Factor VIII, A series of homologous oligomers and a complex of two proteins

Mourik, Jan A. van; Bouma, Bonno N.; Labruyère, Wil T.; Graaf, S. de; Mochtar, I. A.

doi:10.1016/0049-3848(74)90211-4

Cited by 95 publications

(36 citation statements)

References 17 publications

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“…von Willebrand factor exists in plasma as a series of multimers (48,49) composed of a subunit of M, 270,000. In normal plasma, this subunit is present in an -100-fold molar excess over Factor VIII (assuming an M, of 270,000 for Factor VIII and concentrations of 10 and 0.1 tg/ml for von Wiflebrand factor and Factor VIII, respectively).…”

Section: Discussionmentioning

confidence: 99%

Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor.

Koedam¹,

Meijers²,

Sixma³

et al. 1988

J. Clin. Invest.

225

136

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Activated protein C (APC) acts as a potent anticoagulant enzyme by inactivating Factor V and Factor VIII. In this study, protein S was shown to increase the inactivation of purified Factor VIII by APC ninefold. The reaction rate was saturated with respect to the concentration of protein S when protein S was present in a 10-fold molar excess over APC. The heavy chain of Factor VIII was cleaved by APC and protein S did not alter the degradation pattern. Factor VIII circulates in a complex with the adhesive protein von Willebrand factor. When purified Factor VIII was recombined with von Willebrand factor, the inactivation of Factor VIII by APC proceeded at a 10-20-fold slower rate as compared with Factor VIII in the absence of von Willebrand factor. Protein S had no effect on the inactivation of the Factor VIII-von Willebrand factor complex by APC. After treatment of this complex with thrombin, however, the actions of APC and protein S towards Factor VIII were completely restored. In hemophilia A plasma, purified Factor VIII associated with endogenous von Willebrand factor, resulting in a complete protection against APC (4 nM). By mixing hemophilic plasma with plasma from a patient with severe von Willebrand's disease, we could vary the amount of von Willebrand factor. 1 U of von Willebrand factor was needed to provide protection of 1 U Factor VIII. Also in plasma from patients with the IIA-type variant of von Willebrand's disease, Factor VIII was protected. In von Willebrand's disease plasma, which was depleted of protein S., APC did not inactivate Factor VIII. These results indicate that protein S serves as a cofactor in the inactivation of Factor VIII and Factor VIIIa by APC and that von Willebrand factor can regulate the action of these two anticoagulant proteins.

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Section: Discussionmentioning

confidence: 99%

Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor.

Koedam¹,

Meijers²,

Sixma³

et al. 1988

J. Clin. Invest.

225

136

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“…Unreduced, the purified protein does not enter 5% polyacrylamide gels. However, on larger-pore gels of lower acrylamide concentration, purified Factor VIII has the appearance of an aggregating multimeric series (14). Both noncovalent interactions (14) and proteolysis (15) have been suggested as possible causes.…”

Section: Introductionmentioning

confidence: 99%

“…However, on larger-pore gels of lower acrylamide concentration, purified Factor VIII has the appearance of an aggregating multimeric series (14). Both noncovalent interactions (14) and proteolysis (15) have been suggested as possible causes. Austen et al (16,17) and Kirby and Mills (18) have demonstrated the sensitivity of both VWF and AHF to reducing agents and to p-chloromercuribenzoate, suggesting an important role of sulfhydryl groups and disulfide bonding in their biological activity.…”

Section: Introductionmentioning

confidence: 99%

Disulfide Bonds and the Quaternary Structure of Factor VIII/von Willebrand Factor

Counts¹,

Paskell²,

Elgee³

1978

J. Clin. Invest.

156

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A B S T R A C T Human Factor VIII/von Willebrand factor, purified by calcium citrate-cellulose chromatography and 4% agarose gel filtration was subjected to sodium dodecyl sulfate gel electrophoresis on gels containing 2% acrylamide and 0.5% agarose. We find a series of multimers of which the apparent molecular weight of the higher members was -5 million. The higher multimers were isolated by 2% agarose gel filtration. Treatment of the high molecular weight multimers with 2-mercaptoethanol at concentrations of 0.005-0.5% in the presence of 1% dodecyl sulfate resulted in a shift to lower molecular weight multimers. Between mercaptoethanol concentrations of 0.01 and 0.5%, the predominant species was the dimer of the basic subunit. Mercaptoethanol concentrations >0.5% were required to reduce the interchain disulfide bonds of the dimer. An artificial multimeric series was prepared by cross-linking von Willebrand factor subunits with dimethylsuberimidate. Comparison of the multimers produced by reduction with the multimers produced by cross-linking, demonstrated the absence of odd-numbered multimers from the reduced series. Thus, the protomeric unit appears to be the dimer. High molecular weight multimers had both ristocetin cofactor activity and Factor VIII procoagulant activity. Reduction of the protein in the absence of denaturing agents, caused a gradual shift to lower molecular weight species and a concomitant loss ofvon Willebrand factor activity. In contrast, Factor VIII activity was unchanged by reduction. These studies suggest that the moieties having von Willebrand factor activity and those having Factor VIII activities are covalently linked by disulfide bonds.

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“…The use of sodium dodecyl sulfate (SDS), an ionic detergent that dissociates oligomeric molecules into subunits, permitted analysis of VWF without the need for prior denaturation (reduction) of the protein [2]. Plasma (platelet or endothelial cell) VWF multimers are analyzed by visualization of these nonreduced structures subsequent to electrophoresis in agarose gels.…”

Section: Introductionmentioning

confidence: 99%

Analysis of von Willebrand factor structure by multimer analysis

Ledford‐Kraemer¹

2010

American J Hematol

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Analysis of von Willebrand factor (VWF) structure is achieved by performing a highly specialized procedure, VWF multimer analysis. The test is reserved for the reference or specialized laboratory environment. The assay is qualitative (though under some circumstances multimers may be quantified) in that it assesses the overall size distribution of VWF multimers as well as their individual internal structure. The test is used predominantly to type or subtype von Willebrand disease. The analysis of VWF multimers generally consists of four steps: (1) electrophoresis of plasma in an agarose gel, (2) either gel fixation or transfer of the electrophoretic protein product to a membrane, (3) immunodetection of the protein, and (4) evaluation of the protein in the gel or membrane. The assay is complex, time consuming, requires specialized equipment and technical expertise, and is not standardized. Am. J. Hematol. 85:510-514, 2010. V

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Factor VIII, A series of homologous oligomers and a complex of two proteins

Cited by 95 publications

References 17 publications

Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor.

Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor.

Disulfide Bonds and the Quaternary Structure of Factor VIII/von Willebrand Factor

Analysis of von Willebrand factor structure by multimer analysis

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