Clinical impact of glycans in platelet and megakaryocyte biology.

Humans produce and remove 1011 platelets daily to maintain a steady-state platelet count. The tight regulation of platelet production and removal from the blood circulation prevents anomalies in both processes from resulting in reduced or increased platelet count, often associated with the risk of bleeding or overt thrombus formation, respectively. This review focuses on the role of glycans, also known as carbohydrates or oligosaccharides, including N- and O-glycans, proteoglycans, and glycosaminoglycans, in human and mouse platelet and megakaryocyte physiology. Based on recent clinical observations and mouse models, we focused on the pathologic aspects of glycan biosynthesis and degradation and their effects on platelet numbers and megakaryocyte function.

Increased B4GALT1 expression is associated with platelet surface galactosylation and thrombopoietin plasma levels in MPNs.

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 ß4-N-acetyllactosamine (Galß1,4GlcNAc 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 ß-1,4-galactosyltransferase 1 (ß4GalT1). 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 ß4GalT1 as a potential actionable target for therapy.

Immune cells surveil aberrantly sialylated O-glycans on megakaryocytes to regulate platelet count.

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.

Reduced platelet glycoprotein Ibα shedding accelerates thrombopoiesis and COX-1 recovery: implications for aspirin dosing regimen.

Cardiovascular (CV) disease prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet (PLT) cyclooxygenase (COX)-1 activity during the 24-hour dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced PLT glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin (TPO) production and higher rates of newly formed PLT, escaping aspirin inhibition over 24 hours. Two hundred aspirin-treated patients with high CV risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of PLT COX-1 activity recovery during the 10- to 24-hour dosing interval. Whole proteome analysis showed that PLT from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma TPO, megakaryocyte maturation and proplatelet formation, and conversely increased PLT galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin (GC) release. Treatment of HepG2 cells with recombinant GC led to a dose-dependent reduction of TPO mRNA in the liver, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher TPO/GC ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating TPO/GC ratio, reflecting a dysregulation of PLT lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing.

Efficiently Restored Thrombopoietin Production by Ashwell-Morell Receptor and IL-6R Induced Janus Kinase 2/Signal Transducer and Activator of Transcription Signaling Early After Partial Hepatectomy.

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.

RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival.

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.

Circulating platelet count and glycans.

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.

Desialylation of O-glycans on glycoprotein Ibα drives receptor signaling and platelet clearance.

During infection neuraminidase desialylates platelets and induces their rapid clearance from circulation. The underlying molecular basis, particularly the role of platelet glycoprotein (GP)Ibα therein, is not clear. Utilizing genetically altered mice we report that the extracellular domain of GPIbα, but neither von Willebrand factor nor ADAM17 (a disintegrin and metalloprotease 17), is required for platelet clearance induced by intravenous injection of neuraminidase. Lectin binding to platelets following neuraminidase injection over time revealed that the extent of desialylation of O-glycans correlates with the decrease of platelet count in mice. Injection of α2,3-neuraminidase reduces platelet counts in wild-type but not in transgenic mice expressing only a chimeric GPIbα that misses most of its extracellular domain. Neuraminidase treatment induces unfolding of the O-glycosylated mechanosensory domain in GPIbα as monitored by single-molecule force spectroscopy, increases the exposure of the ADAM17 shedding cleavage site in the mechanosensory domain on the platelet surface, and induces ligand-independent GPIb-IX signaling in human and murine platelets. These results suggest that desialylation of O-glycans of GPIbα induces unfolding of the mechanosensory domain, subsequent GPIb-IX signaling including amplified desialylation of N-glycans, and eventually rapid platelet clearance. This new molecular mechanism of GPIbα-facilitated clearance could potentially resolve many puzzling and seemingly contradicting observations associated with clearance of desialylated or hyposialylated platelets.

Refrigeration-Induced Binding of von Willebrand Factor Facilitates Fast Clearance of Refrigerated Platelets.

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 Ca2+, 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.

Circulating blood and platelets supply glycosyltransferases that enable extrinsic extracellular glycosylation.

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(ß3)GlcNAc, Gal(ß4)GlcNAc and Gal(ß3)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 ß4GalT. 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(ß4)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.

Regulating billions of blood platelets: glycans and beyond.

The human body produces and removes 10(11) 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.

Dynamin 2-dependent endocytosis is required for normal megakaryocyte development in mice.

Dynamins are highly conserved large GTPases (enzymes that hydrolyze guanosine triphosphate) involved in endocytosis and vesicle transport, and mutations in the ubiquitous and housekeeping dynamin 2 (DNM2) have been associated with thrombocytopenia in humans. To determine the role of DNM2 in thrombopoiesis, we generated Dnm2(fl/fl) Pf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2(fl/fl) Pf4-Cre mice had severe macrothrombocytopenia with moderately accelerated platelet clearance. Dnm2-null bone marrow MKs had altered demarcation membrane system formation in vivo due to defective endocytic pathway, and fetal liver-derived Dnm2-null MKs formed proplatelets poorly in vitro, showing that DNM2-dependent endocytosis plays a major role in MK membrane formation and thrombopoiesis. Endocytosis of the thrombopoietin receptor Mpl was impaired in Dnm2-null platelets, causing constitutive phosphorylation of the tyrosine kinase JAK2 and elevated circulating thrombopoietin levels. MK-specific DNM2 deletion severely disrupted bone marrow homeostasis, as reflected by marked expansion of hematopoietic stem and progenitor cells, MK hyperplasia, myelofibrosis, and consequent extramedullary hematopoiesis and splenomegaly. Taken together, our data demonstrate that unrestrained MK growth and proliferation results in rapid myelofibrosis and establishes a previously unrecognized role for DNM2-dependent endocytosis in megakaryopoiesis, thrombopoiesis, and bone marrow homeostasis.

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.

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.

Expansion of the neonatal platelet mass is achieved via an extension of platelet lifespan.

The fetal/neonatal hematopoietic system must generate enough blood cells to meet the demands of rapid growth. This unique challenge might underlie the high incidence of thrombocytopenia among preterm neonates. In this study, neonatal platelet production and turnover were investigated in newborn mice. Based on a combination of blood volume expansion and increasing platelet counts, the platelet mass increased sevenfold during the first 2 weeks of murine life, a time during which thrombopoiesis shifted from liver to bone marrow. Studies applying in vivo biotinylation and mathematical modeling showed that newborn and adult mice had similar platelet production rates, but neonatal platelets survived 1 day longer in circulation. This prolonged lifespan fully accounted for the rise in platelet counts observed during the second week of murine postnatal life. A study of pro-apoptotic and anti-apoptotic Bcl-2 family proteins showed that neonatal platelets had higher levels of the anti-apoptotic protein Bcl-2 and were more resistant to apoptosis induced by the Bcl-2/Bcl-xL inhibitor ABT-737 than adult platelets. However, genetic ablation or pharmacologic inhibition of Bcl-2 alone did not shorten neonatal platelet survival or reduce platelet counts in newborn mice, indicating the existence of redundant or alternative mechanisms mediating the prolonged lifespan of neonatal platelets.

Glycans and the platelet life cycle.

Platelet numbers are intricately regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. The growth factor thrombopoietin (TPO) drives platelet biogenesis by inducing megakaryocyte production. A recent study in mice identified a feedback mechanism by which clearance of aged, desialylated platelets stimulates TPO synthesis by hepatocytes. This new finding generated renewed interest in platelet clearance mechanisms. Here, different established and emerging mechanisms of platelet senescence and clearance will be reviewed with specific emphasis on the role of posttranslational modifications.

Novel mechanisms of platelet clearance and thrombopoietin regulation.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: 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.