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.

Further Characterization Of Platelet Membrane Glycoproteins

1981 ◽  
Author(s):  
P Clezardin ◽  
J L McGregor ◽  
K J Clemetson ◽  
M Dechavanne ◽  
E F Lüscher
Keyword(s):  
Ricinus Communis ◽  
Lectin Binding ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Galactose Oxidase ◽  
Membrane Glycoproteins ◽  
Neuraminidase Treatment ◽  
Platelet Surface ◽  
Reducing Conditions ◽  
Surface Labelling

The binding of 125I-labelled lectins to major and minor platelet glycoproteins (GP) and their subunits has been investigated. Human platelets were isolated, washed, solubilized in sodium dodecyl sulphate (SDS) under non-reducing conditions and separated on 5, 7.5 and 10 % non-reduced/reduced 2-D polyacrylamide gels. The gels were incubated with 125I-labelled lectins; Lens culinaris lectin (LCL), concanavalin A (ConA) wheat germ agglutinin (WGA) or Ricinus communis agglutinin (RCA-120), then washed extensively dried and exposed to X-ray film by indirect autoradiography. Surface-labelled platelets were similarly separated. WGA and RCA bound predominantly to GPIbα but also to two minor bands above and below it which were affected by neuraminidase treatment. One of them bound two 125I-lectins (LCL and ConA) while GPIbα did not. Additional GP bands were detected by lectin binding and by surface-labelling beneath GPIIIβ (IV). With platelets labelled by the neuraminidase/galactose oxidase/NaB3H4 method a GP was detected between Ila and Ilia which was not found with periodate/ NaB3H4 labelling (not affected by reduction). Two spots on the diagonal bound LCL and ConA. GP Ibβ bound LCL more strongly than IIbp. GPIbp also bound WGA and RCA. GPIcβ apparently bound only ConA. GPIbβ and IIbβ were labelled equally strongly by surface labelling techniques, Icβ was apparently not labelled. Further GP subunits were detected one below Ibβ and IIbβ and another which originated in the GPVII region. These techniques demonstrate that the platelet surface is even more complex than previously thought.

Comparison Of The Major Membrane Glycoproteins Of Human, Rabbit And Rat Platelets

1981 ◽  
Author(s):  
B Toor ◽  
J L McGregor ◽  
K J Clemetson ◽  
L McGregor ◽  
M Dechavanne ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Surface Composition ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neuraminidase Treatment ◽  
Platelet Surface ◽  
Rabbit Platelets ◽  
Rat Platelets

Rabbit and rat platelets have been extensively investigated under in vitro or in vivo conditions to try to understand the pathology of thrombosis in man. Here, surface-labelling techniques have been used to find out if the platelet surface has a similar composition in these two animals and in man or not. Human, rabbit and rat platelets were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetyl galactosamine residues). Labelled platelets were solubilized in sodium dodecyl sulphate and separated under reducing conditions on 7.5 % Laemmli polyacrylamide gels. Dried gels were exposed to film by fluorography or indirect autoradiography. Terminal Gal/Gal NAc residues (no neuraminidase treatment) were strongly labelled with rat and rabbit platelets compared to human platelets which labelled very poorly. Terminal sialic acid labelling with rat and rabbit platelets showed a weak labelling of a glycoprotein (GP) with the same M.Wt. as GPIb which is the most intensely labelled GP in man. However two GP (with rabbits) and one GP (in rats) were intensely labelled at a M.Wt. similar to that of GPIa in man. These GP had a different M.Wt. with terminal Gal/Gal NAc labelling. Bands with a similar M.Wt. to GPIIb and IIIa in man were strongly iodinated with rabbit platelets but with rat platelets only a single band at the position of GPIIb was strongly iodinated. These results strongly indicate that there are considerable differences in surface composition between rabbit, rat and human platelets.

scholarly journals Calcium-dependent cysteine protease activity in the sera of patients with thrombotic thrombocytopenic purpura

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1683-1687 ◽  
Author(s):  
WG Murphy ◽  
JC Moore ◽  
JG Kelton
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Cysteine Protease ◽  
Platelet Activating Factor ◽  
Aminocaproic Acid ◽  
Human Platelets ◽  
Thrombocytopenic Purpura ◽  
Platelet Surface ◽  
Calcium Dependent

Abstract Plasma and serum from patients with thrombotic thrombocytopenic purpura (TTP) can cause activation and aggregation of normal human platelets in vitro. It is possible that this platelet-activating factor contributes to the disease. In this report we describe studies designed to identify the platelet-activating factor in TTP. Platelet activation by sera from 15 patients with TTP was inhibited by leupeptin, iodoacetamide, and antipain but not by phenylmethylsulphonylfluoride, epsilon-aminocaproic acid, soybean trypsin inhibitor, aprotinin, and D-phenylanyl-1-prolyl-1- arginine chloromethyl ketone. These studies suggested that the platelet- activating factor in TTP serum was a cysteine protease. We confirmed that a calcium-dependent cysteine protease (CDP) was present in the sera of each of the 15 patients when we used an assay based on the ability of CDP to proteolyse platelet membrane glycoprotein 1b (GP1b) and hence to abolish the ability of CDP-treated normal platelets to agglutinate in the presence of ristocetin and von Willebrand factor. This proteolytic activity was inhibited by EDTA, leupeptin, antipain, iodoacetamide, and by N-ethyl-maleamide (NEM) but not by the serine protease inhibitors. Activity was detected in 15 of 15 patients with TTP tested before therapy was begun. In contrast, no activity was detected in the serum of any of five of the TTP patients tested in remission or in any of the sera from 36 patients with thrombocytopenia and 423 nonthrombocytopenic controls. To look for in vivo CDP activity in patients with TTP, we studied platelets from two patients with acute TTP (drawn into acid-citrate-dextrose, NEM, and leupeptin). These platelets showed a loss of GP1b from the platelet surface. Both patients were also studied in remission: GP1b on the platelet surface had returned to normal. These studies provide evidence that CDP is present in the sera of patients with TTP, that it is specific to this disease, and that is is active in vivo as well as in vitro. We postulate that a disorder of CDP homeostasis plays a major role in the pathophysiology of TTP.

REDISTRIBUTION OF PLATELET GLYCOPROTEINS INDUCED BY ALLO- AND AUTOANTIBODIES

1987 ◽  
Author(s):  
S Santoso ◽  
V Kiefel ◽  
C Mueller-Eckhardt
Keyword(s):  
Binding Sites ◽  
Total Radioactivity ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
In Vitro Effects ◽  
Immunoblot Technique ◽  
Antigen Antibody

It is now well established that two of the major membrane glycoproteins (GP) of human platelets, GP lb and Ilb/IIIa, are functionally prominent for adhesion, aggregation and carry the binding sites for allknown types of human platelet specific antibodies (ab). Although a number of in vitro effects of ab on platelet function have been described, the role of the GP specificity of the various ab with regard to membrane mobility and redistribution phenomena is asyet unknown.In this work, we studied the effect on platelet membrane redistribution of allo- ab, auto-aband a quinidine-dependent ab directed against various epitopes on GP lb, lib and Ilia using immunofluorescence and a quantitative radioimmunoassay. The platelet GP's carrying the corresponding epitopes were determined using immunoblot technique or radioimmuno-precipitation. When unfixed platelets were incubated with alio- or auto-ab against epitopes on GP liborGP IlIa cap formation and internalization of antigenantibody complexes were visualized by fluorescence. In contrast, no changes of antigen distribution were seen with auto-ab or quinidine- dependent ab directed against GP lb. To quantitate antigen-antibody complexes internalization a specially designed radioimmunoassay was employed. If unfixed platelets weretreated with allo- or auto-ab against GP lib or GP Ilia precipitous reduction of external radioactivity was found, whereas the total radioactivity remainedessentially unchanged. This indicated that a portionof approximately 50-70% of GP lib or GP Ilia had been removed from the platelet surface and had been internalized. Internalization could not be induced with auto-ab or quinidine dependent ab against GP lb.We conclude that membrane redistribution of human platelets can be induced by various human ab with specificity for GP lib and/or Ilia and is a function of the target GP rather than the source of therespective abSupported by Deutsche Forschungsgemeinschaft (Mu 277/9-6)

Membrane Glycoproteins of Human and Rabbit Platelets: Studies In Vitro and after Circulation in Vivo

1975 ◽  
Author(s):  
J. N. George ◽  
P. C. Lewis ◽  
D. A. Sears
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Age Distribution ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Rabbit Platelets ◽  
Trypsin Hydrolysis ◽  
Initial Recovery

The initial events of hemostasis and thrombosis involve platelet contact interactions and may be mediated by surface glycoproteins. Human and rabbit platelets were labeled with 125I-diazotized diiodosulfanilic acid (I), which reacts covalently with proteins, and proteins were separated by SDS-polyacrylamide gel electrophoresis. Only exposed membrane proteins were labeled because: 1) protein specific activity of membranes was 4-7 times that of whole platelets, 2) different proteins were labeled when I was reacted with isolated membranes, and 3) trypsin-hydrolysis of labeled intact platelets altered the radioactive peaks. Like Phillips (Biochem. 11, 4582, 72) and Nachman et al. (JBC 248, 2928, 73) we found that lactoperoxidase iodinated the 93,000 dalton glycoprotein (GP) of human platelets. In contrast, I labeled both the 93,000 and 118,000 dalton membrane GP of human platelets, and all 3 membrane GP of rabbit platelets.Rabbit platelets labeled simultaneously with I and 51Cr had identical density and therefore age distribution of the 2 labels. After infusion into rabbits, initial recovery of I was 23% of the Cr recovery. After 3 hrs, I disappearance was exponential and more rapid (T/2 = 17 hrs) than the linear Cr disappearance (T/2 = 30 hrs, p < .01). This was due to in vivo removal of I from circulating platelets since 1 did not elute more rapidly from platelets harvested after 3 hrs circulation and incubated in plasma at 37° (T/2 of I elution = 43 hrs, Cr = 33 hrs). Platelets harvested after 14-20 hrs circulation had the same distribution of I on the membrane GP as before circulation. We postulate that this symmetrical label loss indicates uniform loss of membrane GP, suggesting that platelets lose pieces of their plasma membrane during circulation. This could occur during contact interaction in the process of hemostasis.

Anticoagulant and antithrombotic properties of platelet protease nexin-1

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Yacine Boulaftali ◽  
Frédéric Adam ◽  
Laurence Venisse ◽  
Véronique Ollivier ◽  
Benjamin Richard ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Protease Nexin ◽  
Deficient Mice

AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

PLATELET SUBPOPULATION HETEROGENEITY IN IDIOPATHIC THROMBO-CYTOPAENIA AND ESSENTIAL THROMBOCYTHAEMIA STUDIED BY CONTINUOUS FLOW ELECTROPHORESIS: MORPHOLOGICAL AND BIOCHEMICAL CHARACTERISATION

1987 ◽  
Author(s):  
M Crook ◽  
S J Machin ◽  
N Crawford
Keyword(s):  
Sialic Acid ◽  
Continuous Flow ◽  
Thiobarbituric Acid ◽  
Normal Subjects ◽  
Human Platelets ◽  
Essential Thrombocythaemia ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
Disease States ◽  
Biochemical Characterisation

Using continuous flow electrophoresis (CFE) normal human platelets have been separated into a surface-charge dependent profile (roughly gaussian) extending over 15-20 fractions with an electrophoretic mobility range between 0.86 and 0.93 μmetres/sec Volt cm. The technique has also been used to study platelets from adult patients with Stable Essential Thrombocythaemia (platelet counts - 600-800 × 109/L) and chronic Idiopathic Thrombocytopaenia (counts 15-80 × l09/L). Neither group was receiving specific therapy during the investigations. The platelets from both patient groups showed significant anodal shifts in electrophoretic mobilities in comparison with the CFE profiles from normal adults. Sialic acid, a negatively charged sugar moiety, associated with membrane glycoproteins is a major contributor to the platelet surface electronegativity and both total platelet sialic acid and surface neuraminidase-labile sialic acid measurements were made using the thiobarbituric acid procedure. Platelets from the Idiopathic Thrombocytopaenic group showed (with respect to normal levels) significant increases in both total and neuraminidase-labile sialic acid (expressed in terms of platelet number). Platelets from the Essential Thrombocythaemic group did not differ significantly in sialic acid contents from platelets from normal subjects. Platelet sizes (Coulter counter volumes) were also measured across the normal and patient platelet electrophoretic profiles together with morphological monitoring. In platelets from ITP patients these more electronegative platelets were substantially larger and of more bizarre shapes than those of normal patients when viewed with scanning and transmission electron microscopy, whereas platelets from the Essential Thrombocythaemic group showed less significant volume increases. These findings of marked changes in platelet surface electrokinetic and analytical properties in patients with the two disorders suggest that studies in other disease states, involving disturbances of thrombokinetics, may be warranted and particularly should be followed through therapeutic programmes.

Role of Sialic Acid for Platelet Lifespan and Function

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2864-2864
Author(s):  
Anne Louise Sørensen ◽  
Viktoria Rumjantseva ◽  
Sara Nayeb-Hashemi ◽  
Sunita Patel-Hett ◽  
Jennifer Richardson ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Platelet Rich Plasma ◽  
Significant Negative Correlation ◽  
Wild Type ◽  
Platelet Surface ◽  
Lectin Domain ◽  
Liver Macrophages ◽  
Reticulated Platelets

Abstract Although sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet half-life is not fully clarified. We and others have previously provided evidence that thrombocytopenia in mice deficient in the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice) is caused by rapid clearance of the platelets due to recognition of surface galactose by asialoglycoprotein receptor-expressing scavenger cells. Here we report new insight into clearance mechanisms, activation and production of sialic acid deficient platelets. Immunofluorescence staining of organ specimens harvested shortly after platelet transfusion demonstrated the predominant clearance of ST3Gal-IV−/− platelets by liver macrophages and, as previously reported, hepatocytes. The differential cellular clearance pathways were further explored following macrophage depletion in mice using clodronate encapsulated liposomes, a procedure that restored ST3Gal-IV−/− platelet circulation to approximately 40% of normal values, confirming that macrophages play a major role in the clearance of sialic acid deficient platelets and that other clearance pathways are equally important. Ingestion of ST3Gal-IV−/− platelets by hepatocytes was confirmed by in vitro HepG2 phagocytosis assays. We next investigated the function of desialylated platelets. Platelet binding of von Willebrand factor (vWf) upon botrocetin stimulation was 3 fold higher for ST3Gal-IV−/− platelet rich plasma compared to ST3Gal- IV+/+ platelet rich plasma. The circulation time of wild-type platelets transfused into ST3Gal-IV−/− mice was 20% reduced compared to control platelets, indicating that platelet removal could be accelerated due to binding of desialylated vWf to platelets. Loss of sialic acid did not affect platelet production in vitro and in vivo as cultivated megakaryocytes was found to produce proplatelets normally and measurements of reticulated platelets by flow cytometry showed a 3-fold increased thrombocytopoietic activity in the ST3Gal- IV−/− mice compared to wild-type mice. Interestingly, thiazole orange staining revealed a significant negative correlation between platelet size and platelet age for both genotypes, In conclusion, depletion of the ST3Gal-IV gene promotes binding of vWf to platelets, exposes platelet surface galactose residues to the lectin domain of asialoglycoprotein receptors on both hepatocytes and liver macrophages, resulting in rapid platelet clearance from the circulation and supporting previous findings that platelets decrease in size while aging in circulation.

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.

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.

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