment of platelet activation and function. It is used to assess platelet activation, leukocyte-platelet aggregates, platelet aggregation, and measure dense granule release, among others. In addition to the rapid turn around time, this technique is not limited by low platelet counts. Clinical applications of whole blood flow cytometric assays of platelet function in various disease states may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Flow cytometry can also be used for monitoring of glycoprotein IIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.

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.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

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.

Millions of healthy donor platelet (PLT) units are needed each year for therapeutic transfusions of patients with thrombocytopenia and bleeding disorders, but shortages are common. The pandemic worsened donor shortages in the US and Japan. To ensure the predictability of supply, and to mitigate clinical complications associated with conventional donor PLTs, we have developed human induced pluripotent stem cell (hiPSC)-derived megakaryocyte (MK) cell lines, imMKCLs, as a source of in vitro PLT production (iPSC-PLTs)(Nakamura et al., Cell Stem Cell, 2014; Sone et al., Stem Cell Rep, 2021), and manufactured platelets are now in clinical trials using a previously published protocol (Sugimoto et al., Blood, 2022; Sugimoto et al., Blood Adv, 2022). To optimize and improve the in vitro manufacturing protocol of iPSC-PLTs from imMKCLs, we conducted a comprehensive imaging-based compound screening which identified microtubule (MT) destabilizing agents, including the vinca alkaloid vincristine (VCR), that appear to boost proplatelet formation. Combined use of VCR at 10 μM and turbulent hydrodynamic culture conditions (Ito et al., Cell, 2018) allowed production of 23.0 PLTs per imMKCL, 2.5 fold higher than without VCR. This successful result was observed only when VCR was administered at day 3 of 6-days of the maturation phase, when imMKCLs have become polyploid, while exposure at earlier timepoints induced apoptosis. However, PLTs produced in this manner showed poor agonist-induced surface expression of P-selectin or the PAC1 epitope. Therefore, we next examined dose-dependent action of VCR on both yield of iPSC-PLTs and their function. At nanomolar concentrations (10 nM), VCR still exhibited significant PLT biogenesis-boosting activity, but without apparent impairment of PLT in vitro functionality. Interestingly, this low concentration of VCR still resulted in reduced marginal MT band staining in imMKCLs, suggesting a potential role of weakened membrane structure/status in enhanced iPSC-PLT generation. Currently, we are attempting to address if PLTs made from low-dose VCR-exposed imMKCLs have reduced marginal MT bands and whether they restore hemostasis when transfused into immunodeficient thrombocytopenic mice. Our study unveils the potential of VCR as a potent enhancer of PLT biogenesis in the imMKCL system, particularly when combined with optimized turbulent flow production systems. Our results provide a means to make production of iPSC-PLTs more cost-efficient and suggest a mechanism for the previously unexplained observation that vinca alkaloids raise PLT counts in vivo.

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.

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 alpha IIb beta 3 integrin (detected with monoclonal antibody PAC1), P-selectin (CD62P), and lysosomal-associated membrane protein (LAMP3/CD63) in 149 GD patients. Results GD patients had a higher level of unstimulated CD63 expression than healthy subjects, which was mildly correlated with glucosylsphingosine (lyso-Gb1) levels ( r =0.17, p -value=0.042). Splenectomized GD patients had a higher level of unstimulated alpha IIb beta 3 integrin and P-selectin expression. Reduced platelet reactivity (-2 standard deviation of reference range) was found in 79 (53%, 95% confidence interval [CI]: 44-61%) patients, of whom 10 (6.7%, 95% CI: 3.3-12%) had more severe platelet dysfunction. In a multivariate model, only lyso-Gb1 levels were associated with the more severe platelet dysfunction. Fifty-four (49%) of 128 adult patients who completed the bleeding tendency questionnaire reported positive bleeding history. In a multivariate logistic model, older age (odds ratio [OR]: 1.05, 95% CI: 1.01-1.1) and low P-selectin reactivity (OR: 2.03, 95% CI: 1.25-3.35) were associated with more than one bleeding manifestation. Conclusion Flow cytometry enables the study of platelet function in thrombocytopenic GD patients. A platelet degranulation defect, but not alpha IIb beta 3 integrin activation defect, is associated with clinical bleeding. In vivo increased CD63 expression may be related to GD-related inflammation.

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. Using genetic and pharmacologic approaches, as well as iNFATuation, a newly developed mouse model in which NFAT activation can be abrogated in a cell-specific manner, we demonstrated that NFAT inhibition in activated murine and human platelets enhanced their activation and aggregation, as well as their interactions with neutrophils and NET induction. During gram-negative septicemia, NFAT inhibition in platelets promoted disease severity by increasing disseminated coagulation and NETosis. NFAT inhibition also partially restored coagulation ex vivo in patients with hypoactive platelets. Our results define non-transcriptional roles for NFAT that could be harnessed to address pressing clinical needs.

Our objective was to characterize platelet surface glycoprotein (GP)Ib alpha, activated GPIIb-IIIa, and P-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. Platelets from untreated blood samples and samples treated with either adenosine diphosphate (ADP) or thrombin receptor agonist peptide (TRAP) had surface GPIb alpha, activated GPIIb-IIIa, and P-selectin levels measured using flow cytometry. Platelet surface GPIb alpha levels varied significantly by time on ECMO (p = .002) and were significantly higher on ECMO day 5 compared to ECMO day 1 (p = .01). GPIb alpha levels during ECMO did not differ significantly from levels after ECMO decannulation (p = .14). Activated GPIIb-IIIa levels did not change significantly during ECMO, but were significantly higher after ECMO decannulation (p = .04). There were no significant differences in P-selectin levels during ECMO (p = .87) or after ECMO decannulation (p = .41). Platelet surface GPIb alpha and P-selectin levels were similar during and after ECMO whereas activated GPIIb-IIIa levels were lower during ECMO, particularly in response to TRAP stimulation, potentially contributing to ECMO-induced coagulopathy.