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

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.

1979 ◽  
Author(s):  
H.R. Gralnick ◽  
D.K. Morisato

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.


Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 827-831 ◽  
Author(s):  
EJ Harfenist ◽  
MA Packham ◽  
RL Kinlough-Rathbone ◽  
M Cattaneo ◽  
JF Mustard

Abstract To investigate the suggestion that von Willebrand factor (vWf) can substitute for fibrinogen in supporting ADP-induced aggregation of human platelets, we studied platelet reactions in two media: (1) a high calcium medium, Tyrode-albumin solution containing calcium ions in the physiological range of 2 mmol/L, and (2) a low calcium medium, modified Tyrode-albumin solution from which calcium salt was omitted (calcium ion concentration approximately 20 mumol/L). In the high calcium medium vWf even at concentrations up to six times as high as physiological, showed little or no potentiation of ADP-induced platelet aggregation, whereas fibrinogen strongly potentiated reversible aggregation without thromboxane formation or release of granule contents. In the low calcium medium, either vWf or fibrinogen supported biphasic aggregation in response to ADP, with thromboxane formation and release of granule contents. Aspirin and the thromboxane receptor blocker BM 13.177 inhibited these secondary responses to von Willebrand factor, indicating that they require thromboxane A2 formation and feedback amplification by thromboxane A2. A monoclonal antibody, 10E5, to the platelet glycoprotein IIb/IIIa complex inhibited both primary and secondary aggregation. Although vWf supports ADP-induced aggregation when the concentration of ionized calcium is in the micromolar range, it does not support ADP-induced aggregation in the presence of a concentration of ionized calcium in the physiological range, indicating that vWf probably cannot substitute for fibrinogen in supporting ADP- induced aggregation in vivo.


Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 387-397 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
DK Morisato

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.


1981 ◽  
Author(s):  
H Gralnick ◽  
S Williams ◽  
D J Morisato

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.


Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 387-397 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
DK Morisato

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.


Blood ◽  
1980 ◽  
Vol 55 (1) ◽  
pp. 9-15 ◽  
Author(s):  
DK Morisato ◽  
HR Gralnick

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.


Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 827-831 ◽  
Author(s):  
EJ Harfenist ◽  
MA Packham ◽  
RL Kinlough-Rathbone ◽  
M Cattaneo ◽  
JF Mustard

To investigate the suggestion that von Willebrand factor (vWf) can substitute for fibrinogen in supporting ADP-induced aggregation of human platelets, we studied platelet reactions in two media: (1) a high calcium medium, Tyrode-albumin solution containing calcium ions in the physiological range of 2 mmol/L, and (2) a low calcium medium, modified Tyrode-albumin solution from which calcium salt was omitted (calcium ion concentration approximately 20 mumol/L). In the high calcium medium vWf even at concentrations up to six times as high as physiological, showed little or no potentiation of ADP-induced platelet aggregation, whereas fibrinogen strongly potentiated reversible aggregation without thromboxane formation or release of granule contents. In the low calcium medium, either vWf or fibrinogen supported biphasic aggregation in response to ADP, with thromboxane formation and release of granule contents. Aspirin and the thromboxane receptor blocker BM 13.177 inhibited these secondary responses to von Willebrand factor, indicating that they require thromboxane A2 formation and feedback amplification by thromboxane A2. A monoclonal antibody, 10E5, to the platelet glycoprotein IIb/IIIa complex inhibited both primary and secondary aggregation. Although vWf supports ADP-induced aggregation when the concentration of ionized calcium is in the micromolar range, it does not support ADP-induced aggregation in the presence of a concentration of ionized calcium in the physiological range, indicating that vWf probably cannot substitute for fibrinogen in supporting ADP- induced aggregation in vivo.


1975 ◽  
Vol 6 (6) ◽  
pp. 469-480 ◽  
Author(s):  
Barry S. Coller ◽  
Richard J. Hirschman ◽  
Harvey R. Gralnick

1998 ◽  
Vol 79 (06) ◽  
pp. 1191-1198 ◽  
Author(s):  
S. Rabhi-Sabile ◽  
C. de Romeuf ◽  
D. Pidard

SummaryPlasmin triggers a strong metabolic activation in human platelets, leading to shape change and granule exocytosis. However, its capacity to induce cell aggregation remains discussed and, when observed, this aggregation is preceded by a remarkable lag phase. We have thus investigated the effect of plasmin on the adhesive proteins which can be secreted by isolated platelets and mediate cell-to-cell interactions, but are also substrates for the enzyme. Immunoblot analysis of fibrinogen (Fg), thrombospondin-1 (TSP-1), fibronectin (Fn) and von Willebrand factor (vWf) was performed on extracts of platelets exposed under stirring to increasing concentrations of plasmin for up to 10 min at 37° C. Under conditions leading to formation of large aggregates, Fg, Fn and TSP-1 are extensively degraded concomitantly with their secretion, and readily lost from the surface of aggregated cells. Part of the monomers in the platelet vWf are cleaved during secretion into two main fragments with M r ≈180,000 and ≈145,000. However, multimer distribution analysis shows only a slight decrease in the very high molecular weight multimers, and most of the fragmented as well as intact vWf remains associated with the platelet surface when aggregation is maximal. That indeed vWf largely supports plasmin-induced aggregation is suggested by the observation that platelets from a patient with type 3 von Willebrand’s disease, who totally lacks vWf, show little aggregation in response to the enzyme. Finally, plasmin-induced aggregation can be totally inhibited by antagonists of the αIIbβ3 integrin. The present study thus indicates a major role for secreted vWf in platelet aggregation induced by plasmin, through its likely interaction with the multifunctional receptor αIIbβ3.Presented in part at the European Platelet Group Meeting, Erfurt, Germany, May 1996


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