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Changes in surface glycan determinants, specifically sialic acid loss, determine platelet life span. The gradual loss of stored platelet quality is a complex process that fundamentally involves carbohydrate structures. Here, we applied lipophilic extraction and glycan release protocols to sequentially profile N- and O-linked glycans in freshly isolated and seven-day room temperature stored platelet concentrates. Analytical methods including matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), tandem mass spectrometry (MSn), and liquid chromatography (LC) were used to obtain structural details of selected glycans and terminal epitopes. The fresh platelet repertoire of surface structures revealed diverse N-glycans, including high mannose structures, complex glycans with polylactosamine repeats, and glycans presenting blood group epitopes. The O-glycan repertoire was largely comprised of sialylated and fucosylated core-1 and core-2 structures. For both N- and O-linked glycans, we observed a loss in sialylated epitopes with a reciprocal increase in neutral structures as well as increased neuraminidase activity following platelet storage at room temperature. The data indicate that loss of sialylated glycans is associated with diminished platelet quality and untimely removal of platelets following storage.

Abrocitinib is an oral Janus kinase 1 inhibitor with demonstrated efficacy in moderate-to-severe atopic dermatitis (AD). To evaluate the long-term safety profile of abrocitinib and provide relevant information for practitioners. Data from patients who received ≥1 dose of abrocitinib 200 mg or 100 mg in the JADE clinical program were pooled in two cohorts. The consistent-dose (CD) cohort comprised patients who received the same abrocitinib dose during the entire exposure time in parent phase 3 studies and/or the long-term extension study JADE EXTEND (NCT03422822). Parent studies were: JADE MONO-1 (NCT03349060), JADE MONO-2 (NCT03575871), JADE TEEN (NCT03796676), JADE COMPARE (NCT03720470), JADE DARE (NCT04345367; 200 mg only) and JADE REGIMEN (NCT03627767); in JADE REGIMEN only patients from the open-label run-in phase (200 mg only) who did not subsequently enter the randomized phase were included. Patients from the phase 2b study (NCT02780167) were also included in the CD cohort. The variable-dose (VD) cohort comprised patients who received different doses of abrocitinib (200 and 100 mg) throughout exposure time in the parent study JADE REGIMEN and in JADE EXTEND, if enrolled. Patients included in the VD cohort completed the open-label period of JADE REGIMEN (abrocitinib 200 mg only) and entered the randomized phase (abrocitinib 200 mg, abrocitinib 100 mg or placebo); some patients subsequently entered the JADE REGIMEN rescue phase (abrocitinib 200 mg) and/or JADE EXTEND (abrocitinib 200 or 100 mg). The data cutoff was 16 April 2021. A Cox regression analysis evaluated risk factors for specific events of interest. Data from 3582 patients (4313.4 patient-years) were analysed: 2784 patients in the CD cohort (abrocitinib 200 mg, n = 1761, abrocitinib 100 mg, n = 1023) and 798 patients in the VD cohort. Duration of exposure was ≥48 weeks in 43.6%, 66.9% and 86.1% and ≥96 weeks in 18.0%, 23.2% and 45.4% of patients in the CD 200 mg, 100 mg and VD cohorts, respectively. In the CD and VD cohorts, the median age was 30.0 and 29.0 years; 490 (17.6%) and 145 patients (18.2%) were adolescents, and 143 (5.1%) and 30 patients (3.8%) were aged ≥65 years at screening, respectively. Incidence rates (IRs; unique patients with events per 100 patient-years) of serious adverse events (SAEs), deaths, and treatment-emergent adverse events of special interest are shown in Table 1. IRs for SAEs were consistent across abrocitinib doses but were higher in patients aged ≥65 years vs. younger patients. Serious infections were the most frequent SAEs; frequency was not dose related. Herpes zoster (HZ), herpes simplex and pneumonia were the most frequent serious infections. From a Cox regression analysis, potential risk factors for treatment-emergent HZ included abrocitinib dose, baseline age, medical history of HZ, absolute lymphocyte count

PURPOSE OF THE REVIEW This review highlights recent advancements in understanding the regulation of platelet numbers, focusing on mechanisms by which carbohydrates (glycans) link platelet removal with platelet production in the bone marrow in health and disease. RECENT FINDINGS This review is focused on the role of carbohydrates, specifically sialic acid moieties, as a central mediator of platelet clearance. We discuss recently identified novel mechanisms of carbohydrate-mediated platelet removal and carbohydrate-binding receptors that mediate platelet removal. SUMMARY The platelet production rate by megakaryocytes and removal kinetics controls the circulating platelet count. Alterations in either process can lead to thrombocytopenia (low platelet count) or thrombocytosis (high platelet count) are associated with the risk of bleeding or overt thrombus formation and serious complications. Thus, regulation of a steady-state platelet count is vital in preventing adverse events. There are few mechanisms delineated that shed light on carbohydrates' role in the complex and massive platelet removal process. This review focuses on carbohydrate-related mechanisms that contribute to the control of platelet numbers.

Background and aims Thrombocytopenia has been described in most patients with acute and chronic liver failure. Decreased platelet production and decreased half-life of platelets might be a consequence of low levels of thrombopoietin (TPO) in these patients. Platelet production is tightly regulated to avoid bleeding complications after vessel injury and can be enhanced under elevated platelet destruction as observed in liver disease. Thrombopoietin (TPO) is the primary regulator of platelet biogenesis and supports proliferation and differentiation of megakaryocytes. Approach and results Recent work provided evidence for the control of TPO mRNA expression in liver and bone marrow (BM) by scanning circulating platelets. The Ashwell-Morell receptor (AMR) was identified to bind desialylated platelets to regulate hepatic thrombopoietin (TPO) production by Janus kinase (JAK2)/signal transducer and activator of transcription (STAT3) activation. Two-thirds partial hepatectomy (PHx) was performed in mice. Platelet activation and clearance by AMR/JAK2/STAT3 signaling and TPO production were analyzed at different time points after PHx. Here, we demonstrate that PHx in mice led to thrombocytopenia and platelet activation defects leading to bleeding complications, but unaltered arterial thrombosis, in these mice. Platelet counts were rapidly restored by up-regulation and crosstalk of the AMR and the IL-6 receptor (IL-6R) to induce JAK2-STAT3-TPO activation in the liver, accompanied by an increased number of megakaryocytes in spleen and BM before liver was completely regenerated. Conclusions The AMR/IL-6R-STAT3-TPO signaling pathway is an acute-phase response to liver injury to reconstitute hemostasis. Bleeding complications were attributable to thrombocytopenia and platelet defects induced by elevated PGI2 , NO, and bile acid plasma levels early after PHx that might also be causative for the high mortality in patients with liver disease.

Serological classification of individuals as A, B, O, or AB is a mainstay of blood banking. ABO blood groups or ABH antigens, in addition to other surface glycans, act as unique red blood cell (RBC) signatures and direct immune responses. ABO subgroups present as weakened, mixed field, or unexpected reactivity with serological reagents, but specific designations remain complex. Lectins detect glycan motifs with some recognizing ABH antigens. We evaluated a 45-probe lectin microarray to rapidly analyze ABO blood groups and associated unique glycan signatures within complex biological samples on RBC surface glycoproteins. RBC membrane glycoproteins were prepared from donor RBCs, n = 20 for each blood group. ABO blood group was distinguishable by lectin array, including variations in ABH antigen expression not observed with serology. Principal component analysis highlighted broad ABO blood group clusters with unexpected high and low antigen expression and variations were confirmed with ABH antibody immunoblotting. Using a subset of lectins provided an accurate method to predict an ABO serological phenotype. Lectin microarray highlighted the importance of ABO localization on glycoproteins and glycolipids and pointed to increased glycocalyx complexity associated with the expression of A and B antigens including high mannose and branched polylactosamine. Thus, lectins identified subtle surface ABO blood group glycoprotein density variations not detected by routine serological methods. Transfusion services observe alterations in ABH expression during malignancy, and ABO incompatible solid organ transplantation is not without risk of rejection. The presented methods may identify subtle but clinically significant ABO blood group differences for transfusion and transplantation.

Glycosylation is highly prevalent, and also one of the most complex and varied post-translational modifications. This large glycan diversity results in a wide range of biological functions. Functional diversity includes protein degradation, protein clearance, cell trafficking, cell signaling, host-pathogen interactions, and immune defense, including both innate and acquired immunity. Glycan-based ABO(H) antigens are critical in providing compatible products in the setting of transfusion and organ transplantation. However, evidence also suggests that ABO expression may influence cardiovascular disease, thrombosis and hemostasis disorders, including alterations in platelet function and von Willebrand factor (VWF) blood levels. Glycans also regulate immune and hemostasis function beyond ABO(H) antigens. Mutations in glycogenes (PIGA, COSMC) lead to serious blood disorders, including the Tn-syndrome associated with hyperagglutination, hemolysis, and thrombocytopenia. Alterations in genes responsible for sialic acids (Sia) synthesis (GNE) and UDP-galactose (GALE) and lactosamine (LacNAc) (B4GALT1) profoundly affect circulating platelet counts. Desialylation (removal of Sia) is affected by human and pathogenic neuraminidases. This review addresses the role of glycans in transfusion medicine, hemostasis and thrombosis, and red blood cell and platelet survival.

The GNE gene encodes an enzyme that initiates and regulates the biosynthesis of N-acetylneuraminic acid, a precursor of sialic acids. GNE mutations are classically associated with Nonaka myopathy and sialuria, following an autosomal recessive and autosomal dominant inheritance pattern. Reports show that single GNE variants cause severe thrombocytopenia without muscle weakness. Using panel sequencing, we identified two novel compound heterozygous variants in GNE in a young girl with life-threatening bleedings, severe congenital thrombocytopenia, and a platelet secretion defect. Both variants are located in the nucleotide-binding site of the N-acetylmannosamin kinase domain of GNE. Lectin array showed decreased α-2,3-sialylation on platelets, consistent with loss of sialic acid synthesis and indicative of rapid platelet clearance. Hematopoietic stem cell transplantation (HSCT) normalized platelet counts. This is the first report of an HSCT in a patient with an inherited GNE defect leading to normal platelet counts.

Contains fulltext : 282340.pdf (Publisher’s version ) (Closed access) COVID-19-associated coagulopathy (CAC) is a life-threatening complication of SARS-CoV-2 infection. However, the underlying cellular and molecular mechanisms driving this condition are unclear. Evidence supports the concept that CAC involves complex interactions between the innate immune response, the coagulation and fibrinolytic pathways, and the vascular endothelium, resulting in a procoagulant condition. Understanding of the pathogenesis of this condition at the genomic, molecular and cellular levels is needed in order to mitigate thrombosis formation in at-risk patients. In this Perspective, we categorize our current understanding of CAC into three main pathological mechanisms: first, vascular endothelial cell dysfunction; second, a hyper-inflammatory immune response; and last, hypercoagulability. Furthermore, we pose key questions and identify research gaps that need to be addressed to better understand CAC, facilitate improved diagnostics and aid in therapeutic development. Finally, we consider the suitability of different animal models to study CAC.

Platelets are blood components that survive in circulation for 7 to 10 days in humans. Thus, platelet production by bone marrow (BM) megakaryocytes (MKs), and their removal from the blood circulation is precisely orchestrated to maintain an average platelet count. Abnormalities in both processes can result in thrombocytopenia (low platelet count) or thrombocytosis (high platelet count), often associated with the risk of bleeding or overt thrombus formation, respectively. Platelet glycans, particularly sialic acids, are indicators of platelet count. Loss of platelet sialic acids leads to platelet clearance. A State-of-the-Art lecture titled "Platelet and Megakaryocyte Glycobiology" was presented at the ISTH virtual congress 2021 to discuss (i) the loss of O-glycan sialic acid on BM MKs, revealing the Thomsen-Friedenreich (TF) antigen as a new concept of thrombocytopenia; herein, impaired thrombopoiesis is attributed to activation of immune cells with a plasmacytoid dendritic cell signature; and (ii) upregulation of antibodies against the TF antigen in pediatric patients with immune thrombocytopenia (ITP), positing that glycan alterations such as MK asialylation can lead to immune cell responses. Here, we discuss our findings alongside new data presented at the 2020 and 2021 ISTH congresses on the role of sialic acids and glycans in regulating platelet count. Desialylation is a prominent feature in thrombocytopenia, notably in ITP presentation. We compare similarities between ITP mediated with shear-stress and with storage-related asialylation. We also discuss genes involved in sialic acid synthesis leading to thrombocytopenia. Increased awareness in gene-regulating MK and platelet glycans is a giant leap to understanding the underpinning mechanisms of ITP and other forms of thrombocytopenia.

Background Pivotal phase III studies demonstrated that abrocitinib, an oral, once-daily, JAK1-selective inhibitor, is effective treatment for moderate-to-severe atopic dermatitis (AD) as monotherapy and in combination with topical therapy. Objective The aim of this study was to evaluate the long-term safety of abrocitinib 200 mg and 100 mg in an integrated analysis of a phase IIb study, four phase III studies, and one long-term extension study. Methods Two cohorts were analyzed: a placebo-controlled cohort from 12- to 16-week studies and an all-abrocitinib cohort including patients who received one or more abrocitinib doses. Adverse events (AEs) of interest and laboratory data are reported. Results Total exposure in the all-abrocitinib cohort (n = 2856) was 1614 patient-years (PY); exposure was ≥ 24 weeks in 1248 patients and ≥ 48 weeks in 606 (maximum 108 weeks). In the placebo-controlled cohort (n = 1540), dose-related AEs (200 mg, 100 mg, placebo) were nausea (14.6%, 6.1%, 2.0%), headache (7.8%, 5.9%, 3.5%), and acne (4.7%, 1.6%, 0%). Platelet count was reduced transiently in a dose-dependent manner; 2/2718 patients (200-mg group) had confirmed platelet counts of < 50 × 103/mm3 at week 4. Incidence rates (IRs) were 2.33/100PY and 2.65/100 PY for serious infection, 4.34/100PY and 2.04/100PY for herpes zoster, and 11.83/100PY and 8.73/100PY for herpes simplex in the 200-mg and 100-mg groups, respectively. IRs for nonmelanoma skin cancer, other malignancies, and major adverse cardiovascular events were < 0.5/100PY for both doses. Five venous thromboembolism events occurred (IR 0.30/100PY), all in the 200-mg group. There were three deaths due to gastric carcinoma (diagnosed at day 43), sudden death, and COVID-19. Conclusion Abrocitinib, with proper patient and dose selection, has a manageable tolerability and longer-term safety profile appropriate for long-term use in patients with moderate-to-severe AD. Trial Registries ClinicalTrials.gov: NCT02780167, NCT03349060, NCT03575871, NCT03720470, NCT03627767, NCT03422822. Supplementary Information The online version contains supplementary material available at 10.1007/s40257-021-00618-3.

Sickle cell disease is an inherited genetic disorder that causes anemia, pain crises, organ infarction, and infections in 13 million people worldwide. Previous studies have revealed changes in sialic acid levels associated with red blood cell sickling and showed that stressed red blood cells bare surface-exposed clustered terminal mannose structures mediating hemolysis, but detailed glycan structures and anti-glycan antibodies in sickle cell disease remain understudied. Here, we compiled results obtained through lectin arrays, glycan arrays, and mass spectrometry to interrogate red blood cell glycoproteins and glycan-binding proteins found in the plasma of healthy individuals and patients with sickle cell disease and sickle cell trait. Lectin arrays and mass spectrometry revealed an increase in α2,6 sialylation and a decrease in α2,3 sialylation and blood group antigens displayed on red blood cells. Increased binding of proteins to immunogenic asialo and sialyl core 1, Lewis A, and Lewis Y structures was observed in plasma from patients with sickle cell disease, suggesting a heightened anti-glycan immune response. Data modeling affirmed glycan expression and plasma protein binding changes in sickle cell disease but additionally revealed further changes in ABO blood group expression. Our data provide detailed insights into glycan changes associated with sickle cell disease and refer glycans as potential therapeutic targets.

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti–Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK−/−). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK−/− mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H–positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK−/− BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell–like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.

Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies originating from hematopoietic stem cells, leading to an increase in mature blood cells in the peripheral blood. Sialylated derivatives of the glycan structure β4-N-acetyllactosamine (Galβ1,4GlcNAc or type-2 LacNAc, hereafter referred to as LacNAc) regulate platelet life span, hepatic thrombopoietin (TPO) production, and thrombopoiesis. We found increased TPO plasma levels in MPNs with high allele burden of the mutated clones. Remarkably, platelets isolated from MPNs had a significant increase in LacNAc expression that correlated with the high allele burden regardless of the underlying identified mutation. Megakaryocytes derived in vitro from these patients showed an increased expression of the B4GALT1 gene encoding β-1,4-galactosyltransferase 1 (β4GalT1). Consistently, megakaryocytes from MPN showed increased LacNAc expression relative to healthy controls, which was counteracted by the treatment with a Janus kinase 1/2 inhibitor. Altered expression of B4GALT1 in mutant megakaryocytes can lead to the production of platelets with aberrant galactosylation, which in turn promote hepatic TPO synthesis regardless of platelet mass. Our findings provide a new paradigm for understanding aberrant megakaryopoiesis in MPNs and identify β4GalT1 as a potential actionable target for therapy.

Background: Outcomes of adults with relapsed/refractory T-cell acute lymphoblastic leukemia or lymphoma (T-ALL or T-LBL respectively) and acute myeloid leukemia (AML) have remained poor despite the availability of newer agents. CD38 is a 45 kDa transmembrane glycoprotein expressed by lymphoid and myeloid cells with several important functions including a role in immune escape from tumor cells [Chillemi A et al. Front Immunol 2017, Furano A et al. J Immunol 1990]. Several studies have demonstrated that CD38 is expressed in tumor cells of patients with T-ALL and AML and potentially targetable using CD38 blocking agents [Naik J et al. Haematologica 2019, Bride KL et al. Blood 2018, Tembhare et al. J Immunother Cancer 2020, Farber M et al. ASH 2018]. XmAb18968 is structurally distinct CD3-CD38 bi-specific T-cell engager with a full fragment crystallizable (Fc) domain modified to minimize Fcγ receptor binding and non-selective activation of T cells and other immune effector cells. XmAb18968 has optimized relative affinities for both CD3 and CD38 which results in reduced cytokine release without compromising target cell killing. We hypothesize targeting CD38 in relapsed/refractory- T-cell ALL/LBL and AML is safe and feasible using XmAb18968, a novel CD3-CD38 bispecific antibody. Study design and methods: This is an investigator-sponsored multi-institutional phase I study evaluating the safety and tolerability of XmAb18968. Patients aged 18 years or above with T-ALL, T-LBL or AML (including undifferentiated leukemia and bi phenotypic leukemia), in relapsed/refractory status (at least one line of prior therapy) including measurable residual disease relapse, CD38 expression ≥ 20%, and adequate organ function will be eligible. Major exclusion criteria are hematopoietic cell transplantation (HCT) within 6 months of enrollment, active acute GVHD, veno-occlusive disease, and acute promyelocytic leukemia. The study is planned to be conducted at 6 sites in United States. The primary endpoint is to determine the recommended phase 2 dose (RP2D) and toxicity profile of XmAb18968. The secondary endpoints include determination of response rates, duration of response, survival and pharmacokinetics. Exploratory endpoints include correlation of responses with genomic profile, assessment of serum cytokine response, identifying the changes in phenotypic expression of activated T-cells and leukemic cells, correlation of the phenotypic expression with changes in the transcriptome at the single-cell level, proteomics evaluation of cytokine secretion at the single-cell level and correlation of response with N-glycan profiling, quantitative site-occupancy and direct glycopeptide analysis. The study will follow 3+3 design for dose escalation and de-escalation in case of dose limiting toxicity (DLT). Dose escalation will proceed in two separate groups: Group A for subjects with T-ALL and T-LBL and Group B for subjects with AML. Patients will be entered sequentially to each dose level, starting with the first dose level. The DLT observation period for dose-escalation will be 28-days. RP2D will be defined as the highest dose level at which none of the first 3 treated subjects, or no more than 1 of the first 6 treated subjects experience a DLT. A minimum of 24 and a maximum of 60 patients will be needed for the dose escalation phase. Disclosures Guru Murthy: Curio Sciences: Honoraria; Techspert: Consultancy; Qessential: Honoraria; CancerExpertNow: Honoraria; DAVA Oncology: Honoraria; TG Therapeutics: Other: Advisory board meeting; Cardinal health: Honoraria; Guidepoint: Consultancy. Johnson: Miltenyi Biotec: Research Funding. Abedin: AltruBio: Research Funding; Amgen: Honoraria; Agios: Honoraria; Helsinn: Research Funding; Pfizer: Research Funding; Astellas Pharma Inc.: Research Funding; Actinium: Research Funding. Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Shah: Precision Biosciences: Consultancy; Kite, a Gilead Company: Consultancy, Honoraria, Other: Expenses, Research Funding; Pharmacyclics/Janssen: Honoraria, Other: Expenses; Acrotech/Spectrum: Honoraria; BeiGene: Consultancy, Honoraria; Amgen: Consultancy; Pfizer: Consultancy, Other: Expenses; Novartis: Consultancy, Other: Expenses; Bristol-Myers Squibb/Celgene: Consultancy, Other: Expenses; Adaptive Biotechnologies: Consultancy; Servier Genetics: Other; Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding. Leonard: Amgen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees. Pratz: Agios: Consultancy; BMS: Consultancy, Honoraria; Novartis: Consultancy; Astellas: Consultancy, Honoraria, Research Funding; Cellgene: Consultancy, Honoraria; Millenium: Research Funding; University of Pennsylvania: Current Employment; Abbvie: Consultancy, Honoraria, Research Funding. Luger: Syros: Honoraria; Agios: Honoraria; Daiichi Sankyo: Honoraria; Jazz Pharmaceuticals: Honoraria; Brystol Myers Squibb: Honoraria; Acceleron: Honoraria; Astellas: Honoraria; Pfizer: Honoraria; Onconova: Research Funding; Celgene: Research Funding; Biosight: Research Funding; Hoffman LaRoche: Research Funding; Kura: Research Funding. Badar: Pfizer Hematology-Oncology: Membership on an entity's Board of Directors or advisory committees. Atallah: Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy; Abbvie: Consultancy, Speakers Bureau; Takeda: Consultancy, Research Funding; BMS: Honoraria, Speakers Bureau; Pfizer: Consultancy, Research Funding.

Objective— Apheresis platelets for transfusion treatment are currently stored at room temperature because after refrigeration platelets are rapidly cleared on transfusion. In this study, the role of von Willebrand factor (VWF) in the clearance of refrigerated platelets is addressed. Approach and Results— Human and murine platelets were refrigerated in gas-permeable bags at 4°C for 24 hours. VWF binding, platelet signaling events, and platelet post-transfusion recovery and survival were measured. After refrigeration, the binding of plasma VWF to platelets was drastically increased, confirming earlier studies. The binding was blocked by peptide OS1 that bound specifically to platelet glycoprotein (GP)Ibα and was absent in VWF − / − plasma. Although surface expression of GPIbα was reduced after refrigeration, refrigeration-induced VWF binding under physiological shear induced unfolding of the GPIbα mechanosensory domain on the platelet, as evidenced by increased exposure of a linear epitope therein. Refrigeration and shear treatment also induced small elevation of intracellular Ca 2+ , phosphatidylserine exposure, and desialylation of platelets, which were absent in VWF −/− platelets or inhibited by OS1, which is a monomeric 11-residue peptide (CTERMALHNLC). Furthermore, refrigerated VWF −/− platelets displayed increased post-transfusion recovery and survival than wild-type ones. Similarly, adding OS1 to transgenic murine platelets expressing only human GPIbα during refrigeration improved their post-transfusion recovery and survival. Conclusions— Refrigeration-induced binding of VWF to platelets facilitates their rapid clearance by inducing GPIbα-mediated signaling. Our results suggest that inhibition of the VWF–GPIbα interaction may be a potential strategy to enable refrigeration of platelets for transfusion treatment.

The Thomsen-Friedenreich antigen (TF-antigen) occurs during exposure of the underlying Core-1 disaccharide (Gal-beta(1,3)GalNAc) through the loss of its capping sialic acid (Sia). Exposure of the cryptic TF-antigen occurs during inflammation, during acute infections with influenza viruses or bacteria, in malignancies, and is associated with thrombocytopenia. Exposure of the TF-antigen on circulating blood cells, including platelets and red blood cells (RBC), can lead to severe thrombocytopenia or hemolysis in hemolytic uremic syndrome and other immune diseases. Recent data suggest that altered Sia may cause platelet destruction because treatment with the sialidase inhibitor Tamiflu increases platelet count in healthy and thrombocytopenic patients. In humans, genetic mutations involving Sia synthesis and transport, and atypical cell surface sialylation, unrelated to any genetic mutation, are associated with reduced platelet count, supporting the role of Sia in regulating platelet count. Immune cells, including classical dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs), and subsets of T cells (CD8+, CD4+, and Treg cells) can also affect immune thrombocytopenia pathogenesis. Like many other immune cells, cDCs, and pDCs express Siglecs (sialic-acid-binding immunoglobulin-like lectins), which often contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that act as immunosuppressors. Whether BM immune cells monitor MKs via glycan-lectin receptors, including Siglecs and Sia interactions, to control platelet production is unclear. To investigate the role of the TF-antigen in thrombopoiesis, we generated St3gal1MK-KO mice (Pf4-Cre) that display increased TF-antigen specifically in megakaryocytes (MK) and platelets. St3gal1MK-KO mice developed significant thrombocytopenia, but had normal platelet half-life, suggesting that the TF-antigen affected BM thrombopoiesis. In vitro MK maturation and proplatelet production from primary ST3Gal1MK-KO mouse BM cells were also normal, pointing to extrinsic factors in the BM environment affecting thrombopoiesis. Platelet counts of St3gal1MK-KO mice were restored to wild-type levels by 1) crossing St3gal1MK-KO mice with Jak3KO mice that have impaired of lymphoid cell development, 2) by treatment with anti-inflammatory dexamethasone, and 3) treatment with a depleting anti-CD4 antibody. Immunofluorescence staining of the St3gal1MK-KO BM revealed proplatelet structures positive for GPIba+ and the TF-antigen, being infiltrated by mononuclear cells resembling lymphocytes. We speculated that immune cells surveil megakaryocytes to control thrombopoiesis. Bulk RNAseq of CD4+ cells in St3gal1MK-KO BM confirmed a population bias for Type I interferon (IFN-I)-releasing pDCs, a cell type regulated by unique sialic acid binding lectins (Siglecs). Inhibition of IFN-I activity, by a blocking receptor antibody improved platelet counts in St3gal1MK-KO mice. Co-cultures of pDCs with MKs show inhibited pro-platelet formation when TF-antigen is present on MKs with elevated IFN-I levels. Gene set enrichment analysis of BM pDCs single cell RNASeq (scRNAseq) data further confirmed that TF-antigen exposure by MKs up-regulates IFN-I transcripts. scRNAseq also reveals a new population of immune cells with pDC transcript signature and concomitant upregulation of immunoglobulin re-arrangement gene transcripts Igkc and Ighm. In conclusion, the data shows that recognition of aberrant MK sialylation by pDCs regulates thrombopoiesis through IFN-I secretion. Disclosures No relevant conflicts of interest to declare.

G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

The immune response relies on the integration of cell-intrinsic processes with cell-extrinsic cues. During infection, B cells vacate the marrow during emergency granulopoiesis but return upon restoration of homeostasis. Here we report a novel glycosylation-mediated crosstalk between marrow B cells and hematopoietic progenitors. Human B cells secrete active ST6GAL1 sialyltransferase that remodels progenitor cell surface glycans to suppress granulopoiesis. In mouse models, ST6GAL1 from B cells alters the sialylation profile of bone marrow populations, and mature IgD+ B cells were enriched in sialylated bone marrow niches. In clinical multiple myeloma, ST6GAL1 abundance in the multiple myeloma cells negatively correlated with neutrophil abundance. These observations highlight not only the ability of medullary B cells to influence blood cell production, but also the disruption to normal granulopoiesis by excessive ST6GAL1 in malignancy.

The immune response relies on the timely integration of cell-intrinsic processes with cell-extrinsic cues. During infection, B cells vacate the bone marrow for the emergency generation of granulocytes. However, it is unclear if cross-talk between B cells and neutrophils also encourages the return to homeostasis. Here, we report that B cells remodel glycans on hematopoietic progenitors to suppress granulopoiesis. Human B cells secrete active ST6Gal-1 sialyltransferase to modify the sialylation and Gr-1 expression of co-cultured hematopoietic progenitors. After adoptive transfer, total hematopoietic and B cells modified the sialylation of non-self cells and elevated blood ST6Gal-1. Mature IgD+ B cells co-localized with megakaryocytes to sialylated bone marrow niches, suggesting their role in medullary extrinsic sialylation. Finally, ST6Gal-1 expression in multiple myeloma cells negatively correlated with neutrophil abundance in human patients. Our results highlight the growing significance of extracellular glycoslytransferases as mediators of a novel glycan-dependent interaction between B cells and granulocytes.

Congenital amegakaryocytic thrombocytopaenia (CAMT) is a disorder caused by c-MPL mutations that impair thrombopoietin (TPO) signalling, resulting in a near absence of megakaryocytes (MKs). While this phenotype is consistent in adults, neonates with CAMT can present with severe thrombocytopaenia despite normal MK numbers. To investigate this, we characterized MKs and platelets in newborn c-MPL –/– mice. Liver MKs in c-MPL –/– neonates were reduced in number and size compared with wild-type (WT) age-matched MKs, and exhibited ultrastructural abnormalities not found in adult c-MPL –/– MKs. Platelet counts were lower in c-MPL –/– compared with WT mice at birth and did not increase over the first 2 weeks of life. In vivo biotinylation revealed a significant reduction in the platelet half-life of c-MPL –/– newborn mice (P2) compared with age-matched WT pups, which was not associated with ultrastructural abnormalities. Genetic deletion of the pro-apoptotic Bak did not rescue the severely reduced platelet half-life of c-MPL –/– newborn mice, suggesting that it was due to factors other than platelets entering apoptosis early. Indeed, adult GFP+ (green fluorescent protein transgenic) platelets transfused into thrombocytopenic c-MPL –/– P2 pups also had a shortened lifespan, indicating the importance of cell-extrinsic factors. In addition, neonatal platelets from WT and c-MPL –/– mice exhibited reduced P-selectin surface expression following stimulation compared with adult platelets of either genotype, and platelets from c-MPL –/– neonates exhibited reduced glycoprotein IIb/IIIa (GPIIb/IIIa) activation in response to thrombin compared with age-matched WT platelets. Taken together, our findings indicate that c-MPL deficiency is associated with abnormal maturation of neonatal MKs and developmental stage-specific defects in platelet function.

Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(β3)GlcNAc, Gal(β4)GlcNAc and Gal(β3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and β4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(β4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior.

The enzyme β-1-4 galactosyltransferase 1 (β4GalT1) plays a critical role in thrombopoiesis by modulating sialo-glycan (sialyl N-acetyl-lactosamine or LacNAc) content and function of the β1 integrin on megakaryocytes (MKs) (Nat. Commun. 2020;11(1):356). Recent data, however, point to a more complex role for β4GalT1 in hematopoiesis, as the promoter region of its conserved gene, B4galt1, is rich in enhancer sequences for transcription factors associated with cell identity and pro-oncogenic regulatory programs. Here, we investigated the homeostasis of hematopoietic stem and progenitor cells (HSPCs) in B4galt1-/- mice. We demonstrate that lack of Sialylated LacNAc synthesis perturbs HSPCs beyond the homing deficiency associated with lack of homing sialo-glycan motifs. Flow cytometry analysis showed that B4galt1-/- mouse bone marrows have increased numbers of Long-Term HSCs (LT-HSCs, defined as LineageNeg/Sca-1Pos/c-KitPos/CD150Pos/CD48Neg). The increase in HSC numbers led us to investigate their phenotypic and functional features further. While quiescence markers in B4galt1-/- LT-HSCs were indistinguishable relative to controls, LT-HSCs expressed more of the platelet marker CD41 on their surface, supporting a highly expanded CD41+ subset of LT-HSCs. Platelet-bias of LT-HSCs has been associated with inflammation and aging. However, our data do not support an increased cytokine inflammatory profile in the bone marrow. Instead, single-cell RNA sequencing (scRNA seq) of sorted β4galt1-/- LineageNeg/Sca-1Pos/c-KitPos (LSK) cells showed a significantly increased expression of the proto-oncogene Pim-1, its target, Myc, and the heavily O-glycosylated transmembrane receptor mucin 13 (MUC13), compared to control cells. Analysis of LT-HSC glycan expression using lectin microarray showed the expected decrease in N-glycosylation associated with B4galt1 deficiency, but also an increase in O-glycans, consistent with overexpression of MUC13, expression of which was enriched compared to other surface mucins. The data show that B4galt1 deletion leads to overexpression of the proto-oncogenes Pim-1, Myc, and MUC13 in HSPCs. The data suggest that MUC13-associated O-glycans and glyco-synthetic genes are potential therapeutic targets for hematologic malignancies since mucins have anti-inflammatory functions and alterations in mucin expression are with inflammation and cancer. Disclosures No relevant conflicts of interest to declare.

In this issue of Blood, Xu et al unequivocally show by using a series of elegant experiments that platelet GPIbα mediates hepatic thrombopoietin (TPO) production.

The human body produces and removes 1011 platelets daily to maintain a normal steady state platelet count. Platelet production must be regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. Multifaceted and complex mechanisms control platelet production and removal in physiological and pathological conditions. This review will focus on different mechanisms of platelet senescence and clearance with specific emphasis on the role of posttranslational modifications. It will also briefly address platelet transfusion and the role of glycans in the clearance of stored platelets.

The human body produces and removes 10 platelets daily to maintain a normal steady-state platelet count. Platelet production must be tightly regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. Multifaceted and complex mechanisms control platelet removal and production in physiological and pathological conditions. This review will focus on different mechanisms of platelet clearance, with focus on the biological significance of platelet glycans.The Ashwell-Morrell receptor (AMR) recognizes senescent, desialylated platelets under steady state conditions. Desialylated platelets and the AMR are the physiological ligand-receptor pair regulating hepatic thrombopoietin (TPO) mRNA production, resolving the longstanding mystery of steady state TPO regulation. The AMR-mediated removal of desialylated platelets regulates TPO synthesis in the liver by recruiting JAK2 and STAT3 to increase thrombopoiesis.Inhibition of TPO production downstream of the hepatic AMR-JAK2 signaling cascade could additionally contribute to the thrombocytopenia associated with JAK1/2 treatment, which is clinically used in myeloproliferative neoplasms.

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA- PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well- defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA- PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation.

The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count. Platelet production must be tightly regulated to avoid spontaneous bleeding if counts are low (thrombocytopenia) or arterial occlusion and organ damage if counts are high (thrombocytosis). Recent studies have highlighted the role of glycan modifications on platelet surface proteins in mediating platelet clearance. Platelets undergoing cold storage lose sialic acid and β-galactose. Following transfusion, cold-stored platelets are cleared via hepatic Ashwell–Morell receptor (AMR) and the macrophage (Kupffer cell) αMβ2 integrin lectin domain. New data point to the fact that platelets surface loses sialic acid during their circulatory lifetime, suggesting a novel platelet in vivo clearance mechanism via the AMR. Thrombopoietin (TPO) is the primary regulator of platelet production, supporting the survival, proliferation and differentiation of the platelet precursors, bone marrow megakaryocytes. Although it is clear that hepatocytes are the primary source of TPO, the mechanisms regulating circulating TPO levels have been subject to discussion for decades. Two major models for TPO regulation have been proposed. In one model, hepatic TPO production is constitutive and TPO serum levels are maintained solely by its uptake and metabolism by the TPO receptor c-Mpl on platelets and megakaryocytes. In another model, hepatic TPO production is regulated. However, the physiological ligand–receptor pair capable of regulating steady-state TPO production remains unclear. In this review, mechanisms of glycan–lectin-mediated platelet clearance and TPO production will be discussed.

Our understanding of cell biological processes involved in aging has advance greatly over the past decades. Platelets are small cells that circulate for 4-5 days in mice and 7-10 days in humans. And even though, platelets are anucleated cells, a growing body of evidence shows that platelet clearance is a well-regulated mechanism. We have recently demonstrated that platelets lose sialic acid as they circulate and age in blood and are rapidly cleared by the hepatic Ashwell-Morell receptor (AMR). And others have shown, in a series of studies using genetically modified mice or pharmacological inhibitors that platelets undergo apoptosis by triggering the intrinsic mitochondrial apoptotic machinery. Here, we investigate if desialylation and apoptosis are related events. First, using a newly developed state-of-the-art technique called dynamic BH3 profiling (DBP), we investigated the mitochondria readiness to undergo apoptosis on platelets derived from WT and AMR deficient (Asgr2-/-) mice. In our assay, digitonin-permeabilized platelets were exposed to activators signaling peptides (such as Bim, Bid and PUMA), and as cells undergo apoptosis due to peptide treatment, they released Cytochrome C. Our data showed that desialylated platelets derived from Asgr2-/-mice have high background levels of Cytochrome C release when compared to WT platelets in the presence of all activator peptides, indicating that desialylated platelets are highly primed to apoptosis. We also tested the level of dependence on pro-survival protein, by using sensitizer peptides (Bad, Hrk and MS1). We observed that desialylated platelets (Asgr2-/-platelets), and to a certain degree, WT platelets, are extremely sensitive to BCL-xL inhibition, as indicated by the extremely high response to Bad and Hrk peptides even at lower concentrations (0.1 and 1uM). Surprisingly, WT and Asgr2-/-platelets show very little response to the MS1 peptide, indicating that they are not dependent on MCL1 for survival, as otherwise suggested. Flow cytometry analysis revealed desialylated platelets from Asgr2-/-mice have a ~2-fold increase in Phosphatidylserine (PS) surface exposure when compared to WT platelets. In addition, western blot analysis showed increased expression of cleaved caspase 3 in Asgr2-/-platelets, but no changes in Bcl-xL protein expression between WT and Asgr2-/-platelets. Next, WT and Asgr2-/-mice received a single dose of the BH3 mimetic, ABT-737, which binds and inhibits pro-survivor proteins (Bcl-2, Bcl-xL and Bcl-w) inducing apoptosis in vivo. Approximately 2 hours after the injection of ABT-737, we observed a big drop on platelets counts in both WT (~42%) and Asgr2-/-(~59%) mice. Importantly, platelets from Asgr2-/-mouse were cleared more efficiently (~20%) from the circulation when compared to those in WT mice, consistent with the ~20% increment in platelet number observed in this mouse model and supporting the notion that the platelets that circulate longer in the Asgr2-/-mice are more sensitive to apoptotic events. To investigate if apoptosis could be triggering platelet desialylation, WT mice were treated with ABT-737 and 1hour later (time point before platelet count drop), platelets were collected and analyzed by flow cytometry. Interestingly, analysis of galactose exposure by RCA-I lectin showed no differences in desialylation between ABT-737 and PBS control groups. On the other hand, Phosphatidylserine (PS) exposure was significantly elevated on ABT-737 group, indicating that platelets were undergoing apoptosis without changing their sialylated status. To confirm our in vivodata, freshly isolated washed WT platelets were treated with ABT-737 to induce apoptosis or Neuraminidase (NA) to desialylated platelets. NA treatment induced platelet desialylation (increased RCA-I binding) in WT platelets, as expected, and interestingly triggered apoptosis, judge by increased PS exposure in both ABT-737 and NA treated groups. However, ABT-737 treatment wasn't able to induce desialylation as levels of RCA-I binding to platelets was the same when compared to PBS control platelets. Taken together, our data shows that desialylated platelets in circulation are prone to apoptosis. In addition, our findings strongly support the hypothesis that desialylation of platelet surface glycoproteins trigger the intrinsic apoptotic pathway in platelets in vivo. Disclosures No relevant conflicts of interest to declare.

Besides α-granules and dense granules, which play critical roles in and beyond hemostasis, circulating blood platelets and their precursor cells megakaryocytes contain lysosomes, the contents of which are also secreted during platelet activation. In their delivery to the lysosome, acid hydrolases bearing phosphomannosyl residues are trafficked from the trans-Golgi network to the acidic late-endosomal compartment via the mannose 6-phosphate receptor (M6PR) pathway. To determine the role of M6PR-specific targeting of lysosomal enzymes in platelet function, platelet parameters were investigated in M6pr-/- mice lacking the 46-kDa M6PR, the physiological role of which is unclear. M6pr-/- mice had normal platelet count but displayed an increased number of distinct proplatelet-like cells compared to control mice, as determined by immunofluorescent microscopy. Moreover, transmission electron microscopy revealed the presence of abnormal membrane tubulations, elongated and electron-dense granules, and large vacuole-like structures within resting M6pr-/- platelets. M6pr-/- platelets expressed normally major glycoproteins on their surface and von Willebrand factor and fibrinogen in their α-granules. M6pr-/- mice were hyper-thrombotic, as assessed by tail bleeding time, and M6pr-/- platelets adhered to type I collagen with a significantly greater propensity than control platelets under arterial shear in in vitro flow experiments. Heparanase, an endo-β-glucuronidase that cleaves extracellular matrix heparan sulfate proteoglycans, is the most abundant lysosomal enzyme in platelets. Thus, its contribution to the phenotype of M6pr-/- mice was investigated further. Heparanase expression was decreased in the bone marrow megakaryocytes and blood platelets of M6pr-/- mice and increased in M6pr-/- plasma, as evidenced by immunoblot and fluorescent microscopy analysis, consistent with its mistargeting in the absence of M6PR. Interestingly, pharmacological inhibition of heparanase with OGT 2115 normalized the adhesion of M6pr-/- platelets to collagen in vitro, indicating that increased plasma heparanase contributes to the thrombotic phenotype of M6pr-/- mice. Taken together, the data suggest that the M6PR-specific targeting of lysosomal heparanase plays a critical role in platelet function, thereby regulating hemostasis. Disclosures No relevant conflicts of interest to declare.

The thrombopoietin receptor Mpl plays a critical role in thrombopoiesis, as it is the primary signaling constituent in megakaryocyte (MK) differentiation from hematopoietic stem and progenitor cells (HSPCs) and maintains circulating blood thrombopoietin levels via clathrin-mediated endocytosis. Thus, thrombopoiesis is tightly controlled by Mpl cell-surface expression on HSPCs, MKs, and platelets. Previous work by our group has shown that Dnm2fl/flPF4-Cre (Dnm2Plt-/-) mice, lacking the large endocytic GTPase dynamin 2 (DNM2) specifically within MKs and platelets, develop a variety of clinical phenotypes that closely resemble myelofibrosis, such as bone marrow fibrosis, marked HSPC expansion, MK hyperplasia, extramedullary hematopoiesis, and severe splenomegaly (Bender, Giannini, et al. Blood. 2015;125(6):1014-1024). Dnm2Plt-/- mice also displayed elevated plasma TPO levels and constitutive JAK2 activation in platelets, due to defective Mpl endocytosis. Here, the role of Mpl endocytosis in the maintenance of normal hematopoiesis was assessed by generating Dnm2Plt-/- mice in the Mpl-/- background. At 3 weeks of age, Mpl-/- Dnm2Plt-/- mice displayed significantly reduced HSPCs, a near complete depletion of bone marrow MKs, similar to Mpl-/- mice, indicating that Mpl is the primary receptor contributing to the aberrant hyperproliferative phenotype of Dnm2Plt-/- mice. However, Mpl-/- Dnm2Plt-/- mice also showed severe anemia, defects in erythroblast maturation, grossly elevated plasma erythropoietin (EPO) levels, and splenomegaly, resulting in early fatality by 3 to 4 weeks of age, suggesting that Mpl contributes to normal erythropoiesis in young mice. Mpl-/- and Mpl+/- Dnm2Plt-/- mice displayed reduced erythroblast development at 3 weeks of age, which returned to normal with adulthood. Taken together, the data shows that DNM2-dependent Mpl endocytosis is required for steady-state hematopoiesis and provides novel insights into a developmentally-controlled role for Mpl in normal erythropoiesis. Disclosures Sola-Visner: Sysmex America, Inc.: Other: Laboratory equipment on loan, Research Funding.

One of the key unmet needs to improve long-term outcomes of heart transplantation is to develop accurate, noninvasive, and practical diagnostic tools to detect transplant rejection. Early intragraft inflammation and endothelial cell injuries occur prior to advanced transplant rejection. We developed a novel diagnostic imaging platform to detect early declines in microvascular perfusion (MP) of cardiac transplants using contrast-enhanced ultrasonography (CEUS). The efficacy of CEUS in detecting transplant rejection was tested in a murine model of heart transplants, a standard preclinical model of solid organ transplant. As compared to the syngeneic groups, a progressive decline in MP was demonstrated in the allografts undergoing acute transplant rejection (40%, 64%, and 92% on days 4, 6, and 8 posttransplantation, respectively) and chronic rejection (33%, 33%, and 92% on days 5, 14, and 30 posttransplantation, respectively). Our perfusion studies showed restoration of MP following antirejection therapy, highlighting its potential to help monitor efficacy of antirejection therapy. Our data suggest that early endothelial cell injury and platelet aggregation contributed to the early MP decline observed in the allografts. High-resolution MP mapping may allow for noninvasive detection of heart transplant rejection. The data presented have the potential to help in the development of next-generation imaging approaches to diagnose transplant rejection.

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This platelet-glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions.

In recent years, it has become increasingly apparent that the life span of transfused platelets in circulation is regulated, at least in part, by glycan-lectin mediated mechanisms. There is clear evidence that refrigerated platelets are cleared by glycan-lectin mediated clearance mechanisms. Acute platelet cooling clusters glycoprotein (GP) Ibα receptors bearing uncovered N-acetylglucosamine (GlcNAc), and α(M) β(2) integrins on hepatic macrophages recognise clustered GlcNAc to rapidly clear these platelets from circulation. With prolonged refrigeration GPIbα clustering bearing uncovered galactose increases, which mediates the removal of long-term refrigerated platelets via hepatic Ashwell-Morell receptors (AMR), originally named as asialoglycoprotein receptors. In contrast, little is known about the molecular mechanisms of transfused room temperature platelet clearance. This review examines the role of glycan-lectin mediated clearance of exogenous, that is transfused chilled platelet clearance and briefly addresses the current knowledge of stored platelet function, degradation and its relation to platelet clearance.

Blood platelets are involved in primary and secondary hemostasis and thus maintain the integrity of the vasculature. They circulate with an average lifespan of 5-9 days in humans. Thus, the body must generate and clear platelets daily to maintain normal physiological blood platelet counts. Known platelet clearance mechanisms include antibody-mediated clearance by spleen macrophages, as in immune thrombocytopenia, and platelet consumption due to massive blood loss.New concepts in the clearance mechanisms of platelets have recently emerged. New evidence shows that platelets desialyted due to chilling or sepsis are cleared in the liver by macrophages, that is Kupffer cells, as well as hepatocytes, through lectin-mediated recognition of platelet glycans. On the contrary, platelet-associated antibodies normalize the clearance of platelets in a mouse model for Wiskott-Aldrich syndrome.The goal of this review is to summarize the latest findings in platelet clearance mechanisms with a focus on lectin-mediated recognition of platelet glycans. Transfusion medicine and treatments of hematopoietic disorders associated with severe thrombocytopenia may benefit from a better understanding of these mechanisms.

The role of the Wiskott-Aldrich syndrome protein (WASp) in platelet function is unclear because platelets that lack WASp function normally. WASp constitutively associates with WASp-interacting protein (WIP) in resting and activated platelets. The role of WIP in platelet function was investigated using mice that lack WIP or WASp. WIP knockout (KO) platelets lack WASp and thus are double deficient. WIP KO mice have a thrombocytopenia, similar to WASp KO mice, resulting in part from enhanced platelet clearance. Most WIP KO, but not WASp KO, mice evolved platelet-associated immunoglobulins (Ig) of the IgA class, which normalize their platelet survival but diminish their glycoprotein VI (GPVI) responses. Protein tyrosine phosphorylation, including that of phospholipase C-γ2, and calcium mobilization are impaired in IgA-presenting WIP KO platelets stimulated through GPVI, resulting in defects in α-granule secretion, integrin αIIbβ3 activation, and actin assembly. The anti-GPVI antibody JAQ1 induces the irreversible loss of GPVI from circulating platelets in wild-type mice, but not in WIP KO mice that bear high levels of platelet-associated IgAs. Together, the data indicate that platelet-associated IgAs negatively modulate GPVI signaling and function in WIP KO mice.

Although surface sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet circulation lifetime is not fully clarified. We show that thrombocytopenia in mice deficient in the St3gal4 sialyltransferase gene (St3Gal-IV−/− mice) is caused by the recognition of terminal galactose residues exposed on the platelet surface in the absence of sialylation. This results in accelerated platelet clearance by asialoglycoprotein receptor-expressing scavenger cells, a mechanism that was recently shown to induce thrombocytopenia during Streptococcus pneumoniae sepsis. We now identify platelet GPIbα as a major counterreceptor on ST3Gal-IV−/− platelets for asialoglycoprotein receptors. Moreover, we report data that establish the importance of sialylation of the von Willebrand factor in its function.

Platelet products are currently stored at room temperature, because refrigeration causes their rapid clearance from the circulation upon transfusion. Glycans have recently been emphasized as important determinants for the clearance of refrigerated platelets. The present review addresses the current knowledge of platelet glycans and the potential of glycosylation for improving platelet storage.Removal of refrigerated platelets from the circulation is partly mediated by recognition of clustered beta-N-acetylglucosamine on platelet surface glycoproteins by the alphaMbeta2 hepatic lectin receptor. Capping the exposed beta-N-acetylglucosamine residues by enzymatic galactosylation restored the circulation of short-term chilled murine platelets, introducing a novel method that allows for cold storage of platelet. Recent studies have, however, shown that galactosylation is not sufficient to restore circulation of long-term refrigerated platelets. Additional data indicate that differential carbohydrate-mediated mechanisms may exist for clearance of short-term and long-term cold-stored platelets.Room temperature storage of platelet products increases the risk of transfusion-mediated sepsis and accelerates platelet deterioration, limiting platelet shelf life. Recent evidence suggests that glycoengineering of platelets might allow for their cold storage, significantly improving the quality of platelet products.

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (± 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (± 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP–expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states.

BACKGROUND Short-term refrigeration of platelets (PLTs) in the absence of plasma results in their rapid clearance after transfusion. Blocking beta-N-acetylglucosamine (beta-GlcNAc) residues of glycoprotein Ibalpha (GPIbalpha) with galactose prevents binding of refrigerated human and mouse PLTs to macrophages and prolongs the circulation times of refrigerated mouse PLTs. PLT-associated galactosyltransferase efficiently galactosylates chilled PLTs in the presence of its substrate UDP-galactose is added to PLT-rich plasma. STUDY DESIGN AND METHODS To characterize the hemostatic function of refrigerated and galactosylated human PLTs processed in the blood bank, PLT aggregation was studied in vitro under static and flow conditions and expression of integrin beta3 (CD61), CD62P (P-selectin), GPIbalpha (CD42b), annexin V binding, and integrin alphaIIbeta3 activation with flow cytometry. Affinity of macrophages for galactosylated refrigerated PLTs was evaluated with THP-1 cells, which recognize and phagocytize refrigerated PLTs. RESULTS PLTs refrigerated and galactosylated for 14 days 1) maintained their ability to aggregate when exposed to agonists in a standard aggregometry assay, 2) showed less pronounced changes in surface expression of GPIbalpha compared with room temperature (RT)-stored PLTs, 3) increased P-selectin expression, and 4) were poorly phagocytized by differentiated THP-1 cells in vitro. In addition, it is shown that refrigeration of PLTs does not affect their adhesive properties under in vitro flow conditions. CONCLUSION It is shown that refrigerated human PLTs retain in vitro function better than RT PLTs during storage and demonstrate that galactosylation prevents recognition of stored refrigerated PLTs by macrophages in vitro.

Transfusion of human platelets, stored in blood banks as concentrates, is the treatment of choice for thrombocytopenia and bleeding. To be clinically effective, transfused platelets must circulate

Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa and/or the GPIb complex. Current theory suggests that antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc–FcγR interactions. However, we and others have demonstrated that anti-GPIbα (but not GPIIbIIIa)-mediated ITP is often refractory to therapies targeting FcγR pathways. Here, we generate mouse anti-mouse monoclonal antibodies (mAbs) that recognize GPIbα and GPIIbIIIa of different species. Utilizing these unique mAbs and human ITP plasma, we find that anti-GPIbα, but not anti-GPIIbIIIa antibodies, induces Fc-independent platelet activation, sialidase neuraminidase-1 translocation and desialylation. This leads to platelet clearance in the liver via hepatocyte Ashwell–Morell receptors, which is fundamentally different from the classical Fc–FcγR-dependent macrophage phagocytosis. Importantly, sialidase inhibitors ameliorate anti-GPIbα-mediated thrombocytopenia in mice. These findings shed light on Fc-independent cytopenias, designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of refractory ITP., Immune thrombocytopenia (ITP) is caused by autoantibody-mediated platelet clearance, but refractoriness to current immunomodulatory therapies is common. Here the authors show that desialylated platelets can be cleared via hepatic Ashwell–Morell receptor, a process that can be attenuated by sialidase inhibitors, suggesting a new therapy for ITP.