The Inactivation of Factor VIII in Vitro

Summary

When normal citrated plasma is stored at 37° C and pH 7.8 the factor VIII activity drops to about 50% of its initial value during the first 8-12 h. In the following 4 days practically no further drop in activity is found. If the logarithm of the factor VIII activity is plotted against time a curve is obtained which can be described as biphasic. To explore the underlying mechanism of this phenomenon the influence of temperature, pH, Ca⁺⁺ concentration and some other clotting factors was investigated. Between temperatures of 21° C and 45° C the inactivation of factor VIII was biphasic, the decrease of factor VIII being faster in both phases at higher temperatures. The inactivation at these temperatures showed a Q₁₀ of about 2. At 52° C nearly all factor VIII activity disappeared within 8 h. Possibly the precipitation of fibrinogen at this temperature is of influence. Between pH 6.4 and 8.5 the decrease in factor VIII in the first phase was obviously slower at lower pH and the level of the second phase maintained at a higher factor VIII activity. No alteration of the normal inactivation pattern was seen in plasma from patients with congenital deficiencies of factors XII, IX or V or in normal plasma adsorbed with BaS0₄ which has factors II, VII, IX and X markedly decreased, nor was there any difference between platelet rich and platelet poor plasma. Low calcium concentrations (Resinplasma) markedly increased the rate of inactivation in the first phase, but did not influence the second phase.

Four hypotheses are given to explain the biphasic inactivation of factor VIII: a) The presence of an inactivating substance in the first phase or a stabilizing factor in the second phase of factor VIII inactivation. b) The existence of two independent substances with factor VIII activity with different inactivation rates. c) Reversible denaturation of factor VIII in one or more steps. d) Factor VIII exists in plasma in two interdependent molecular forms. It is discussed that in view of the results of the experiments hypotheses a and b are not very likely. At present we cannot differentiate experimentally between c and d.

• References

• 1 Barrow E. M, Graham J. B. Von Willebrand’s Disease. Progr. Hemat 04: 203 1964;
• 2 Blombäck M. Purification of anti-hemophilic globulin. Archiv. Kemi 12: 387 1958;
• 3 Bounameaux J. Dosage des facteurs de coagulation continus dans l’atmosphère plasmatique des plaquettes humaines. Rev. frang. Étud. clin. biol 02: 32 1957;
• 4 Bowie E. J. W, Thompson J. H, Ch A.Owen. The stability of anti-hemophilic globulin and labile factor in human blood. Mayo Clin. Proc 39: 145 1964;
• 5 Ellis B. G, Stransky A. A quick and accurate method for the determination of fibrinogen in plasma. J. Lab. clin. Med 58: 477 1961;
• 6 Fantl P, Marr A. G. The preservation of coagulation factors in human plasma. Aust. J. exp. Biol. med. Sei 34: 433 1956;
• 7 Feissley R, Lüdin H. Action of cocain on blood platelets. Helv. physiol, pharmacol. Acta 07: 69 1949;
• 8 Joly M. A physico-chemical approach to the denaturation of proteins. Academic Press; London and New York: 1965
• 9 Kekwick R. A, Walton P. L. Studies on the purification and stability of human anti-haemo- philic factor (factor VIII). Brit. J. Haemat 11: 537 1965;
• 10 Kettenborg H. K, de Vries S. I, van der Pol E. T. Modification in the behaviour of coagulation factors during storage of blood and its importance for blood transfusion. Rev. beige Path 24: 136 1955;
• 11 Mammen E. F. Investigations on the nature of haemophilia-A. Thrombos. Diathes. haemorr. (Stuttg) 09: 30 1963;
• 12 den Ottolander G. JH, Bleyenberg A. Le dosage du facteur VIII. Hémostase 01: 281 1961;
• 13 Penick G. D, Brinkhous K. M. Relative stability of plasma Antihemophilic factor (AHF) under different conditions of storage. Amer. J. med. Sei 232: 434 1956;
• 14 Quick A. J. Haemorrhagic diseases. Philadelphia; Pa: 1957
• 15 Rapaport S. J, Schiffman I, Patch M. J, Ames S. B. The importance of activation of antihemophilic globulin and pro-accelerin by traces of thrombin in the generation of intrinsic prothrombinase activity. Blood 21: 221 1963;
• 16 Rapaport S. I, Schiffman S, Ames S. B. Activation of antihemophilic globulin by traces of thrombin: An essential step in intrinsic prothrombinase generation. In: Brinkhous K. M. The haemophilias. 49 Chapel Hill, U.S. A; 1964
• 17 Rizza G. R, Walker W. Inactivation of AHG by thrombin. Nature (Lond) 180: 143 1957;
• 18 Shinowara G. Y. Kinetic studies on the thermal denaturation of Human anti-haemophilic globulin (AHG). Thrombos. Diathes. haemorrh. (Stuttg) 15: 636. 1966;
• 19 Stibbe J. Experiments on the spontaneous inactivation of blood clotting factor VIII in vitro. Short Communication. Fourth meeting FEBS; Oslo: 1967
• 20 Stibbe J. De inactivatie van factor VIII in vitro. Thesis, Amsterdam; 1967
• 21 Straub F. B. Formation of the secondary and tertiary structure of enzymes. Advanc. Enzymol 26: 89 1964;
• 22 Surgenor D. M, Wilson H. enry. Factor V from human plasma. Thrombos. Diathes. haemorrh 05: 1 1960;
• 23 Thelin G. M, Wagner R. H. Sedimentation of plasma antihemophilic factor. Arch. Bioch 95: 70 1961;
• 24 Waaler B. A. A simple one-stage method for the assay of anti-haemophilic-A factor. Scand. J. clin. Lab. Invest 11: 194 1959;
• 25 Wagner R. H, Thelin G. M. Haemophilia and Haemophiloid Diseases. International Symposium, New York. 3 Ed. Brinkhous, K.M. University of North Carolina Press; Chapel Hill, NC: 1957
• 26 Weiss H. J. A study of the cation-and pH dependent stability of factors V and VIII in plasma. Thrombos. Diathes. haemorrh. (Stuttg) 14: 32 1965;
• 27 Weiss H. J, Kochwa J. Molecular forms of antihemophilic globulin in plasma, cryoprecipitate and after thrombin activation. Brit. J. Haemat 18: 89 1970;
• 28 Wolf P. Studies of temperature and pH stability of human AHF in plasma and concentrate. Brit. J. Haemat 05: 169 1959;