Effect Of The Multimeric Structure Of The Factor VIII/Von Willebrand Factor Protein On Binding To Human Platelets

1981 ◽  
Author(s):  
H Gralnick ◽  
S Williams ◽  
D J Morisato
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Competitive Inhibition ◽  
High Capacity ◽  
Molecular Size ◽  
Human Platelets ◽  
Affinity Binding ◽  
Platelet Receptor ◽  
Von Willebrand ◽  
Willebrand Factor

We have studied the characteristics of binding of intact factor VIII/von Willebrand factor (f. VIII/vWf) protein and 2-mercaptoethanol (2ME) treated f. VIII/vWf protein to human platelets. The purified f. VIII/vWf was radiolabelled with tritiated 3H potassium borohydride; 4.5 × 103 molecules of the intact radiolabelled material bound per platelet. Of these some molecules bound with a high affinity/low capacity (Kd 0.21 nM and 1.5 × 103 molecules) and another with a low affinity/high capacity (Kd 2.5 nM and 3 × 103 molecules). When the material was reduced with 2ME at 0.01%, it bound with an intermediate affinity of 1.6 nM with a capacity of 4.0 × 103 and a low affinity binding of 12.5 nM and a capacity of 4.0 × 103 . The 0.1% 2ME-treated material revealed only low affinity binding with a Kd of 15 nM and the number of molecules bound 13 × 103. Studies of competitive inhibition of the intact f. VIII/vWf binding to human platelets by the reduced materials revealed that the smallest f. VIII/vWf protein (i.e., 0.1% 2ME) was the least effective while the 0.01% 2ME material was intermediate between the 0.1% and the intact material. The differences noted in the ability to displace the intact material as well as in binding to the human platelet were paralleled by decreases in the vWf activity of these proteins.These studies aid in our understanding of the binding of the f. VIII/vWf to platelets in that the binding sites on platelets may be homogeneous while the ligand is heterogenous. These studies reinforce the structure/function relationships of f. VIII/vWf proteins which have been defined using ristocetin-induced platelet aggregation (i.e., the minimum molecular size of the f. VIII/vWf protein and the penultimate galactose residues on the carbohydrate side chain). We conclude that these defects of the f. VIII/vWf protein also interfere with the protein binding to its platelet receptor and that f. VIII/vWf binding to platelets is an important primary step in hemostasis.

scholarly journals Effect of multimeric structure of the factor VIII/von Willebrand factor protein on binding to platelets

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 387-397 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
DK Morisato
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Competitive Inhibition ◽  
Human Platelets ◽  
Factor Vii ◽  
Variant Form ◽  
Factor Binding ◽  
Plasma Factor ◽  
Von Willebrand ◽  
Willebrand Factor

The characteristics of the intact factor VIII/von Willebrand factor protein binding to human platelets was compared to 2-mercaptoethanol- treated factor VIII/von Willebrand factor protein and to fractions of plasma factor VIII/von Willebrand factor protein that elute after the void volume. These studies indicate that the factor VIII/von Willebrand factor protein larger size oligomers bind preferentially with high affinity to low capacity sites on human platelets. The intermediate and smaller size oligomers bind with intermediate or low affinity to sites with a much greater capacity. The results from binding analysis are also paralleled by the competitive inhibition of the intact factor VIII/von Willebrand factor protein by the various 2-mercaptoethanol- treated materials. These studies indicate that the two classes of binding sites seen in previous reports of factor VII/von Willebrand factor binding reflect heterogeneity in the oligomer size of the factor VIII/von Willebrand factor protein used in these assays. This study provides a model for understanding some of the normal structure- function relationships of the normal factor VIII/von Willebrand factor protein and the defect(s) in a variant form of von Willebrand's disease. In this form of the disease, decreased factor VIII/von Willebrand factor binding to platelets is reflected in decreased von Willebrand factor activity but coagulant and/or antigen levels are normal or only slightly decreased.

scholarly journals Effect of multimeric structure of the factor VIII/von Willebrand factor protein on binding to platelets

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 387-397 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
DK Morisato
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Competitive Inhibition ◽  
Human Platelets ◽  
Factor Vii ◽  
Variant Form ◽  
Factor Binding ◽  
Plasma Factor ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The characteristics of the intact factor VIII/von Willebrand factor protein binding to human platelets was compared to 2-mercaptoethanol- treated factor VIII/von Willebrand factor protein and to fractions of plasma factor VIII/von Willebrand factor protein that elute after the void volume. These studies indicate that the factor VIII/von Willebrand factor protein larger size oligomers bind preferentially with high affinity to low capacity sites on human platelets. The intermediate and smaller size oligomers bind with intermediate or low affinity to sites with a much greater capacity. The results from binding analysis are also paralleled by the competitive inhibition of the intact factor VIII/von Willebrand factor protein by the various 2-mercaptoethanol- treated materials. These studies indicate that the two classes of binding sites seen in previous reports of factor VII/von Willebrand factor binding reflect heterogeneity in the oligomer size of the factor VIII/von Willebrand factor protein used in these assays. This study provides a model for understanding some of the normal structure- function relationships of the normal factor VIII/von Willebrand factor protein and the defect(s) in a variant form of von Willebrand's disease. In this form of the disease, decreased factor VIII/von Willebrand factor binding to platelets is reflected in decreased von Willebrand factor activity but coagulant and/or antigen levels are normal or only slightly decreased.

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.

scholarly journals Characterization of the defect of the factor VIII/von Willebrand factor protein in von Willebrand's disease

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 542-548 ◽  
Author(s):  
HR Gralnick ◽  
MC Cregger ◽  
SB Williams
Keyword(s):  
Sialic Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Molecular Forms ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Platelet Receptor ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The factor VIII/von Willebrand factor (f.VIII/vWf) protein was purified from the plasma of a patient with von Willebrand's disease (vWd). The patient had all of the classic laboratory findings of vWd except for the ristocetin-induced platelet aggregation of his own platelet-rich plasma. The disease has been documented in three generations. Comparison of the purified normal and vWd f.VIIi/vWf protein revealed several abnormalities, including decreased concentration of f.VIII/vWf antigen; decreased specific vWf activity; absence of the larger molecular forms of the f.VIII/vWf protein; carbohydrate deficiencies affecting the sialic acid, penultimate galactose and N- acetylglucosamine moieties; and decreased binding of the f.VIII/vWf protein to its platelet receptor. These studies indicate the multiplicity of biochemical and functional abnormalities associated with the f.VIII/vWf protein in vWd. f.VIII/vWf protein to normal f.VIII/vWf protein that had been treated with 2-mercaptoethanol (2-ME) to reduce the multimer size and then treated with specific exoglycosidases to remove the sialic acid and penultimate galactose residues revealed similar biologic properties.

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.

Presence Of Receptor For Activated Factor VIII On The Surface Of Released Platelets

1981 ◽  
Author(s):  
K Brodén ◽  
L-O Andersson
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Factor X ◽  
Factor Viii Activity ◽  
Platelet Receptor ◽  
Plasma Factor ◽  
Centrifuge Tube ◽  
Dilute Suspension ◽  
Von Willebrand ◽  
Willebrand Factor

In normal plasma Factor VIII activity is associated with a series of high molecular weight glycoprotein complexes also containing von Willebrand Factor related activities. To study the possible binding of various forms of Factor VIII to released platelets, a solution containing Factor VIII was mixed with a dilute suspension of platelets, which were released by addition of collagen. After 10 minutes of incubation the mixture was layered over 1.5 ml of 30% human serum albumin solution in a centrifuge tube and subjected to centrifugation at 7,000xg. Fractions were collected and analyzed for Factor VIII activity and phospholipid-related procoagulant activity. When purified Factor VUI/von Willebrand Factor complex was studied no significant association between the Factor VIII activity and the platelets were found. When purified Factor VUI/von Willebrand Factor complex was activated with 10-3 units/ml of thrombin and then tested, the main part of the Factor VIII activity became associated with the platelets. Even at very low platelet counts this binding was clearly detectable. The binding occurred both in the presence and in the absence of Ca2+. Thus released platelets bind thrombin-activated Factor VIII but not the Factor VUI/von Willebrand Factor complex. It is known that activation of Factor VIII by thrombin causes dissociation of the Factor VIII from the von Willebrand Factor part of the complex. The data obtained indicate that this dissociation is necessary in order to get the Factor VIII to bind to the platelet receptor. It may work as an amplification mechanism where the first traces of Thrombin formed upon initiation of coagulation dissociates Factor VIII from von Willebrand Factor, followed by binding to receptor on released platelets and formation of Factor X activator complex on the surface of the platelets.

scholarly journals The interaction between human blood-coagulation factor VIII and von Willebrand factor. Characterization of a high-affinity binding site on factor VIII

1989 ◽  
Vol 257 (3) ◽  
pp. 679-683 ◽  
Author(s):  
A Leyte ◽  
M P Verbeet ◽  
T Brodniewicz-Proba ◽  
J A Van Mourik ◽  
K Mertens
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Coagulation Factor ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

The interaction between human Factor VIII and immobilized multimeric von Willebrand Factor (vWF) was characterized. Equilibrium binding studies indicated the presence of multiple classes of Factor VIII-binding sites on vWF. The high-affinity binding (Kd = 2.1 x 10(-10) M) was restricted to only 1-2% of the vWF subunits. Competition studies with monoclonal antibodies with known epitopes demonstrated that the Factor VIII sequence Lys1673-Arg1689 is involved in the high-affinity interaction with vWF.

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.

Studies on the factor VIII/von Willebrand factor antigen on human platelets

1975 ◽  
Vol 6 (6) ◽  
pp. 469-480 ◽  
Author(s):  
Barry S. Coller ◽  
Richard J. Hirschman ◽  
Harvey R. Gralnick
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Platelets ◽  
Von Willebrand Factor Antigen ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen
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