Chilling rapidly (<4 h) clusters Glycoprotein - (GP)Ib receptors on blood platelets, and ß2-integrins of hepatic macrophages bind ßGlcNAc residues in the clusters leading to rapid clearance of acutely chilled platelets following transfusion. Although capping the ßGlcNAc moieties by galactosylation prevents clearance, this strategy is ineffective after prolonged (>24 h) refrigeration. We report here that prolonged refrigeration increases the density/concentration of exposed galactose residues such that hepatocytes become increasingly involved in the removal of platelets using their Ashwell-Morell receptors. Macrophages always rapidly remove a large fraction of transfused platelets (~40%). With platelet cooling, hepatocyte-dependent clearance further diminishes their recoveries following transfusion.

The MUC1 mucin represents a prime target antigen for cancer immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas. Attempts to generate strong humoral immunity to MUC1 by immunization with peptides have generally failed partly because of tolerance. In this study, we have developed chemoenzymatic synthesis of extended MUC1 TR glycopeptides with cancer-associated O-glycosylation using a panel of recombinant human glycosyltransferases. MUC1 glycopeptides with different densities of Tn and STn glycoforms conjugated to KLH were used as immunogens to evaluate an optimal vaccine design. Glycopeptides with complete O-glycan occupancy (five sites per repeat) elicited the strongest antibody response reacting with MUC1 expressed in breast cancer cell lines in both Balb/c and MUC1.Tg mice. The elicited humoral immune response showed remarkable specificity for cancer cells suggesting that the glycopeptide design holds promise as a cancer vaccine. The elicited immune responses were directed to combined glycopeptide epitopes, and both peptide sequence and carbohydrate structures were important for the antigen. A MAb (5E5) with similar specificity as the elicited immune response was generated and shown to have the same remarkable cancer specificity. This antibody may hold promise in diagnostic and immunopreventive measures.

The cell membrane mucin MUC1 is over-expressed and aberrantly glycosylated in many cancers, and cancer-associated MUC1 glycoforms represent potential targets for immunodiagnostic and therapeutic measures. We have recently shown that MUC1 with GalNAcalpha1-O-Ser/Thr (Tn) and NeuAcalpha2-6GalNAcalpha1-O-Ser/Thr (STn) O-glycosylation is a cancer-specific glycoform, and that Tn/STn-MUC1 glycopeptide-based vaccines can override tolerance in human MUC1 transgenic mice and induce humoral immunity with high specificity for MUC1 cancer-specific glycoforms (Sorensen AL, Reis CA, Tarp MA, Mandel U, Ramachandran K, Sankaranarayanan V, Schwientek T, Graham R, Taylor-Papadimitriou J, Hollingsworth MA, et al. 2006. Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology. 16:96-107). In order to further characterize the immune response to Tn/STn-MUC1 glycoforms, we generated monoclonal antibodies with specificity similar to the polyclonal antibody response found in transgenic mice. In the present study, we define the immunodominant epitope on Tn/STn-MUC1 glycopeptides to the region including the amino acids GSTA of the MUC1 20-amino acid tandem repeat (HGVTSAPDTRPAPGSTAPPA). Most other MUC1 antibodies are directed to the PDTR region, although patients with antibodies to the GSTA region have been identified. A panel of other MUC1 glycoform-specific monoclonal antibodies was included for comparison. The study demonstrates that the GSTA region of the MUC1 tandem repeat contains a highly immunodominant epitope when presented with immature short O-glycans. The cancer-specific expression of this glycopeptide epitope makes it a prime candidate for immunodiagnostic and therapeutic measures.

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 receptorexpressing scavenger cells, a mechanism that was recently shown to induce thrombocytopenia during Streptococcus pneumoniae sepsis. We now identify platelet GPIb␣ 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. (Blood. 2009;114:1645-1654)

Cold storage of platelets for transfusion is desirable to extend platelet storage times and to prevent bacterial growth. However, the rapid clearance of coldstored platelets prevents their use. A novel method for preventing the rapid clearance of cold-stored platelets has previously been developed in a murine model. Cold storage induces the clustering and recognition of exposed ␤-Nacetylglucosamine (␤GlcNAc) on platelet surfaces. Glycosylation of ␤GlcNAc residues with uridine 5-diphosphogalactose (UDP-galactose) results in the normal survival of short-term (2 h) 0°C-stored murine platelets. Based on this finding, we developed a similar glycosylation process by adding UDP-galactose to human apheresis platelets. A phase 1 clinical trial was conducted transfusing radiolabeled autologous apheresis platelets stored for 48 hours at 4°C with or without pretreatment with UDP-galactose. In contrast to the murine study, galactosylation of human platelets did not prevent the accelerated platelet clearance routinely observed after 4°C storage. We next developed a murine model of platelet storage for 48 hours at 4°C and showed that UDP-galactose treatment of murine platelets also did not prevent their rapid clearance, in agreement with the human platelet study. We conclude that different mechanisms of clearance may exist for short-and long-term cold-stored platelets. (Blood. 2008;111:3249-3256)

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver proand 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 plateletglycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions. (Blood. 2012;120(3):626-635) IntroductionGlycosylation of proteins and lipids has a wide range of biologic functions. 1 The glycosylation apparatus of nucleated cells is primarily located in the secretory pathway throughout the endoplasmic reticulum-Golgi stacks and consists of more than 200 glycosyltransferases, most of which are type II transmembrane-anchored proteins with distinct localization. Their topology within these compartments is directed by signal motifs contained in their stem, transmembrane, and cytoplasmic domains. 2,3 Golgi-located membrane-bound glycosyltransferases are believed to be retained in the appropriate compartments by coat protein I-mediated retrograde transport 3 and to be released and secreted only after proteolytic cleavage in their stalk regions to yield soluble, catalytically active truncated enzymes lacking their N-terminal transmembrane segment. 4 Several previous studies have described unusual subcellular localization of glycosyltransferases outside of their normal confinement to the endoplasmic reticulum-Golgi (referred to herein as ectopic localization). Methodologic ambiguities, however, have cast doubt on these findings. 5 One notable exception concerns reports of glycosyltransferase activities associated with blood platelets. [6][7][8][9][10][11] Platelets are anucleated megakaryocyte subfragments that participate in hemostatic, inflammatory, and host defense reactions. 12 Moreover, platelets deliver pro-and antiangiogenic factors throughout the vascular system. 12 Glycosyltransferase activities in platelets were described 30 years ago and are propo...

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Recently, reports of severe thromboses, thrombocytopenia, and hemorrhage in persons vaccinated with the chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19, AZD1222, Vaxzevria; Oxford/AstraZeneca) against severe acute respiratory syndrome coronavirus 2 have emerged. We describe an otherwise healthy 30-year-old woman who developed thrombocytopenia, ecchymosis, portal vein thrombosis, and cerebral venous sinus thrombosis the second week after she received the ChAdOx1 nCoV-19 vaccine. Extensive diagnostic workup for thrombosis predispositions showed heterozygosity for the prothrombin mutation, but no evidence of myeloproliferative neoplasia or infectious or autoimmune diseases. Her only temporary risk factor was long-term use of oral contraceptive pills (OCPs). Although both the prothrombin mutation and use of OCPs predispose to portal and cerebral vein thrombosis, the occurrence of multiple thromboses within a short time and the associated pattern of thrombocytopenia and consumption coagulopathy are highly unusual. A maximum 4T heparin-induced thrombocytopenia (HIT) score and a positive immunoassay for anti-platelet factor 4/heparin antibodies identified autoimmune HIT as a potential pathogenic mechanism. Although causality has not been established, our case emphasizes the importance of clinical awareness. Further studies of this potentially new clinical entity have suggested that it should be regarded as a vaccine-induced immune thrombotic thrombocytopenia.