Bririshjoumal ofhlaematology, 1979,42,637446

Factor VIII Procoagulant Activity, Factor VIII Related Antigen and von Willebrand Factor in Newborn Cord Blood H. FUKUI,T. TAKASE, H. IKARI,* Y. MURAKAMI,* Y. oKUBO* AND K. NAKAMURAt Department of Pediatrics, Nara Medical College, and *Pediatric Clinic and t C l i n i c of Gynaecology and Obstetrics of Senboku National Hospital (Received

10

August 1978; accepted f o r publication

18 October

1978)

SUMMARY. The mean level of factor VIII procoagulant activity (V1II:C) and factor VIII related antigen (VI1IR:AG) was normal in IOO newborn cord plasmas, whereas that of von Willebrand factor (VIIIR:WF) activity was slightly lower than normal. O n crossed immunoelectrophoresis, 20 of 50 newborn infants had an increased anodal mobility of VII1R:AG. When the cord plasma showing an abnormal electrophoretic pattern was mixed with normal plasma, two precipitation peaks with a broad base were found. Similar mixing experiments with the abnormal cord plasma and plasma from a patient with atypical von Willebrand’s disease did not normalize the electrophoretic mobility of VI1IR:AG. Gel filtration of the cord plasma with an abnormal electrophoretic pattern of VIIIR:AG, showed that the three activities were all detected at the position corresponding to a molecular weight of about 800 000. The results suggest the presence of qualitative abnormalities of the factor VIII molecule in half of full-term newborn cord plasma. It is generally accepted that the level of both factor V and VIII activity is within the normal range in the newborn period, whereas that of vitamin K-dependent clotting factors such as prothrombin, factor VII, IX and X is at a lower level (Fresh et al, 1956; von Kunzer & Stroder, 1957; Aballi et al, 1959; Preston, 1964; Nossel et al, 1966; Yagi et al, 1969; Hathaway, 1970; Biland & Duckert, 1973; Sell & Corrigan, 1973; Foley et al, 1977). Recent studies have indicated that human factor VIII may be a complex of two components, consisting of a higher molecular weight subunit responsible for both factor VIII-related antigen (VI1IR:AG) and ristocetin platelet aggregation cofactor (von Willebrand factor, VIIIR: WF) activity and of a lower molecular weight subunit responsible for factor VIII-procoagulant activity (VII1:C) (Weiss et a l , 1973; Rick & Hoyer, 1973; Meyer et al, 1974; Bouma et al, 1975; Peake & Bloom, 1976; Koutts et al, 1977). These three activities of factor VIII-complex have not been studied systematically on the newborn cord blood. Hence, these were studied in this work, using the cord plasma of IOO newborn infants and their mothers’ plasma. The results suggest the presence Correspondence:Professor H. Fukui, Department of Pediatrics, Nara Medical College, 840, Shijo-cho, Kashihara, Japan. 000’/-1048/79/0800-0637$02.0001979 Blackwell Scientific Publications

637

638

H . Fukui et a1

of some qualitative differences in the factor VIII of the newborn cord blood compared to that of adults. MATERIALS AND METHODS Subjects and blood collection. The blood of 100full-term newborn infants was drawn from the umbilical vein immediately after delivery, using a disposable syringe. Their mothers' blood was collected from the cubital vein shortly before delivery. In a plastic tube, 9 ml of the whole blood was mixed at once with I ml of 0 . 1 M sodium oxalte. Plasma was separated by centrifugation for I S min at 3000 rpm and 4'C. It was tested without delay or stored at -8o'C until used. Assay o f f c t o r VllI-procoagulant activity was performed by the one-stage method. Assay of von Willebrandfactor activity. I t was determined by a modification of the macroscopic platelet aggregation test described by Allain et al (1975). Instead of paraformaldehydefixed platelets, a formalin-fixed human platelet suspension was prepared according to the method of Macfarlane et a1 (1975). In a well of a microtitre plate, 20 p1 of the platelet suspension (5 x I O l/l) ~ were mixed with 20 p1 of a test sample, 20 gl ristocetin (4mg/ml) added, and then the aggregation time determined. The results were expressed in per cent activity compared with that of a pooled normal plasma. Factor VlIl-related antigen activity was estimated by the Laurell technique (1966) using rabbit antisera to human factor VIII. Crossed immunoelectrophoresis was performed according to the technique described by Laurell (1965). A sample (20 pl) was allowed to migrate on I % agarose plate (8 x 8 cm) for 3 h in the first dimension and then on I % agarose containing 0.3% rabbit antiserum to human factor VIII for 16 h in the second dimension. Electrophoretic mobility of factor VIII-related antigen was determined by measuring the distances from the well (do), to the beginning of precipitation arc (dl), to the peak of arc ( d J , and the end of arc (d2). RESULTS Level of Factor VIII-Procoagulant ( V I I I : C )Activity, Factor VIll-Related Antigen ( V I I 1 R : A G )and von Willebrand Factor ( V I I I R : W F ) Activity in Maternal Plasma and in Newborn Cord Plasma The averages and ranges (lowest-highest) of the values obtained from maternal, cord, and normal plasma are summarized in Table I. A significant increase in the level of VIII:C, VI1IR:AG and VII1R:WF was observed in the maternal plasma. But the mean level of VI1I:C and VII1R:AG was normal in the newborn cord plasma, whereas that of VII1R:WF was slightly lower than normal. Thus, a value less than 60% was obtained for VIII:C, VII1R:AG and V1IIR:WF in 28, 28 and 56, respectively, out of 100 newborn cord plasmas examined. Relationship between Factor VIII-Procoagulant ( V 1 I I : C ) and von Willebrand Factor ( V I l I R : W F ) Activity and between Factor VIII-Related Antigen ( V l I 1 R : A G ) and von Willebrand Factor ( V I l I R : W F ) in Maternal Plasma and Newborn Cord Plasma In IOO maternal plasmas, VIII:C and VII1R:AG activity were excellently correlated, respect-

Factor VZZI in Cord Blood

639

TABLE I. Factor VIII procoagulant activity (VllI:C), factor VIII-related antigen (VII1R:AG) and von Willebrand factor (VII1R:WF) in maternal blood and in newborn cord blood No. of samples V1II:C (range) V1IIR:AC (range) VIIIR: WF (range) Maternal blood Newborn cord blood Control blood

IOO

100

20

198.7+66.4% (70-300%) 76.5_+42.8% (20- I 8 0 Yo) 98.8?42.0% (50-1 72 %)

210.1 +70.0%

(6@300%) 75.0*48.2% (22-200%)

97.IIf:43.0% (6+176%)

188.0+69.9% (56-280%) 5 5.6 f48.3 % (5-160%)

99.4+41.0% (62-148%)

ively, to VIIIR:WF, giving a correlation index of 0.82 and 0.81. In contrast, the correlation between VII1:C and VII1R:WF in the newborn infant was not close, showing an index of 0.46. Out of these newborn infants, 47 were outside the 99% confidence range of normal as judged by the ratio of V1IIR:WF to VIII:C (Fig I , left). The correlation index between VII1R:WF and VII1R:AG in the newborn infants was 0.36, and 3 I infants were not normal a t 99% confidence, as estimated by the ratio of VI1IR:WF to VII1R:AG (Fig I , right).

//.

0

50

100

150 VIII:C 6 1

200

250

300 0

50

100

150 VIIIR:AC 6 1

zoo

ZM

FIG I . Relationship of VII1:C to VII1R:WF and that of VI1IR:AG to VI1IR:WF in newborn cord blood (0)and maternal blood (0).The centre line is the regression line for the data obtained from so normal women, the outermost lines represent the 99% confidence belt, and the other two lines represent the 95% confidence belt. Regression line for VI1IR:WF ( y ) to VI1I:C (x) was y=o.78x+t1.6, and that for VII1R:WF (y) to VII1R:AG (x) was y=0.88x+13.4.

ux)

640

H . Fukui et a1

Crossed Immunoelectrophoretic Pattern of Factor VIII-Related Antigen (VlI1R:AG) of the Plasma from Newborn Infants The electrophoretic mobilities of VII1R:AG of the plasmas from 20 normal subjects, 20 mothers and 50 newborn infants are shown in Table 11. Both of the mean values of d o - d l (distancefrom the well to the peak of arc) and do -dp (distance from the well to the peak of arc) were greater in the newborn cord plasma than in the normal and maternal plasma. Twenty of 50 newborn cord plasmas showed an increased mobility exceeding 15 mm of d o - d , distance. Of these 20 newborn cord plasmas, 10were low (less than 60%) in VIII:C, six in VII1R:AG and 18in VII1R:WF activity. Of a total of 50 infants, 3 0 showed a normal electrophoretic mobility of VII1R:AG and a low value was observed of VIII:C in seven, of VII1R:AG in eight and of VII1R:WF in 17 (Table 111). Fig 2 shows different types of migration pattern of newborn cord plasma. TABLE 11. Distances of migration of VII1R:AG in crossed immunoelectrophoresis of plasmas from 20 normal subjects, 20 mothers and 50 newborn infants

NO.of Sample

samples

Maternal plasma Newborn cord plasma Normal plasma

20

50 20

do-dl (mm)

do-d,

dl-d2

(mm)

(mm)

do-dp (mm)

1 . 6 f 1 . 3 2 0 . 1 k 1 . 7 1 8 . 4 k 1 . 4 12.4-t-3.1 3 . 7 2 3 . 2 21.5+4.0 18.2k2.4 I4.5k2.9 1 . 7 k 1 . 5 19.9+2.1 1 7 . 6 k 2 . 9 11.4f3.3

TABLE 111. Levels of VIII:C, VI1IR:AG and VI1IR:WF in cord plasmas showing normal electrophoretic pattern and those in cord plasmas with an increased electrophoretic mobility of VII1R:AG

VIII:c

V1IIR:AG

VIIIR: WF

No. of

Normal electrophoretic pattern Abnormal electrophoreticpattern

samples

260%

30

23

20

I0

260%

Factor VIII procoagulant activity, factor VIII related antigen and von Willebrand factor in newborn cord blood.

Bririshjoumal ofhlaematology, 1979,42,637446 Factor VIII Procoagulant Activity, Factor VIII Related Antigen and von Willebrand Factor in Newborn Cord...
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