Binding of Ristocetin to Platelets

1979 ◽  
Author(s):  
C.S.P. Jenkins ◽  
K.J. Clemetson ◽  
E.F. Lüscher
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Direct Relationship ◽  
Reductive Methylation ◽  
Presence And Absence ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Normal Pool Plasma

Ristocetin induces platelet agglutination in the presence of von Willebrand factor (VIIIR:WF), part of the factor VIII complex. The role of ristocetin in platelet agglutination is not yet. Clear. To follow the interaction of ristocetin with platelets and/or VIIIR:WF, ristocetin was labelled with[3H] by reductive methylation. [3H]-Ristocetin agglutinates platelets in the presence of VIIIR:WF in a manner indistinguishable from unlabelled ristocetin. The binding of[3H]-ristocetin to normal and chymotrypsin-treated platelets (which ore not agglutinated by ristocetin/VIIIR:WF) was studied in the presence and absence of VIIIR:WF (normal pool plasma and plasmas from patients with von Willebrand’s disease) and at non-agglutinating and agglutinating concentrations of ristocetin. Virtually no difference in binding was seen whether VIIIR:WF was present or not. Chymotrypsin-treated platelets do not bind less ristocetin than control platelets. A pronounced direct relationship was found between [3H]-ristocetin bound by normal platelets and total ristocetin concentration. This implies that at the higher concentrations of ristocetin either more binding sites are exposed or that aggregation of ristocetin occurs.

scholarly journals Regulation of plasma von Willebrand factor

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 96 ◽  
Author(s):  
Karl C Desch
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Coagulation Factor ◽  
Vascular Wall ◽  
Healthy Human ◽  
Venous Thromboembolic ◽  
Artery Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a central role in the initiation of blood coagulation. Through interactions between its specific functional domains, the vascular wall, coagulation factor VIII, and platelet receptors, VWF maintains hemostasis by binding to platelets and delivering factor VIII to the sites of vascular injury. In the healthy human population, plasma VWF levels vary widely. The important role of VWF is illustrated by individuals at the extremes of the normal distribution of plasma VWF concentrations where individuals with low VWF levels are more likely to present with mucocutaneous bleeding. Conversely, people with high VWF levels are at higher risk for venous thromboembolic disease, stroke, and coronary artery disease. This report will summarize recent advances in our understanding of environmental influences and the genetic control of VWF plasma variation in healthy and symptomatic populations and will also highlight the unanswered questions that are currently driving this field of study.

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 Factor VIII-von Willebrand factor requires calcium for facilitation of platelet adherence

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 996-103 ◽  
Author(s):  
KS Sakariassen ◽  
M Ottenhof-Rovers ◽  
JJ Sixma
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Divalent Cations ◽  
Platelet Adherence ◽  
Human Arteries ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.

Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 529-538 ◽  
Author(s):  
Ana Victoria Bendetowicz ◽  
Jill A. Morris ◽  
Robert J. Wise ◽  
Gary E. Gilbert ◽  
Randal J. Kaufman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Free System ◽  
N Terminus ◽  
A Cell ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

Effective Factor VIII Endocytosis Is under Conformational Control of von Willebrand Factor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1767-1767
Author(s):  
Alexander B. Meijer ◽  
Sigrid D. Roosendaal ◽  
Bas de Laat ◽  
Maartje van den Biggelaar ◽  
Vincent Limburg ◽  
...  
Keyword(s):  
Cell Surface ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Ldl Receptor ◽  
Active Conformation ◽  
Binding Studies ◽  
Early Endosomes ◽  
Presence And Absence ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Von Willebrand Factor (VWF) protects Factor VIII (FVIII) from rapid clearance of the cofactor from the circulations. This notion may agree with the observation that VWF blocks FVIII binding to LDL receptor (LDLR) and LDL receptor-related protein (LRP) in vitro. In spite of the presence of VWF, however, we have previously demonstrated that LRP and LDLR are involved in the catabolism of FVIII in vivo. This suggests that dissociation of the FVIII-VWF complex in plasma or at the cell surface drives the LRP/LDLR dependent clearance of FVIII. We therefore first assessed the effect of VWF on the binding of FVIII to the cell surface employing confocal microscopy. To this end, a functional FVIII derivative containing the yellow fluorescent protein (FVIIIYFP) was incubated with LRP-expressing or LDLR-expressing CHO cells at 4 °C in the presence and absence of VWF. The result showed that there was a distinct yellow fluorescent staining of the cell surface irrespective of the presence of VWF. When the same experiment was performed at 37 °C, however, yellow fluorescent focal spots rapidly appeared inside the cells but only in the absence of VWF. Co-localization studies demonstrated that these spots originated from FVIIIYFP present in early endosomes. These findings suggest that VWF blocks the transfer of FVIII to its endocytic receptors but not the binding of FVIII to the cell surface. Intriguingly, in the presence of ristocetin, FVIIIYFP was localized inside the cells not only in the absence of VWF but also in its presence. In agreement with this finding, flow cytometric analysis confirmed that VWF was no longer able to prevent endocytosis of FVIIIYFP in the presence of ristocetin. These results suggest that switching VWF in the VWF-FVIIIYFP complex into its active conformation triggers the endocytic uptake of FVIII. The restored endocytosis of FVIII was not the consequence of a reduced affinity of “active-VWF” for FVIII. The latter was concluded from solid phase binding studies that showed that FVIII-VWF complex formation is indistinguishable in the presence and absence of ristocetin. We propose that switching VWF in the VWF-FVIII complex into its active conformation initiates at the cell surface a sequence of molecular events, which ultimately lead to the endocytic uptake of FVIII by cells expressing LRP or LDLR.

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.

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