scholarly journals Interaction of factor VIII-von Willebrand Factor with phospholipid vesicles

1981 ◽  
Vol 200 (1) ◽  
pp. 161-167 ◽  
Author(s):  
L O Andersson ◽  
J E Brown
Keyword(s):  
Factor Viii ◽  
Density Gradient ◽  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Factor Viii Activity ◽  
Von Willebrand ◽  
Willebrand Factor

The interaction of Factor VIII-von Willebrand Factor with phospholipid vesicles has been studied by using sucrose-density-gradient ultracentrifugation. When purified Factor VIII-von Willebrand Factor was run alone. Factor VIII activity and Factor VIIIR-Ag sedimented together to the lower half of the tube. Addition of phosphatidylserine/phosphatidylethanolamine vesicles at concentrations above 250 microgram/ml resulted in complete separation of Factor VIII activity and Factor VIIIR-Ag, the former appearing with the phospholipid on the top of the tube and the latter sedimenting as before. This separation was obtained even in the presence of proteinase inhibitors. Activation of Factor VIII-von Willebrand Factor by thrombin resulted in formation of a slow sedimenting component containing essentially all the Factor VIII activity, whereas the Factor VIIIR-Ag sedimented towards the bottom of the tube as before. The thrombin-induced Factor VIII activity was strongly bound to phospholipid vesicles as determined by density-gradient centrifugations at various Factor VIII concentrations and low concentrations of phospholipid. Based on certain assumptions a dissociation constant of 2.5 nM was calculated, a mechanism for the formation in vivo of the Factor X-activator complex is suggested.

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.

Interaction Of Factor VIII/Von Willebrand Factor With Phospholipid Vesicles

1981 ◽  
Author(s):  
J E Brown ◽  
L O Andersson
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Sucrose Density Gradient ◽  
Complete Separation ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Phospholipid Complex ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Activator Complex

The interaction of factor VIII/von Willebrand factor with phospholipid vesicles was studied using sucrose density gradient ultracentrifugation. Purified VIII/vWf (Kabi) sediments as a complex in the lower third of a 5-30% sucrose gradient centrifuged for 18 hours at 160,000 g. Addition of sonicated phosphatidylserine-phosphatidylethanolamine vesicles at concentrations above 250 μg/ml results in complete separation of VIII:C from vWf, the former appearing with the phospholipid in the top of the gradient and the latter sedimenting as before. At lower levels of phospholipid, vWf competes for binding of VIII:C. The separation at higher levels of phospholipid is obtained in the presence of DFP and Trasylol, does not require calcium and occurs with plasma as well as purified preparations. Activation (7-10-fold) of purified VUI/vWf by thrombin (10-3 units/ml) results in the formation of a slowly sedimenting component containing essentially all of the VIII:C activity with little or no associated vWf. The thrombin-activated VIII:C is strongly bound to phospholipid vesicles and is more stabile than uncomplexed VIII:C (thrombin-activated). By varying the concentration of thrombin activated VIII:C with a constant (5 μg/ml) concentration of phospholipid, a Scatchard binding plot was obtained and a dissociation constant of 2.5 x 10-9 M estimated (assuming a molecular weight for VIII:C of 90,000) for the thrombin-activated VIII:C-phospholipid complex. These studies suggest that thrombin activated VIII:C binds to exposed phospholipid on released platelets concentrating the factor X activator complex.

Abnormal Structure of von Willebrand Factor in Myeloproliferative Syndrome Is Associated to Either Thrombotic or Bleeding Diathesis

1987 ◽  
Vol 58 (02) ◽  
pp. 753-757 ◽  
Author(s):  
M F López-Fernández ◽  
C López-Berges ◽  
R Martín ◽  
A Pardo ◽  
F J Ramos ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Relative Proportion ◽  
Variable Degree ◽  
Bleeding Diathesis ◽  
Myeloproliferative Syndrome ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe multimeric and subunit patterns of plasma von Willebrand factor (vWF) were analyzed in eight patients with myeloproliferative syndrome (MS) in order to investigate the possible existence of heterogeneity in the “in vivo” proteolytic cleavage of the protein, previously observed in this entity. Six patients lacked large vWF multimers, five of them having normal bleeding times (BT) and clinically documented episodes of thrombotic origin, whereas one patient had long BT and bleeding symptoms. Seven patients showed a relative increase in the 176 kDa subunit fragment while the 189 kDa polypeptide was increased in only one. In addition, another patient (and prior to any therapy) showed the presence of a new fragment of approximately 95 kDa which disappeared after Busulfan therapy. The collection of blood from these patients with proteinase inhibitors did not correct the abnormalities.The infusion of DDAVP to two patients with abnormal vWF was accompanied by: the appearance of larger vWF multimers which disappeared rapidly from plasma; an increase in the relative proportion of the satellite bands of each multimer and a further increase of the 176 kDa fragment. These data point to some heterogeneity in the vWF abnormality present in MS which may be related in part to a variable degree of proteolysis of vWF occurring “in vivo” rather than “in vitro”, and which may be associated to either a thrombotic or a bleeding diathesis. They also suggest that despite the presence of abnormal, already proteolyzed vWF, DDAVP-enhanced proteolysis occurs in MS to a similar extent to what is described in normal individuals.

scholarly journals Direct radioimmune detection in human plasma of the association between factor VIII procoagulant protein and von Willebrand factor, and the interaction of von Willebrand factor-bound procoagulant VIII with platelets

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1163-1173 ◽  
Author(s):  
JL Moake ◽  
MJ Weinstein ◽  
JH Troll ◽  
LE Chute ◽  
NM Colannino
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Von Willebrand Factor ◽  
Sodium Dodecylsulfate ◽  
Coagulation Factors ◽  
Cysteine Protease Inhibitors ◽  
Proteolytic Activation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The predominant procoagulant factor VIII (VIII:C) form in normal human plasma containing various combinations of anticoagulants and serine/cysteine protease inhibitors is a protein with mol wt 2.6 +/- 0.2 X 10(5). This protein can be detected by 125I-anti-VIII:C Fab binding and gel electrophoresis in the presence and absence of sodium dodecylsulfate (SDS) and is distinct from the subunit of factor VIII/von Willebrand factor (VIII:vWF) multimers. No larger VIII:C form is present in plasma from patients with severe congenital deficiencies of each of the coagulation factors, other than VIII:C. The mol wt approximately 2.6 X 10(5) VIII:C form is, therefore, likely to be the in vivo procoagulant form of VIII:C, rather than a partially proteolyzed, partially activated derivative of a larger precursor. About 60% of this procoagulant mol wt approximately 2.6 X 10(5) VIII:C form in plasma is present in noncovalent complexes with larger VIII:vWF multimers, which attach reversibly to platelet surfaces in the presence of ristocetin. This VIII:vWF-bound protein of mol wt approximately 2.6 X 10(5) may be the plasma procoagulant form of VIII:C which, after proteolytic activation, accelerates the IXa-mediated cleavage and activation of X postulated to occur on platelet surfaces.

scholarly journals Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells.

1989 ◽  
Vol 9 (3) ◽  
pp. 1233-1242 ◽  
Author(s):  
R J Kaufman ◽  
L C Wasley ◽  
M V Davies ◽  
R J Wise ◽  
D I Israel ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Translation Product ◽  
Factor Viii Activity ◽  
Ovary Cells ◽  
Von Willebrand ◽  
Willebrand Factor

In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide. This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF). Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF. Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium. In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated. vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells. Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm. In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF. These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.

ABNORMAL STRUCTURE OF VON WILLEBRAND FACTOR IN MYELOPROLIFERATIVE SYNDROME IS ASSOCIATED WITH EITHER THROMBOTIC OR BLEEDING DIATHESIS

1987 ◽  
Author(s):  
M F López-Fernández ◽  
C López-Berges ◽  
R Martín ◽  
A Pardo ◽  
F J Romos ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Relative Proportion ◽  
Variable Degree ◽  
Myeloproliferative Syndrome ◽  
Normal Individuals ◽  
Von Willebrand ◽  
Willebrand Factor

The multimeric and subunit patterns of plasma von Willebrand factor (vWF) were analyzed in eight patients with myeloproliferative syndrome (MS) in order to investigate the possible existence of heterogeneity in the "in vivo" proteolytic cleavage of the protein, previously observed in this entity. Six patients lacked large vWF multimers, five of them having normal bleeding times (BT) and clinically documented episodes of thrombotic origin, whereas one patient had long BT and bleeding symptoms. Seven patients showed an increase 176 kDa subunit fragment while the 189 kDa polypeptide was increased in only one. In addition, another patient (and prior to any therapy) showed the presence of a new fragment of aproximately 95 kDa which disappeared after Busulfan therapy. The collection of blood from these patients with proteinase inhibitors did not correct the abnormalities.The infusion of DDAVP to two patients with abnormal vWF was accompanied by: the appearence of larger vWF multimers which disappeared rapidly from plasma; an increase in the relative proportion of the satellite bands of each multimer and a further increase in the 176 kDa fragment. These data show some heterogeneity in the vWF abnormality present in MS which may be related in part to a variable degree of proteolysis of vWF ocurring "in vivo" rather than "in vitro", and which may be associated to either a bleeding or (even) a thrombotic diathesis. They also suggest that despite the presence of abnormal, already proteolyzed vWF, DDAVP-enhanced proteolysis occurs in MS to a similar extent as described in normal individuals.

Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells

1989 ◽  
Vol 9 (3) ◽  
pp. 1233-1242
Author(s):  
R J Kaufman ◽  
L C Wasley ◽  
M V Davies ◽  
R J Wise ◽  
D I Israel ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Translation Product ◽  
Factor Viii Activity ◽  
Ovary Cells ◽  
Von Willebrand ◽  
Willebrand Factor

In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide. This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF). Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF. Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium. In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated. vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells. Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm. In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF. These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.

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