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.

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.

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.

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.

scholarly journals Factor VIII-induced superaggregation of human platelets

Blood ◽  
1982 ◽  
Vol 60 (6) ◽  
pp. 1359-1369 ◽  
Author(s):  
EP Kirby ◽  
DC Mills ◽  
H Holmsen ◽  
M Russo
Keyword(s):  
Factor Viii ◽  
Cytochalasin B ◽  
Dextran Sulfate ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
High Concentrations ◽  
Induced Aggregation ◽  
Very High ◽  
Bovine Factor

Abstract High concentrations of bovine factor VIII cause clumping of platelets into a few very large aggregates. This response is termed superaggregation. It is distinct from factor-VIII-induced agglutination but is also independent of both extracellular calcium ions and platelet energy metabolism. Neither agglutinating lectins nor aggregating agents, including thrombin, ADP, the ionophore A23187, and U46619, a prostaglandin analog, can induce superaggregation, even at very high concentrations. Washed platelets undergo superaggregation, and superaggregation does not increase the amounts of fibrinogen or albumin trapped by agglutinated platelets. It is not inhibited by membrane- stabilizing drugs or by colchicine or cytochalasin-B. Formaldehyde and glutaraldehyde prevent superaggregation without affecting the binding of radiolabeled factor VIII to the platelets. Superaggregated platelets are separated by approximately 50 nm and are not shape-changed or degranulated. In adenosine diphosphate (ADP) induced aggregation, the platelets are distorted and only 30 nm apart. Superaggregation is reversed by dextran sulfate, and the dispersed platelets are still able to respond to ADP. Our observations are consistent with the binding of high molecular weight multimers of bovine factor VIII to more than one receptor on each platelet, with superaggregation occurring through recruitment of additional receptors. This process may be interrupted by protein crosslinking reagents, such as formaldehyde and glutaraldehyde.

The Effects Of Chymotrypsin And Of ADP On The Binding Site For Bovine Factor VIII On Human Platelets

1981 ◽  
Author(s):  
Edward P Kirby ◽  
David C B Mills
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Shape Change ◽  
Human Platelets ◽  
Simple Process ◽  
Affecting Factor ◽  
Platelet Binding ◽  
Bovine Factor

The aggregation of human platelets by bovine Factor VIII (Platelet Agglutinating Factor-PAF) is inhibited by exposure of the cells to ADP or Chymotrypsin. We have investigated the mechanism of these effects using washed platelets. The washing procedure was modified from the method of Mustard et al. (Brit. J. Haematol. 22:193, 1972), omitting heparin and using a protein-free Tyrode’s solution for the final resuspension. The washed platelets were stable and responded to ADP (0.1-1 μM) with a shape change and, if fibrinogen was added, with aggregation. Bovine Factor VIII was purified to >90% homogeneity and was labeled with 125I (approx. 1 atom/subunit) by the IodoGen procedure, with no loss of activity. Aggregation was measured in the aggregometer in the presence of 7 mM EDTA. Binding was measured after incubation of labeled Factor VIII with washed platelets in the presence of 7 mM EDTA for 5 min at 37° without stirring.Treatment of washed platelets with Chymotrypsin progressively destroyed their ability to bind Factor VIII and to be agglutinated by it. Responsiveness to Factor VIII disappeared before any alteration was detected in the ability of platelets to undergo ADP-induced shape change. Treatment of platelets with ADP, however, inhibited agglutination induced by Factor VIII without affecting the binding of Factor VIII to the platelets. Agglutination by wheat germ agglutinin or phytohemagglutinin was not inhibited by ADP treatment. We conclude that Chymotrypsin probably inhibits Factor VIII- induced agglutination by destroying the platelet binding site for Factor VIII, but that ADP must act at a point distal to Factor VIII binding. Agglutination of metabolically intact platelets by Factor VIII may not be a simple process, because ADP can specifically inhibit it without affecting Factor VIII binding or aggregation of the platelets by lectins.

scholarly journals The Binding of Bovine Factor VIII of Human Platelets

1977 ◽  
Author(s):  
Edward P. Kirby ◽  
Sha May Tang
Keyword(s):  
Factor Viii ◽  
Evans Blue ◽  
Sodium Iodide ◽  
Dextran Sulfate ◽  
Human Platelets ◽  
Procoagulant Activity ◽  
Human Fibrinogen ◽  
Total Binding ◽  
Antihemophilic Factor ◽  
Bovine Factor

Highly purified bovine Factor VIII (FVIII) was iodinated by the lactoperoxidase method. Subsequent chromatography on agarose and extensive dialysis against sodium iodide solutions was required to remove non-covalently bound 1-125 which adhered tightly to FVIII. Iodination destroyed the procoagulant activity (Antihemophilic Factor-AHF) of FVIII, but did not affect its Platelet Aggregating Factor (PAF) activity. Binding to human platelets was determined by incubating radio!abelled FVIII with formalin-treated platelets, centrifuging, and measuring both bound and free radioactivity. Results obtained by this method were much more precise than those obtained by measuring disappearance of unlabelled AHF, PAF, or FVIII-related antigen from the supernatant, although the estimates of total binding obtained were comparable. Binding of FVIII to formal in-treated platelets was approximately the same as to unfixed platelets, and the binding could be saturated by adding an excess of unlabelled FVIII. Maximal binding occurred within 1-2 minutes at 37° and binding could be blocked by dextran sulfate, Evans Blue or other inhibitors of FVIII-induced platelet agglutination. Treatment of platelets with trypsin inhibited binding of labelled F-VIII. Binding was not affected by the presence of plasma, or high levels of purified human fibrinogen or FVIII.

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.

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.)

scholarly journals Antibodies as a Cause for Platelet Functional Defects

1977 ◽  
Author(s):  
J. P. Caen ◽  
S. Levy-Toledano ◽  
C. Tobelem ◽  
H. Michel ◽  
A. T. Nurden ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Factor Viii ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Rabbit Aorta ◽  
Human Platelets ◽  
Platelet Membranes ◽  
Normal Human ◽  
Functional Defects ◽  
Bovine Factor

The Luc. antibody occurring in a polytransfused thrombasthenic patient is an IgG which agglutinates all human platelets except thrombasthenic ones. At sub-agglutinating doses, ADP, Adrenaline, Collagen, Thrombin and arachidonic acid-induced aggregations but not ristocetin and bovine factor VIII mediated platelet aggregations are inhibited. It does not modify ADP binding to normal human platelet membranes. The antibody strongly reduced thrombus formation on rabbit aorta subendothelium. Its activity is consumed after incubation with normal human platelet membranes but not with thrombasthenic ones. It inhibits thromboxane formation with all inducers but not with arachidonic acid and PGG2. This antibody reacts with a component present on human platelet membranes 120.000 MW.The Por. antibody occurring in a polytransfused Bernard-Soulier patient is an IgG which agglutinates all human platelets except those of Bernard-Soulier and thrombasthenia. The activity is consumed after incubation with normal and thrombasthenic platelet membranes but not with platelet membranes of Bernard-Soulier patients. At sub-agglutinating doses only ristocetin, bovine factor VIII aggregation and adhesion of human platelets to rabbit aorta subendothelium are inhibited. Purified neuraminidase, chymotrypsin or trypsin destroy the reaction of normal platelets with this antibody. This IgG reacts with a component of human platelet membrane 155.000 MW.

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