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DOI: 10.1055/s-0038-1646433
A Method for Measuring Activated Factor VIII in Plasma
Publikationsverlauf
Received 08. August 1990
Accepted 21. März 1991
Publikationsdatum:
25. Juli 2018 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/199104/lookinside/thumbnails/10-1055-s-0038-1646433-1.jpg)
Summary
A method is described which enables a quantitative measurement of the concentration of activated factor VIII (VIIIa) in plasma. Based on the ability of factor VIIIa to accelerate the activation of factor X by factor IXa, phospholipid and calcium ions, the course of factor X activation in time is measured using a chromogenic substrate. Free factor Xa is able to activate non-activated factor VIII present in a plasma sample, which increases the factor X activation velocity, and thus disturbs the measurement of factor VIIIa. Furthermore, factor Xa was found to be inactivated by serine protease inhibitors from the plasma sample. By adding surplus chromogenic substrate these reactions of factor Xa are inhibited and at the same time the rate of substrate conversion is a measure of the amount of factor Xa present. Factor X activation and amidolysis of chromogenic substrate then take place simultaneously. It is shown that under proper conditions the factor X activation velocity is linearly proportional to the factor VIIIa concentration. This causes the optical density to increase as a parabolic function of time. The concentration of factor VIIIa can be obtained from the quadratic coefficient of the equation describing the parabola. The method is specific for factor VIIIa in that the extrinsic factor X activator is shown to have no influence on the measurement of factor VIIIa in thromboplastin activated plasma. We conclude that a sensitive and reliable method for assessing factor VIIIa concentrations in plasma has been developed on the basis of simultaneous inhibition and measurement of factor Xa by a high concentration of chromogenic substrate.
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References
- 1 Hurlet-Birk Jensen A, Beguin S, Josso F. Factor V and VIII activation "in vivo" during bleeding. Evidence of thrombin formation at the early stage of hemostasis. Pathol Biol 1976; 24: 6-10
- 2 Colman RW. The effect of proteolytic enzymes on bovine factor V. Kinetics of activation and inactivation by bovine thrombin. Biochemistry 1969; 4: 1438-44
- 3 Lindhout MJ, Jackson CM. Activation of bovine factor V by thrombin and a protease from Russell's viper venom (RVV). Thromb Haemostas 1979; 42: 491 (Abstr)
- 4 Pieters J, Hemker HC, Lindhout T. In Situ Generated Thrombin is the only enzyme that effectively activates factor VIII and factor V in plasma. Blood 1989; 74: 1021-4
- 5 Rapaport SI, Hjort PF, Patch MJ. Further evidence that thrombin activation of factor VIII is an essential step in intrinsic clotting. Scand J Clin Lab Invest Suppl 1965; 17: 84-8
- 6 Biggs R, Macfarlane RG, Denson WE, Ash BJ. Thrombin and the interaction of factors VIII and IX. Br J Haematol 1965; 11: 276-95
- 7 Hemker HC, Kahn MJP. Reaction sequence of blood coagulation. Nature 1967; 215: 1201
- 8 Osterud B, Rapaport SI, Schiffman S, Chong MMY. Formation of intrinsic factor X activator with special reference to the role of thrombin. Br J Haematol 1971; 21: 643-60
- 9 Hultin MB, Nemerson Y. Activation of factor X by factors IXa and VIII; a specific assay for factor IXa in the presence of thrombin-activated factor VIII. Blood 1978; 52: 928-40
- 10 Davey MG, Luscher EF. Actions of thrombin and other proteolytic enzymes on blood platelets. Nature 1967; 216: 857-8
- 11 Bevers EM, Comfurius P, van Rijn JLML, Hemker HC, Zwaal RFA. Generation of prothrombin converting activity and the exposure of phosphatidylserine at the outer surface of platelets. Eur J Biochem 1982; 122: 429-36
- 12 Soulier P, Larrieu MJ. Déficit en 3ème facteur prothromboplastique plasmatique. Rapports entre le PTA et le facteur Hageman. Thromb Diathes Haemorrh 1958; 2: 1-23
- 13 Rapaport SI, Schiffman S, Patch MJ, Ware AG. A simple, specific one-stage assay for plasma thromboplastin antecedent activity. J Lab Clin Med 1961; 57: 771-80
- 14 Hardisty RM, Macpherson JC. A one-stage factor VIII (antihemophilic globulin) assay and its use on venous and capillary plasma. Thromb Diathes Haemorrh 1962; 7: 215-29
- 15 Veltkamp JJ, Drion EF, Loeliger EA. Detection of the carrier state in hereditary coagulation disorders-I, II. Thromb Diathes Haemorrh 1968; 19 279-303 and 403-22
- 16 Suomela H, Blombôck B. The activation of factor X evaluated by using synthetic substrates. Thromb Res 1977; 1: 267-81
- 17 Van Dieijen G, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor Villa in the activation of bovine factor X. J Biol Chem 1981; 256: 3433-42
- 18 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-10
- 19 Hultin MB. Role of human factor VIII in factor X activation. J Clin Invest 1982; 69: 950-5
- 20 Lollar P, Knutson GJ, Fass DN. Activation of porcine factor VIII by thrombin and factor Xa. Biochemistry 1985; 24: 8056-64
- 21 Neuenschwander P, Jesty J. A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X. Blood 1988; 72: 1761-70
- 22 Jesty J, Spencer AK, Nemerson Y. The mechanism of activation of factor X. J Biol Chem 1974; 249: 5614-22
- 23 Wagenvoord R, Hendrix H, Hemker HC. Development of a simple chromogenic factor VIII-assay for clinical use. Haemostasis 1989; 19: 196-204
- 24 Fujikawa K, Legaz ME, Davie EW. Bovine factor X1 and X2 (Stuart factor). Isolation and characterization. Biochemistry 1972; 11: 4882-91
- 25 Fujikawa K, Legaz ME, Davie EW. Bovine factor X1 (Stuart factor). Mechanism of activation by a protein from Russel's viper venom. Biochemistry 1972; 11: 4892-9
- 26 Wagenvoord R, Hendrix H, Soria C, Hemker HC. Localization of the inhibitory site(s) of pentosan polysulphate in blood coagulation. Thromb Haemostas 1988; 60: 220-5
- 27 Van Dieijen G, Van Rijn J, Govers-Riemslag J, Hemker HC, Rosing J. Assembly of the intrinsic factor X activating complex – Interactions between factor IXa, factor VIIIa and phospholipid. Thromb Haemostas 1985; 53 (3) 396-400
- 28 Rosing J, Tans G, Govers-Riemslag JWP, Zwaal RFA, Hemker HC. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-83
- 29 Hemker HC, Willems GM, Béguin S. A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb Haemostas 1986; 56: 9-17
- 30 Owren PA, Aas K. The control of dicumarol therapy and the quantitative determination of prothrombin and proconvertin. Scand J Clin Lab Invest 1951; 3: 201-18
- 31 Lollar P, Parker CG. pH-dependent denaturation of thrombin-activated porcine factor VIII. J Biol Chem 1990; 265: 1688-92
- 32 Lollar P, Knutson GJ, Fass DN. Stabilization of thrombin-activated porcine factor VIII by factor IXa and phospholipid. Blood 1984; 63: 1303-8
- 33 Kane WH, Davie EW. Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 1988; 71: 539-55
- 34 Hoyer LW. The factor VIII complex. Structure and function. Blood 1981; 58: 1-13