O-linked sialic acid residues on platelet membrane glycoprotein IIb mask the human HPA-9b alloepitope.

Sialic acids occupy the terminal position of glycan chains and have the potential to influence the antigenicity of glycoproteins (GP). The polymorphisms of human platelet alloantigens (HPA)-3 and HPA-9, located near the C-terminus of the extracellular domain of platelet membrane GPIIb, are adjacent to sialyl-core 1 O-glycans emanating from serines 845 and 847. Whether the nearby O-glycans affect the antigenicity of HPA-9b or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of neonatal alloimmune thrombocytopenia is unknown. To address this issue, we generated a series of O-glycan mutant HPA-9 allele-specific induced pluripotent stem cell lines, differentiated them to megakaryocytes (MKs), and examined their ability to bind HPA-9b-specific alloantibodies. We found that both wild-type MKs treated with neuraminidase and those genetically modified to lack the sialidases ST3GAL1 and ST3GAL2 dramatically increased anti-HPA-9b alloantibody binding, indicating that the HPA-9b epitope is partially masked by terminal sialic acids on nearby O-glycans of GPIIb. Interestingly, mutating the serine residues that carry these glycan chains to alanine actually reduced the binding of anti-HPA-9b alloantibodies, indicating that these 2 O-glycan chains contribute to the presentation of the HPA-9b epitope-perhaps by stabilizing the conformation of the GP in this region. Collectively, our data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues but retain the glycan chains to which they are attached.

Mechanosensing via a GpIIb/Src/14-3-3ζ axis critically regulates platelet migration in vascular inflammation.

Platelets are not only the first responders in thrombosis and hemostasis but also central players in inflammation. Compared with platelets recruited to thrombi, immune-responsive platelets use distinct effector functions including actin-related protein complex 2/3-dependent migration along adhesive substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cellular level is incompletely understood. Here, we use time-resolved morphodynamic profiling of individual platelets to show that migration, in contrast to clot retraction, requires anisotropic myosin IIa-activity at the platelet rear which is preceded by polarized actin polymerization at the front to initiate and maintain migration. Integrin GPIIb-dependent outside-in signaling via Gα13 coordinates polarization of migrating platelets to trigger tyrosine kinase c-Src/14-3-3ζ-dependent lamellipodium formation and functions independent of soluble agonists or chemotactic signals. Inhibitors of this signaling cascade, including the clinically used ABL/c-Src inhibitor dasatinib, interfere predominantly with the migratory capacity of platelets, without major impairment of classical platelet functions. In murine inflammation models, this translates to reduced migration of platelets visualized by 4D intravital microscopy, resulting in increased inflammation-associated hemorrhage in acute lung injury. Finally, platelets isolated from patients with leukemia treated with dasatinib who are prone to clinically relevant hemorrhage exhibit prominent migration defects, whereas other platelet functions are only partially affected. In summary, we define a distinct signaling pathway essential for migration and provide novel mechanistic insights explaining dasatinib-related platelet dysfunction and bleeding.

A mechanism of platelet integrin αIIbß3 outside-in signaling through a novel integrin αIIb subunit-filamin-actin linkage.

The communication of talin-activated integrin αIIbß3 with the cytoskeleton (integrin outside-in signaling) is essential for platelet aggregation, wound healing, and hemostasis. Filamin, a large actin crosslinker and integrin binding partner critical for cell spreading and migration, is implicated as a key regulator of integrin outside-in signaling. However, the current dogma is that filamin, which stabilizes inactive αIIbß3, is displaced from αIIbß3 by talin to promote the integrin activation (inside-out signaling), and how filamin further functions remains unresolved. Here, we show that while associating with the inactive αIIbß3, filamin also associates with the talin-bound active αIIbß3 to mediate platelet spreading. Fluorescence resonance energy transfer-based analysis reveals that while associating with both αIIb and ß3 cytoplasmic tails (CTs) to maintain the inactive αIIbß3, filamin is spatiotemporally rearranged to associate with αIIb CT alone on activated αIIbß3. Consistently, confocal cell imaging indicates that integrin α CT-linked filamin gradually delocalizes from the ß CT-linked focal adhesion marker-vinculin likely because of the separation of integrin α/ß CTs occurring during integrin activation. High-resolution crystal and nuclear magnetic resonance structure determinations unravel that the activated integrin αIIb CT binds to filamin via a striking α-helix→ß-strand transition with a strengthened affinity that is dependent on the integrin-activating membrane environment containing enriched phosphatidylinositol 4,5-bisphosphate. These data suggest a novel integrin αIIb CT-filamin-actin linkage that promotes integrin outside-in signaling. Consistently, disruption of such linkage impairs the activation state of αIIbß3, phosphorylation of focal adhesion kinase/proto-oncogene tyrosine kinase Src, and cell migration. Together, our findings advance the fundamental understanding of integrin outside-in signaling with broad implications in blood physiology and pathology.

P2Y12 Inhibition Suppresses Proinflammatory Platelet-Monocyte Interactions.

BACKGROUND: Monocyte-platelet aggregates (MPAs) represent the crossroads between thrombosis and inflammation, and targeting this axis may suppress thromboinflammation. While antiplatelet therapy (APT) reduces platelet-platelet aggregation and thrombosis, its effects on MPA and platelet effector properties on monocytes are uncertain. OBJECTIVES: To analyze the effect of platelets on monocyte activation and APT on MPA and platelet-induced monocyte activation. METHODS: Agonist-stimulated whole blood was incubated in the presence of P-selectin, PSGL1, PAR1, P2Y12, GP IIb/IIIa, and COX-1 inhibitors and assessed for platelet and monocyte activity via flow cytometry. RNA-Seq of monocytes incubated with platelets was used to identify platelet-induced monocyte transcripts and was validated by RT-qPCR in monocyte-PR co-incubation ± APT. RESULTS: Consistent with a proinflammatory platelet effector role, MPAs were increased in patients with COVID-19. RNA-Seq revealed a thromboinflammatory monocyte transcriptome upon incubation with platelets. Monocytes aggregated to platelets expressed higher CD40 and tissue factor than monocytes without platelets (p < 0.05 for each). Inhibition with P-selectin (85% reduction) and PSGL1 (87% reduction) led to a robust decrease in MPA. P2Y12 and PAR1 inhibition lowered MPA formation (30 and 21% reduction, p < 0.05, respectively) and decreased monocyte CD40 and TF expression, while GP IIb/IIIa and COX1 inhibition had no effect. Pretreatment of platelets with P2Y12 inhibitors reduced the expression of platelet-mediated monocyte transcription of proinflammatory SOCS3 and OSM. CONCLUSIONS: Platelets skew monocytes toward a proinflammatory phenotype. Among traditional APTs, P2Y12 inhibition attenuates platelet-induced monocyte activation.

An Updated Review on Glycoprotein IIb/IIIa Inhibitors as Antiplatelet Agents: Basic and Clinical Perspectives.

The glycoprotein (GP) IIb/IIIa receptor is found integrin present in platelet aggregations. GP IIb/IIIa antagonists interfere with platelet cross-linking and platelet-derived thrombus formation through the competition with fibrinogen and von Willebrand factor. Currently, three parenteral GP IIb/IIIa competitors (tirofiban, eptifibatide, and abciximab) are approved for clinical use in patients affected by percutaneous coronary interventions (PCI) in the location of acute coronary syndrome (ACS). GP IIb/IIIa antagonists have their mechanism of action in platelet aggregation prevention, distal thromboembolism, and thrombus formation, whereas the initial platelet binding to damage vascular areas is preserved. This work is aimed to provide a comprehensive review of the significance of GP IIb/IIIa inhibitors as a sort of antiplatelet agent. Their mechanism of action is based on factors that affect their efficacy. On the other hand, drugs that inhibit GP IIb/IIIa already approved by the FDA were reviewed in detail. Results from major clinical trials and regulatory practices and guidelines to deal with GP IIb/IIIa inhibitors were deeply investigated. The cardiovascular pathology and neuro-interventional surgical application of GP IIb/IIIa inhibitors as a class of antiplatelet agents were developed in detail. The therapeutic risk/benefit balance of currently available GP IIb/IIa receptor antagonists is not yet well elucidated in patients with ACS who are not clinically evaluated regularly for early cardiovascular revascularization. On the other hand, in patients who have benefited from PCI, the antiplatelet therapy intensification by the addition of a GP IIb/IIIa receptor antagonist (intravenously) may be an appropriate therapeutic strategy in reducing the occurrence of risks of thrombotic complications related to the intervention. Development of GP IIb/IIIa inhibitors with oral administration has the potential to include short-term antiplatelet benefits compared with intravenous GP IIb/IIIa inhibitors for long-term secondary preventive therapy in cardiovascular disease. But studies showed that long-term oral administration of GP IIb/IIIa receptor inhibitors has been ineffective in preventing ischemic events. Paradoxically, they have been linked to a high risk of side effects by producing prothrombotic and pro-inflammatory events.

Inflammation-mediated changes in haemostatic variables of pulmonary tuberculosis patients during treatment.

Tuberculosis (TB) disease is usually marked by inflammation which is closely linked to haemostasis both in health and disease. Close monitoring of haemostatic response to inflammatory changes during treatment is important to improve TB management. Here we studied associations between haemostatic markers and inflammatory cytokines in 60 TB-infected individuals, aged 18-65 years who received anti-TB therapy. They were recruited before commencement of therapy and followed up till completion of therapy after 6-months. The TNF-α, IL-6, IL-2 (pro-inflammatory cytokines) and P-selectin, GP IIb/IIIa, thrombopoietin (haemostatic variables) were significantly increased at 2 month into therapy compared to pre-treatment values and decreased at 6 month into therapy. Also at 6 month into therapy in comparison to 2-month into therapy, there were significant increase in IL-10 and TGF-ß (anti-inflammatory cytokines) as well as a significant decline in PF-4. There were significant positive correlations between GP IIb/IIIa and TNF-α, IL-6 and PSEL, IL-6 and TPO, PF4 and TGF-ß. Conclusively, the changes in the TNF-α, IL-6, IL-2 aligned with changes in the levels of P-selectin, GP IIb/IIIa, and TPO in the course of TB therapy. This may suggest that the levels of inflammatory cytokines are linked to the levels of these haemostatic variables in TB individuals.

Hemostatic Effect of 20(S)-Panaxadiol by Induced Platelet Aggregation Depending on Calcium Signaling Pathway.

Panax notoginseng (Burk.) F.H. Chen is the most traditional hemostatic herb in China. Our previous research found that 20(S)-protopanaxadiol showed the hemostatic effect. And 20(S)-panaxadiol (PD) has a similar structure to 20(S)-protopanaxadiol with a dammarane skeleton. So, this article mainly studies the hemostatic effect of PD. The mouse tail amputation and liver scratch models were used to detect the hemostatic effect of PD. Blood routine and plasma coagulation parameters were measured by using a blood analyzer. The platelet aggregometer analyzed the platelet aggregation rate and adenosine triphosphate (ATP) concentration. Moreover, the intracellular calcium concentration ([Ca2+] i ), P-selectin (CD62P), PAC-1 (GP IIb/IIIa receptor marker), and cyclic adenosine monophosphate (cAMP) of platelets were also detected. The results showed that PD obviously shortened the bleeding time of the model mouse, affected the RBC and PLT parameters of rats, reduced APTT and TT, elevated FIB concentration, and promoted human/rat-washed platelet aggregation in vitro. PD promoted the release of ATP and [Ca2+] i and slightly increased the expression of CD62P and PAC-1 of platelets without 1 mM Ca2+. After adding 1 mM Ca2+, PD obviously increased ATP releasing and CD62P and GP IIb/IIIa expression rate and decreased the cAMP level of platelets. These parameter changes of PD-caused platelet were inhibited by vorapaxar. Besides, PD increased the phosphorylation of phosphoinositide 3-kinase/protein kinase B/glycogen synthase kinase 3ß (PI3K/Akt/GSK3ß) of human platelets. PD is an important hemostatic ingredient in Panax notoginseng, which induced platelet aggregation by affecting the calcium signaling and activating the PI3K/Akt/GSK3ß signaling pathway.

Effects of Momordica charantia exosomes on platelet activation, adhesion, and aggregation.

The platelets play a crucial role in the progression of multiple medical conditions, such as stroke and tumor metastasis, where antiplatelet therapy may be a boon for treating these diseases. In this study, we have attempted to study the effects of extracted Momordica charantia exosomes (MCEs) on platelet activation, adhesion, and aggregation. Adult platelets isolated from healthy individuals were dose-dependently treated with MCEs (0.1, 40, and 200 µg/ml). We performed flow cytometry to detect the expression of platelet activation protein marker-activated GP IIb/IIIa (PAC-1) and P-selectin (CD62P). Platelet adhesion was analyzed through fluorescence labeling assays. The effect of MCEs on platelet-mediated cell migration of HCT116 cells was observed by transwell. Furthermore, the MCAO model of Sprague-Dawley rats was used to observe the effect of MCEs (200, 400, and 800 µg/kg) on platelet aggregation and maximum thrombotic agglutination in vivo . The results showed that 200 µg/ml MCEs exerted the most pronounced effect on platelet activation, adhesion, and aggregation. Experiments on animals showed that MCEs significantly inhibited platelet aggregation and attenuated the maximum thrombus agglutination. We concluded that MCEs inhibited platelet activation, adhesion, aggregation, and platelet-mediated migration of HCT116 cells, indicating the potential role MCEs may play in the treatment of stroke and tumor metastasis.

The role of platelet glycoprotein IIb / IIIa inhibitors in current treatment of acute coronary syndrome.

Current management of patients with acute coronary syndrome (ACS) includes a dual antiplatelet therapy with acetylsalicylic acid and a platelet P2Y12 receptor inhibitor. For patients without a high risk of bleeding, prasugrel and ticagrelor are preferred, since their effect is more pronounced, less dependent on metabolism of a specific patient, and occurs faster that the effect of clopidogrel. The prescription rate of platelet glycoprotein IIb/IIIa (GP IIb / IIIa) receptor inhibitors has considerably decreased. However, these drugs remain relevant in percutaneous coronary interventions in patients with a high risk of coronary thrombosis or a massive coronary thrombus, in thrombotic complications of the procedure, and in the "no-reflow" phenomenon. The intravenous route of GP IIb / IIIa inhibitor administration provides their effectiveness in patients with difficulties of drug intake or with impaired absorption of oral medications. This review presents clinical and pharmacological characteristics of various GP IIb / IIIa inhibitors and data of randomized clinical studies and registries of recent years that evaluated results of their use in patients with ACS.

Genetic Deletion of HLJ1 Does Not Affect Blood Coagulation in Mice.

HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.

Analysis of Integrin αIIb Subunit Dynamics Reveals Long-Range Effects of Missense Mutations on Calf Domains.

Integrin αIIbß3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbß3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIbCalf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf-1 + Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.

Binding of phosphatidylserine-positive microparticles by PBMCs classifies disease severity in COVID-19 patients.

Infection with SARS-CoV-2 is associated with thromboinflammation, involving thrombotic and inflammatory responses, in many COVID-19 patients. In addition, immune dysfunction occurs in patients characterised by T cell exhaustion and severe lymphopenia. We investigated the distribution of phosphatidylserine (PS), a marker of dying cells, activated platelets and platelet-derived microparticles (PMP), during the clinical course of COVID-19. We found an unexpectedly high amount of blood cells loaded with PS+ PMPs for weeks after the initial COVID-19 diagnosis. Elevated frequencies of PS+ PMP+ PBMCs correlated strongly with increasing disease severity. As a marker, PS outperformed established laboratory markers for inflammation, leucocyte composition and coagulation, currently used for COVID-19 clinical scoring. PS+ PMPs preferentially bound to CD8+ T cells with gene expression signatures of proliferating effector rather than memory T cells. As PS+ PMPs carried programmed death-ligand 1 (PD-L1), they may affect T cell expansion or function. Our data provide a novel marker for disease severity and show that PS, which can trigger the blood coagulation cascade, the complement system, and inflammation, resides on activated immune cells. Therefore, PS may serve as a beacon to attract thromboinflammatory processes towards lymphocytes and cause immune dysfunction in COVID-19.

An alternate covalent form of platelet αIIbß3 integrin that resides in focal adhesions and has altered function.

The αIIbß3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbß3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbß3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis.

Megakaryocytes (MKs), the platelet progenitor cells, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N) and performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within 3 distinct subpopulations that possess gene signatures related to platelet generation, HSC niche interaction, and inflammatory responses. In situ immunostaining of mouse BM demonstrated that platelet generation and the HSC niche-related MKs were in close physical proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under blood shear forces, were predominantly formed on a platelet generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented ∼5% of total MKs in the BM. These MKs could specifically respond to pathogenic infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a preexisting PU.1- and IRF-8-associated monocytic-like transcriptional program involved in pathogen recognition and clearance as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable of engulfing and digesting bacteria and stimulating T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.

Protein analysis of extracellular vesicles to monitor and predict therapeutic response in metastatic breast cancer.

Molecular profiling of circulating extracellular vesicles (EVs) provides a promising noninvasive means to diagnose, monitor, and predict the course of metastatic breast cancer (MBC). However, the analysis of EV protein markers has been confounded by the presence of soluble protein counterparts in peripheral blood. Here we use a rapid, sensitive, and low-cost thermophoretic aptasensor (TAS) to profile cancer-associated protein profiles of plasma EVs without the interference of soluble proteins. We show that the EV signature (a weighted sum of eight EV protein markers) has a high accuracy (91.1 %) for discrimination of MBC, non-metastatic breast cancer (NMBC), and healthy donors (HD). For MBC patients undergoing therapies, the EV signature can accurately monitor the treatment response across the training, validation, and prospective cohorts, and serve as an independent prognostic factor for progression free survival in MBC patients. Together, this work highlights the potential clinical utility of EVs in management of MBC.

Platelet-enhanced plasma: Characterization of a novel candidate resuscitation fluid's extracellular vesicle content, clotting parameters, and thrombin generation capacity.

BACKGROUND: Platelet transfusion is challenging in emergency medicine because of short platelet shelf life and stringent storage conditions. Platelet-derived extracellular vesicles (PEV) exhibit platelet-like properties. A plasma generated from expired platelet units rich in procoagulant PEV may be able to combine the benefits of plasma and platelets for resuscitation while increasing shelf life and utilizing an otherwise wasted resource. STUDY DESIGN AND METHODS: Freeze-thaw cycling of platelet-rich plasma (PRP) followed by centrifugation to remove platelet remnants was utilized to generate platelet-enhanced plasma (PEP). An in vitro model of dilutional coagulopathy was also designed and used to test PEP. Rotational thromboelastometry and calibrated automated thrombography were used to assess clotting and extracellular vesicles (EV) procoagulant activity. Capture arrays were used to specifically measure EV subpopulations of interest (ExoView™, NanoView Biosciences). Captured vesicles were quantified and labeled with Annexin-V-FITC, CD41-PE, and CD63-AF647. Platelet alpha granule content (platelet-derived growth factor AB, soluble P-selectin, vascular endothelial growth factor A, and neutrophil activating peptide 2-chemokine (C-X-C motif) ligand 7) was measured. Commercially available platelet lysates were also characterized. RESULTS: PEP is highly procoagulant, rich in growth factors, exhibits enhanced thrombin generation, and restores hemostasis within an in vitro model of dilutional coagulopathy. The predominant vesicle population were PEV with 7.0 × 109 CD41+PS+ EV/ml compared to 4.7 × 107 CD41+PS+ EV/ml in platelet-free plasma (p = .0079). Commercial lysates show impaired but rescuable clotting. DISCUSSION: PEP is a unique candidate resuscitation fluid containing high PEV concentration with preliminary evidence, indicating a potential for upscaling the approach using platelet concentrates. Commercial lysate manufacturer workflows may be suitable for this, but further optimization and characterization of PEP is required.

Platelet association with leukocytes in active eosinophilic esophagitis.

We previously demonstrated that the percentage of blood eosinophils that are associated with platelets and thus positive for CD41 (integrin αIIb-subunit) correlates with and predicts peak eosinophil count (PEC) in biopsies of eosinophilic esophagitis (EoE) patients after treatment. Thus, flow cytometric determination of CD41+ eosinophils is a potential measure of EoE disease activity. Determinants of association of platelets with eosinophils and other leukocytes in EoE are largely unknown. The objectives of this study were to test the hypotheses that platelets associate with blood leukocytes other than eosinophils in EoE and that such associations also predict EoE activity. Whole blood flow cytometry was performed on samples from 25 subjects before and after two months of standard of care EoE treatment. CD41 positivity of cells within gates for eosinophils, neutrophils, monocytes, lymphocytes, and natural killer cells was compared. We found that percent CD41+ neutrophils, monocytes, and eosinophils correlated with one another such that principal component analysis of the five cell types identified "myeloid" and "lymphoid" factors. Percent CD41+ neutrophils or monocytes, or the myeloid factor, like CD41+ eosinophils, correlated with PEC after treatment, and CD41+ neutrophils or the myeloid factor predicted PEC < 6/high power field after treatment, albeit with lower area under the curve than for CD41+ eosinophils. We conclude that the processes driving platelets to associate with eosinophils in EoE also drive association of platelets with neutrophils and monocytes and that association of platelets with all three cell types is related to disease activity. Clinicaltrials.gov identifier: NCT02775045.

JAK2-V617F and interferon-α induce megakaryocyte-biased stem cells characterized by decreased long-term functionality.

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.