Characterization of Factor VIII Related Protein Synthesized by Human Endothelial Cell: A Study of Structure and Function

1982 ◽  
Vol 48 (02) ◽  
pp. 177-181 ◽  
Author(s):  
Vivian Chan ◽  
T K Chan
Keyword(s):  
Endothelial Cell ◽  
Factor Viii ◽  
Culture Medium ◽  
Sodium Dodecyl ◽  
Von Willebrand Disease ◽  
Human Endothelial Cell ◽  
Molecular Forms ◽  
Related Protein ◽  
Covalent Bonds ◽  
And Function

SummaryCrossed immunoelectrophoresis showed that factor VIII related protein (VIII R) synthesized in the human endothelial cell (EC-VIII R) had faster electrophoretic mobility than that secreted into the culture medium (MED-VIII R). Sodium dodecyl sulphate agarose gel electrophoresis followed by radioimmunofixation showed that EC-VIII R consisted of molecules varying from 0.26 × 106 to 3.76 × 106 daltons while MED-VIII R had molecules ranging from 0.93 × 106 to greater than 10 × 106 daltons, similar to that present in plasma. The smallest VIII R molecule present in normal plasma or spent culture medium (0.93 × 106 daltons) corresponded to a tetramer of subunits of 0.22-0.24 × 106 daltons. Only molecular forms greater than 3.76 × 106 daltons possessed ristocetin cofactor activity. Sonication (15 μ. amplitued for 30 secs) effectively broke the non-covalent bonds of the VIII R multimers resulting in smaller molecules. Thus endothelial cells in culture synthesized VIII R subunits and assembled them into the higher multimeric forms on secretion. Different types of von Willebrand disease could result from defects of either of the two processes.

Serum enhancement of human endothelial cell attachment to and spreading on collagens I and IV does not require serum fibronectin or vitronectin

1990 ◽  
Vol 95 (2) ◽  
pp. 255-262
Author(s):  
W.D. Norris ◽  
J.G. Steele ◽  
G. Johnson ◽  
P.A. Underwood
Keyword(s):  
Endothelial Cell ◽  
Culture Medium ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Human Endothelial Cell ◽  
Collagen Type Iv ◽  
Type I ◽  
Type Iv ◽  
Endothelial Cell Attachment

The initial attachment and spreading of endothelial cells from human umbilical artery onto type I collagen, type IV collagen or gelatin substrata was shown to be enhanced by inclusion of serum in the culture medium. To test whether this serum effect was mediated by adsorption of serum fibronectin or vitronectin onto the collagen, these adhesive glycoproteins were selectively removed from the serum prior to addition to the culture medium. The stimulatory effect of serum on human endothelial cell spreading on collagens I and IV was also observed with serum from which either fibronectin or vitronectin, or both, had been selectively removed. The stimulatory effect for cell spreading on gelatin was diminished by selective removal of serum fibronectin, but unaffected by removal of vitronectin. Human endothelial cell attachment and spreading onto tissue culture plastic was abolished by removal of vitronectin from the serum in the culture medium. These results emphasize that the native structure of collagens is required for serum-enhancement of human endothelial cell attachment and spreading on native collagen types I and IV, and show that on these substrata the stimulated adhesion and spreading are not dependent upon adsorption of serum fibronectin or vitronectin onto the collagen substratum.

scholarly journals The factor VIII complex: structure and function

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 1-13 ◽  
Author(s):  
LW Hoyer
Keyword(s):  
Factor Viii ◽  
Complex Structure ◽  
Related Protein ◽  
Molecular Defects ◽  
Normal Human ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Antihemophilic Factor ◽  
Immunologic Properties

Abstract Normal human plasma contains a complex of two proteins that are important in hemostasis and coagulation. The factor VIII procoagulant protein (antihemophilic factor) and the factor VIII-related protein (von Willebrand factor) are under separate genetic control, have distinct biochemical and immunologic properties, and have unique and essential physiologic functions. While the nature of their interaction and the details of the biochemical structures remain to be determined, the information now available permits a preliminary understanding of the molecular defects in hemophilia and von Willebrand's diseases.

scholarly journals The factor VIII complex: structure and function

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 1-13 ◽  
Author(s):  
LW Hoyer
Keyword(s):  
Factor Viii ◽  
Complex Structure ◽  
Related Protein ◽  
Molecular Defects ◽  
Normal Human ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Antihemophilic Factor ◽  
Immunologic Properties

Normal human plasma contains a complex of two proteins that are important in hemostasis and coagulation. The factor VIII procoagulant protein (antihemophilic factor) and the factor VIII-related protein (von Willebrand factor) are under separate genetic control, have distinct biochemical and immunologic properties, and have unique and essential physiologic functions. While the nature of their interaction and the details of the biochemical structures remain to be determined, the information now available permits a preliminary understanding of the molecular defects in hemophilia and von Willebrand's diseases.

scholarly journals Immunologic Approach to the Study of Factor VIII

1977 ◽  
Author(s):  
Leon W. Hoyer
Keyword(s):  
Factor Viii ◽  
Carrier State ◽  
Procoagulant Activity ◽  
Related Protein ◽  
Low Protein ◽  
Biochemical Analyses ◽  
And Function ◽  
Deficiency Diseases

In contrast to direct biochemical analyses, immunologic studies of factor VIII are less limited by that factor’s lability and low protein concentration. Both human and heterologous antifactor VIII have been used in these studies. Human antibodies inactivate factor VIII procoagulant. activity but they do not fix complement or form immunoprecipitates. Their interaction appears to involve the formation of an immune complex with a univalent component distinct from that which has been designated factor VIII-related protein. This component is synthesized—but remains nonfunctional—in hemophilia, and at least two different defects have been distinguished. Very different reactions can be identified using heterologous antibodies. Although many of these antibodies do not react directly with the procoagulant site, they do fix complement, precipitate with factor VIII-related protein, and inactivate ristocetin cofactor activity. These antibodies are useful for classification of factor VIII-deficiency diseases according to the relative levels of factor VIII procoagulant activity and factor VIII-related antigen; identification of the hemophilic carrier state; characterization of heterogeneity in factor VIII structure; and purification of factor VIII procoagulant activity by immunoadsorbent chromatography. In general, the information obtained by immunologic studies has complemented that from direct biochemical analyses. Both approaches contribute to our evolving understanding of factor VIII structure and function.

scholarly journals Degradation of Factor VIII-Related Protein in Factor VIII Concentrates

1977 ◽  
Author(s):  
T. Jakab ◽  
R. Pflugshaupt ◽  
M. Furlan ◽  
E.A. Beck
Keyword(s):  
Factor Viii ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Relative Increase ◽  
Related Protein ◽  
Normal Plasma ◽  
Human Factor Viii ◽  
Factor Viii Concentrates ◽  
Weight Protein ◽  
Von Willebrand

Lipolytic and/or proteolytic treatment of highly purified human factor VIII results in a progressive disappearance of high-molecular weight protein as shown by electrophoresis on 3% Polyacrylamide gels in the presence of sodium dodecyl sulphate. Partial degradation of factor VIII is typically accompanied by a relative increase of factor VIII-related antigen (VIII R:AG)whereas coagulant activity (VIII:C) or von Willebrand activity (VIII R:WF) may still appear intact. VIII R:AG was measured by crossed Immunoelectrophoresis against specific heterologous antibody in agarose gels. VIII R:WF was expressed as ristocetin cofactor activity in comparison with a normal plasma pool. Assuming a VIII R:AG/ VIII R:WF ratio of 1.0 in our normal plasma, we found a similar ratio using intact purified factor VIII. This ratio increased regularly following proteolytic degradation of factor VIII concentrate by contaminating proteases or plasmin. Our findings suggest that a high VIII R:AG/VIII R:WF ratio could indicate undesirable alterations of VIII-related protein(s) during plasma fractionation.

scholarly journals Studies on the Structure and Subunit Composition of Human Antihaemophilic Factor

1977 ◽  
Author(s):  
J. J. Gorman
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Protein S ◽  
Molecular Forms ◽  
Major Band ◽  
Human Factor Viii ◽  
Von Willebrand

Human antihaemophilic factor has been purified by hydroxylapatite chromatography following precipitation from plasma and gel filtration on Sepharose 6B.Application to hydroxyiapatite was in 0.02 M tris HCl (pH 7.35) – 0.14 M NaCl and after washing with 5mM phosphate (pH 6.8) – 0.1 M NaCI the antihaemophilic factor was eluted with 0. 1M phosphate (pH 6.8) – 0.1M NaCl. Factor VIII coagulant activity, factor VIII related antigen and von Willebrand factor activity eluted simultaneously.The protein(s) had a molecular weight in excess of 500,000 and multiple subunits as shown by electrophoresis in 5% acrylamide gels containing sodium dodecyl sulphate;without reduction the protein failed to enter these gels but following reduction multiple bands were observed, the major band had a molecular weight around 200,000.Thin layer peptide mapping demonstrated structural inter-relationship between the 200,000 dalton protein and three of the smaller species, however, two other unrelated smaller species were evident.It is apparent from these findings that human factor VIII may exist as multiple molecular forms due to heterogeneity of one subunit (MW around 200,000) and the molecular structure may include other smaller non-identical subunits. The structure-function relationships of these subunits remains to be elucidated.

scholarly journals Factor VIII-related protein circulates in normal human plasma as high molecular weight multimers

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 1056-1059 ◽  
Author(s):  
LW Hoyer ◽  
JR Shainoff
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Sodium Dodecyl ◽  
Related Protein ◽  
Calcium Chelation ◽  
Human Factor Viii ◽  
Agarose Electrophoresis ◽  
Normal Human ◽  
Purification Methods ◽  
High Molecular Weight Multimers

Abstract The size of human factor VIII-related protein in plasma has been determined by sodium dodecyl sulfate (SDS) glyoxyl agarose electrophoresis. The protein was immobilized after the electrophoresis by coupling it to the modified agarose, and it was identified by autoradiography using purified rabbit anti-factor VIII-related antigen (VIIR:Ag). A series of multimeric forms was identified with Mr of 0.85- 12 x 10(6). The distribution of VIIR:Ag multimers was the same in heparin and citrate anticoagulated plasmas and in serum, and the pattern was the same after freezing as in plasma kept at 37 degrees C from the time of venipuncture until the electrophoresis was complete. These observations indicate that VIIR:Ag circulates in normal plasma as a population of very large multimers and that the size distribution is not an artifact induced by purification methods, freezing, or calcium chelation.

scholarly journals von Willebrand factor and von Willebrand disease [published erratum appears in Blood 1988 Mar;71(3):830]

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 895-904 ◽  
Author(s):  
ZM Ruggeri ◽  
TS Zimmerman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Current Knowledge ◽  
Von Willebrand Disease ◽  
The Past ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Functional Alteration

Progress has occurred in the past several years in the understanding of the structure and function of von Willebrand factor (vWF). This multimeric glycoprotein exhibits a dual role, that of mediating platelet adhesion and aggregation onto thrombogenic surfaces, and that of functioning as carrier in plasma for the factor VIII procoagulant protein. New insights into the nature of the several functional domains of vWF have led to the identification of the regions of the molecule that interact with factor VIII, heparin, the glycoprotein lb of platelets, and collagen. Alterations of vWF are the cause of von Willebrand disease (vWD), a congenital bleeding disorder. In the majority of patients, the plasma levels of vWF are decreased, but there is no demonstrable structural or functional alteration of the protein. In other patients, however, the structure of vWF is abnormal. This review summarizes the current knowledge on vWF and vWD.

scholarly journals Factor VIII-related protein circulates in normal human plasma as high molecular weight multimers

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 1056-1059 ◽  
Author(s):  
LW Hoyer ◽  
JR Shainoff
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Sodium Dodecyl ◽  
Related Protein ◽  
Calcium Chelation ◽  
Human Factor Viii ◽  
Agarose Electrophoresis ◽  
Normal Human ◽  
Purification Methods ◽  
High Molecular Weight Multimers

The size of human factor VIII-related protein in plasma has been determined by sodium dodecyl sulfate (SDS) glyoxyl agarose electrophoresis. The protein was immobilized after the electrophoresis by coupling it to the modified agarose, and it was identified by autoradiography using purified rabbit anti-factor VIII-related antigen (VIIR:Ag). A series of multimeric forms was identified with Mr of 0.85- 12 x 10(6). The distribution of VIIR:Ag multimers was the same in heparin and citrate anticoagulated plasmas and in serum, and the pattern was the same after freezing as in plasma kept at 37 degrees C from the time of venipuncture until the electrophoresis was complete. These observations indicate that VIIR:Ag circulates in normal plasma as a population of very large multimers and that the size distribution is not an artifact induced by purification methods, freezing, or calcium chelation.

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