Phosphatidylserine Expression Research Articles

Ponatinib Induces a Procoagulant Phenotype in Human Coronary Endothelial Cells via Inducing Apoptosis.

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective drugs in the treatment of patients with chronic myeloid leukemia. However, based on clinical studies, ponatinib was associated with the development of thrombotic complications. Since endothelial cells (ECs) regulate blood coagulation, their abnormal phenotype may play a role in the development of thrombotic events. We here aimed to investigate the effect of ponatinib on the procoagulant activity of cultured endothelial cells in vitro. Human coronary artery endothelial cells (HCAECs) were incubated with 50, 150, and 1000 nM of ponatinib. Subsequently, phosphatidylserine (PS) exposure and endothelial microvesicles (EMVs) were measured by flow cytometry. In addition, EC- and EMV-dependent thrombin generation was analyzed. To investigate pro-apoptotic effects of ponatinib, the level of Bax and Bcl-xL proteins were studied using Western blot and F3, THBD, and VCAM1 mRNAs were quantified by qPCR. Therapeutic concentrations of ponatinib significantly increased PS expression on ECs and the amount of EMVs which significantly shortened the time parameters of thrombin generation. In addition, these changes were associated with an increased ratio of Bax and Bcl-xL proteins in the presence of the decreased THBD mRNA level. Overall, ponatinib enhances the procoagulant activity of ECs via inducing apoptosis, which may contribute to thrombotic events.

Whole Blood Storage Duration Alters Fibrinogen Levels and Thrombin Formation.

Whole blood resuscitation for hemorrhagic shock in trauma represents an opportunity to correct coagulopathy in trauma while also supplying red blood cells. The production of microvesicles in stored whole blood and their effect on its hemostatic parameters have not been described in previous literature. We hypothesized that microvesicles in aged stored whole blood are procoagulant and increase thrombin production via phosphatidylserine. Whole blood was obtained from male C57BL/6 male mice and stored in anticoagulant solution for up to 10 days. At intervals, stored whole blood underwent examination with rotational thromboelastography and platelet poor plasma was prepared for analysis of thrombin generation. Microvesicles were prepared from 10 day old whole blood aliquots and added to fresh whole blood or platelet poor plasma to assess changes in coagulation and thrombin generation. Microvesicles were treated with recombinant mouse lactadherin prior to addition to plasma to inhibit phosphatidylserine's role in thrombin generation. Aged murine whole blood had decreased fibrin clot formation compared to fresh samples with decreased plasma fibrinogen levels. Thrombin generation in plasma from aged blood increased over time of storage. The addition of microvesicles to fresh plasma resulted in increased thrombin generation compared to controls. When phosphatidylserine on microvesicles was blocked with lactadherin, there was no difference in the endogenous thrombin potential but the generation of thrombin was blunted with lower peak thrombin levels. Cold storage of murine whole blood results in decreased fibrinogen levels and fibrin clot formation. Aged whole blood demonstrates increased thrombin generation and this is due in part to microvesicle production in stored whole blood. One mechanism by which microvesicles are procoagulant is by phosphatidylserine expression on their membranes. Basic Science.

Comprehensive functional characterization of a novel ANO6 variant in a new patient with Scott syndrome

BackgroundScott syndrome is a mild platelet-type bleeding disorder, first described in 1979, with only 3 unrelated families identified through defective phosphatidylserine (PS) exposure and confirmed by sequencing. The syndrome is distinguished by impaired surface exposure of procoagulant PS on platelets after stimulation. To date, platelet function and thrombin generation in this condition have not been extensively characterized. ObjectivesGenetic and functional studies were undertaken in a consanguineous family with a history of excessive bleeding of unknown cause. MethodsA targeted gene panel of known bleeding and platelet genes was used to identify possible genetic variants. Platelet phenotyping, flow adhesion, flow cytometry, whole blood and platelet-rich plasma thrombin generation, and specialized extracellular vesicle measurements were performed. ResultsWe detected a novel homozygous frameshift variant, c.1943del (p.Arg648Hisfs∗23), in ANO6 encoding Anoctamin 6, in a patient with a bleeding history but interestingly with normal ANO6 expression. Phenotyping of the patient’s platelets confirmed the absence of PS expression and procoagulant activity but also revealed other defects including reduced platelet δ granules, reduced ristocetin-mediated aggregation and secretion, and reduced P-selectin expression after stimulation. PS was absent on spread platelets, and thrombi formed over collagen at 1500/s. Reduced thrombin generation was observed in platelet-rich plasma and confirmed in whole blood using a new thrombin generation assay. ConclusionWe present a comprehensive report of a patient with Scott syndrome with a novel frameshift variant in AN06, which is associated with no platelet PS exposure and markedly reduced thrombin generation in whole blood, explaining the significant bleeding phenotype observed.

Red Blood Cell Contribution to Thrombosis in Polycythemia Vera and Essential Thrombocythemia.

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPN) characterized by clonal erythrocytosis and thrombocytosis, respectively. The main goal of therapy in PV and ET is to prevent thrombohemorrhagic complications. Despite a debated notion that red blood cells (RBCs) play a passive and minor role in thrombosis, there has been increasing evidence over the past decades that RBCs may play a biological and clinical role in PV and ET pathophysiology. This review summarizes the main mechanisms that suggest the involvement of PV and ET RBCs in thrombosis, including quantitative and qualitative RBC abnormalities reported in these pathologies. Among these abnormalities, we discuss increased RBC counts and hematocrit, that modulate blood rheology by increasing viscosity, as well as qualitative changes, such as deformability, aggregation, expression of adhesion proteins and phosphatidylserine and release of extracellular microvesicles. While the direct relationship between a high red cell count and thrombosis is well-known, the intrinsic defects of RBCs from PV and ET patients are new contributors that need to be investigated in depth in order to elucidate their role and pave the way for new therapeutical strategies.

Quantification of Phosphatidylserine Molecules on the Surface of Individual Cells Using Single-Molecule Force Spectroscopy.

Identification of the phosphatidylserine (PS) discrepancies occurring on the cellular membrane during apoptotic processes is of the utmost importance. However, monitoring the quantity of PS molecules in real-time at a single-cell level currently remains a challenging task. Here, we demonstrate this objective by leveraging the specific binding and reversible interaction exhibited by the zinc(II) dipyridinamine complex (ZnDPA) with PS. Lipoic acid-functionalized ZnDPA (LP-ZnDPA) was subsequently immobilized onto the surface of an atomic force microscopy cantilever to form a force probe, ALP-ZnDPA, enabling a PS-specific dynamic imaging and detection mode. By utilizing this technique, we can not only create a heat map of the expression level of PS with submicron resolution but also quantify the number of molecules present on a single cell's surface with a detection limit of 1.86 × 104 molecules. The feasibility of the proposed method is demonstrated through the analysis of PS expression levels in different cancer cell lines and at various stages of paclitaxel-induced apoptosis. This study represents the first application of a force probe to quantify PS molecules on the surface of individual cells, providing insight into dynamic changes in PS content during apoptosis at the molecular level and introducing a novel dimension to current detection methodologies.

Examination of Bovine Red Blood Cell Death in Vitro in Response to Pathophysiologic Proapoptotic Stimuli.

Interspecies variations in mammalian red blood cells (RBCs) are observed in circulating RBC lifespan, cell size, fluidity, aggregation, water permeability, metabolism, lipid composition, and the overall proteome. Bovine RBC cell membrane is deficient in phosphatidylcholine and exhibits anomalies in the arrangement of phosphatidylethanolamine within the lipid bilayer. However, like human RBCs, virtually all the aminophospholipid phosphatidylserine (PS) is found within the cytoplasmic side of the cell membrane of intact circulating bovine RBCs. During apoptotic cell death of human and murine RBCs, PS translocates to the outer leaflet of the cell membrane via Ca2+-dependent and -independent signaling mechanisms. However, little is known about this process in bovine RBCs. Using cytofluorometry analyses, we characterized and compared the cell death responses in bovine and human RBCs in vitro exposed to various pathophysiologic cell stressors. Ionic stress, by ionophore treatment, and oxidative stress enhanced cytoplasmic Ca2+ levels and cell membrane PS expression in both bovine and human RBCs. Fever-grade hyperthermia and energy starvation promoted Ca2+ influx and elevated reactive oxygen species levels in both human and bovine RBCs. However, bovine RBCs displayed minimal increases in PS expression elicited by hyperthermia, energy starvation, and extracellular hypertonicity as compared to human RBCs. In response to decreased extracellular osmolality, bovine RBCs exhibited significantly enhanced fragility as compared to human RBCs. Bovine RBCs display differential cell death patterns as compared to human RBCs, only partly explained by increased Ca2+ influx and oxidative stress. Premature removal of circulating RBCs could potentially contribute to the pathogenesis of anemia in cattle caused by a wide range of factors such as systemic diseases, parasitic infections, and nutritional deficiencies.

Turbulence Enhances Fine-Tuning of Mitochondria Delivery in Megakaryocyte Maturation Contributing to Biogenesis of High Quality Platelets

Platelet (PLT) products have a short shelf life of 4-5 days but blood donations are limited. As a solution, we established induced pluripotent stem cell (iPSC)-derived immortalized megakaryocyte cell lines (imMKCLs) (Nakamura, Cell Stem Cell, 2014), which generated platelet-like particles (iPSC-PLTs) under static conditions, albeit with low yield and function. Subsequently, by developing turbulence-flow bioreactors, imMKCLs generated high yield of competent iPSC-PLTs (Ito, Cell, 2018). Meanwhile, it is still unclear the reason why turbulence stimulation into imMKCLs allow higher production efficiency and improved function. Herein we found turbulence increases CD42b (GPIbα) expression in iPSC-PLTs, which was inversely correlated with phosphatidylserine (PS) expression levels (as represented by Annexin V binding). Only CD42b + population of iPSC-PLTs responded to agonist stimulation, detected by P-selectin expression or PAC1 binding. We demonstrated functional CD42b + iPSC-PLTs are determined by mitochondria activity in iPSC-PLTs, but not in imMKCLs. Regarding CD42b expression, it is known that ADAM17 causes CD42b shedding upon exposure of phosphatidylserine on the cell surface (Sommer, Nature communications, 2016). Therefore, we examined whether mitochondria activity influence CD42b shedding. To test this, we added FCCP, a mitochondrial uncoupler depleting mitochondria membrane potential, in the absence or presence of shedding inhibitors in imMKCL culture. Depleting mitochondria activity decreased CD42b expression levels on iPSC-PLTs, independently of culture conditions. Pre-incubation with ADAM17 inhibitor or different type shedding inhibitors prior to FCCP treatment allowed to retain CD42b levels, suggesting that mitochondria function might regulate downstream ADAM17 activity or shedding mechanisms. This was confirmed by GPIX deficiency in imMKCLs, as no difference in mitochondria activity was found compared to the control. Differences in CD42b levels were not seen in MKs during maturation, independent of culture conditions, therefore we suspected turbulence promote intact mitochondria delivery in imMKCLs. To examine this hypothesis, we used Mdivi-1, which inhibit Drp1 activity and thereby suppress mitochondria fission, to mimic impaired mitochondria delivery. Administration of Mdivi-1 to turbulence-exposed imMKCLs decreased CD42b expression in subsequently generated iPSC-PLTs. Using confocal microscopy, we also visualized fused mitochondria in imMKCLs upon addition of Mdivi-1, strongly suggesting that negative CD42b expression in iPSC-PLTs is due to impaired mitochondria delivery. Next, to gain mechanistic insight at molecular levels, we differentiated imMKCLs in static or turbulence conditions and swapped the culture conditions on day 3 of the total 6 days of imMKCL maturation. It revealed that turbulence is required only after day 3 onwards, but not before. scRNA analysis and pharmacological examination results strongly suggested that increased HDAC5, 6, and 9 levels by turbulence stimulation are crucial for efficient mitochondria delivery into iPSC-PLTs. We conclude that turbulence increases ‘fine-tuning’ of mitochondria distribution in imMKCLs and efficient delivery to PLTs with relatively sufficient activity via an HDAC mediated mechanism, where mitochondria activity is required for inhibition of phosphatidylserine exposure on the cell surface and of ADAM17 activity.

BIOM-15. CIRCULATING EXOSOMES CORRELATE WITH RADIOGRAPHIC PROGRESSION IN GLIOBLASTOMA PATIENTS AFTER ULTRASOUND-BASED OPENING OF THE BLOOD-BRAIN BARRIER

Abstract Non-invasive methods to detect progression of glioblastoma (GBM) are needed to optimize patient management. Circulating blood-based biomarkers for GBM progression are limited by the blood-brain barrier, which restricts leakage of tumor-related elements into circulation. To address this, we investigated circulating GBM-associated exosomes using samples obtained as part of a clinical trial where the blood-brain barrier was temporarily opened with a skull-implantable ultrasound system (NCT04528680). Samples were collected after an intra-operative sonication procedure at multiple time points, as well as longitudinally before and after subsequent outpatient sonication cycles. Plasma exosomes were isolated using a newExoChip microfluidic device, and enrichment of tumor-derived exosomes was based on expression of Phosphatidylserine (which binds to Annexin V on the device). Tumoral origin of the exosomes was validated with GBM cell lines as positive controls, using Western blots and variant-specific ddPCR to confirm tumor contents. We observed a 2.1-fold increase in exosome concentration in plasma samples obtained intraoperatively between 2 and 45 minutes after sonication, relative to pre-operative baseline (n = 4 patients; 19 plasma samples; one-tailed t-test p = 0.02). In a longitudinal analysis of eight patients, volumetric MRI analysis of enhancement was compared with changes in exosome concentration after sonication. Among patients with short progression-free survival (PFS) (n = 4; PFS < 90 days), multi-subject analysis yielded a repeated-measures correlation of -0.59 (p = 0.06), compared to 0.01 among long PFS patients (p = 0.96). In both cohorts, use of raw pre-sonication exosome concentrations had less association with MRI enhancement (p = 0.67 and 0.98 respectively). Considering each sample independently, we found the average Pearson correlation among short PFS patients was 0.73 (avg. p = 0.39), while no correlation was seen among long PFS patients (avg. r value = 0.10; avg. p-value = 0.54). Circulating exosomes hold promise as a non-invasive method to track GBM progression upon opening of the blood-brain barrier. Further refinement and validation in larger cohorts is warranted.

Lyophilized apoptotic vesicle-encapsulated adhesive hydrogel sponge as a rapid hemostat for traumatic hemorrhage in coagulopathy

Rapid hemostasis of uncontrolled bleeding following traumatic injuries, especially accompanied by coagulopathies, remains a significant clinical challenge. Extracellular vesicles (EVs) show therapeutic effects for fast clotting. However, low yield, specific storage conditions, and lack of proper carriers have hindered EVs’ clinical application. Herein, we establish an optimized procedure method to generate lyophilized mesenchymal stem cell-derived apoptotic vesicles (apoVs) with adhesive hydrogel sponge to show superior procoagulant activity for traumatic hemorrhage. Mechanistically, apoVs’ procoagulant ability stems from their high tissue factor (TF) and phosphatidylserine (PS) expression independent of hemocytes and circulating procoagulant microparticles (cMPs). Their stable hemostatic capability was maintained after 2-month room temperature storage. Subsequently, we mixed apoVs with both phenylboronic acid grafted oxidized hyaluronic acid (PBA-HA) and poly(vinyl alcohol) (PVA) simultaneously, followed by lyophilization to construct a novel apoV-encapsulated hydrogel sponge (apoV-HS). Compared to commercial hemostats, apoV-HS exhibits rapid procoagulant ability in liver-laceration and femoral artery hemorrhage in rat and rabbit models of coagulopathies. The combination of high productivity, physiological stability, injectability, plasticity, excellent adhesivity, biocompatibility, and rapid coagulant property indicates that apoV-HS is a promising therapeutic approach for heavy hemorrhage in civilian and military populations.Graphical

Heparin-activated procoagulant platelet assay: a flow cytometry-based functional test for heparin-induced thrombocytopenia

BackgroundFunctional platelet activation assays are required for the diagnosis of heparin-induced thrombocytopenia (HIT). Due to their sophisticated methodology, they are only available in reference centers. ObjectivesTo evaluate the diagnostic accuracy of the flow cytometry-based heparin-activated procoagulant platelet (HAPP) assay in the laboratory diagnosis of HIT. MethodsProcoagulant platelets (PCP), defined by the expression of phosphatidylserine and CD62-P, were evaluated by flow cytometry in platelet-rich plasma from healthy donors after incubation with patient sera in the absence and presence of heparin. A sample was considered positive in HAPP assay, if the following 3 criteria were met: 1) the percentage of PCPs was ≥10.3% after incubation with 0.2 IU/mL heparin, 2) the fold increase in presence of 0.2 IU/mL heparin compared with buffer was ≥1.5, and 3) 100 IU/mL of heparin resulted in ≥50% inhibition of PCP. HAPP assay was validated in a prospective cohort (n = 202) of consecutive specimens submitted to our laboratory for serologic diagnosis of HIT. Heparin-induced platelet activation (HIPA) assay was used as the reference standard. ResultsHIT-positive sera induced PCPs in the presence of 0.2 IU/mL heparin, which was inhibited with 100 IU/mL of heparin. In the prospective validation cohort, there were 15 HIPA+ and 187 HIPA- sera. HAPP was positive in 20 samples in this cohort. Using optimized cut-offs, HAPP assay had a sensitivity of 93.3% and specificity of 96.8%. ConclusionHAPP assay is promising as a simple and reliable functional assay for HIT; however, further studies are needed to confirm our results in larger cohorts.

Platelet and endothelial cell responses under concurrent shear stress and tensile strain

Thrombosis can lead to significant mortality and morbidity. Both platelets and vascular endothelial cells play significant roles in thrombosis. Platelets' response to blood flow-induced shear stress can vary greatly depending on shear stress magnitude, pattern and shear exposure time. Endothelial cells are also sensitive to the biomechanical environment. Endothelial cell activation and dysfunction can occur under low oscillatory shear stress and low tensile strain. Platelet and endothelial cell interaction can also be affected by mechanical conditions. The goal of this study was to investigate how blood flow-induced shear stress, vascular wall tensile strain, platelet-endothelial cell stress history, and platelet-endothelial cell interaction affect platelet thrombogenicity. Platelets and human coronary artery endothelial cells were pretreated with physiological and pathological shear stress and/or tensile strain separately. The pretreated cells were then put together and exposed to pulsatile shear stress and cyclic tensile strain simultaneously in a shearing-stretching device. Following treatment, platelet thrombin generation rate, platelet and endothelial cell activation, and platelet adhesion to endothelial cells was measured. The results demonstrated that shear stress pretreatment of endothelial cells and platelets caused a significant increase in platelet thrombin generation rate, cell surface phosphatidylserine expression, and adhesion to endothelial cells. Shear stress pretreatment of platelets and endothelial cells attenuated endothelial cell ICAM-1 expression under stenosis conditions, as well as vWF expression under recirculation conditions. These results indicate that platelets are sensitized by prior shearing, while in comparison, the interaction with shear stress-pretreated platelets may reduce endothelial cell sensitivity to pathological shear stress and tensile strain.

Effect of hydroxyurea on erythrocyte apoptosis in hemoglobinopathy patients

ABSTRACT Background Hydroxyurea (HU) therapy improves the clinical severity of patients with hemoglobinopathies. Few studies have documented some mechanisms of HU, but the exact mechanism of action is unknown. Phosphatidylserine on erythrocytes is responsible for apoptosis. In this study, we investigate the expression of phosphatidylserine on the erythrocytes surface of hemoglobinopathies before and after HU treatment. Research Designs and Methods Blood samples from 45 thalassemia intermedia and 40 SCA and 30 HbE-b-thalassemia patients were analyzed before and after 3 and 6 months of HU treatment. The profile of phosphatidylserine was determined by flow-cytometry using the Annexin V-RBC apoptosis kit. Results Hydroxyurea proved effective in improving clinical severity of hemoglobinopathies. After treatment with hydroxyurea, the percentage of phosphatidylserine-positive cells was significantly reduced in all 3 patient groups (p < 0.0001). Correlation analysis using different hematological parameters as independent variables and % phosphatidylserine as dependent variable showed a negative relationship with HbF, RBC, and hemoglobin in all 3 patient groups. Conclusion Hydroxyurea reduces the expression of phosphatidylserine on erythrocytes, contributing to the beneficial effects of this therapy. We suggest that the use of such a biological marker in conjunction with HbF levels may provide valuable insights into the biology and consequences of early RBC apoptosis.

Feasibility evaluation of two novel systems for the automated preparation and extended storage of DMSO cryopreserved platelets.

Manufacturing methods for dimethyl sulfoxide (DMSO)-cryopreserved platelets (CPPs) are manual and labor intensive. Thawing and prepare-for-transfusion steps are in an open system that requires transfusion within 4 h. A fill-and-finish system (CUE) can automate the manufacturing process. A newly configured bag system allows freezing, thawing, and use of resuspension solutions while maintaining the functionally closed system, and extending the post-thaw shelf life beyond 4 h. Our objective is to evaluate the feasibility of the CUE system and the functionally closed bag system. DMSO was volumetrically added to double-dose apheresis platelets, concentrated, and delivered to a 50- or 500-mL ethylene-vinyl acetate (EVA) bag by the CUE (n = 12). The functionally closed bag system contained 25 mL platelet additive solution 3 (PAS-3) in a 50-mL EVA bag. Control CPP (n = 2) were manually prepared. PAS-3 and CPP were thawed together. CPP were stored up to 98 h (20-24°C) and tested using a standard assay panel. CUE prepared CPP met the design targets: volume, platelet content, and DMSO concentration. CUE CPP P-selectin was high. CD42b, phosphatidylserine (PS) expression, and live cell percentage were favorable compared to controls and favorably maintained over storage. The thrombin generation potency was slightly reduced compared to controls. The 50 mL EVA bag maintained pH for up to 30 h, and the 500 mL EVA bag beyond 76 h. The CUE system presents a technically feasible method to prepare CPP. A functionally closed bag system with resuspension solution was successful and can extend the post-thaw storage time of CPP.

C3/CD11b-Mediated Leishmania major Internalization by Neutrophils Induces Intraphagosomal NOX2-Mediated Respiratory Burst but Fails to Eliminate Parasites and Induces a State of Stalled Apoptosis.

Recruited neutrophils are among the first phagocytic cells to interact with the phagosomal pathogen Leishmania following inoculation into the mammalian dermis. Analysis of Leishmania-infected neutrophils has revealed alterations in neutrophil viability, suggesting that the parasite can both induce or inhibit apoptosis. In this study, we demonstrate that entry of Leishmania major into murine neutrophils is dependent on the neutrophil surface receptor CD11b (CR3/Mac-1) and is enhanced by parasite opsonization with C3. Infected neutrophils underwent robust NADPH oxidase isoform 2 (NOX2)-dependent respiratory burst based on detection of reactive oxygen species within the phagolysosome but largely failed to eliminate the metacyclic promastigote life cycle stage of the parasite. Infected neutrophils displayed an "apoptotic" phosphatidylserine (PS)-positive phenotype, which was induced by both live and fixed parasites but not latex beads, suggesting that PS expression was parasite specific but does not require active infection. In addition, neutrophils from parasite/neutrophil coculture had increased viability, decreased caspase 3, 8, and 9 gene expression, and reduced protein levels of both the pro and cleaved forms of the classical apoptosis-inducing executioner caspase, Caspase 3. Our data suggest that CD11b-mediated Leishmania internalization initiates respiratory burst and PS externalization, followed by a reduction in both the production and cleavage of caspase 3, resulting in a phenotypic state of "stalled apoptosis."

Abstract 611: Effect Of Tumor-derived Extracellular Vesicles On Platelet Function

Tumor-derived extracellular vesicles (EVs) have been described as pro-coagulant particles causing cancer-associated thrombosis. Tumor-derived EVs circulating in the bloodstream may interact with platelets or trigger the coagulation cascade, leading to venous and arterial thrombosis. In order to better understand the interaction of EVs with the hemostatic components, we aim to determine the mechanistic aspects of their interaction with platelets and their potential role in initiating coagulation. Given the wide variation of protein and integrin expression between different cancer cell types and the expectation of varying mechanistic effects, we have selected to study lung (A549), breast (MDA-MB-231) and glioblastoma (LN18) cell lines. We have characterized the effect of EVs derived from these cell lines on platelet function of aggregation and activation. Our data shows that both A549 and MDA-MB-231 EVs at 50 ug/mL significantly increase platelet aggregation as compared to control when incubated with the platelets for 10 min and in the presence of an agonist (ADP 20 uM) (Fig. 1). We confirmed the expression of phosphatidylserine on the surface of EVs using Annexin V and flow cytometry, indicating that they are pro-coagulant in nature. Furthermore, we will be investigating interaction of EVs with platelet receptor GP IIb-IIIa, agonist release, and presence of proteins such as Tissue Factor, as possible mechanisms for EV-induced platelet aggregation. Thus, the understanding of the mechanistic aspects behind the interaction between EVs and the hemostatic components could prove beneficial in the design of next generation agents for cancer-associated thrombosis. Figure 1. Platelet aggregation measured by light transmission aggregometry with ADP 20uM in the presence of A549 and MDA-MB-231 EVs (n=8 to 11)

A case of hyperhemolysis syndrome in sickle cell disease and concomitant COVID-19

BackgroundHyperhemolysis syndrome (HHS) is an uncommon transfusion reaction described in several hematologic disorders, including sickle cell disease (SCD). HHS is characterized by a decline in hemoglobin (Hb) values below pre-transfusion levels following transfusion of red blood cells (RBCs), coupled with laboratory markers consistent with hemolysis. The proposed pathophysiologic mechanisms underlying HHS include increased phosphatidylserine expression, macrophage activation, and complement dysregulation. Many pathophysiologic mechanisms thought to contribute to HHS have been similarly described in cases of severe COVID-19. Case reportA 28-year-old male with a history of HbSS presented with shortness of breath, right-sided chest pain, and a two-day history of fever. Polymerase chain reaction (PCR) detected SARS-CoV-2 infection with the omicron variant. The patient required an RBC transfusion (pre-transfusion hemoglobin [Hb]5.8 g/dL) with an immediate post-transfusion Hb of 6.3 g/dL. However, Hb rapidly declined to 1.7 g/dL, and lactate dehydrogenase (LDH) rose to 8701 u/L. The absolute reticulocyte count of 538 × 109/L correspondingly fell to 29 × 109/L. Despite additional RBC transfusions and initiation of immunosuppressive therapy, he expired on Day 9(D9). ConclusionGiven the similarities in their proposed pathophysiology, patients with SCD and concomitant SARS-CoV-2 infection may be predisposed to developing HHS.

Inhibiting efferocytosis reverses macrophage-mediated immunosuppression in the leukemia microenvironment.

Previous studies show that the spleen and bone marrow can serve as leukemia microenvironments in which macrophages play a significant role in immune evasion and chemoresistance. We hypothesized that the macrophage driven tolerogenic process of efferocytosis is a major contributor to the immunosuppressive leukemia microenvironment and that this was driven by aberrant phosphatidylserine expression from cell turnover and cell membrane dysregulation. Since MerTK is the prototypic efferocytosis receptor, we assessed whether the MerTK inhibitor MRX2843, which is currently in clinical trials, would reverse immune evasion and enhance immune-mediated clearance of leukemia cells. We found that inhibition of MerTK decreased leukemia-associated macrophage expression of M2 markers PD-L1, PD-L2, Tim-3, CD163 and Arginase-1 compared to vehicle-treated controls. Additionally, MerTK inhibition led to M1 macrophage repolarization including elevated CD86 and HLA-DR expression, and increased production of T cell activating cytokines, including IFN-β, IL-18, and IL-1β through activation of NF-κB. Collectively, this macrophage repolarization had downstream effects on T cells within the leukemia microenvironment, including decreased PD-1+Tim-3+ and LAG3+ checkpoint expression, and increased CD69+CD107a+ expression. These results demonstrate that MerTK inhibition using MRX2843 altered the leukemia microenvironment from tumor-permissive toward immune responsiveness to leukemia and culminated in improved immune-mediated clearance of AML.

ДИНАМИКА ФУНКЦИОНАЛЬНОЙ АКТИВНОСТИ ТРОМБОЦИТОВ У НЕДОНОШЕННЫХ НОВОРОЖДЕННЫХ ДЕТЕЙ С ИНФЕКЦИОННО-ВОСПАЛИТЕЛЬНЫМИ ЗАБОЛЕВАНИЯМИ В РАННЕМ НЕОНАТАЛЬНОМ ПЕРИОДЕ

Platelet involvement in inflammatory and immune responses and its functional activity in various pathological conditions are actively studied. Despite there are scientific publications characterizing platelets of newborns vs. adults the data on the features of the expression of activation markers on platelets of premature infants with a complicated course of the early neonatal period are not enough. The purpose of the research was to analyze the expression of glycoproteins and activation molecules on the surface of peripheral blood platelets in newborns with congenital infection depending on gestational age (GA). Materials and methods of research: a prospective single-center case-control study was conducted in the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after V.I. Kulakov (Moscow, Russia) in March 2018 - September 2019. The expression of activation markers on resting and activated platelets on the 1st and 3rd days of life (DOL) in 158 newborns aged 24 to 38 weeks was analyzed using the flow cytometry. The patients were divided into 3 groups: group 1 (n=20) of newborns with GA 24 to 28 weeks; group 2 (n=52) of newborns with GA of 29 to 33 weeks; and group 3 (n=86) of newborns with GA of 34 weeks and older. Results: in severely preterm (G1) compared with infants of older GA on the 3rd DOL platelets had higher MPV: 11.3 (10.4-12.3) vs. 10.1 (9.6-10.3), p=0.011, and PDW: 13.1 (12.2-15.6) vs. 11.3 (10.6-12.3), p&lt;0.001. In infectious diseases in children with GA of 29 to 33 weeks there is a lower expression of phosphatidylserine on activated platelets in the 1st DOL: 8.8 (7.5-11.1) vs. 11.2 (10.3-12.1), p=0.046, and lower the content of activated platelets expressing P-selectin on the 3rd DOL: 89.5 (87.1-92.7) vs. 93.1 (90.4-94.4), p=0.048. In newborns with GA of 34 weeks and older with congenital infection in the 1st DOL there was a higher expression of von Willebrand Factor receptor revealed: 13.9 (12.3-16.4) vs. 12.4 (10.9-14.1), p=0.028, and a higher content of dense granules in activated platelets: 0.73 (0.65-0.79) vs. 0.67 (0.60-0.71), p=0.008. By the 3rd DOL there was an increase in MPV and PDW in all groups of children (p&lt;0.05) recorded. Conclusion: in premature infants with congenital infection the expression of structural and activation molecules on platelets undergoes greater changes during the first three days of life than in children of similar GA without infectious pathology.

Investigation of Platelet Apoptosis in Patients after Surgical Myocardial Revascularization.

Platelets are one of the main participants in vascular accidents in cases of coronary heart disease (CHD). In this study, we sought to detect platelet apoptosis in patients with coronary artery disease who underwent scheduled myocardial revascularization surgery. To identify apoptotic events, we analyzed phosphatidylserine (PS) expression on the surface of platelets and mitochondrial membrane potential (ΔΨm) by flow cytometry in two groups of 30 patients aged 45-60 years: Group 1-patients before myocardial revascularization surgery and group 2-patients after myocardial revascularization surgery. The control group consisted of 10 healthy volunteers aged 45-60 years. According to our data, the percentage levels of PS expression in patients greatly decreased after surgery. We confirmed platelet apoptosis by recording depolarization of ΔΨm in pre- and postoperative patients. ΔΨm readings were considerably improved after surgery. Our data indicated that the functional parameters of platelets in patients with coronary heart disease differed from the characteristics of platelets in patients who underwent myocardial revascularization, and from those of patients in a control group. Future studies of platelet phenotypic characteristics and platelet apoptosis biomarkers should greatly advance our understanding of the pathophysiology of coronary heart disease, and further promote the development of methods for predicting adverse outcomes after surgery.

Abstract B025: CD28-negative T cells from head and neck cancer patients are functionally frail but not senescent

Abstract Introduction: T cells lacking costimulatory receptors CD27/28, with high levels of differentiation markers CD57 and KLRG1, may accumulate with aging. These T cells have been described as “senescent” (unable to proliferate) with high cytolytic enzyme expression. However, robust functional studies on these cells are lacking. Further, emerging studies on the implications of these cells in the context of prognosis and responses to immunotherapy have shown mixed results. To better understand the role of these cells in the pathogenesis of cancer, a better understanding of their functional potential is needed. Methods and Results: We collected blood from 46 patients with previously untreated, locally advanced head and neck squamous cell carcinoma (HNSCC), aged 47-89, undergoing surgery. Matched tumor tissue was collected from 29 patients. Samples were analyzed by flow cytometry. There was correlation between the percentage of CD27/28-CD57+ (CD28neg) T cells in the blood vs. tumor (R2 = 0.51 for CD8+, 0.41 for CD4+; p&amp;lt;0.001). The CD28neg proportion among CD8+ T cells was significantly higher in patients aged 70 or greater vs. patients under age 70. CD28neg cells from blood and tumors expressed very high levels of granzyme B and perforin but were not enriched for senescence markers p16 and beta-galactosidase. Additional CD8+ cells from the peripheral blood were sorted into CD28neg (CD27/28-CD57+) vs. CD27/28+ for comparative functional studies. Proliferation assays showed a stepwise reduction in CellTrace Violet in both the CD28neg and CD27/28+ populations over 5 days in culture with IL-2 and CD2/3/28 stimulation cocktail. While the percentage of CD28neg cells that had completed 3 or more cell divisions was significantly reduced from that of the CD27/28+ population, approximately 40% of the CD28neg cells had divided at least three times over 5 days in culture. Sorted CD28neg cells retained high levels of granzyme B/perforin, and PMA/ionomycin stimulation induced increased CD107a expression and granzyme B secretion, suggesting that these cells do possess enhanced cytolytic capacity. CD28neg cells also expressed higher levels of surface phosphatidylserine, which has been associated with cellular stress in viable cells. CD28neg cells stimulated with CD3 beads secreted higher levels of TNF-a, but not IFN-g or IL-2, vs. CD27/28+ cells. Conclusions: CD28neg T cells sampled from HNSCC patients have altered functionality, including a reduced (but intact) capacity to proliferate and increased phosphatidylserine expression. However, they also have enhanced capacity to secrete cytolytic enzymes and TNF-a, suggesting they may have differentiated to perform those specific functions. We propose that these T cells are more appropriately described as “frail,” and “immunofrailty” may be a more appropriate term than “immunosenescence.” Additional prospective clinical studies are needed to elucidate the effects of these cells on prognosis and treatment responses in cancer patients. Citation Format: Brendan L.C. Kinney, Connor Parrish, Vikash Kansal, Zachary S. Buchwald, Haydn T. Kissick, Amy Y. Chen, Nicole C. Schmitt. CD28-negative T cells from head and neck cancer patients are functionally frail but not senescent [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B025.