European Journal of
Eur. J. Pediatr. 128, 283--289 (1978)
Pediatrics 9 by Springer-Verlag 1978
Factor VIII Activity and Factor VIII Related Antigen in Newborns* B. Maak**, B. Scheidt, and J. Frenzel Department of Neonatology (Head: Prof. Dr. Frenzel), Childrens Hospital (Director: OMR Prof. Dr. Plenert) of the University of Jena, DDR-6900 Jena, German Democratic Republic
Abstract. Factor VIII procoagulant activity and factor VIII related antigen were examined in 20 full-term and preterm newborn infants during the first days of life. The control group involved 15 adults volunteers. Factor VIII activity was estimated by a one-stage test and factor VIII related antigen was determined by immunelectrophoresis according to Laurell, using our own rabbit antiserum. The following results were obtained: - - Factor VIII activity during the first 3 days of life did not differ from the normal range of the adult controls. - - The concentration of factor VIII related antigen in newborns was markedly higher than in adults on the first, and to a lesser extent on the second, day of life. - - The antigen concentration decreases on the second and following days of life to adult levels. The cause of this discrepancy cannot be completely explained but possible reasons are discussed.
Key words: Factor VIII procoagulant activity - Factor VIII related antigen Newborns.
Zusammenfassung. Faktor VIII-Gerinnungsaktivitfit und Faktor VIII-assoziiertes Antigen wurden bei 20 reifen und unreifen Neugeborenen wfihrend der ersten Lebenstage bestimmt. Die Kontrollgruppe bestand aus 15 Erwachsenen. Die Faktor VIII-AktivitM wurde mit einem Einstufentest bestimmt. Das Faktor VIII-assoziierte Antigen wurde mit der Immunelektrophorese nach Laurell unter Verwendung eines selbst hergestellten Antiserums bestimmt. Folgende Ergebnisse wurden erhalten: - - Die Faktor VIII-Aktivit~it wfihrend der ersten 3 Lebenstage unterschied sich nicht signifikant yore Normalbereich, der durch die Untersuchungen an Erwachsenen festgelegt wurde. * In honour of Prof. Dr. Ingeborg Rapoport on the occasion of her 65th birthday ** Address for offprint requests: Univ.-Kinderklinik, Kochstr. 2, DDR-6900 Jena/Thtir.
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B. Maak et al. - - Die K o n z e n t r a t i o n des F a k t o r V I I I - a s s o z i i e r t e n A n t i g e n s ist bei den N e u g e b o r e n e n , i n s b e s o n d e r e a m 1. u n d in g e r i n g e r e m A u s m a g auch a m 2. Leb e n s t a g , erh6ht. - - Die K o n z e n t r a t i o n des F a k t o r V I I I - a s s o z i i e r t e n A n t i g e n s verringert sich im Verlaufe der ersten 3 L e b e n s t a g e bis a u f die E r w a c h s e n e n w e r t e . Die U r s a c h e n dieser D i s k r e p a n z k 6 n n e n gegenw[irtig nicht vollstfindig erklfirt werden. I n der D i s k u s s i o n wird a u f m6gliche G r i i n d e eingegangen.
Introduction T h e b e h a v i o u r o f f a c t o r V I I I activity in m a t u r e a n d p r e m a t u r e n e w b o r n s has been dealt with in a n u m b e r o f p u b l i c a t i o n s (Kiinzer a n d S t r 6 d e r , 1957; R o g n e r , 1960) a n d reviewed b y Bleyer et al. (1971). In the m a j o r i t y o f m a t u r e n e w b o r n s , the e s t i m a t e d values do n o t deviate significantly f r o m a d u l t levels. P r e m a t u r e n e w b o r n s exhibit distinctly lower values. O n the o t h e r h a n d , v i t a m i n K d e p e n d e n t factors show significantly decreased activities in b o t h f u l l - t e r m a n d p r e t e r m infants. F o r f a c t o r V I I , it has been shown t h a t n o t only is the p r o c o a g u l a n t activity decreased, b u t the a n t i b o d y neutralizing m a t e r i a l is also r e d u c e d ( M a a k a n d Frenzel, 1977). The p u r p o s e o f this s t u d y was to e v a l u a t e w h e t h e r a similar p a t t e r n exists for f a c t o r V I I I .
Material and Methods 1. Plasma from 20 newborns was tested on 28 occasions in relation to monitoring of coagulation. Blood was obtained by scalp vein puncture, using a siliconized No. 1 needle. The blood was collected in 3.13% sodium citrate solution in a siliconized glass tube (1 part citrate solution and 9 parts of venous blood). 11 samples were taken on the 1st day of life, 8 on the 2nd day, and the others on the 3rd or following days. Six of the infants were of low birth weight (below 2500 g). Four newborns were delivered by caesarean section, 1 by forceps and 1 by vacuum extraction. In all the cases the clinical condition was good, without any signs of delayed or disturbed adaptation to extrauterine life. The control group consists of 15 healthy adult volunteers (6 male, and 9 female). 2. The procoagulant activity of factor VIII was estimated by a one-stage test, using an artificial factor VIII deficient plasma (Perlick and Bergmann, 1971). Factor V in the artificial substrate plasma was replaced by 10% bovine serum. Estimations were performed within 1 hour of blood sampling. 3. Factor VIII related antigen was determined by immunelectrophoresis according to Laurell (1966), using an antiserum produced in our laboratory. Factor VIII antigen was prepared by cryoprecipitation and successive chromatography on Sepharose 4B R and injected repeatedly into rabbits. Details of the procedure have been published in a previous paper (Maak et al., 1977). 4. Means and confidence limits for a 5% probability were calculated. The relationship between factor VIII activity and factor VIII related antigen was analyzed by calculation of the regression line and correlation coefficient.
Results T h e results are given in F i g u r e s 1 - - 3 . F i g u r e 1 shows the f a c t o r V I I I activity a n d f a c t o r V I I I r e l a t e d antigen values for the c o n t r o l g r o u p (points) a n d the n e w b o r n s (circles). T h e regression line for
Factor VIII Activity and Factor VIII Related Antigen
285
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,
,
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,
.
.
.
.
i
200
300
%Fector Viii-related Antigen
Fig. 1, Relationship of factor VIII clotting activity (abscissa) to factor VIII related antigen (ordinate). The solid line is the regression line for adults. Individual values for adults are shown as points whereas those for newborns are shown as circles
%
200
t
1.0
I00
T
1.
2. 3, Day of life
>3.
Adults
1.
2. ,t., >3. D a y of l i f e
Adults
Fig. 2. Mean values and 5% confidence limits for factor VIII clotting activity (circles) and factor VIII related antigen (points). The ordinate represents the clotting activity and antigen concentration respectively Fig. 3. Ratio between factor VIII clotting activity and factor VIII related antigen, Mean values (points) and 5% confidence limits are presented
286
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the adult levels is also depicted. The equation for the line is: y = 0.6094 x + 35.7. The coefficient of correlation between factor VIII procoagulant activity and factor VIII related antigen was 0.7332, significant at less than the 1% level. The mean value for the factor VIII activity of the adult controls was 85.9 + 11.7%, and the mean for factor VIII related antigen 82.2 + 14%. The average ratio between factor VIII procoagulant activity and factor VIII related antigen was 1.09 + 0.12. The values for some of the newborns are in the normal adult range. However, the majority of the newborns investigated exhibit a different pattern. Almost normal procoagulant activity contrasts with markedly increased antigen concentration. Figure 2 shows the mean values for the newborn group on different days of life. A clear age-dependency can be seen with respect to the concentration of factor VIII related antigen (points). On the first day, and to lesser extent on the second day of life, the antigen concentration was markedly higher than on the following days. The mean value for the first day was 182%, nearly twice the mean for adults. By the second day of life, a decrease has taken place and this continues on the following days. The mean for the first day differs significantly from that for the third day, and from the mean values for the control group. On the other hand, factor VIII activity (circles) shows no significant differences from the normal adult range. The slight differences between the different days of life in the newborn group are without statistical significance. The ratio between factor VIII activity and factor VIII related antigen is given in Figure 3. Because of the decrease of antigen concentration, this ratio increases during the first days of life.
Discussion
In contrast to factor VII, which shows a close correlation between procoagulant activity and antibody neutralizing material (Maak and Frenzel, 1977) a similar relationship cannot be found for factor VIII during the first days of life. On the contrary, the relation between both parameters is characterized on the one hand by an almost normal procoagulant activity and on the other by a markedly increased antigen concentration, especially on the first day of life. On the second day the concentration has already decreased to near the adult level, indicating relatively rapid elimination of antigen. This pattern is difficult to explain. First, the methods used in our investigation have to be considered critically with respect to their possible effects on the results. It is well known that the value for factor VIII activity depends on the test which is chosen. For example, investigations on adult patients with liver disease have shown that factor VIII activity measured by different tests differs markedly in the same sample. In particular, estimation by one-stage methods results in lower activity than that obtained with the thromboplastin generation test (TGT). It should be noted that only the values determined by T G T correlated well with the concentration of factor VIII related antigen (Rogers and Eyster, 1976). It is assumed by the authors that the action of a serum factor which is supplied with the normal serum reagent in the TGT-mixture converts precursor material, and
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thus results in higher activity than in the one-stage tests. Further it is also known that the one-stage test may yield higher values than the T G T in certain conditions. This phenomenon takes place when active intermediate products of coagulation exist. These products do not influence the T G T method because they are removed by the necessary absorption with barium sulfate or aluminium hydroxide, whereas incubation of streptokinase with normal plasma results in reduced one-stage and two-stage factor VIII activities (Niemetz and Nossel, 1960). However, no change could be seen in the concentration of factor VIII related antigen before and after treatment of plasma with streptokinase, or serum with plasmin (Castillo et al., 1976). Simultaneous determinations using both methods were not performed in our study. However, some authors have published normal values for factor VIII activity, obtained by T G T in the newborn period. Comparison with their results shows that the activity was generally lower than in our study. When these divergent facts are taken into consideration, it seems unlikely that the factor VIII activity in our study was low as a result of the one-stage test which was chosen. Any influence of spontaneous fibrinolysis appears unlikely because of the short interval between blood sampling and estimation of factor VIII procoagulant activity. The high concentration of factor VIII related antigen is difficult to explain and this cannot be resolved by the methods chosen. However, one may speculate that the high antigen concentrations result from tissue stores. Tuddenham et al. (1974) found factor VIII related antigen was already present in fetal serum between the 8th and 20th weeks of gestation. Moreover, factor VIII related antigen could be demonstrated in the fetal vascular intima and in splenic cells. Hoyer et al. (1973) found antigen only in the intima. Thus, it seems likely that the intima of blood vessels stores--and possibly synthesizes--factor VIII related antigen. Bennett and Ratnoff (1972) have shown that stress increases factor VIII procoagulant activity, as well as antigen concentration. In this way, the high concentration of factor VIII related antigen in the newborn might be caused by perinatal stress. It is so far not clear whether the increase as a result of stress is caused by increased synthesis or by liberation from stores. A materno-fetal transfer across the placenta seems unlikely because of the high molecular weight of the antigen (Review by Vermylen, 1975). However, another explanation seems possible. Investigations have shown that during the normal coagulation process, factor VIII procoagulant activity is consumed whereas antigen concentration remains unchanged and can still be found in serum. Thus, the discrepancy between factor VIII procoagulant activity and factor VIII related antigen on the first and second days of-life may reflect a coagulation phenomenon. A similar relationship between factor VIII procoagulant activity and factor VIII related antigen was reported by Bennett et al. (1974). These authors investigated patients with diseases complicated by disseminated intravascular coagulation (DIC) and women immediately after labour. There is also some evidence in newborns that DIC takes place during delivery but in most cases does not lead to clinical symptoms. Such evidence arose from monitoring platelet counts during the postnatal period (Maak et al., 1972). The way in which this process might be caused could be by the liberation of tissue thromboplastin, which has been found in high concentrations in the vascular intima (Astrup, 1965). Cord blood leucocytes also contain considerable amounts
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o f t h r o m b o p l a s t i n which is liberated by acidosis or bacterial endotoxins (Rivers and H a t h a w a y , 1975). Several authors have suggested that tissue thromboplastin takes part in the pathogenesis o f perinatal bleeding and most o f the t h r o m b o plastin is liberated f r o m the placenta (Bonnar et al., 1971; Nielsen, 1970). Against the b a c k g r o u n d o f these experimental observations, one can speculate that the low factor V I I I p r o c o a g u l a n t activity reflects c o n s u m p t i o n which is initiated by liberation o f tissue t h r o m b o p l a s t i n into the fetal circulation during delivery. As a consequence, the greatest difference between p r o c o a g u l a n t activity and antigen concentration is observed immediately after birth. The antigen excess is then eliminated by a slower process lasting until the third day of life. O u r results were obtained f r o m a small n u m b e r o f newborns. The newborns were i n h o m o g e n o u s in respect to birth weight and mode of delivery. Further studies are needed to prove the above speculations, and to reveal the possible influences o f different perinatal conditions.
Addendum After completion of this paper we found the work of Muntean, W., B. H. Belohradsky, H. J. Klose, K. Riegel: "Faktor-VIII-Aktivit~it und Faktor-VIII-assoziiertes Antigen bei Neugeborenen" which appeared in "Klin. P~idiat. 189, 412--416 (1977)". Their results agree partly with our findings.
References Astrup, T.: Assay and content of tissue thromboplastin in different organs. Thromb. Diath. haemorrh. 14, 401--406 (1965) Bennett, B., Ratnoff, O. D.: Antihemophilic factor (AHF, factor VIII) procoagulant activity and AHF-like antigen in normal pregnancy, and following exercise and pneumencephalography. J. Lab. clin. Med. 80, 256--263 (1972) Bennett, B., Oxnard, S. C., Douglas, A. S., Ratnoff, O. D.: Studies on antihemophilic factor (AHF, factor VIII) during labor in normal women, in patients with premature separation of the placenta, and in a patient with yon Willebrand's disease. J. lab. clin. Med. 84, 851--860 (1974) Bleyer, W. A., Hakami, N., Shepard, Th. H.: The development of hemostasis in the human fetus and newborn infant. J. Pediat. 79, 838--853 (1971) Bonnar, J., McNicol, G. P., Douglas, A. S.: The blood coagulation and fibrinolytic systems in the newborn and the mother at birth. J. Obstet. Gynaecol. Brit. Commonw. 78,355--360 (1971) Castillo, R., Maragall, S., Guisasola, J. A., Casals, F., Profilos, J., Ordinas, A.: Inhibition of human platelet aggregation by the proteolytic effect of streptokinase. Role of the human factor VIII - - related protein. Haemostasis 5, 306--317 (1976) Hoyer, L. W., de los Santos, R. P., Hoyer, J. R.: Antihemophilic factor antigen. Localization in endothelial cells by immunofluorescent microscopy. J. clin. Invest. 52, 2737--2744 (1973) Ktinzer, W., Str6der, J.: Gerinnungsstudien bei Kindern. I. Mitteilung: Untersuchungen zur Thromboplastinbildung im Nabelschnurblut. Ann. paediat. 188, 147--152 (1957) Laurell, C. B.: Quantitative estimation of proteins by electrophoresis in agarose gels containing antibodies. Anal. Biochem. 16, 45--52 (1966) Maak, B., Rogner, G., Frenzel, J.: Untersuchungen der Thrombozytenadh~isivitM und der Thrombozytenzahl beim reifen und unreifen Neugeborenen. Z. Kinderheilk. 111, 193--204 (1972) Maak, B., Frenzel, J.: Antibody neutralizing material of factor VII during the first weeks of life. Europ. J. Pediat. 125,255--258 (1977)
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Maak, B., Scheidt, B., Frenzel, J.: Erfahrungen bei der Herstellung eines Antiserums gegen das Faktor VIII-assoziierte Antigen. Dtsch. Ges.-Wesen 32, 1335--1338 (1977) Nielsen, N. Chr.: Coagulation and fibrinolysis in mothers and their newborn infants following premature separation of the placenta. Acta Obstet. Gynecol. Scand. 49, 77--83 (1970) Niemetz, J., Nossel, H. L.: Activated coagulation factors: In-vivo and in-vitro studies. Brit. J. Haemat. 16, 337--351 (1969) Perlick, E., Bergmann, A.: Gerinnungslaboratorium in Klinik und Praxis. Leipzig: VEB Georg Thieme 1971 Rivers, R. P. A., Hathaway, W. E.: Studies on tissue factor activity and production by leucocytes of human umbilical cord and adult origin. Pediat. Res. 9, 167--171 (1975) Rogers, J. S., Eysters, M. E.: Relationship of factor VIII-like antigen (VIII AGN) and clot promoting activity (VIII AHF) as measured by one - - and two - - stage assays in patients with liver disease. Brit. J. Haemat. 34, 655--661 (1976) Rogner, G.: Die Blutgerinnung bei Frtihgeburten. Z. Kinderheilk. 84, 197--212 (1960) Tuddenham, E. G. D., Shearn, S. A. M., Peake, I. R., Giddings, J. C., Bloom, A.L.: Tissue localization and synthesis of factor VIII-related antigen in the human fetus. Brit. J. Haemat. 26, 669--677 (1974) Vermylen, J.: Physical and chemical properties of normal and haemophilic factor VIII. Path. Biol. 23, Suppl. 5--10 (1975)
Received November 21, 1977
Eur. J. Pediatr. 128, 283--289 (1978)
Pediatrics 9 by Springer-Verlag 1978
Factor VIII Activity and Factor VIII Related Antigen in Newborns* B. Maak**, B. Scheidt, and J. Frenzel Department of Neonatology (Head: Prof. Dr. Frenzel), Childrens Hospital (Director: OMR Prof. Dr. Plenert) of the University of Jena, DDR-6900 Jena, German Democratic Republic
Abstract. Factor VIII procoagulant activity and factor VIII related antigen were examined in 20 full-term and preterm newborn infants during the first days of life. The control group involved 15 adults volunteers. Factor VIII activity was estimated by a one-stage test and factor VIII related antigen was determined by immunelectrophoresis according to Laurell, using our own rabbit antiserum. The following results were obtained: - - Factor VIII activity during the first 3 days of life did not differ from the normal range of the adult controls. - - The concentration of factor VIII related antigen in newborns was markedly higher than in adults on the first, and to a lesser extent on the second, day of life. - - The antigen concentration decreases on the second and following days of life to adult levels. The cause of this discrepancy cannot be completely explained but possible reasons are discussed.
Key words: Factor VIII procoagulant activity - Factor VIII related antigen Newborns.
Zusammenfassung. Faktor VIII-Gerinnungsaktivitfit und Faktor VIII-assoziiertes Antigen wurden bei 20 reifen und unreifen Neugeborenen wfihrend der ersten Lebenstage bestimmt. Die Kontrollgruppe bestand aus 15 Erwachsenen. Die Faktor VIII-AktivitM wurde mit einem Einstufentest bestimmt. Das Faktor VIII-assoziierte Antigen wurde mit der Immunelektrophorese nach Laurell unter Verwendung eines selbst hergestellten Antiserums bestimmt. Folgende Ergebnisse wurden erhalten: - - Die Faktor VIII-Aktivit~it wfihrend der ersten 3 Lebenstage unterschied sich nicht signifikant yore Normalbereich, der durch die Untersuchungen an Erwachsenen festgelegt wurde. * In honour of Prof. Dr. Ingeborg Rapoport on the occasion of her 65th birthday ** Address for offprint requests: Univ.-Kinderklinik, Kochstr. 2, DDR-6900 Jena/Thtir.
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B. Maak et al. - - Die K o n z e n t r a t i o n des F a k t o r V I I I - a s s o z i i e r t e n A n t i g e n s ist bei den N e u g e b o r e n e n , i n s b e s o n d e r e a m 1. u n d in g e r i n g e r e m A u s m a g auch a m 2. Leb e n s t a g , erh6ht. - - Die K o n z e n t r a t i o n des F a k t o r V I I I - a s s o z i i e r t e n A n t i g e n s verringert sich im Verlaufe der ersten 3 L e b e n s t a g e bis a u f die E r w a c h s e n e n w e r t e . Die U r s a c h e n dieser D i s k r e p a n z k 6 n n e n gegenw[irtig nicht vollstfindig erklfirt werden. I n der D i s k u s s i o n wird a u f m6gliche G r i i n d e eingegangen.
Introduction T h e b e h a v i o u r o f f a c t o r V I I I activity in m a t u r e a n d p r e m a t u r e n e w b o r n s has been dealt with in a n u m b e r o f p u b l i c a t i o n s (Kiinzer a n d S t r 6 d e r , 1957; R o g n e r , 1960) a n d reviewed b y Bleyer et al. (1971). In the m a j o r i t y o f m a t u r e n e w b o r n s , the e s t i m a t e d values do n o t deviate significantly f r o m a d u l t levels. P r e m a t u r e n e w b o r n s exhibit distinctly lower values. O n the o t h e r h a n d , v i t a m i n K d e p e n d e n t factors show significantly decreased activities in b o t h f u l l - t e r m a n d p r e t e r m infants. F o r f a c t o r V I I , it has been shown t h a t n o t only is the p r o c o a g u l a n t activity decreased, b u t the a n t i b o d y neutralizing m a t e r i a l is also r e d u c e d ( M a a k a n d Frenzel, 1977). The p u r p o s e o f this s t u d y was to e v a l u a t e w h e t h e r a similar p a t t e r n exists for f a c t o r V I I I .
Material and Methods 1. Plasma from 20 newborns was tested on 28 occasions in relation to monitoring of coagulation. Blood was obtained by scalp vein puncture, using a siliconized No. 1 needle. The blood was collected in 3.13% sodium citrate solution in a siliconized glass tube (1 part citrate solution and 9 parts of venous blood). 11 samples were taken on the 1st day of life, 8 on the 2nd day, and the others on the 3rd or following days. Six of the infants were of low birth weight (below 2500 g). Four newborns were delivered by caesarean section, 1 by forceps and 1 by vacuum extraction. In all the cases the clinical condition was good, without any signs of delayed or disturbed adaptation to extrauterine life. The control group consists of 15 healthy adult volunteers (6 male, and 9 female). 2. The procoagulant activity of factor VIII was estimated by a one-stage test, using an artificial factor VIII deficient plasma (Perlick and Bergmann, 1971). Factor V in the artificial substrate plasma was replaced by 10% bovine serum. Estimations were performed within 1 hour of blood sampling. 3. Factor VIII related antigen was determined by immunelectrophoresis according to Laurell (1966), using an antiserum produced in our laboratory. Factor VIII antigen was prepared by cryoprecipitation and successive chromatography on Sepharose 4B R and injected repeatedly into rabbits. Details of the procedure have been published in a previous paper (Maak et al., 1977). 4. Means and confidence limits for a 5% probability were calculated. The relationship between factor VIII activity and factor VIII related antigen was analyzed by calculation of the regression line and correlation coefficient.
Results T h e results are given in F i g u r e s 1 - - 3 . F i g u r e 1 shows the f a c t o r V I I I activity a n d f a c t o r V I I I r e l a t e d antigen values for the c o n t r o l g r o u p (points) a n d the n e w b o r n s (circles). T h e regression line for
Factor VIII Activity and Factor VIII Related Antigen
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%Fat#or VIii- A c t i v i t y 200"
o
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9
i e
o
o
o
o o
oo~
O~o
r
o
,
,
lO0
,
o
,
.
.
.
.
i
200
300
%Fector Viii-related Antigen
Fig. 1, Relationship of factor VIII clotting activity (abscissa) to factor VIII related antigen (ordinate). The solid line is the regression line for adults. Individual values for adults are shown as points whereas those for newborns are shown as circles
%
200
t
1.0
I00
T
1.
2. 3, Day of life
>3.
Adults
1.
2. ,t., >3. D a y of l i f e
Adults
Fig. 2. Mean values and 5% confidence limits for factor VIII clotting activity (circles) and factor VIII related antigen (points). The ordinate represents the clotting activity and antigen concentration respectively Fig. 3. Ratio between factor VIII clotting activity and factor VIII related antigen, Mean values (points) and 5% confidence limits are presented
286
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the adult levels is also depicted. The equation for the line is: y = 0.6094 x + 35.7. The coefficient of correlation between factor VIII procoagulant activity and factor VIII related antigen was 0.7332, significant at less than the 1% level. The mean value for the factor VIII activity of the adult controls was 85.9 + 11.7%, and the mean for factor VIII related antigen 82.2 + 14%. The average ratio between factor VIII procoagulant activity and factor VIII related antigen was 1.09 + 0.12. The values for some of the newborns are in the normal adult range. However, the majority of the newborns investigated exhibit a different pattern. Almost normal procoagulant activity contrasts with markedly increased antigen concentration. Figure 2 shows the mean values for the newborn group on different days of life. A clear age-dependency can be seen with respect to the concentration of factor VIII related antigen (points). On the first day, and to lesser extent on the second day of life, the antigen concentration was markedly higher than on the following days. The mean value for the first day was 182%, nearly twice the mean for adults. By the second day of life, a decrease has taken place and this continues on the following days. The mean for the first day differs significantly from that for the third day, and from the mean values for the control group. On the other hand, factor VIII activity (circles) shows no significant differences from the normal adult range. The slight differences between the different days of life in the newborn group are without statistical significance. The ratio between factor VIII activity and factor VIII related antigen is given in Figure 3. Because of the decrease of antigen concentration, this ratio increases during the first days of life.
Discussion
In contrast to factor VII, which shows a close correlation between procoagulant activity and antibody neutralizing material (Maak and Frenzel, 1977) a similar relationship cannot be found for factor VIII during the first days of life. On the contrary, the relation between both parameters is characterized on the one hand by an almost normal procoagulant activity and on the other by a markedly increased antigen concentration, especially on the first day of life. On the second day the concentration has already decreased to near the adult level, indicating relatively rapid elimination of antigen. This pattern is difficult to explain. First, the methods used in our investigation have to be considered critically with respect to their possible effects on the results. It is well known that the value for factor VIII activity depends on the test which is chosen. For example, investigations on adult patients with liver disease have shown that factor VIII activity measured by different tests differs markedly in the same sample. In particular, estimation by one-stage methods results in lower activity than that obtained with the thromboplastin generation test (TGT). It should be noted that only the values determined by T G T correlated well with the concentration of factor VIII related antigen (Rogers and Eyster, 1976). It is assumed by the authors that the action of a serum factor which is supplied with the normal serum reagent in the TGT-mixture converts precursor material, and
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thus results in higher activity than in the one-stage tests. Further it is also known that the one-stage test may yield higher values than the T G T in certain conditions. This phenomenon takes place when active intermediate products of coagulation exist. These products do not influence the T G T method because they are removed by the necessary absorption with barium sulfate or aluminium hydroxide, whereas incubation of streptokinase with normal plasma results in reduced one-stage and two-stage factor VIII activities (Niemetz and Nossel, 1960). However, no change could be seen in the concentration of factor VIII related antigen before and after treatment of plasma with streptokinase, or serum with plasmin (Castillo et al., 1976). Simultaneous determinations using both methods were not performed in our study. However, some authors have published normal values for factor VIII activity, obtained by T G T in the newborn period. Comparison with their results shows that the activity was generally lower than in our study. When these divergent facts are taken into consideration, it seems unlikely that the factor VIII activity in our study was low as a result of the one-stage test which was chosen. Any influence of spontaneous fibrinolysis appears unlikely because of the short interval between blood sampling and estimation of factor VIII procoagulant activity. The high concentration of factor VIII related antigen is difficult to explain and this cannot be resolved by the methods chosen. However, one may speculate that the high antigen concentrations result from tissue stores. Tuddenham et al. (1974) found factor VIII related antigen was already present in fetal serum between the 8th and 20th weeks of gestation. Moreover, factor VIII related antigen could be demonstrated in the fetal vascular intima and in splenic cells. Hoyer et al. (1973) found antigen only in the intima. Thus, it seems likely that the intima of blood vessels stores--and possibly synthesizes--factor VIII related antigen. Bennett and Ratnoff (1972) have shown that stress increases factor VIII procoagulant activity, as well as antigen concentration. In this way, the high concentration of factor VIII related antigen in the newborn might be caused by perinatal stress. It is so far not clear whether the increase as a result of stress is caused by increased synthesis or by liberation from stores. A materno-fetal transfer across the placenta seems unlikely because of the high molecular weight of the antigen (Review by Vermylen, 1975). However, another explanation seems possible. Investigations have shown that during the normal coagulation process, factor VIII procoagulant activity is consumed whereas antigen concentration remains unchanged and can still be found in serum. Thus, the discrepancy between factor VIII procoagulant activity and factor VIII related antigen on the first and second days of-life may reflect a coagulation phenomenon. A similar relationship between factor VIII procoagulant activity and factor VIII related antigen was reported by Bennett et al. (1974). These authors investigated patients with diseases complicated by disseminated intravascular coagulation (DIC) and women immediately after labour. There is also some evidence in newborns that DIC takes place during delivery but in most cases does not lead to clinical symptoms. Such evidence arose from monitoring platelet counts during the postnatal period (Maak et al., 1972). The way in which this process might be caused could be by the liberation of tissue thromboplastin, which has been found in high concentrations in the vascular intima (Astrup, 1965). Cord blood leucocytes also contain considerable amounts
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o f t h r o m b o p l a s t i n which is liberated by acidosis or bacterial endotoxins (Rivers and H a t h a w a y , 1975). Several authors have suggested that tissue thromboplastin takes part in the pathogenesis o f perinatal bleeding and most o f the t h r o m b o plastin is liberated f r o m the placenta (Bonnar et al., 1971; Nielsen, 1970). Against the b a c k g r o u n d o f these experimental observations, one can speculate that the low factor V I I I p r o c o a g u l a n t activity reflects c o n s u m p t i o n which is initiated by liberation o f tissue t h r o m b o p l a s t i n into the fetal circulation during delivery. As a consequence, the greatest difference between p r o c o a g u l a n t activity and antigen concentration is observed immediately after birth. The antigen excess is then eliminated by a slower process lasting until the third day of life. O u r results were obtained f r o m a small n u m b e r o f newborns. The newborns were i n h o m o g e n o u s in respect to birth weight and mode of delivery. Further studies are needed to prove the above speculations, and to reveal the possible influences o f different perinatal conditions.
Addendum After completion of this paper we found the work of Muntean, W., B. H. Belohradsky, H. J. Klose, K. Riegel: "Faktor-VIII-Aktivit~it und Faktor-VIII-assoziiertes Antigen bei Neugeborenen" which appeared in "Klin. P~idiat. 189, 412--416 (1977)". Their results agree partly with our findings.
References Astrup, T.: Assay and content of tissue thromboplastin in different organs. Thromb. Diath. haemorrh. 14, 401--406 (1965) Bennett, B., Ratnoff, O. D.: Antihemophilic factor (AHF, factor VIII) procoagulant activity and AHF-like antigen in normal pregnancy, and following exercise and pneumencephalography. J. Lab. clin. Med. 80, 256--263 (1972) Bennett, B., Oxnard, S. C., Douglas, A. S., Ratnoff, O. D.: Studies on antihemophilic factor (AHF, factor VIII) during labor in normal women, in patients with premature separation of the placenta, and in a patient with yon Willebrand's disease. J. lab. clin. Med. 84, 851--860 (1974) Bleyer, W. A., Hakami, N., Shepard, Th. H.: The development of hemostasis in the human fetus and newborn infant. J. Pediat. 79, 838--853 (1971) Bonnar, J., McNicol, G. P., Douglas, A. S.: The blood coagulation and fibrinolytic systems in the newborn and the mother at birth. J. Obstet. Gynaecol. Brit. Commonw. 78,355--360 (1971) Castillo, R., Maragall, S., Guisasola, J. A., Casals, F., Profilos, J., Ordinas, A.: Inhibition of human platelet aggregation by the proteolytic effect of streptokinase. Role of the human factor VIII - - related protein. Haemostasis 5, 306--317 (1976) Hoyer, L. W., de los Santos, R. P., Hoyer, J. R.: Antihemophilic factor antigen. Localization in endothelial cells by immunofluorescent microscopy. J. clin. Invest. 52, 2737--2744 (1973) Ktinzer, W., Str6der, J.: Gerinnungsstudien bei Kindern. I. Mitteilung: Untersuchungen zur Thromboplastinbildung im Nabelschnurblut. Ann. paediat. 188, 147--152 (1957) Laurell, C. B.: Quantitative estimation of proteins by electrophoresis in agarose gels containing antibodies. Anal. Biochem. 16, 45--52 (1966) Maak, B., Rogner, G., Frenzel, J.: Untersuchungen der Thrombozytenadh~isivitM und der Thrombozytenzahl beim reifen und unreifen Neugeborenen. Z. Kinderheilk. 111, 193--204 (1972) Maak, B., Frenzel, J.: Antibody neutralizing material of factor VII during the first weeks of life. Europ. J. Pediat. 125,255--258 (1977)
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Maak, B., Scheidt, B., Frenzel, J.: Erfahrungen bei der Herstellung eines Antiserums gegen das Faktor VIII-assoziierte Antigen. Dtsch. Ges.-Wesen 32, 1335--1338 (1977) Nielsen, N. Chr.: Coagulation and fibrinolysis in mothers and their newborn infants following premature separation of the placenta. Acta Obstet. Gynecol. Scand. 49, 77--83 (1970) Niemetz, J., Nossel, H. L.: Activated coagulation factors: In-vivo and in-vitro studies. Brit. J. Haemat. 16, 337--351 (1969) Perlick, E., Bergmann, A.: Gerinnungslaboratorium in Klinik und Praxis. Leipzig: VEB Georg Thieme 1971 Rivers, R. P. A., Hathaway, W. E.: Studies on tissue factor activity and production by leucocytes of human umbilical cord and adult origin. Pediat. Res. 9, 167--171 (1975) Rogers, J. S., Eysters, M. E.: Relationship of factor VIII-like antigen (VIII AGN) and clot promoting activity (VIII AHF) as measured by one - - and two - - stage assays in patients with liver disease. Brit. J. Haemat. 34, 655--661 (1976) Rogner, G.: Die Blutgerinnung bei Frtihgeburten. Z. Kinderheilk. 84, 197--212 (1960) Tuddenham, E. G. D., Shearn, S. A. M., Peake, I. R., Giddings, J. C., Bloom, A.L.: Tissue localization and synthesis of factor VIII-related antigen in the human fetus. Brit. J. Haemat. 26, 669--677 (1974) Vermylen, J.: Physical and chemical properties of normal and haemophilic factor VIII. Path. Biol. 23, Suppl. 5--10 (1975)
Received November 21, 1977
