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.

Generation Of Platelet Aggregating Activity By Adsorption Of Low-Molecular-Weight Factor VIII- Related Protein To Colloidal Gold

1981 ◽  
Author(s):  
M Furlan ◽  
M Horisberger ◽  
B A Perret ◽  
E A Beck
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Platelets ◽  
Reduction Factor ◽  
Low Molecular Weight ◽  
Weight Factor ◽  
Related Protein ◽  
Gold Particles ◽  
Disulfide Reduction ◽  
Human Factor Viii

Both the ristocetin cofactor (VIII:Rcof) and the platelet aggregating factor (PAF) are preferentially associated with the largest complexes of human and bovine factor VIII, respectively. It is not known whether the functional deficiency of smaller species of factor VIII is due to their size or quality. Binding of small oligomers of factor VIII-related protein, isolated by gel filtration from cryoprecipitates or prepared by disulfide reduction of multimeric factor VIII, onto gold granules (diameter 20-30 nm) generated platelet aggregating activity; on the other hand, adsorption of large factor VIII multimers onto gold particles impaired the initially high VIII: Rcof and PAF. Aggregation of human platelets by human factor VIII-coated gold particles required ristocetin and was not competitively inhibited by unbound low-molecular-weight factor VIII. We conclude that essential properties of factor VIII, necessary for its “von Willebrand activity”, are present potentially in the smallest species recovered from plasma or even after disulfide reduction. Factor VUI-coated gold granules can be used as an electron-dense label for surface receptors for factor VIII on human platelets.

Impaired Prothrombin Consumption in Bernard-Soulier Syndrome Is Corrected In Vitro by Human Factor VIII

1997 ◽  
Vol 77 (02) ◽  
pp. 383-386 ◽  
Author(s):  
S Bellucci ◽  
J P Girma ◽  
M Lozano ◽  
D Meyer ◽  
J P Caen
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Platelet Membrane ◽  
Giant Platelets ◽  
Prolonged Bleeding Time ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Bernard Soulier Syndrome

SummaryThe Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein lb (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII.'C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.

Effects of Bovine Factor VIII-Related Protein on Human Platelets and Isolated Human Platelet Membranes

1975 ◽  
Author(s):  
N. O. Solum
Keyword(s):  
Factor Viii ◽  
Human Platelet ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Human Platelets ◽  
Related Protein ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Bovine Factor

The first phase of aggregation of human platelets induced by bovine factor VIII-related protein does not require an intact energy metabolism and probably represents a passive agglutination phenomenon due to receptors for the protein on the outside of the platelet cytoplasmic membrane. However, washed human platelets lost their ability to be aggregated by bovine factor VIH-related protein after freezing and thawing of the platelets. Furthermore, isolated human platelet membranes were not flocculated by the bovine protein in the absence of added calcium ions. Additions of calcium chloride (2.1 mM) alone flocculated the isolated membranes. The membranes used represented the material sedimenting between 10,000 and 100,00 g from a homogenate obtained by homogenizing washed platelets suspended in 0.27 M sucrose in an Aminco-French pressure cell (1.361 atm. 2 × 1 min). Subsequent sucrose density gradient centrifugation of the preparations did not reveal bands of particulate matter at a higher sucrose density than 1.12. In contrast, freezing and thawing of formalin-treated human platelets (4 mg/ml formaldehyde) did not destroy their ability to be aggregated by bovine factor Vlll-related protein. Application of the method described above to the formalin-treated platelets resulted in a low yield of 10,000-100,000 g sedimenting material and to more particulate bands on subsequent sucrose density gradient centrifugation. The stabilizing effect of the formalin treatment (through protein polymerization ?) may also be seen from the fact that such platelets are aggregated by bovine factor VHI-related protein even after their ability to aggregate with thrombin has been lost.

The Largest Isoform of Platelet Membrane Glycoprotein Iba Is Commonly Distributed in Eastern Asian Populations

1996 ◽  
Vol 76 (02) ◽  
pp. 245-247 ◽  
Author(s):  
Fumihiro Ishida ◽  
Kenichi Furihata ◽  
Kimitaka Ishida ◽  
Hiroshi Kodaira ◽  
Kyou Sup Han ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Chinese Population ◽  
Gene Frequency ◽  
Tandem Repeats ◽  
Platelet Membrane ◽  
The Other ◽  
Membrane Glycoprotein ◽  
Eastern Asia ◽  
Platelet Membrane Glycoprotein ◽  
Asian Populations

SummaryPlatelet membrane glycoprotein Iba has at least two polymorphisms which affect phenotype. One is the dimorphism at codon 145, and the other is a molecular weight polymorphism due to variable numbers of tandem repeats (TR) in the macroglycopeptide region. These two polymorphisms are in linkage disequilibrium. The frequencies of these polymorphisms differ considerably depending on race, and the largest variant with four TR is almost exclusively present in the Japanese population. We examined the genotypes of HPA-2 and TR polymorphism in three different races from Eastern Asia; the Japanese (n = 103), Korean (n = 101) and Chinese population (n = 177). The gene frequency of HPA-2 differed significantly among these three populations. Among HPA-2b-positive individuals, the A isoform with four TR and B with three TR were present in all three populations and A dominated over B. Individuals homozygous for the A isoform were found in both Japanese and Korean populations. These findings indicate that the largest haplotype is common in the Eastern Asian region.

scholarly journals Thrombin-induced platelet membrane glycoprotein IIb and IIIa complex formation. An electron microscope study.

1981 ◽  
Vol 154 (4) ◽  
pp. 1058-1068 ◽  
Author(s):  
M J Polley ◽  
L L Leung ◽  
F Y Clark ◽  
R L Nachman
Keyword(s):  
Complex Formation ◽  
Keyhole Limpet Hemocyanin ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Macromolecular Complex ◽  
Membrane Glycoprotein ◽  
Platelet Surface ◽  
Platelet Membrane Glycoprotein ◽  
Normal Platelet ◽  
Glycoprotein Iiia

The topographic relationships of platelet membrane glycoprotein IIb and glycoprotein IIIa have been studied in stimulated and unstimulated human platelets using immunoelectron microscopy. An indirect approach with ferritin-conjugated goat anti-rabbit gamma-globulin was used to localize the rabbit antibody to glycoprotein IIIa. The second ultrastructural label was keyhole limpet hemocyanin conjugated directly to antibody to glycoprotein IIb. Using the double labels, it was demonstrated that glycoprotein IIb and glycoprotein IIIa were distributed randomly in the unstimulated platelet membrane. After platelet stimulation with thrombin, large clusters of glycoprotein IIb-glycoprotein IIIa complexes were formed. No complex formation between glycoprotein Ib and glycoprotein IIb was observed in control experiments. These observations suggest that thrombin stimulation initiates the specific glycoprotein IIb-glycoprotein IIIa macromolecular complex formation on the platelet surface, which may act as the active fibrinogen-binding site required for normal platelet aggregation.

Inhibition of Bovine Factor VIII and Ristocetin Plus Human Factor VIII Induced Platelet Agglutination by Small Peptides

1975 ◽  
Author(s):  
D. E. Macfarlane
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Human Platelets ◽  
Small Peptides ◽  
Platelet Surface ◽  
Muramyl Peptide ◽  
Human Factor Viii ◽  
Peptide Precursor ◽  
Von Willebrand ◽  
Bovine Factor

The antibiotic ristocetin induces platelet agglutination in the presence of human factor VIII. A similar agglutination is induced by bovine factor VIII without ristocetin. This antibiotic binds C-terminal D-alanine peptides including the nucleotide-N-acetyl-muramyl-peptide precursor of bacterial cell walls. The effect of several peptides on the agglutination of formalin fixed washed human platelets was investigated. At 1.25 mM acetyl-glycyl-D-alanine (AGDA) and diacetyl-L-lysine-D-alanine-D-alanine (ALAA) increased the amount of ristocetin required for agglutination in the presence of purified human factor VIII; both these peptides bind to ristocetin (Bichochem. J. 124, 845). Glycyl-L-alanine (GLA) glycyl-D-alanine (GDA), acetyl-glycyl-L-alanine (AGLA) were inactive. AGLA (0.25 mM) and AGDA (1.25 mM) inhibited purified bovine factor VIII induced agglutination, ALAA, GLA and GDA (3.2 mM) were inactive.These results suggest that ristocetin acts in part by binding to peptides on the platelet surface, and that bovine VIII combines to the same site but has a different specificity. Suitable small peptides may be able to induce a von Willebrand like state for therapeutic purposes.(Dr. Kirby, Temple University provided the purified factor VIII’s and Prof. Perkins, Liverpool University provided ALAA; supported by N. I. H. HL 14217.)

Membrane Glycocalicin and Platelet Agglutination by Bovine Factor VIII Related Protein

1979 ◽  
Author(s):  
N.O. Solum ◽  
I. Hagen ◽  
T. Stabæk ◽  
C. Filion-Myklebust
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Soluble Fraction ◽  
Human Platelets ◽  
Related Protein ◽  
Monospecific Antiserum ◽  
Bovine Factor

Membrane glycocalicin was extracted from human platelets in 3 M KCl and purified using affinity chromatography on WGA-Sepharose as the most efficient step. Antibodies to the purified material were raised in rabbits. Monospecific antiserum was obtained either directly or after adsorption with soluble fraction from platelets which had been frozen and thawn in ED TA-buff er and therefore contained very little glycocalicin. Such anti serum agglutinated human platelets, and in low amounts it inhibited agglutination by bovine factor VIII. This inhibition could be reversed by adsorption with platelet “ghosts” dependent on their glycocalicin content. Immunoelectrophoretic quantitation (Laurell) gave values of 47-67{U 9 of glycocalicin per 10 platelets corresponding to around 3% of tota plateLet prote in and accounting for most of the neuraminidase-sensitive plateletsialic acid. Whereas glycocalicin was quantitatively removed from whole platelets by 3M KCl, this procedure could remove only 39% of glycocalicin from glycocalicin-containing platelet ghosts prepared in the presence of EDTA. Such KCl treated ghosts could still agglutinate by bovine factor VIII whereas the KCl-treated whoie platelets were not agglutinated.

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.

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