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.

scholarly journals Identification of a Thrombin Proteolytic Receptor on Human Platelets

1977 ◽  
Author(s):  
David R. Phillips ◽  
Patricia Poh Agin
Keyword(s):  
Membrane Surface ◽  
Human Platelets ◽  
Complete Loss ◽  
Surface Proteins ◽  
Galactose Oxidase ◽  
Membrane Glycoproteins ◽  
Hydrolytic Product ◽  
A 23187 ◽  
Labeling Method ◽  
Kinetics Of

Thrombin is one of the most potent physiological agents causing platelet stimulation. It would appear that proteolysis is intimately linked to stimulation because trypsin, but not thrombin inactivated with PMSF, also stimulates platelets. Our approach to identifying the proteolytic substrate was to radioactively label the membrane surface proteins and determine which of these were hydrolyzed by thrombin. A glycoprotein labeling method (neuraminidase/galactose oxidase/(3H)-NaBH4) was employed. Twelve membrane glycoproteins were labeled, including most of those labeled by 1actoperoxidase-catalyzed iodination. Secretion and aggregation experiments showed that platelets labeled by this procedure are equally responsive to thrombin, collagen, and ADP as unlabeled platelets.Of the glycoproteins labeled by this procedure, only glycoprotein V (Mr = 89,000) was decreased as a result of thrombin action. Although low thrombin concentrations (0.2 U/ml) were sufficient to obtain significant hydrolysis, the complete loss of glycoprotein V occurred at the ratio of 1 U thrombin per 109 platelets; no further changes were observed when the thrombin concentration was increased to 10 U/109 platelets. A soluble glycopeptide hydrolytic product (Mr = 70,000) was released into solution. The kinetics of glycoprotein V hydrolysis were comparable to those of secretion and aggregation. Glycoprotein V hydrolysis was not observed when platelets were aggregated by collagen, ADP, or the Ca++ ionophore A-23187. It is proposed that glycoprotein V is a proteolytic receptor of thrombin.

scholarly journals Platelet release reaction and intracellular cGMP

Blood ◽  
1978 ◽  
Vol 52 (3) ◽  
pp. 524-531 ◽  
Author(s):  
A Weiss ◽  
NL Baenziger ◽  
JP Atkinson
Keyword(s):  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Serotonin Release ◽  
Secretory Process ◽  
Release Reaction ◽  
Intracellular Cgmp ◽  
Exogenous Addition ◽  
Human Thrombin ◽  
Platelet Release

Abstract Enchanced cAMP concentrations inhibit the aggregation and release reaction of isolated human platelets and platelet-rich plasma to all known inducing agents. An opposing role for cGMP in this phenomenon has been proposed by some but not by others, and the function of cGMP in this secretory process is unclear. To further elucidate the role of cGMP in the release reaction, the effect of increased concentrations of this cyclic nucleotide on 14C-serotonin release was evaluated utilizing isolated human platelets and highly purified human thrombin or commercially available bovine thrombin. Several recently described stimulators of guanylate cyclase, including sodium nitroprusside, sodium azide, nitrosoquanidines, and ascorbic acid, were found to markedly augment platelet cGMP levels. Enhanced platelet cGMP concentrations produced by these drugs or by the exogenous addition of cGMP and its analogues neither caused these cells to secrete nor modulated the thrombin-induced serotonin release reaction. The inhibition of serotonin release by increased cAMP concentrations was not counteracted by increased cGMP levels. Platelet cGMP concentrations were unaltered by thrombin. These data indicate that cGMP is not an obligatory signal or a modulator of the thrombin-induced platelet release reaction.

The Reduced Aggregation Response Of Bernard-Soulier Platelets To Thrombin May Be Related To An Abnormal Glycoprotein V

1981 ◽  
Author(s):  
A T Nurden ◽  
D Dupuis
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Surface Proteins ◽  
Galactose Oxidase ◽  
Blue Staining ◽  
Lag Phase ◽  
Coomassie Blue Staining ◽  
Aggregation Response ◽  
Coomassie Blue ◽  
Sds Page

Both platelet membrane GP Ib and GP V have been proposed as receptors for the activation of human platelets by thrombin. Bernard-Soulier (B-S) platelets exhibit a reduced aggregation response to thrombin with a lag phase that precedes aggregation. When B-S platelets, whose surface proteins had been labelled with (125I), were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by PAS-staining, Coomassie blue staining or autoradiography, the apparent absence of GP Ib and the normal presence of GP IIb, IIIa and IIIb was demonstrated. On the basis of such studies several authors have stated that “GP I” is the thrombin receptor. However, GP V is not located by the above procedure, requiring more sensitive analytical methods for its detection. To meet this requirement washed platelets isolated from 3 B-S patients have been treated sequentially with neuraminidase, galactose oxidase and sodium(3H,)-boro- hydride. The labelled platelets were analysed by SDS-PAGE using 7-12% gradient acrylamide gels and the (3H,)-labelled GP’s located by fluorography. In addition to the GP Ib defect the platelets of each B-S patient were lacking the band corresponding to GP V of normal platelets. In agreement with previous studies we observed that when (3H,)-labelled normal human platelets were incubated with thrombin GP V (Mr=82,000) was hydrolysed,and that this was accompanied by the appearance of a labelled glycopeptide (Mr=69,500) in the supernatant. When (3H)-labelled B-S platelets were treated with thrombin no labelled glycopeptide was located. GP V could therefore be either absetit from B-S platelets or have a modified carbohydrate composition rendering it insensitive to the analytical procedure used. Interpretations into the reduced aggregation response of B-S platelets to thrombin should be extended to include a possible GP V defect.

Platelet Reactions and Immune Processes

1970 ◽  
Vol 23 (01) ◽  
pp. 110-119 ◽  
Author(s):  
F Jobin ◽  
France Tremblay ◽  
M Morissette
Keyword(s):  
Platelet Aggregation ◽  
Platelet Aggregation Inhibitors ◽  
Human Platelets ◽  
Present Knowledge ◽  
Release Reaction ◽  
Cellular Processes ◽  
Cell Release ◽  
Aggregation Inhibitors ◽  
Platelet Release

SummaryWe have studied the effect of chymotrypsin substrates and inhibitors on the aggregation of human platelets by collagen, latex, and epinephrine :1. We have found that platelet aggregation was inhibited by most chymotrypsin substrates and inhibitors which we studied.2. In general, there was a positive correlation between the effectiveness of the compounds as chymotrypsin substrates or inhibitors on one hand, and as platelet aggregation inhibitors on the other hand. However aromatic amino acid derivatives acetylated on the α-amine group were much less effective with platelets than they are with chymotrypsin.3. Chymotrypsin substrates and inhibitors also inhibit the anaphylactic release of histamine. The view is presented that the platelet release reaction and the mast cell release reaction have several common biological and biochemical features.4. The possible role of platelet esterases in platelet thrombogenetic reactions is discussed in the light of the present knowledge of the role of cell bound esterase in several inflammatory or immune cellular processes.

scholarly journals Platelet release reaction and intracellular cGMP

Blood ◽  
1978 ◽  
Vol 52 (3) ◽  
pp. 524-531
Author(s):  
A Weiss ◽  
NL Baenziger ◽  
JP Atkinson
Keyword(s):  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Serotonin Release ◽  
Secretory Process ◽  
Release Reaction ◽  
Intracellular Cgmp ◽  
Exogenous Addition ◽  
Human Thrombin ◽  
Platelet Release

Enchanced cAMP concentrations inhibit the aggregation and release reaction of isolated human platelets and platelet-rich plasma to all known inducing agents. An opposing role for cGMP in this phenomenon has been proposed by some but not by others, and the function of cGMP in this secretory process is unclear. To further elucidate the role of cGMP in the release reaction, the effect of increased concentrations of this cyclic nucleotide on 14C-serotonin release was evaluated utilizing isolated human platelets and highly purified human thrombin or commercially available bovine thrombin. Several recently described stimulators of guanylate cyclase, including sodium nitroprusside, sodium azide, nitrosoquanidines, and ascorbic acid, were found to markedly augment platelet cGMP levels. Enhanced platelet cGMP concentrations produced by these drugs or by the exogenous addition of cGMP and its analogues neither caused these cells to secrete nor modulated the thrombin-induced serotonin release reaction. The inhibition of serotonin release by increased cAMP concentrations was not counteracted by increased cGMP levels. Platelet cGMP concentrations were unaltered by thrombin. These data indicate that cGMP is not an obligatory signal or a modulator of the thrombin-induced platelet release reaction.

Release, Aggregation and Lysis of Human Platelets by Antilymphocyte Globulin and Antiplatelet Serum

1976 ◽  
Vol 36 (02) ◽  
pp. 411-423 ◽  
Author(s):  
Nicholas Lekas ◽  
J. C Rosenberg
Keyword(s):  
Platelet Aggregation ◽  
Gamma Globulin ◽  
Human Platelets ◽  
Plasma Medium ◽  
Release Reaction ◽  
Clinical Events ◽  
Thrombotic Complications ◽  
Platelet Release ◽  
Free Media

SummaryHuman platelets labeled with 51Cr were used to determine the contribution made by platelet lysis to the platelet release reaction and platelet aggregation induced by rabbit antihuman platelet serum (APS) and equine antihuman thymocyte globulin (ATG). Platelets were tested in both plasma (PRP) and non-plasma containing media. Antibodies directed against platelets, either as APS or ATG, induced significant amounts of platelet release and aggregation, as well as some degree of lysis, in the absence of complement. The presence of complement increased platelet lysis and aggregation, but not the release reaction. Non-immune horse gamma globulin produced different responses depending upon whether platelets were investigated in PRP or non-plasma containing media. Aggregation was seen in the latter but not the former. These differences can be explained by the presence of plasma components which prevent non-specific immune complexes from causing platelet aggregation. Since platelets in vivo are always in a plasma medium, one must be wary of utilizing data from platelet studies in synthetic plasma-free media as the basis of explaining clinical events. These observations demonstrate at least two, and possibly three, different mechanisms whereby ATG could activate platelets causing thrombotic complications and thrombocytopenia, i.e., via 1) specific and, 2) non-specific non-lytic pathways and 3) a lytic pathway.

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.

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)

Factors Influencing Thrombin Induced Platelet Release Reaction

1979 ◽  
Author(s):  
E. Hattey ◽  
B.R. Binder
Keyword(s):  
Platelet Count ◽  
Human Platelets ◽  
Serotonin Release ◽  
Major Influence ◽  
Release Reaction ◽  
Platelet Counts ◽  
Ph Values ◽  
Factors Influencing ◽  
Platelet Release ◽  
Phosphate Buffered Saline

To study the effect of pH and platelet counts on thrombin induced platelet release reaction, washed human platelets labeled with 14C-serotonin were suspended in phosphate buffered saline of varying pH values for 15 minutes with thrombin concentrations between 1,1 and 0,068 NIH U/ml of suspension. The amount of serotonin released caused by the thrombin added was dependent on the pH of the incubation medium with an optimum in the range of pH 7,4 -7,6. This effect was more marked with higher thrombin concentrations. The serotonin release controles without thrombin were not influenced by the different pH values and were always less than 10%. The amount of platelets in the reaction mixture influenced the release values not significantly at thrombin concentrations of 1,1 and 0,27 NIH U/ml While with 0,068 NIH U/ml a significant dependence of the release on the number of platelets was observed, resulting in higher release values in platelet mixtures with lower platelet counts.Therefore it can be concluded that the pH is of major influence on the release reaction especially at high thrombin concentrations while the platelet count is of importance only at low thrombin concentrations.

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.

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