Detection of the Carrier State in Hereditary Coagulation Disorders I

Jj, Veltkamp; Ef, Drion; Ea, Loeliger

doi:10.1055/s-0038-1651206

Cited by 96 publications

(58 citation statements)

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“…Radioiodination of Factor VIII was carried out with the lactoperoxidase/glucose oxidase method (29). Factor VIII activity was determined using a onestage clotting assay (30,31) (23). The specific activity of the preparation was 100 VWF:Ag U/mg.…”

Section: Methodsmentioning

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: Methodsmentioning

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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“…(18) and modified by Bouma and Starkenburg (19). Frozen (-70°C) reference plasma pooled from four normal donors was standardized against fresh pooled plasma of 40 normal donors (mean age 31.5 yr, ratio women/men = 1, nonpregnant women not taking oral contraceptives).…”

Section: Methodsmentioning

confidence: 99%

“…In this way the biphasic nature of the disappearance curve was discovered. The early data showed some discordance, but more recent studies have shown that the t1/2s are in the range of about [12][13][14][15][16][17][18] h. The introduction of the quantitative assay for VIIIR:Ag using heterologous antisera (2) allowed the analysis of the disappearance of Factor VIII on the basis of VIIIR:Ag. It was then reported that this VIIIR:Ag decreased more slowly than VIII:C after transfusion of normal cryoprecipitate into hemophiliacs (15)(16)(17).…”

Section: Introductionmentioning

confidence: 99%

Survival of 125iodine-labeled Factor VIII in normals and patients with classic hemophilia. Observations on the heterogeneity of human Factor VIII.

Over¹,

Sixma²,

Bruïne³

et al. 1978

J. Clin. Invest.

100

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“…In addition, the radiolabeled factor VIII contained 10 mM 6-aminohexanoic acid, 10 pg/ml soybean trypsin inhibitor and 0.1 % bovine serum albumin (BSA). Factor VIII activity was measured with a one-stage clotting assay [25,26] using kaolin/cephalin from Boehringer Mannheim GmbH (Mannheim, FRG) and factor-VIII-deficient plasma from a severe hemophilia A patient.…”

Section: Proteinsmentioning

confidence: 99%

The effect of von Willebrand factor on activation of factor VIII by factor Xa

Koedam¹,

Hamer²,

Beeser‐Visser³

et al. 1990

European Journal of Biochemistry

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Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 Ujml) with 0.1 pg/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 pg/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 pg/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12,20/21,23,27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.Factor VIII is an important plasma protein which acts as a cofactor in the intrinsic pathway of coagulation. It does so by dramatically increasing the V,,, for the activation of factor X by the enzyme factor IXa in the presence of calcium and a phospholipid surface [I]. The critical role of factor VIII in hemostasis is illustrated by the severe bleeding tendency (known as hemophilia A) in its absence [2]. It is a key protein in the regulation of the coagulation cascade, since its cofactor activity can be enhanced through feed-back mechanisms by thrombin and factor Xa. Factor VIII activity is subsequently destroyed by the regulatory-enzyme-activated protein C. Both activation and inactivation of factor VIII occur through specific proteolytic cleavages within the molecule [3 -51.Factor VIII is synthesized as a single-chain precursor molecule of 2332 amino acids [6, 71 and purified from plasma as a carboxy-terminal light chain of 80 kDa which is associated with an amino-terminal heavy chain ranging in molecular mass between 90 -210 kDa [4, 8 -111. In many studies, the kinetics of factor-VIII and factor-X activation [I, 12-181, as well as the changes that occur within the factor VIII molecule upon activation by thrombin or factor Xa [3,4, 17, 191, have been described. These studies have ignored, however, that factor VIII circulates as a non-covalent complex with von Willebrand factor [20]. Formation of this complex is of great physiological importance for factor VIII, since it prolongs its half-life in the circulation [21,22]. Since von Willebrand factor is a very large glycoprotein which represents 99% of the mass of the factor-VIII -von-Willebrand-factor complex, it is conceivable that it modulates the activation and inactivation of factor VIII.In a previous paper ...

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Detection of the Carrier State in Hereditary Coagulation Disorders I

Cited by 96 publications

References 0 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.

Survival of 125iodine-labeled Factor VIII in normals and patients with classic hemophilia. Observations on the heterogeneity of human Factor VIII.

The effect of von Willebrand factor on activation of factor VIII by factor Xa

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