The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin

1981 ◽  
Author(s):  
P Silber ◽  
T H Finlay
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Binding Constant ◽  
Specific Binding ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Binding Data ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.

The Importance of the Carbohydrate Moiety of the Factor VIII/Von Willebrand Factor Protein in Binding to and Causing Agglutination of Human Platelets

1979 ◽  
Author(s):  
H.R. Gralnick ◽  
D.K. Morisato
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Human Platelets ◽  
Galactose Oxidase ◽  
Scatchard Analysis ◽  
Human Fibrinogen ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the binding of radiolabelled factor VIII/von Willebrand factor (f. VIII/vWf) protein to human platelets (P) in the presence of ristocetin (R). In these atudies we have delineated the importance of the carbohydrate (CHO) moiety(s) in both the binding to the P and in cauaing agglutination of P. Binding of the f.VIII/vWf protein to human P was time and temperature dependent and dependent on the concentration of R. Binding was specific in that it could not be blocked by human fibrinogen but was inhibited by unlabelled f.VIII/vWf protein. In studies utilizing varying amounts of the f.VIII/vWf protein or by varying the number of P in the assay, the number of binding sites for the f. VIII/vWf protein were estimated at 9,500-9,800 per platelet. Scatchard analysis revealed 11,000 binding sites with 3,600 of high affinity and 7,400 of low affinity. Removal of the sialic acid of the f.VIII/vWf protein resulted in no significa nt change in its ability to bind to the P surface or cause agglutination in the presence, IR. Removal of the galactose by 6-galactosijase resulted in a 75% reduction of binding of the f.VIII/vWf protein and a 91% decrease in the agglutination of human P. Similar studies with galactose oxidase showed that oxidation of the penultimate galactose residue s results in a decrease in agglutination comparable to that seen with 6-galactosidase treatment. These studies indicate that the CHO moiety of the f.VIII/vWf protein is important in both binding to the P surface as well as causing agglutination of human P.

scholarly journals Selective binding of the factor VIII/von Willebrand factor protein to human platelets

Blood ◽  
1980 ◽  
Vol 55 (1) ◽  
pp. 9-15 ◽  
Author(s):  
DK Morisato ◽  
HR Gralnick
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Human Platelets ◽  
Galactose Oxidase ◽  
Scatchard Analysis ◽  
Potassium Borohydride ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The factor VIII/von Willebrand factor protein was radiolabeled after modification by galactose oxidase and reduction with tritiated potassium borohydride. This mild efficient method for labeling resulted in retention of over 90% of the biologic activities of the factor VIII/von Willebrand factor protein. Binding of this protein to platelets was found to be specific, and binding sites could be saturated in the presence of ristocetin. However, binding was highly dependent on ristocetin concentration, as the number of human factor VIII/von Willebrand factor molecules bound per platelet was a function of the ristocetin concentration. At a ristocetin concentration of 0.55 mg/ml, each platelet binds approximately 11,000 factor VIII/von Willebrand factor molecules per platelet. Scatchard analysis of the concentration-dependent binding sites yielded a hyperbolic plot that appeared to be related to the existence of two classes of binding sites. The higher affinity class had a Kd of 3.7 x 10(-10) M 3500 sites/platelet, while the lower affinity class had a Kd of 2.35 x 10(- 9) M and a capacity of 7500 sites/platelet. As with ristocetin-induced platelet agglutination, the carbohydrate content plays a significant role in the binding of the factor VIII/von Willebrand factor protein to the platelet.

scholarly journals Selective binding of the factor VIII/von Willebrand factor protein to human platelets

Blood ◽  
1980 ◽  
Vol 55 (1) ◽  
pp. 9-15 ◽  
Author(s):  
DK Morisato ◽  
HR Gralnick
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Human Platelets ◽  
Galactose Oxidase ◽  
Scatchard Analysis ◽  
Potassium Borohydride ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

The factor VIII/von Willebrand factor protein was radiolabeled after modification by galactose oxidase and reduction with tritiated potassium borohydride. This mild efficient method for labeling resulted in retention of over 90% of the biologic activities of the factor VIII/von Willebrand factor protein. Binding of this protein to platelets was found to be specific, and binding sites could be saturated in the presence of ristocetin. However, binding was highly dependent on ristocetin concentration, as the number of human factor VIII/von Willebrand factor molecules bound per platelet was a function of the ristocetin concentration. At a ristocetin concentration of 0.55 mg/ml, each platelet binds approximately 11,000 factor VIII/von Willebrand factor molecules per platelet. Scatchard analysis of the concentration-dependent binding sites yielded a hyperbolic plot that appeared to be related to the existence of two classes of binding sites. The higher affinity class had a Kd of 3.7 x 10(-10) M 3500 sites/platelet, while the lower affinity class had a Kd of 2.35 x 10(- 9) M and a capacity of 7500 sites/platelet. As with ristocetin-induced platelet agglutination, the carbohydrate content plays a significant role in the binding of the factor VIII/von Willebrand factor protein to the platelet.

scholarly journals Related binding mechanisms for fibrinogen, fibronectin, von Willebrand factor, and thrombospondin on thrombin-stimulated human platelets

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 724-727 ◽  
Author(s):  
EF Plow ◽  
RP McEver ◽  
BS Coller ◽  
VL Jr Woods ◽  
GA Marguerie ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Dose Range ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Inhibitory Antibodies ◽  
Fixed Cell ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Fibrinogen, fibronectin, von Willebrand factor, and thrombospondin are four large glycoproteins that bind to thrombin-stimulated platelets and influence cellular adhesive functions. The effects of five monoclonal antibodies that react with platelet membrane glycoproteins (GP) IIb and/or IIIa on the binding of these four molecules to stimulated platelets were assessed. Tab and PMI-1, antibodies recognizing GPIIb, had no effect, whereas 10E5 and 2G12, antibodies that immunoprecipitate both GPIIb and IIIa in the presence of calcium, inhibited binding of all four ligands by greater than 85%. T10, an antibody specific for the GPIIb-IIIa complex, produced partial inhibition (60% to 80%) of the binding of each ligand. Inhibitory antibodies were effective in the same dose range for all four proteins and also inhibited binding of fibrinogen, fibronectin, and von Willebrand factor to receptors fixed in an induced state (thrombin-stimulated platelets fixed with paraformaldehyde). Thrombospondin did not bind to these fixed cell preparations. The results suggest that these four adhesive proteins have a related mechanism of binding to thrombin-stimulated platelets. This related mechanism may entail the sharing of some, but not necessarily all, binding sites for the four ligands or a proximal relationship between these binding sites.

scholarly journals Related binding mechanisms for fibrinogen, fibronectin, von Willebrand factor, and thrombospondin on thrombin-stimulated human platelets

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 724-727 ◽  
Author(s):  
EF Plow ◽  
RP McEver ◽  
BS Coller ◽  
VL Jr Woods ◽  
GA Marguerie ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Dose Range ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Inhibitory Antibodies ◽  
Fixed Cell ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

Fibrinogen, fibronectin, von Willebrand factor, and thrombospondin are four large glycoproteins that bind to thrombin-stimulated platelets and influence cellular adhesive functions. The effects of five monoclonal antibodies that react with platelet membrane glycoproteins (GP) IIb and/or IIIa on the binding of these four molecules to stimulated platelets were assessed. Tab and PMI-1, antibodies recognizing GPIIb, had no effect, whereas 10E5 and 2G12, antibodies that immunoprecipitate both GPIIb and IIIa in the presence of calcium, inhibited binding of all four ligands by greater than 85%. T10, an antibody specific for the GPIIb-IIIa complex, produced partial inhibition (60% to 80%) of the binding of each ligand. Inhibitory antibodies were effective in the same dose range for all four proteins and also inhibited binding of fibrinogen, fibronectin, and von Willebrand factor to receptors fixed in an induced state (thrombin-stimulated platelets fixed with paraformaldehyde). Thrombospondin did not bind to these fixed cell preparations. The results suggest that these four adhesive proteins have a related mechanism of binding to thrombin-stimulated platelets. This related mechanism may entail the sharing of some, but not necessarily all, binding sites for the four ligands or a proximal relationship between these binding sites.

Calcium and the Fc Receptor on Human Platelets

1987 ◽  
Vol 57 (03) ◽  
pp. 298-301
Author(s):  
William F Clark ◽  
Gerald J M Tevaarwerk ◽  
Bruce D Reid ◽  
Suzanne Hall ◽  
Anita Caveney ◽  
...  
Keyword(s):  
Specific Binding ◽  
Normal Subjects ◽  
Human Platelets ◽  
Fc Receptor ◽  
Normal Male ◽  
Scatchard Analysis ◽  
Normal Female ◽  
Apparent Difference ◽  
Binding Data ◽  
Direct Binding

SummaryWe have described the calcium dependence of the IgG Fc receptor (Fc-R) on human platelets by analyzing the direct binding of radiolabelled Fc fragments, monomers and dimers of IgG. Specific binding to platelets was undetectable at 37° C in a calcium-free preparation but readily detected when calcium was restored. Scatchard analysis of the binding data for the calcium-restored platelets permitted calculation of the available Fc-R and the Ka of binding for the different IgG ligands. The mean Ka of binding for 12 normal subjects varied from 107 to 108 L/M, with an equal receptor number measured by Fc fragments and dimers of IgG, but a lesser amount for monomeric IgG. There was no apparent difference in Fc-R number for platelets from 6 normal male versus 6 normal female subjects.At 4° C binding was detectable for dimers and polymers of IgG in a calcium-free preparation and this was markedly increased with recalcification. Thus, our data are consistent with an Fc receptor population on human platelets whose avidity for binding is significantly enhanced in a calcium-restored medium.

Interactions of Purified Rat Factor VIII/von Willebrand Factor with Rat and Human Platelets – Effect of Albumin and Ristocetin

1984 ◽  
Vol 52 (01) ◽  
pp. 057-059 ◽  
Author(s):  
E Dejana ◽  
M Furlan ◽  
B Barbieri ◽  
M B Donati ◽  
E A Beck
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Rat Plasma ◽  
Human Platelets ◽  
High Concentrations ◽  
Low Sensitivity ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Rat Platelets

SummaryRat platelets do not respond to ristocetin in their own plasma nor do they aggregate in the presence of bovine or porcine factor VIII von Willebrand factor (F VIII R:WF) or human F VIII R:WF in presence of ristocetin. However, rat plasma supports ristocetin induced aggregation of washed human platelets. In this study we report on purification of rat F VIII R:WF from cryoprecipitate. Similarly to porcine or bovine material, purified rat F VIII R:WF induced aggregation of human washed fixed platelets. This effect was enhanced by addition of ristocetin and was not modified by addition of albumin. Rat washed platelets were aggregated by ristocetin in the presence of rat or human F VIII R:WF provided that high concentrations of ristocetin are added in a system essentially free of extraneous proteins. Increasing concentrations of albumin dramatically reduced the ability of ristocetin to aggregate rat platelets while human platelet aggregation by human or rat F VIII R:WF was only moderately affected.These studies show that rat F VIII R:WF can interact with rat and human platelets. The lack of response of rat platelets to ristocetin in their own plasma is most likely due to a low sensitivity of rat platelets to this drug and to an inhibitory activity of plasma proteins on this reaction.

Sign in / Sign up

Export Citation Format

Share Document