Studies on the Organisation of Glycoproteins and Proteins in Human Platelet Membranes

1979 ◽  
Author(s):  
A.T. Nurden ◽  
D. Dupuis ◽  
H. de la Baume ◽  
J.P. Caen
Keyword(s):  
Human Platelet ◽  
Membrane Vesicles ◽  
Human Platelets ◽  
Cross Linking ◽  
Platelet Response ◽  
Membrane Glycoproteins ◽  
Sh Groups ◽  
Sds Page ◽  
The Cross ◽  
Neighbour Analysis

Addition of wheat germ agglutinin (WGA) (50 ug/ml) to washed human platelets (3 x 108/ml) resulted in platelet activation and the release of l4C-5HT within the same time scale as 0.05 units/ml thrombin. In contrast, succinyl-WGA (100 ug/ml) induced no platelet response. The increased valency of WGA (4) compared with succinyl-WGA (2) suggests that the activation is induced through the cross-linking (immobilisation ?) of closely associated receptors in the membrane. This finding induced us to attempt to cross-link and thereby identify adjacent molecules in the membrane by “near-neighbour” analysis. Constituent -SH groups were oxidised employing Cu2+/phenanthroline or diamide as catalysts, and polymers formed as a result of intermolecular -S-S- formation between adjacent molecules were identified by SDS-PAGE. Although previous reports have shown that the major human platelet membrane glycoproteins contain -SH groups, no apparent cross-linking of the glycoproteins was located following the incubation of either washed platelets or isolated membranes with Cu2+/phenanthroline or diamide. However bidimensional SDS-PAGE (1st dimension non-reduced, 2nd dimension reduced) showed the presence of several protein polymers including complexes formed by the cross-linking of 3 large polypeptides of M. Wt. 250 000, 220 000 and 200 000. These components were easily eluted from membrane vesicles at pH 10 and may represent closely associated constituents at the cytoplasmic surface of the plasma membrane.

scholarly journals Further studies on the interaction between human platelet membrane glycoproteins IIb and IIIa in triton X-100

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 894-904 ◽  
Author(s):  
D Pidard ◽  
JP Rosa ◽  
TJ Kunicki ◽  
AT Nurden
Keyword(s):  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Indirect Evidence ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Nonionic Detergent ◽  
Triton X

Abstract Analysis of human platelet membrane proteins by crossed immunoelectrophoresis (CIE) in the presence of Triton X-100 (TX-100) has previously shown that glycoproteins (GP) IIb and IIIa are located in a single immunoprecipitate, band 16.2 To investigate whether IIb and IIIa are associated in a complex, we have analyzed TX-100-solubilized 125I-labeled membrane proteins by density gradient ultracentrifugation using 10%-40% sucrose gradients containing the nonionic detergent. studies were performed using soluble proteins derived from membranes isolated in the presence or absence of EDTA. Analysis of gradient fractions by SDS-polyacrylamide gel electrophoresis showed that in the absence of divalent cation chelation, GP IIb and IIIa penetrated well into the gradient (fractions 15–17). Analysis of fractions 15–17 by CIE revealed the presence of band 16. In contrast, when the membrane proteins were incubated with EDTA prior to or after TX-100 solubilization, IIb and IIIa remained near the top of the gradient (fractions 8–11) and gave separate immunoprecipitates during CIE. Incubation of washed platelet lysates with leupeptin, an inhibitor of the Ca2+-dependent protease of human platelets, had no effect on the shape of the band 16 immunoprecipitate. Thus, for the first time, direct evidence has been obtained that GP IIb and IIIa may form a divalent cation-mediated complex. Calibration of the sedimentation profiles using proteins of known molecular weight suggests that the complex is of limited size. Indirect evidence suggests that the complex is a heterodimer.

The Role Of Glycoprotein V (GPV) In Thrombin Activation Of Human Platelets

1981 ◽  
Author(s):  
K Fujimura ◽  
S Maehama ◽  
A Kuramoto
Keyword(s):  
Membrane Surface ◽  
Human Platelets ◽  
Surface Proteins ◽  
Galactose Oxidase ◽  
Membrane Glycoproteins ◽  
Release Reaction ◽  
Sds Page ◽  
Thrombin Activation ◽  
Platelet Release

The analysis of platelet membrane glycoproteins and platelet functions was conducted to disclose the role of GPI and V in the thrombin activation of platelet. Our previous study proved that native and HNB thrombin hydrolyzed GPV(M. W.8-9 × 104) selectively and released new glycoprotein fragment (M.W. 6.2-6.8 × 104 ) of GPV, resulting in the development of 14C-5HT release reaction and platelet MDA production. But DIP thrombin could not induce these phenomena.Membrane surface proteins of intact platelets were labeled with Na[3H]BH4 by neuraminidase and galactose oxidase method and analyzed by fluorography after SDS-PAGE.The high molecular weight glycoproteins, GPI, GPIII and GPV were diminished by trypsin treatment in correlation with the concentration and incubation time. In correspond to the diminution of these membrane glycoproteins, platelet release reaction was increased .Chymotrypsin treatment in various concentrations, release reaction and MDA production were not induced in spite of long incubation times. But the ristocetin aggregation was decreased in Chymotrypsin treated platelets whose membrane glycoproteins did not change significantly. The Chymotrypsin treated platelets whose GPI was modified functionally, showed normal release reaction and MDA production by thrombin stimulation. On the other hand, the thrombin treated platelets in low concentration previously whose GPV was hydrolyzed partially, demonstrated little release reaction and MDA production by thrombin or trypsin stimulation. From these results, the GPV was hydrolyzed specifically by thrombin and nonspecifically by trypsin but was not hydrolyzed by Chymotrypsin. It was concluded that the thrombin binds to the GPI and hydrolyzed GPV specifically, and hydrolysis of GPV might act as a signal to induce the platelet release reaction and prostaglandin metabolism.

The effect of cryoprotective agents on proteins of the erythrocyte membrane-cytoskeleton complex

2020 ◽  
Vol 66 (6) ◽  
pp. 456-463
Author(s):  
N.G. Zemlianskykh
Keyword(s):  
Actin Binding ◽  
Cross Linking ◽  
Protein Modifications ◽  
Sh Groups ◽  
Cryoprotective Agents ◽  
Membrane Cytoskeleton ◽  
Sds Page ◽  
Structural Abnormalities ◽  
Good Biocompatibility ◽  
Polypeptide Profile

The aim of the study was to evaluate of the effects of glycerol and DMSO, belonging to the endocellular type of cryoprotective agents (CPAs), as well as polyethylene glycol, dextran, sucrose, and mannitol, related to exocellular CPAs, on proteins of the membrane-cytoskeleton complex (MCC) of human erythrocytes at the stage preceding freezing. The assessment of protein modifications was performed by SDS-PAGE using different approaches when preparing samples for analysis. The use of β-mercaptoethanol in the solubilizing buffer showed no changes in the MCC polypeptide profile of erythrocytes preincubated with CPAs thus suggesting good biocompatibility of the studied substances. The use of the cross-linking reagent diamide for assessment of protein modifications did not reveal structural abnormalities that would result in significant changes in the localization of −SH groups and an increase in the production of high-molecular-weight polypeptide complexes identified by SDS-PAGE without β-mercaptoethanol. However, the recognized changes in the electrophoretic mobility of proteins in the area of band 5 in erythrocytes incubated with CPA in the presence of diamide suggest a reorganization of the structural state of actin protofilaments, which can be caused by alterations of actin monomers themselves or initiated by modifications of actin-binding proteins in the presence of CPAs. In addition, an increase in the amount of the protein fraction located between bands 5 and 6 in the MCC profiles of erythrocytes incubated with CPA and diamide was revealed. Despite the similarity of the reaction of erythrocyte proteins to different CPAs, the properties of cells depending on MCC, may differ due to modifications in the macromolecule structures, which are not associated with changes in the localization of the −SH-groups of proteins. The results obtained indicate that CPAs may have a significant impact on the erythrocyte MCC, and this requires further research.

scholarly journals Ultrasound-Assisted Transglutaminase Catalysis of the Cross-Linking and Microstructure of αs-Casein, β-Casein and κ-Casein

Processes ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1630
Author(s):  
Chun-Chi Chen ◽  
Liang-Yu Chen ◽  
Wen-Tai Li ◽  
Ken-Lin Chang ◽  
Hsien-Wei Tseng ◽  
...  
Keyword(s):  
Particle Size Analysis ◽  
Size Analysis ◽  
Cross Linking ◽  
Comprehensive Utilization ◽  
Transmission Electron ◽  
Microscopy Investigation ◽  
Electron Microscopy Investigation ◽  
Sds Page ◽  
The Cross ◽  
The Individual

The effects of ultrasonic treatment (UT)-assisted transglutaminase (TGase) catalysis on the physicochemical properties of individual αs-casein (αs-CN), β-casein (β-CN), and κ-casein (κ-CN) were investigated. After 60 min of incubation at 30 °C, αs-CN, β-CN, and κ-CN were cross-linked with TGase (6.0 units/mL), and high molecular weight polymers (>200 kDa) were formed. The use of TGase in conjunction with UT (20 kHz, power of 400 W, and amplitude 20%) led to an increase in the rate of αs-CN, β-CN, and κ-CN polymerization compared to the individual casein that contained TGase but did not undergo UT. SDS-PAGE scrutiny showed that the intensities of αs-CN, β-CN, and κ-CN incubation with regard to TGase and UT at 30 °C for 60 min noticeably decreased to 5.66 ± 0.39, 3.97 ± 0.43, and 26.07 ± 1.18%, respectively (p < 0.05). Particle size analysis results indicated that the molecule size appropriation for the cross-linking of αs-CN, β-CN, and κ-CN ranged from 6000 to 10,000 nm after 60 min incubation with TGase and UT. Transmission electron microscopy investigation showed network structures of cross-linking αs-CN, β-CN, and κ-CN were formed from αs-CN, β-CN, and κ-CN, respectively. As our results show, the comprehensive utilization of TGase and UT will be a superior method for the polymerization of αs-CN, β-CN, and κ-CN.

Absence of Myosin on Platelet Membranes Supported by Membrane Iodination, Immuno-Fluorescence and Functional Studies

1979 ◽  
Author(s):  
J.C. Mattson ◽  
W.J. Esselman ◽  
M.L. Schwarz ◽  
C.A. Zuiches
Keyword(s):  
Membrane Proteins ◽  
Protein Complex ◽  
Human Platelet ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Clot Retraction ◽  
Platelet Surface ◽  
Functional Studies ◽  
Sds Page ◽  
Formalin Fixed

Immunofluorescence and functional studies were performed utilizing the immunoglobulin fraction of antisera raised against chromatographically purified human platelet myosin. When non-permeable, formalin fixed platelets were used, no FITC staining of the platelet membrane occurred indicating the myosin is not present on the platelet surface. When similar studies were performed on formalin fixed platelets using antibodies raised against the contractile protein complex, thrombosthenin, membrane fluorescence occurred. Autoradiographs of SDS-PAGE gels of the immune precipitate produced by reacting 125I labeled human platelet thrombosthenin with antithrombosthenin demonstrated that anti-thrombosthenin antisera was capable of reacting with at least three iodinated proteins In addition to myosin. 125I lactoperoxidase catalyzed labeling of the external membrane proteins of resting and ADP stimulated platelets indicated no external labeling of myosin although actin appeared to be labeled. In functional studies, incubation of human platelets with antimyosin did not produce aggregation nor did it Inhibit subsequent aggregation by ADP, collagen or epinephrine. Similarly, treatment of intact platelets with antimyosin did not cause Inhibition of clot retraction. These studies support the thesis that myosin is not localized on the external platelet membrane.

scholarly journals Further studies on the interaction between human platelet membrane glycoproteins IIb and IIIa in triton X-100

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 894-904 ◽  
Author(s):  
D Pidard ◽  
JP Rosa ◽  
TJ Kunicki ◽  
AT Nurden
Keyword(s):  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Indirect Evidence ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Nonionic Detergent ◽  
Triton X

Analysis of human platelet membrane proteins by crossed immunoelectrophoresis (CIE) in the presence of Triton X-100 (TX-100) has previously shown that glycoproteins (GP) IIb and IIIa are located in a single immunoprecipitate, band 16.2 To investigate whether IIb and IIIa are associated in a complex, we have analyzed TX-100-solubilized 125I-labeled membrane proteins by density gradient ultracentrifugation using 10%-40% sucrose gradients containing the nonionic detergent. studies were performed using soluble proteins derived from membranes isolated in the presence or absence of EDTA. Analysis of gradient fractions by SDS-polyacrylamide gel electrophoresis showed that in the absence of divalent cation chelation, GP IIb and IIIa penetrated well into the gradient (fractions 15–17). Analysis of fractions 15–17 by CIE revealed the presence of band 16. In contrast, when the membrane proteins were incubated with EDTA prior to or after TX-100 solubilization, IIb and IIIa remained near the top of the gradient (fractions 8–11) and gave separate immunoprecipitates during CIE. Incubation of washed platelet lysates with leupeptin, an inhibitor of the Ca2+-dependent protease of human platelets, had no effect on the shape of the band 16 immunoprecipitate. Thus, for the first time, direct evidence has been obtained that GP IIb and IIIa may form a divalent cation-mediated complex. Calibration of the sedimentation profiles using proteins of known molecular weight suggests that the complex is of limited size. Indirect evidence suggests that the complex is a heterodimer.

Platelet Ca2+ Homeostasis: Na+-Ca2+ Exchange in Plasma Membrane Vesicles

1987 ◽  
Vol 57 (03) ◽  
pp. 337-340 ◽  
Author(s):  
Appavoo Rengasamy ◽  
Soudabeh Soura ◽  
Harold Feinberg
Keyword(s):  
Plasma Membrane ◽  
Human Platelet ◽  
Membrane Vesicles ◽  
Human Platelets ◽  
Plasma Membrane Vesicles

SummaryA vesicular plasma membrane-enriched fraction obtained from human platelets exhibited 45Ca2+ uptake in exchange for intravesicular Na+. The rate of Ca2+ uptake was linear up to 4 sec. The apparent Km for Ca2+ was 22 pM and the Vmax 280 pmol/mg/ sec. Ca2+ efflux from Ca2+ loaded vesicles was obtained upon dilution into a NaCl but not a KC1 medium. The extent of Ca2+uptake was monotonically increased as the pH increased from 6 to 9. Na+-Ca2+ exchange was shown to be electrogenic. Ca2+ uptake was distinguished from binding by the induction of Ca2+ release after A23187 addition. These findings support a role for Na+-Ca2+ exchange in human platelet Ca2+ transport.

Further Observations On The Possible Association Of Human Platelet Membrane Glycoproteins IIb And IIIa

1981 ◽  
Author(s):  
J-P Rosa ◽  
D Pidard ◽  
T Kunicki ◽  
A T Nurden
Keyword(s):  
Platelet Aggregation ◽  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Normal Human ◽  
Triton X

Studies are described which represent a continuation of our investigation into the role of membrane glycoproteins (GP’s) IIb and IIIa during human platelet aggregation. The surface proteins of washed platelets were labelled with 125I by the lactoperoxidase-catalysed method prior to membrane isolation by the glycerol lysis procedure. Solubilisation of the membrane proteins by triton X-100 was followed by their analysis by crossed immunoelectrophoresis (CIE) using a rabbit antibody prepared against normal human platelets. In the absence of divalent cation chelation GP IIb and Ilia were contained within a single 125I-labelled immunoprecipitate. When the isolated membranes were solubilised by triton X-100 in the presence of 5mM EDTA, GP IIb and IIIa formed distinctand separate immunoprecipitates during CIE. In order to further investigate this finding 125I-labelled membrane proteins solubilised by triton X-100 in the presence or absence of EDTA were subjected to centrifugation for 18 h at 100,000 g over a 10-40% sucrose gradient containing the nonionic detergent. The results confirmed that in the presence of divalent cations lib and Ilia were associated in a complex, and that this complex is dissociated by EDTA. The IgG..L is an alloantibody isolated from a polytransfused thrombasthenic patient that has been shown in previous studies to inhibit ADP-induced platelet aggregation and the binding of 125I-fibrinogen to normal human platelets in the presence of ADP. When the IgG..L was incorporated in an intermediate gel during CIE it was shown to precipitate the complex containing IIb/IIIa but under identical conditions it did not precipitate the individual glycoproteins dissociated by EDTA. Divalent cation-mediated changes in the orientation of lib and Ilia in the platelet membrane should be considered in assessing the role of these GP’s in platelet function.

Two Regions of the Human Platelet F11-Receptor (F11R) Are Critical for Platelet Aggregation, Potentiation and Adhesion

2002 ◽  
Vol 87 (04) ◽  
pp. 712-721 ◽  
Author(s):  
Anna Babinska ◽  
Mamdouh Kedees ◽  
Humra Athar ◽  
Tomasz Sobocki ◽  
Malgorzata Sobocka ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Recombinant Protein ◽  
Adhesion Molecule ◽  
Human Platelet ◽  
3D Structure ◽  
Human Platelets ◽  
Platelet Response ◽  
External Domain ◽  
A Cell ◽  
Induced Aggregation

SummaryThe F11 receptor (F11R) was first identified on the surface of human platelets as a target for a stimulatory monoclonal antibody (M.Ab.F11) that induces secretion, followed by exposure of fibrinogen receptors and aggregation. Cloning of the gene of F11R has revealed that this protein is a cell adhesion molecule (CAM), a member of the Ig superfamily and an ortholog of the murine protein called junctional adhesion molecule (JAM). The present study has identified two domains through which M.Ab.F11 triggers a platelet response culminating with aggregation. M.Ab.F11-mediated platelet adhesion, and the potentiation of collagen and ADP-induced platelet aggregation by M.Ab.F11, were found to involve the same two domains. A F11R recombinant protein (sF11R) completely inhibited platelet aggregation, adhesion and potentiation induced by M.Ab.F11, indicative that the active conformation of the external domain of F11R is present in the soluble, secreted recombinant protein. Furthermore, a specific peptide containing the sequence of the N-terminal amino acids S-1 to C-23 of F11R, and a peptide with the sequence of K-70 to C-82 in the 1st immunoglobulin-like (Ig) fold of F11R, both inhibited M.Ab.F11-induced aggregation, adhesion and potentiation of the aggregation of human platelets. Modeling of the 3D structure of the extracellular domain of the human platelet F11R suggests that these two regions form an active site within the conformation of this CAM. The sequence of these functional domains of F11R (in the N-terminus and 1st Ig-fold) provide the basis for new drug development in the treatment of certain types of thrombocytopenia and inflammatory thrombosis.

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