scholarly journals Sugar Matters: Improving In Vivo Clearance Rate of Highly Glycosylated Recombinant Plasma Proteins for Therapeutic Use

2021 ◽  
Vol 14 (1) ◽  
pp. 54
Author(s):  
Sacha Zeerleder ◽  
Ruchira Engel ◽  
Tao Zhang ◽  
Dorina Roem ◽  
Gerard van Mierlo ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Relative Abundance ◽  
Clearance Rate ◽  
Ricinus Communis ◽  
Protein Solubility ◽  
Chinese Hamster ◽  
Screening Tools ◽  
Terminal Sialic Acid ◽  
Terminal Galactose

Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. N-glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA120) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.

scholarly journals Modulation of human thyrotropin oligosaccharide structures--enhanced proportion of sialylated and terminally galactosylated serum thyrotropin isoforms in subclinical and overt primary hypothyroidism

1998 ◽  
Vol 158 (3) ◽  
pp. 359-365 ◽  
Author(s):  
J Trojan ◽  
M Theodoropoulou ◽  
KH Usadel ◽  
GK Stalla ◽  
L Schaaf
Keyword(s):  
Sialic Acid ◽  
Human Serum ◽  
Ricinus Communis ◽  
Primary Hypothyroidism ◽  
Metabolic Clearance ◽  
Terminal Galactose ◽  
Before And After ◽  
Few Data ◽  
Serum Tsh

Enhanced sialylation of thyrotropin (TSH) prolongs its metabolic clearance rate and thus increases the hormone's in vivo bioactivity. This has been shown for hypothyroid rats and for recombinant human TSH, but there are few data on the sialylation of human serum TSH. The aim of this work was to further study sialylated human serum TSH, its precursors bearing terminal galactose residues, and the role of pharmacological doses of thyrotropin-releasing hormone (TRH) on their secretion under different degrees of primary hypothyroidism. We analyzed serum TSH in patients with subclinical (n = 9) and overt primary hypothyroidism (n = 13) compared with euthyroid individuals (n = 12) and human standard pituitary TSH (IRP 80/558). Blood was drawn before and 30 min after intravenous administration of 200 micrograms TRH, and TSH was purified by immunoaffinity concentration. The content of sialylated (sialo-) TSH and isoforms bearing terminal galactose (Gal-TSH, asialo-Gal-TSH) was measured by Ricinus communis (RCA 120) affinity chromatography in combination with enzymatic cleavage of sialic acid residues. TSH immunoreactivity was measured by an automated second generation TSH immunoassay. Pituitary TSH contained 16.5 +/- 0.8% Gal-TSH. In euthyroid individuals the proportion of Gal-TSH was 14.6 +/- 1.9%, whereas TSH in patients with subclinical and overt primary hypothyroidism contained 23.9 +/- 3.5% (P < 0.05 vs euthyroid individuals) and 21.1 +/- 1.7% Gal-TSH respectively. The mean ratio of asialo-Gal TSH was 23.8 +/- 0.6% for pituitary TSH, 35.7 +/- 4.2% in euthyroid individuals, 48.0 +/- 3.3% in patients with subclinical, and 61.5 +/- 3.8% (P < 0.001 vs euthyroid individuals) in patients with overt primary hypothyroidism. For pituitary TSH the calculated proportion of sialo-TSH was 6.5 +/- 0.2%, for euthyroid individuals 20.3 +/- 2.8%, for patients with subclinical hypothyroidism 24.1 +/- 3.0%, and for patients with overt primary hypothyroidism 40.7 +/- 3.0% (P < 0.001 vs euthyroid individuals). The proportions of Gal-TSH, asialo-Gal-TSH, and sialo-TSH did not differ significantly before and after TRH administration in the individuals studied. Our data show that patients with subclinical and overt primary hypothyroidism have a markedly increased proportion of serum TSH isoforms bearing terminal galactose and sialic acid residues, which may represent a mechanism for the further stimulation of thyroid function. Pharmacological doses of TRH cause an increased quantity of TSH to be released, but do not significantly alter the proportion of sialylated or terminally galactosylated TSH isoforms.

Loss Of Platelet Membrane Glycoproteins Or Terminal Sialic Acid Detected By Fitc-Lectins

1981 ◽  
Author(s):  
L McGregor ◽  
J L McGregor ◽  
K J Clemetson ◽  
M Dechavanne ◽  
E F Lüscher
Keyword(s):  
Sialic Acid ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Binding Studies ◽  
Platelet Surface ◽  
Terminal Sialic Acid

Pre-thrombic conditions in certain individuals resulting from enhanced platelet-vessel wall or platelet-platelet interactions are perhaps characterized by a reduction in certain membrane glycoproteins or loss of terminal sialic acid. In order to investigate if such changes are detectable, the binding of FITC-lectins to human platelets treated under in vitro conditions with certain proteases to mimic possible in vivo changes occuring on the platelet surface, has been examined. Human platelets were isolated, washed and either treated with neuraminidase (10 U) or plasmin (1 CU) before fixing with formaldehyde. Binding studies were performed by the method of Monsigny et al. using FITC labelled wheat germ agglutinin (WGA), Lens culinaris lectin (LCL), Ricinus communis agglutinin (RCA) and concanavalin A (ConA). The number of lectin-binding sites (n) and the dissociation constant (Kd) were obtained by Steck and Wallach reciprocal plots. After neuraminidase or plasmin treatment n was reduced but Kd remained approximately the same with WGA. FITC-RCA-60 gave a slight fluorescence with untreated and very strong fluorescence with neuraminidase treated platelets. Platelet glycoproteins separated by 2-dimensional gel electrophoresis were identified by binding of fluorescent lectins. Plasmin decreased the intensity of GP Ib and IIb and removed Ia completely. Neuraminidase decreased the labelling of Ib by WGA. These techniques show promise as methods of detecting pre-thrombotic conditions.

scholarly journals Differential Glycosylation Between Recombinant Factor VIII Produced in Baby Hamster Kidney and Chinese Hamster Ovary Cells Confers Differences in Immunogenicity in a Humanized Hemophilia Α Mouse Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 326-326 ◽  
Author(s):  
Jesse Lai ◽  
Laura L Swystun ◽  
Dominique Cartier ◽  
Cunjie Zhang ◽  
Kate Nesbitt ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Factor Viii ◽  
Research Funding ◽  
Clinical Evidence ◽  
Lectin Binding ◽  
Chinese Hamster ◽  
Specific Igg ◽  
Ifn Γ ◽  
High Mannose

Abstract Introduction The formation of factor VIII (FVIII)- neutralizing antibodies is the most critical complication in the treatment of hemophilia A (HA). Recent clinical evidence suggests that recombinant FVIII (rFVIII) produced in baby hamster kidney (BHK-rFVIII) cells is more immunogenic than that produced in Chinese hamster ovary (CHO-rFVIII) cells. This difference in FVIII immunogenicity may be attributed to differences in protein glycosylation, which can impact the removal of FVIII from circulation through mechanisms leading to clearance and antigen presentation. Here, we document significant differences among the 25 potential N-linked glycans between these products, and we provide in vivo animal model-based evidence that supports these clinical observations. Methods Factor VIII lectin binding was assessed by ELISA to detect exposed glycans. Commercially-available rFVIII products were adsorbed at 1 ug/mL and specific glycan moieties were detected using a panel of biotinylated lectins and HRP-conjugated streptavidin. Confirmation of differences and determination of N-linked glycan structures was conducted by LC-MS/MS. Eight to 12 week old transgenic C57BL/6 HA mice were used in these studies. This model contains a murine f8 exon 16 KO and additionally harbors a human F8 transgene with a R593C point mutation. While these mice have undetectable plasma levels of human FVIII antigen and activity, they are tolerant to intravenously infused human rFVIII. Clearance was assessed following a 6 IU (~240 IU/kg) infusion of each rFVIII product, and FVIII activity was measured by chromogenic assay and normalized to a 5-minute time point. The number of interferon (IFN)-γ secreting splenocytes from rFVIII-primed naïve mice was determined by ELISPOT. FVIII immune responses were elicited by subcutaneous infusion (6 IU twice-weekly for 2 weeks) and adjuvant-coupled intravenous infusion (1 ug lipopolysacharride with the first infusion as described above) with either rFVIII product. Week 5 plasma samples were assessed for FVIII-specific IgG by ELISA, and FVIII inhibitors by one-stage clotting assay. Results Lectin binding showed that rFVIII produced in BHK cell lines exhibit lower proportions of high-mannose glycans (p<0.01), and greater levels of sialic acid capping (p<0.01) and fucosylated glycans (p<0.01). Mass spectra confirmed higher levels of sialic acid and identified two additional N-linked sites bearing high mannose glycans on CHO-rFVIII. In this mouse model we observed that BHK-rFVIII had a circulating half-life of 6.06 hours compared to the 10.01 hour half-life of CHO-rFVIII (p<0.0001). The immunogenicity of the BHK- and CHO-rFVIII products was next evaluated in vivo. In mice primed with a single 6 IU dose of BHK-rFVIII, we identified a higher proportion of FVIII-specific IFN-γ secreting splenocytes after seven days. Furthermore, long-term studies showed that 100% of mice subcutaneously exposed to BHK-rFVIII developed anti-FVIII IgG compared to the 47% that received CHO-rFVIII (p<0.01). Coincidently, when FVIII inhibitors were measured, we observed an incidence of 100% vs 37% (p<0.01), respectively. While the titres of FVIII-specific IgG were higher in mice exposed to BHK-rFVIII (p<0.01), there were no significant differences in the inhibitor concentrations. Similarly, we observed increased titres of FVIII-specific IgG in mice exposed intravenously (1 ug LPS with the first infusion) to BHK-rFVIII compared to CHO-rFVIII. However, there were no differences in the incidence of FVIII-specific IgG, nor in the incidence and concentration of inhibitors between the intravenously-infused mice. Conclusions Our results demonstrate that BHK-rFVIII exhibits altered pharmacokinetic and immunogenic properties compared to CHO-rFVIII in humanized hemophilia A mice. The observed early increase in the proportion FVIII-specific IFN-γ producing cells in the spleen suggests an intrinsic immunogenic element of BHK-rFVIII. Similarly, the substantially increased immunogenicity of BHK-rFVIII in mice when treated subcutaneously complements the previously-reported clinical evidence. These differences may be attributed to the significant disparities in N-linked glycosylation, most notably high mannose and sialic acid containing glycans. Additional studies are underway to directly address the role of the these specific glycans and their potential impact on immunogenicity of rFVIII. Disclosures Lillicrap: Octapharma: Research Funding; Baxalta: Research Funding; Biogen-Idec: Research Funding; Bayer: Research Funding.

scholarly journals Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26)

2021 ◽  
Author(s):  
Jack R Hemsath ◽  
Manuel Liaci ◽  
Jeffrey Rubin ◽  
Brian Parrett ◽  
Shao-Chia Lu ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Ex Vivo ◽  
Chinese Hamster Ovary Cells ◽  
Ectopic Expression ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Adenovirus Serotype ◽  
Cell Counts

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against SARS-CoV-2 and HIV-1. Yet, its primary receptor portfolio remains controversial, potentially including sialic acid, CAR, integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned by the inability to co-crystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domains CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wt and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (IM) injection, but not after intranasal (IN) administration. Ad26 transduction was 10-fold lower than Ad5 after intratumoral (IT) injection of CD46-expressing tumors. Ad26 transduction of liver was 1000-fold lower than Ad5 after intravenous (IV) injection. These data demonstrate the use of CD46 by Ad26 under certain situations, but also show that the receptor has little consequence by other routes of administration. Finally, IV injection of high doses of Ad26 into CD46 mice induced release of liver enzymes in the bloodstream and reduced white blood cell counts, but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during COVID-19 vaccination with this serotype of adenovirus.

scholarly journals Broad requirement for terminal sialic acid residues and FcγRIIB for the preventive and therapeutic activity of intravenous immunoglobulins in vivo

2014 ◽  
Vol 44 (5) ◽  
pp. 1444-1453 ◽  
Author(s):  
Inessa Schwab ◽  
Sidonia Mihai ◽  
Michaela Seeling ◽  
Michael Kasperkiewicz ◽  
Ralf J. Ludwig ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Intravenous Immunoglobulins ◽  
Therapeutic Activity ◽  
Terminal Sialic Acid

Isoelectric properties, lectin binding characteristics and biological activities of neuraminidase-treated rat LH components

1988 ◽  
Vol 117 (1) ◽  
pp. 73-79
Author(s):  
Masa-aki Hattori ◽  
Kazunori Ozawa ◽  
Katsumi Wakabayashi
Keyword(s):  
Sialic Acid ◽  
Ricinus Communis ◽  
Biological Activities ◽  
Lectin Binding ◽  
Maximum Activity ◽  
Adenylate Cyclase System ◽  
Neuraminidase Treatment ◽  
Binding Characteristics ◽  
Terminal Sialic Acid ◽  
Lentil Lectin

Abstract. The present study was performed to evaluate the different carbohydrate structure of rat LH isoelectric components related to their intrinsic biological activities. Terminal sialic acid residues were essential to the formation of multiple LH components observed in the isoelectric focussing profile, which was proved by their interaction with Ricinus communis agglutinin-120 following neuraminidase treatment, and the conversion of component F (pI, 10.0) to less alkaline components after incubation with liver Golgi membrane fraction in the presence of CMP-NeuNAc. The affinity studies using lentil lectin indicated that component F was not an asialo form of component A (pI 8.4). The serial removal of sialic acid residues from these components led to increases in the steroidogenic activity, owing to increases in the activation of the receptor-adenylate cyclase system. The enhancement of the steroidogenic activity by desialylation was very great in component A'(pI, 8.0) (751% increase), and decreased with increasing pI. It can be concluded that the different biological potencies of intact LH components are attributable principally to terminal sialic acid residues. However, the peripheral chains of asialo oligosaccharides of less alkaline components (pI, 8.0, 8.4) seem to prevent the maximal cellular responses, since their desialylated forms did not attain the maximum activity.

scholarly journals Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26)

2021 ◽  
Author(s):  
Jack R. Hemsath ◽  
A. Manuel Liaci ◽  
Jeffrey D. Rubin ◽  
Brian J. Parrett ◽  
Shao-Chia Lu ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Ex Vivo ◽  
Chinese Hamster Ovary Cells ◽  
Ectopic Expression ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Adenovirus Serotype ◽  
Cell Counts ◽  
Hiv 1

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against SARS-CoV-2 and HIV-1. Yet, its primary receptor portfolio remains controversial, potentially including sialic acid, CAR, integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned by the inability to co-crystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domains CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (IM) injection, but not after intranasal (IN) administration. Ad26 transduction was 10-fold lower than Ad5 after intratumoral (IT) injection of CD46-expressing tumors. Ad26 transduction of liver was 1000-fold lower than Ad5 after intravenous (IV) injection. These data demonstrate the use of CD46 by Ad26 under certain situations, but also show that the receptor has little consequence by other routes of administration. Finally, IV injection of high doses of Ad26 into CD46 mice induced release of liver enzymes in the bloodstream and reduced white blood cell counts, but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during COVID-19 vaccination with this serotype of adenovirus. IMPORTANCE Human species D Ad26 is being pursued as a low seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection, and its role in virus biology, vaccine efficacy, and importantly, in vaccine safety.

Differences in carbohydrate composition of FSH preparations detected with lectin-ELISA systems

1995 ◽  
Vol 145 (3) ◽  
pp. 527-533 ◽  
Author(s):  
B Rafferty ◽  
J A Mower ◽  
H L Ward ◽  
M Rose
Keyword(s):  
Ricinus Communis ◽  
Sugar Composition ◽  
Sambucus Nigra ◽  
Simple Method ◽  
Maackia Amurensis ◽  
Terminal Galactose ◽  
Different Sources ◽  
Elisa Data

Abstract FSH is a glycoprotein containing N-linked carbohydrates which exhibit a variety of forms ranging from mono- to multibranched structures. Variation in glycosylation, particularly the degree of terminal sialylation, determines the half-life of the hormone and hence its in vivo bioactivity. The glycoform content of FSH preparations can differ according to the source (e.g. pituitary, urine), cell line (for rDNA-derived material) and selectivity of purification procedures, and may create difficulties in the preparation and characterization of standards and therapeutic products. In order to develop a simple method to detect changes in glycocomposition, an FSH ELISA was modified by the incorporation of lectins of recognized sugar specificity, and used to examine the terminal sugar composition of ampouled preparations of pituitary, urinary and rDNA-derived FSH. FSH was captured with a specific monoclonal antibody (MAb) and detected with either biotinylated anti-FSH MAb (ELISA) or the sugar-specific lectins (L-ELISA) from Triticum vulgaris (sialic acid, SA), Sambucus nigra (α2,6-linked SA), Maackia amurensis (α2,3-linked SA) or Ricinus communis (free terminal galactose; GAL). Relative estimates of the amounts of terminal SA, its different forms and GAL were derived from the L-ELISA/ELISA data compared with the highly sialylated 1st International Standard for pituitary FSH (IS) 83/575. All the FSH preparations had less SA than the IS with the ratio of α2,3- and α2,6-linked SA varying between preparations. The amounts of α2,6-linked SA relative to the IS were not significantly different in the urinary and pituitary preparations whereas α2,3-linked SA in all preparations was generally less than that of the standard. The rDNA FSH material, produced in CHO cells, was confirmed as containing only α2,3-linked SA. The degree of asialylation revealed by the Ricinus lectin showed that the more heavily sialylated IS had less exposed GAL than the other preparations. The method provides a simple and rapid technique for assessing changes in terminal sugar composition and allows comparison of the patterns of terminal glycosylation between preparations from different sources and, when assayed against the appropriate standard, between batches of therapeutic products. Journal of Endocrinology (1995) 145, 527–533

The Glycosaminoglycan of Recombinant Human Soluble Thrombomodulin Affects Antithrombotic Activity in a Rat Model of Tissue Factor-Induced Disseminated Intravascular Coagulation

1992 ◽  
Vol 67 (03) ◽  
pp. 366-370 ◽  
Author(s):  
Katsuhiko Nawa ◽  
Teru Itani ◽  
Mayumi Ono ◽  
Katsu-ichi Sakano ◽  
Yasumasa Marumoto ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Disseminated Intravascular Coagulation ◽  
Rat Model ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Soluble Thrombomodulin ◽  
Intravascular Coagulation ◽  
Platelet Counts ◽  
Recombinant Human Soluble Thrombomodulin

SummaryPrevious studies on recombinant human soluble thrombomodulin (rsTM) from Chinese hamster ovary cells revealed that rsTM was expressed as two proteins that differed functionally in vitro due to the presence (rsTMp) or absence (rsTMa) of chondroitin-4-sulfate. The current study evaluates the in vivo behavior of rsTM in rats and in a rat model of tissue factor-induced disseminated intravascular coagulation (DIC). rsTMp was more potent than rsTMa for prolongation of the activated partial thromboplastin time (APTT) and their in vivo half-lives determined by ELISA were 20 min for rsTMp and 5.0 h for rsTMa. Injection of a tissue factor suspension (5 mg/kg) resulted in DIC as judged by decreased platelet counts and fibrinogen concentrations, prolonged APTT, and increased fibrin and fibrinogen degradation products (FDP) levels. A bolus injection of either rsTM (0.2 mg/kg) 1 min before induction of DIC essentially neutralized effects on platelets, fibrinogen, and FDP levels, and had only a moderate effect on APTT prolongation. The dose of anticoagulant to inhibit the drop in platelet counts by 50% (ED50) was 0.2 mg/kg rsTMa, 0.07 mg/kg rsTMp, and 7 U/ kg heparin. The effect of increasing concentrations of rsTM and heparin on bleeding times were compared in experiments involving incision of the rat tail. Doubling of the bleeding times occurred at 5 mg/kg rsTMa, 3 mg/kg rsTMp or 90 U/kg heparin. These values represent a 25-fold increase over the ED50 for rsTMa, 43-fold for rsTMp and 13-fold for heparin. These results suggest that rsTMp is a potent anticoagulant to inhibit the platelet reduction when injected prior to the induction of DIC in rats.

Influence of Platelet Membrane Sialic Acid and Platelet-Associated IgG on Ageing and Sequestration of Blood Platelets in Baboons

1993 ◽  
Vol 70 (04) ◽  
pp. 676-680 ◽  
Author(s):  
H F Kotzé ◽  
V van Wyk ◽  
P N Badenhorst ◽  
A du P Heyns ◽  
J P Roodt ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Life Span ◽  
Blood Platelets ◽  
Senescent Cell ◽  
Platelet Membrane ◽  
Antigen Complex ◽  
The Mean ◽  
Aggregation Of Platelets

SummaryPlatelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of Sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the Sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of Sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/108 platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p <0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p <0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/108 platelets was removed (p <0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane. The antibody-antigen complex may then provide a signal to the macrophages that the platelet is old, and can be phagocytosed and destroyed.

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