Platelet Membrane Glycoproteins and the Interaction between Bovine Factor VIII Related Protein and Human Platelets

1977 ◽  
Vol 38 (04) ◽  
pp. 0914-0923 ◽  
Author(s):  
Nils Olav Solum ◽  
Inger Hagen ◽  
Torbjørn Gjemdal
Keyword(s):  
Membrane Protein ◽  
Factor Viii ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Polyacrylamide Gel ◽  
Related Protein ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
Bovine Factor

SummaryThe “GP I fraction” seen on polyacrylamide gel electrophoresis of reduced samples of whole normal platelets contains three glycopolypeptides corresponding to the integral membrane protein GP I (GP Ia), the easily solubilized membrane protein GPS (GP Ib, glycocalicin) and a granule-located glycoprotein.Freezing and thawing of platelets in tris-buffered saline leads to a lysis of platelets and platelet granules with the result that both GPS and the granule glycoprotein is found in the soluble fraction. The two glycoproteins can be separated by SDS polyacrylamide gel electrophoresis both in reduced and unreduced samples when urea and EDTA is incorporated into the gels. This permitted electrophoretic studies of GPS using the granule glycoprotein as a control and marker substance.A working hypothesis stating that the presence of GPS on the platelet surface is a prerequisite to the agglutination of human platelets with bovine factor VIII related protein, has been investigated. The hypothesis was supported by the observation that storage of platelets in tris-buffered saline at 4° C led to the elution of GPS and loss of agglutination, as was also the case when platelets were frozen and thawed in tris-buffered saline, or preincubated in 3 M KCl and resuspended in either tris-buffered saline or the EDTA-containing medium A. GPS was not, or only slightly, solubilized when platelets were frozen and thawed in the EDTA-containing medium and the resulting platelet ghosts still agglutinated.Platelets from 1 patient of the Bernard-Soulier type did not agglutinate with the bovine factor VIII-related protein, nor did the platelets contain GPS. An improved technique for the isolation of such platelets is described.

Studies on Some Thrombin-Induced Platelet Membrane Alterations

1975 ◽  
Author(s):  
I. Hagen
Keyword(s):  
Factor Viii ◽  
Gel Electrophoresis ◽  
Membrane Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Radioactivity ◽  
Human Platelets ◽  
Drastic Reduction ◽  
Release Reaction ◽  
Ristocetin Cofactor ◽  
Bovine Factor

Membrane alterations in connection with thrombin-induced release reaction have been studied with washed, human platelets. The release reaction was induced by 0.01–1.0 NIH units of thrombin, incubated for 3 min in the presence of 3 mM EDTA. Membrane proteins exposed on the surface were labeled with 125I-by the lactoperoxidase-iodination technique.Three radioactive peaks (approx. MW 97.000, 120.000 and 150.000) were seen on subsequent SDS Polyacrylamide gel electrophoresis of control platelets. Treatment of prelabeled platelets with thrombin did not reveal any changes in the “specific” radioactivity of the three peaks, indicating that no labeled fragments had been split off.Treatment with thrombin before iodination under conditions where the release reaction occurred, resulted in a drastic reduction in the incorporation of radioactivity. When the release reaction was inhibited by antimycin and 2-deoxyglucose before thrombin treatment and subsequent iodination, very little or no effect could be observed on the incorporation of radioactivity.Aggregation of these platelet suspensions with bovine factor VIII, and ristocetin plus human ristocetin cofactor indicate that platelets which have undergone the release reaction aggregate poorly with these agents. The membrane conformation necessary for iodination of the three membrane polypeptides and the ability of the platelets to aggregate in the factor VIII-dependant systems thus seem to be altered during thrombin-induced release reaction, possibly because of alterations in the membrane structure.

scholarly journals Degradation of Bovine Factor VIII by Plasmin and Trypsin

Blood ◽  
1974 ◽  
Vol 43 (5) ◽  
pp. 629-640 ◽  
Author(s):  
Edward P. Kirby ◽  
Neil Martin ◽  
Victor J. Marder
Keyword(s):  
Acetic Acid ◽  
Factor Viii ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Antigenic Determinants ◽  
Major Protein ◽  
Acid System ◽  
Coagulant Activity ◽  
Bovine Factor

Abstract The degradation of bovine Factor VIII by plasmin, trypsin, and thrombin was monitored with polyacrylamide gel electrophoresis in a urea—acetic acid system. The sequences of plasmic and tryptic proteolysis were very similar, proceeding via several intermediate, enzyme-sensitive products to smaller derivatives which were relatively resistant to further degradation. The intermediate degradation products were similar for both enzymes, although prolonged digestion with trypsin produced final products which were smaller than those observed with plasmin. Stages of digestion which reflect the degree of completeness of proteolysis were defined on the basis of the presence or absence of these distinct components in the electrophoretic profiles. Thrombin treatment of Factor VIII caused a loss of coagulant activity but did not yield products small enough to be characterized in this electrophoretic system. During the course of plasmic and tryptic digestion, there was a rapid loss of all coagulant activity without the generation of anticoagulant activity. Agarose chromatography of a 24-hr plasmic digest revealed three major protein peaks, which corresponded to individual electrophoretic bands on polyacrylamide gel electrophoresis in a nondenaturing system at pH 8.8. Upon polyacrylamide gel electrophoresis in the urea—acetic acid system, the components of the first two of these peaks dissociated into two and three separate fragments, respectively. Immunoelectrophoresis demonstrated two major distinct antigenic determinants in these first two elution peaks.

Glanzmann’s Thrombasthenia : Surface Labelling by Various Techniques and Analysis by Gel Electrophoresis.

1979 ◽  
Author(s):  
J.L. McGregor ◽  
K.J. Clemetson ◽  
E. James ◽  
M. Dechavanne
Keyword(s):  
Sialic Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Polyacrylamide Gel ◽  
Galactose Oxidase ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

The surface membrane glycoproteins of glanzmann’s thrombasthenia and normal whole platelets were labelled by techniques specific for either sugar or protein moieties. The labelled platelets were solubilized and electrophoresed in reduced or unreduced state by discontinuous SDS-polyacrylamide gel electrophoresis. Galactose oxidase + NaB3H4, labelling, showed with reduced samples 4 glycoprotein bands : a high M.W. glycoprotein and GP Ia, GP Ib, GP IIIb, more intensely labelled than with control platelets but with similar M.W. After treatment with neuraminidase + galactose oxidase + NaB3H4 to remove terminal sialic acid and label penultimate galactose residues the gels showed on both unreduced and reduced samples the absence of PG IIb and IIIa and a relatively broad and intensely labelled GPIb band compared with control platelets. The use of sodium periodate + NaB3H4, to label predominantly sialic acid moieties gave essentially the same number of GP bands in both reduced and unreduced samples as in normal platelets. Lactoperoxidase iodination showed in thrombasthenic platelets both in the reduced and unreduced states the absence of GPIIb, GPIIIa and more intensely labelled GPIb and CPIIIb than with control platelets. The combination of multilabelling and discontinuous Polyacrylamide gel system provides a reliable method for investigating the platelet surface.

scholarly journals Studies on Platelet Glycoproteins by Surface Labeling and SDS Polyacrylamide Gel Electrophoresis

1977 ◽  
Author(s):  
I. Hagen ◽  
N.O. Solum ◽  
M. Peterka
Keyword(s):  
Electrophoretic Mobility ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Conformational State ◽  
Polyacrylamide Gel ◽  
The Other ◽  
Metabolic Inhibitors ◽  
Conventional System ◽  
Platelet Surface ◽  
Release Reaction

Platelet surface (glyco)proteins, and alterations in these in connection with the thrombin-induced release reaction has been studied. Platelets were labeled by lactoperoxidase-catalyzed iodination, and examined by SDS gel electrophoresis in two different gel systems, one conventional(J. Biol. Chem.1969 244 4406), and the other containing urea and EDTA in the gels. In the conventional system the bulk of radioactivity coincided with a PAS band (GP III) of MW about 100, 000. In the other system, the main radioactive peak appeared in the GP II area (MW 120,000), and a shift in the PAS stain intensity from GP III to GP II was seen. Thrombasthenic platelets showed only traces of the GP II band in both systems. The bulk of radioactivity was associated with the surface glycopolypeptide GPS, which is present, but not labeled in normal platelets. In thrombin-released platelets, GPS in its unreduced state has an altered electrophoretic mobility compared to control platelets and platelets which have been incubated with metabolic inhibitors to prevent secretion. The findings indicate that the GP III band consists of two different polypeptides, one of which appears in the GP II area in gels containing urea and EDTA. Further, that thrombasthenic platelet membrane exists in a conformational state different from that of normal platelets. And finally, GPS is in some way involved in, or influenced by, the thrombin-induced release reaction.

scholarly journals Isolation from human chronic-lymphocytic-leukaemia cells of membrane glycoproteins associated with Fc-receptor functions. Physical parameters and production of polyclonal antibodies

1987 ◽  
Vol 245 (1) ◽  
pp. 75-83 ◽  
Author(s):  
G Gorini ◽  
G A Medgyesi ◽  
M Garavini ◽  
K J Dorrington ◽  
J Down
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Gel Electrophoresis ◽  
Lymphocytic Leukaemia ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fc Receptor ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Membrane Glycoproteins

Two membrane glycoproteins that bound immune complexes and inhibited Fc-receptor- (FcR-)mediated functions in vitro were purified from human FcR+ chronic-lymphocytic-leukaemia cells. A multi-step purification was developed, consisting essentially in: (i) Tween 40 extraction of crude cell membranes; (ii) solubilization of membrane fragments by Renex-30; (iii) isolation of glycoproteins by affinity chromatography on Lens culinaris haemagglutinin-Sepharose; (iv) papain treatment of the eluted glycoproteins followed by gel-filtration chromatography; (v) purification by polyacrylamide-gel electrophoresis of two molecular species from the protein-size fraction enriched for immune-complex-binding activity. The two electrophoretically isolated components displayed apparent molecular masses of 70 and 45 kDa by SDS/polyacrylamide-gel electrophoresis and restricted charge heterogeneity by two-dimensional analysis. Two-dimensional peptide mapping revealed the presence of many peptides in common between the two proteins and the absence of a number of peptides in the 45 kDa component. These two polypeptides were used as immunogens to produce polyclonal antibodies that cross-reacted with both proteins and specifically inhibited FcR-mediated reactions in vitro. Furthermore, FcR-related components from detergent-extracted lysates of the human K562 and U937 cell lines or human placental membranes were revealed by the putative anti-FcR antibodies adsorbed on Protein A-Sepharose.

Absence of the 145,000 Molecular Weight, Soluble Platelet Membrane Glycoprotein - Lack of Platelet Agglutination

1979 ◽  
Vol 42 (05) ◽  
pp. 1626-1629 ◽  
Author(s):  
Nils Olav Solum ◽  
Inger Hagen ◽  
Miroslav Peterka ◽  
Torbjørn Gjemdal
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Related Protein ◽  
Giant Platelets ◽  
Platelet Membrane Glycoprotein ◽  
Human Factor Viii ◽  
Bovine Factor

SummaryOne step in the function of platelets in hemostasis is their adhesion to subendothelial tissue. The human factor VIII related protein (von Willebrand factor) is considered to be involved in the adhesion phenomenon (Baumgartner et al. 1977). One manifestation of the protein-cell interaction can be observed as a platelet agglutination after addition to the human platelets of a combination of the human protein and the glycopeptide ristocetin, or after addition of the bovine protein alone. The bovine factor VIII related protein as such directly binds to the platelet membrane (Kirby and Sha May Tang 1977) and thus represents a simpler system than ristocetin plus the human cofactor which may have to interact with each other before excerting their effect on the platelet membrane. The present paper concerns the se.One of the characteristics of the agglutination of human platelets brought about by the bovine factor VIII related protein (as well as by ristocetin plus the human cofactor) is that it is independent of the energy metabolism and the internal organization of the platelet. One would therefore expect that modified platelets and platelet “ghosts” would agglutinate as long as certain structures on the outer cell surface are chemically and sterically intact. Because of the hydrophilic character of the carbohydrate side chains, the membrane glycoproteins are considered of special importance for cell contact phenomena. Thus it has already been known for some years that giant platelets of the Bernard-Soulier type which do not agglutinate with the bovine protein (Bithell et al. 1972), contain a reduced amount of sialic acid related to protein content and surface area (Grottum and Solum 1969), and show a reduced glycoprotein stain in the GP I region on SDS polyacrylamide gel electrophoresis (Nurden and Caen 1975).This paper presents five observations which support a working hypothesis stating that the presence on the platelet membrane of the 145,000 molecular weight, soluble platelet membrane glycoprotein called GPS or glycocalicin is a prerequisite to the agglutination of human platelets by bovine factor VIII related protein.

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