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Varied immunological reactivity of factor viii from animal plasmas
TRROHSOSIS RESEARCH 17; 473-480 OPergamon Press Ltd. 1980. Printed
in the United States oo49-3848/80/0215-0473
VARIED IMMUNOLOGICAL REACTIVITY OF FACTOR FROM ANIMAL PLASMAS Raffaella
Coppola,
Rossana
Lombardi, Christine P. M,. Mannucci
Hawkey,
$02.00/0
VIII
Z. M. Ruggeri
Hemophilia and Thrombosis Centre Angelo Bianchi Bonomi, University Milano, Italy; and the Hematology Department, Nuffield Laboratories Comparative Medicine, London, United Kingdom (Received
,
of of
17.9.1979; in revised form 5.11.1979 Accepted by Editor I.M. Nilsson)
ABSTRACT Two-step immunoradiometric assays (1RMA)wit.h human or rabbit antibodies (Ab) have been employed to investigate the antigenic reactivity of Factor VIII (FVIII) from a large group of animals belonging to different species. Chimpanzee and gorilla plasmas gave IRMA dose-response curves barely distinguishable from those of human plasma. Old World primates showed a consistent decrea se of maximum bound radioactivity (MBR) with rabbit Ab and, toa lesser extent, also with human Ab These findings suggest that FVIII from these primates share a lower number of antigenic sites with human FVIII. Plasma from a New World monkey showed more striking differences (lack,of parallelism, low MBR), which were even more evident in the remaining mammalian plasmas tested. All these samples were more reactive with human Ab then with rabbit Ab. The only tested bird plasma was poorly reactive with both the Abs. This study shows that the antigenic properties of FVIII from various mammalian plasmas are partially similar to those of human FVIII particularly when human Abs are employed. Such similarity is much more evident in primates more closely related to man in the zoological scale. INTRODUCTION The properties of precipitating antibodies (Abs) developing in multivon Willebrand’s disease transfused patients with severe, homozygous-like (VWD) are generally similar to those of Abs produced in animals (rabbit,
Key words
: Factor
VIII-Factor
VIII related 473
antigen.
474
FACTOR VIII IN ANIMAL
SPECIES
Vo1.17,No.3/4
goat) against human Factor VIII (FVIII)(1).However, substantialdifferences between homologous and heterologous Abs have been observed when their reactivity with FVIII of various animal species was compared in double diffusion (1). Although the precipitating titer of rabbit and goat Abs against monkey plasma was substantially comparable to that against human plasma, the affinity of heterologous Abs for FVIII of other mammals was more restricted than that of human Abs (l), suggesting that heterologous Abs might be employed to characterize the antigenic differences existing betweenFVII1 of various animal species. Solid-phase immunoradiometric assays (IRMA) utilise low concentrations of Abs and appear to be more useful than precipitin assays to demonstrate subtle differences in the immunological reactivity of FVIII, as shown by the experience in the characterization of VWD “variants” (2, 3). Therefore, we elected to employ two-step IRMA based on the use of human and rabbit Abs to study in more details the antigenic character istics of FVIII among a large group of animals belonging to different species. MATERIAL
AND
METHODS
Collection of animal samples - Blood was obtained from ostensibly healthy animals admitted to the Animal Hospital at the Zoological Society of London for routine clinical examination. Most animals were anesthetised for this procedure; the drugs used varied for different species but none are known to influence the level of circulating FVIII. Blood was obtained from a suitable vein using disposable plastic syringes and disposable needles and was mixed in plastic tubes with either trisodium citrate (3. 2% w/v, one part plus 9 parts of blood) or lithium heparin (15 I. U. per ml. of blood). Samples were centrifuged immediately at 6000 g for 15 min at 4OC to obtain platelet-poor plasma (PPP). This was stored in aliquots in plastic tubes at -35’C. Plasma samples were transported from London to Milan in dry ice. All mammals included in the study had high levels of factor-VIII coagulant activity assessed by their ability to correct the clotting time of human factor VIII-deficient plasma in a one-stage assay system (4). Factor VIII coagulant activity in the demoiselle crane plasma was not measured because of lack of a suitable assay system. Collection of human samples - Human titrated PPP obtained from 10 healthy subjects of both sexes was pooled, frozen at -7OOC and used as reference plasma for comparison with animal plasma in IRMA. Aliquots of the individual plasmas were also frozen and tested in this assay. Rabbit Ab IRMA was carried out in a solid phase, two-site system as pre: polystyrene tubes viously described (5) with the following modifications were coated with rabbit anti-FVIII antiserum and either used immediately or stored at -2OOC after addition of 0. 5 ml Tris buffer (0. 05 M Tris-HCl, 0. 1 M NaCl, pH 7.4) for as long as one month without appreciable loss of activity. Preparation of rabbit anti-FVIII antiserum, separation of the IgG fraction and radiolabelling with carrier-free 1251 were performed as preanti-FVIII from the viously reported (5). Purification of the 1251,labelled bulk of labelled IgG was obtained by solid-phase immunoadsorption using activated sepharose as described in FVIII bound to cyanogen-bromide
Vol.i7,No.3/4
detail elsewhere
FACTOR
VIII IN ANIMAL
(1). The same Ab preparation
SPECIES
475
was used throughout the study.
Human Ab IRMA was similarly carried out. The only difference was that -. plasma from a patient with severe VWD who developed a precipitating antiF VI I I A b (1) was substituted for rabbit anti - F VII I to coat polystyrene tubes, and 125 I labelled a n t i - FVIII IgG from the same patient were employed in the second step. The same Ab preparation was used throughout the study. (B) was calculated for each Analysis of the results - Bound radioactivity plasma dilution as the percentage of total radioactivity (T). Dose response curves were drawn by plotting the values of B/T (after logit transformation) against the logs of plasma dilutions. A regression equation was used to calculate the slopes of reference (pooled human) and test (human and animal) plasmas. Parallelism of the dose-response curves was assessed by means of a slope distribution test exploring the hypothesis of identity of slopes of animal and pooled human plasma (6). The maximum bound radioactivity(MBR) was also used to compare the antigenic reactivity of human and animal plasmas. RESULTS Human plasma - The IRMA dose-response curve of pooled human plasma had a sigmoid shape with both human and rabbit Ab, being characterized by and ascending linear part and a plateau (Fig. l-2). The slopes of the regression lines of human plasmas from 10 healthy subjects ranged between 0. 57 and 0.69 with human Ab, whereas the rabbit Ab IRMA resulted in lower values (range : 0.48-o. 54). The values of MBR showed similar differences between human and rabbit Abs (Table ). - Striking differences were found when plasmas from monkeys of Primates three different families were tested in the IRMA (Fig. 1). With both human and rabbit Ab, plasmas from gorilla and chimpanzee (Pongidae) gave results very similar to human plasma in term of slopes, MBR and parallelism of the dose-response curves. Samples from moor and patas monkeys (Cercopithecidae) did not significantly differ from human plasma with the human Ab IRMA but resulted in a marked decrease of MBR employing the rabbit Ab, though the slopes were significantly similar to those of human plasma (Table). Finally, plasma from a monkey of the Cebidae family (brown capuchin) gave low values of slope and MBR as well as non-parallel curves with rabbit Ab, whereas these measurements were not markedly different from those of human plasma using human Ab. Other mammalian orders - When plasma from 21 mammals belonging to six different orders were tested in the IRMA, values of slopes and MBR were generally lower than those of human plasma (Table). The differences were more marked with rabbit than human Ab IRMA. This was particularly evident when parallelism of the dose-response curves was considered. Only Suidae (pig ‘and wild boar) gave significantly parallel lines with the rabbit Ab, whereas the majority of mammalian plasmas gave parallel lines with human Ab only (Fig. 2). MBR values, however, were much lower than in human and other primate plasmas(Table).
FACTOR VIII
476
Human
.
.
IN ANIMAL SPECIES
Vo1.17,No.3/4
Antibody
.
.
.
_
.
.
.
.
.
.
-
60, c)
so.
3
40.
E d
30.
s
20.
z
10.
g
5.
is m
2.
Rabbit
Antibody Chimpuaa
BmwnCupuchin
B
r
1, 7,
409eo
1
10240
r
1
.
RECIPROCAL
I
I
640
2560 OF
I
160
PLASMA FIG.
.
.
1
40
.
1
10
DILUTION
1
IRMA dose-response curves of pooled human plasma ( 0 1, Chimpanzee ( ??) Moor Monkey ( A ) and Brown Capuchin ( 0) plasma using human antibody (upper graph) or rabbit antibody (lower graph). Reciprocal of plasma dilutions are reported logarithmically on the horizontal axis and logit of percentage bound radioactivity on the vertical axis.
Vo1.17,No.3/4
FACTOR
Human
VIII IN ANIMAL
SPECIES
477
Antibody
60, 50. 40.
M8n
30.
Pig
20. OX
10. 5.
--+.t
1c r8ne
2. 1, ,
.
,
60, 60,
Rabbit
Ant ibody
40, 30. 20.
~--Y-A
40660 - 10240 - 2&o RECIPROCAL
. 640 OF
Crme
Ii0
PLASMA
-
40
.
,b
’
DILUTION
FIG. 2 IRMA dose-response curves of pooled human plasma ( 0 ), Pig ( I 1, Ox ( A ) and Demoiselle Crane ( 0) plasma using human antibody (upper graph) or rabbit antibody (lower graph). Reciprocal of plasma dilutions are reported logarithmically on the horizontal axis and logit of percentage bound radioactivity on the vertical axis.
FACTOR VIII IN ANIMAL
478
TABLE -
Slopes and maximum bamd radioactivity
SPECIES
in plasma of different animal species
Source of plasma Order
Human Ab Family
Primates
Vo1.17,No.3/4
Species
MBR%
Rabbit
Slope 69
Ab
MBRW
Slope
49-53
0.48-Q. 5
Hominoidea
Man (Homo sapiens)
56-64
0.57-o.
Pongidae
Cbimpansae
60
0.59+++
47
0.57*
61
0.61++t
41
0.51+t+
51
0.55+++
32
0.40++
57
0.57+++
35
0.40++-
Brown caplchin (Cebus anella)
47
0.52++
12
0.23
Dog (Cams familiaris)
40
0.48+
23
0.36
coyote
(C.lawlq)
35
0.46+
21
0.35
(Pan troKlodytes) Gorilla (Gailla Cercopithecedae
xorilla)
Moor mon,ey (Macaca
maurus)
Patas monkey (Ewhrocebur Cebidae
Carnivaa
Canidae
Felidae
Perissodactyla
vatas)
Cat (Felis catus)
42
0.42+
9
0.25
Lynx (F. lynx)
38
0.42+
13
0.27
Jaguar (Panthera onca)
34
0.44+
10
0.17
Umidae
Bear (Tremarcta
13
0.31
13
0. 16
Equidae
Hme
33
0.41+
10
0.35
30
0.42+
7
0.35
0.50+
17
0.31
ccnatus)
(Eouus caballus)
Zebra (E. sebm) Tapiridae &tiodpCtyla
Suidae
Camelidae
Cervidae Bovidae
Caviidae
Taf
(Taniristemesth) 32
Pig (Sus so&a)
36
0.52*
22
0.37+
Wild boar (Sus scrofal,
33
0. Sl++
23
0.41+
Camel (Camelius bactrianur)
10
0.42+
9
0.27
Guanaco (Ipma xuanacoe)
18
0.42+
9
0.25
Red deer (Cervuaeleuhar)
27
0.41+
13
0.34
Siha deer (C. ninpon)
28
0.46+
12
0.33
Ox (Bee taurug)
16
0.33
Sheep (Ovis pries )
26
Kudu (Tranclcuhas sQweiceroQ Guinea pig (Cavia
9
0.29
0.44+
11
0.31
17
0.31
12
0.32
24
0.33
9
0.14
Rabbit (Oryctolaxus cuniculus)
26
0.37
0
0
Wallaby arisce)
24
0.’44+
6
0.12
PorceDus) bgomm@=
Leporidae
klarsupialia
Asterisk
(Protemnodon
indicate the degree of parallelism of the doss-reqonse
curves assessed by means of slop
dishibution
tes& exploring the hypothesis of identity of sloPes of test and Pooled human plasma. Tha values for human plasma are the ranges observed in plasmas dram 10 healthy subjectx of bah sexes. ++t P
4
++
P
< 0.01
0.001
+
P
< 0.05
Vo1.17,No.3/4
FACTOR VIII
IN ANII?AL SPECIES
479
Bird - Only one avian plasma (demoiselle crane) could be tested. With rabbit kb,the bound radioactivity was not different from that of the blank samples, whereas human Ab IRMA reached a MBR of 8 per cent ( Fig. 2 1. DISCUSSION Immunoradiometric assays are useful tools for studying quantitative and qualitative abnormalities of the F VI II protein in humans (2,3). No specific attempt was made in this study to quantitate F VI I I in. animal plasma by IRMA., However, inspection of the dose-response curves clearly demonstrates that in all the tested mammals the plasma concentration of FVIII is much higher than in human plasma.IRMA with human and rabbit Ab was employed to provide informations on the antigenic structure of animal FVIII and to allow a qualitative comparison with the human protein. In general, the human Ab had a wider spectrum of reactivity with F VIII of various animal species than rabbit Ab. This may be explained by the fact that when’ normal rabbits, whose plasma already contains F VI II ,. are immunized with human .F VI !I , the antibody response is restricted to the specific sites of the human molecule : there is therefore a limited degree of cross-reac tivity with antigenic sites shared by other species. In patients with severe VWD, lacking F VI 1 I in their plasma, multiple antigenic stimulations with a totally foreign protein introduced by replacement therapy are more likely to generate antibodies with broader specificity. Thus, rabbit Ab IRMA was more useful than the method employing human Ab to assess the varied antigenie reactivity of animal F VI I I , a 6 well as any difference or similarity with the human protein. In this regard, the study of non-human primate plasma was of particular interest. The chimpanzee and gorilla, which are most closely related to man in the phylogenetic classification, gave dose-response curves barely distinguishable from human plasma in terms of steepness of the ascending part of MBR. Old World primates, such as moor and the dose-response curve and patas monkeys (Cercopithecidae), showed a consistent decrease of MBR with rabbit Ab, and, to a lesser extent, also with human Ab. Since the decrease of antibody binding capacity could not be improved by increasing the incubation time in the two successive reaction phases of the assay , this difference cannot be accounted for by lack of equilibrium attainement but rather by a lower number of antigenic sites shared with human FVIII . Fitily, plasma from the brown capuchin, the only studiea rJew World monkey (Cebidae), showed more striking differences of the dose response-curve (lack of parallelism, low MBR), suggesting that the antigenic properties of F VI I I diverge markedly from those of human plasma. The anomalies of the IRMA dose response curves of thecebidae and Cercopithecidae are similar to those reported by Peake and Bloom (2) and Ardaillou et al (3) in human “variant” VWD, which are thought to be related to a deficiency of high-molecul ar weight oligomers of the FVIII multimeric series. Itwouldbe of interest to see if the similar antigenic behaviour of these monkey plasmas in the IRMA system also reflects the degree of polymerization of the F VI I I protein Human Ab showed some degree of cross-rectivity with all mammalian and, to a lesser extent, with plasma of the only studied bird specimens,
480
FACTOR VIII
Vo1.17,No.3/4
IN ANIMAL SPECIES
(demoiselle crane). The rabbit Ab did not give any cross-class antigenic reac tivity with demoiselle crane plasma and, as expected, with rabbit plasma. _ The most primitive mammal examined, the red-necked wallaby, was also poorly reactive.There is no simple zoological explanation for the degree of antigenic similarity or difference found among F VI I I of the remaining mammalian orders using either human or rabbit Abs. On the whole, the IRMA slope values suggest that the antigenic properties of F VI I I from various mammalian plasmas were reasonally similar, particularly when the human Ab was employed. This is in contrast with the finding that the platelet F VI I I reactions involved in aggregation with or without ristocetin showed a great deal of species specificity in mammals (7). It is likely that subtle differences in F VI I I structure and composition existing among species are sufficient to modify profaandy the functional properties of the protein, whereas the antigenic sites are probably less affected. REFERENCES 1.
RUGGERI, Z. M., CIAVARELLA, N., MANNUCCI,P.M., MOLINARI, A., DAMMACCO, F., LAVERGNE, J. M., MEYER, D. Familial incidence of precipitating antibodies in von Willebrand’s disease. A study of four cases. J. Lab. Clin.Med. 94, 60-75, 1979.
2.
assay PEAKE, I. R. , B,LOOM, A. L. The use of an immunoradiometric for Factor VIII related antigen in the study of atypical von Willebrand’s disease. Thromb. Res. 10, 27-37,1977.
3.
ARDAILLOU, N. , GIRMA, J. P. , MEYER, D , LAVERGNE, J. M. , disease. SHOA’I, I. , LARRIEU, M. J., Variants oi von Willebrand’s Demonstration of decreased antigenic reactivity by immunoradiometric assay. Thromb. Res. 12, 817-830,1978.
4.
HAWKEY, C. M. in Comparative Medical Books, London, 1975.
5.
RUGGERI, Z. M. , MANNUCCI, P.M., JEFFCOATE, S. L., INGRAM, Immunoradiometric assay of Factor VIII related antigen with G. I. C., observations in 32 patients with von Willebrand’s disease. Br. J. Haemats. 33, 221-232,1976.
6.
BALAAM, L. N., in Fundamentals London, 19 72.
7.
BRINKHOUS, K. M., THOMAS, B. D., IBRAHIM, S. A. , READ, M. S., Plasma levels of platelet aggregating factor/van Willebrand factor in various species. Thromb. Res. 11, 345-355,1977.
Mammalian
Haematology,
of biometry,
George
Heinneman
Allen & Unwin,
in the United States oo49-3848/80/0215-0473
VARIED IMMUNOLOGICAL REACTIVITY OF FACTOR FROM ANIMAL PLASMAS Raffaella
Coppola,
Rossana
Lombardi, Christine P. M,. Mannucci
Hawkey,
$02.00/0
VIII
Z. M. Ruggeri
Hemophilia and Thrombosis Centre Angelo Bianchi Bonomi, University Milano, Italy; and the Hematology Department, Nuffield Laboratories Comparative Medicine, London, United Kingdom (Received
,
of of
17.9.1979; in revised form 5.11.1979 Accepted by Editor I.M. Nilsson)
ABSTRACT Two-step immunoradiometric assays (1RMA)wit.h human or rabbit antibodies (Ab) have been employed to investigate the antigenic reactivity of Factor VIII (FVIII) from a large group of animals belonging to different species. Chimpanzee and gorilla plasmas gave IRMA dose-response curves barely distinguishable from those of human plasma. Old World primates showed a consistent decrea se of maximum bound radioactivity (MBR) with rabbit Ab and, toa lesser extent, also with human Ab These findings suggest that FVIII from these primates share a lower number of antigenic sites with human FVIII. Plasma from a New World monkey showed more striking differences (lack,of parallelism, low MBR), which were even more evident in the remaining mammalian plasmas tested. All these samples were more reactive with human Ab then with rabbit Ab. The only tested bird plasma was poorly reactive with both the Abs. This study shows that the antigenic properties of FVIII from various mammalian plasmas are partially similar to those of human FVIII particularly when human Abs are employed. Such similarity is much more evident in primates more closely related to man in the zoological scale. INTRODUCTION The properties of precipitating antibodies (Abs) developing in multivon Willebrand’s disease transfused patients with severe, homozygous-like (VWD) are generally similar to those of Abs produced in animals (rabbit,
Key words
: Factor
VIII-Factor
VIII related 473
antigen.
474
FACTOR VIII IN ANIMAL
SPECIES
Vo1.17,No.3/4
goat) against human Factor VIII (FVIII)(1).However, substantialdifferences between homologous and heterologous Abs have been observed when their reactivity with FVIII of various animal species was compared in double diffusion (1). Although the precipitating titer of rabbit and goat Abs against monkey plasma was substantially comparable to that against human plasma, the affinity of heterologous Abs for FVIII of other mammals was more restricted than that of human Abs (l), suggesting that heterologous Abs might be employed to characterize the antigenic differences existing betweenFVII1 of various animal species. Solid-phase immunoradiometric assays (IRMA) utilise low concentrations of Abs and appear to be more useful than precipitin assays to demonstrate subtle differences in the immunological reactivity of FVIII, as shown by the experience in the characterization of VWD “variants” (2, 3). Therefore, we elected to employ two-step IRMA based on the use of human and rabbit Abs to study in more details the antigenic character istics of FVIII among a large group of animals belonging to different species. MATERIAL
AND
METHODS
Collection of animal samples - Blood was obtained from ostensibly healthy animals admitted to the Animal Hospital at the Zoological Society of London for routine clinical examination. Most animals were anesthetised for this procedure; the drugs used varied for different species but none are known to influence the level of circulating FVIII. Blood was obtained from a suitable vein using disposable plastic syringes and disposable needles and was mixed in plastic tubes with either trisodium citrate (3. 2% w/v, one part plus 9 parts of blood) or lithium heparin (15 I. U. per ml. of blood). Samples were centrifuged immediately at 6000 g for 15 min at 4OC to obtain platelet-poor plasma (PPP). This was stored in aliquots in plastic tubes at -35’C. Plasma samples were transported from London to Milan in dry ice. All mammals included in the study had high levels of factor-VIII coagulant activity assessed by their ability to correct the clotting time of human factor VIII-deficient plasma in a one-stage assay system (4). Factor VIII coagulant activity in the demoiselle crane plasma was not measured because of lack of a suitable assay system. Collection of human samples - Human titrated PPP obtained from 10 healthy subjects of both sexes was pooled, frozen at -7OOC and used as reference plasma for comparison with animal plasma in IRMA. Aliquots of the individual plasmas were also frozen and tested in this assay. Rabbit Ab IRMA was carried out in a solid phase, two-site system as pre: polystyrene tubes viously described (5) with the following modifications were coated with rabbit anti-FVIII antiserum and either used immediately or stored at -2OOC after addition of 0. 5 ml Tris buffer (0. 05 M Tris-HCl, 0. 1 M NaCl, pH 7.4) for as long as one month without appreciable loss of activity. Preparation of rabbit anti-FVIII antiserum, separation of the IgG fraction and radiolabelling with carrier-free 1251 were performed as preanti-FVIII from the viously reported (5). Purification of the 1251,labelled bulk of labelled IgG was obtained by solid-phase immunoadsorption using activated sepharose as described in FVIII bound to cyanogen-bromide
Vol.i7,No.3/4
detail elsewhere
FACTOR
VIII IN ANIMAL
(1). The same Ab preparation
SPECIES
475
was used throughout the study.
Human Ab IRMA was similarly carried out. The only difference was that -. plasma from a patient with severe VWD who developed a precipitating antiF VI I I A b (1) was substituted for rabbit anti - F VII I to coat polystyrene tubes, and 125 I labelled a n t i - FVIII IgG from the same patient were employed in the second step. The same Ab preparation was used throughout the study. (B) was calculated for each Analysis of the results - Bound radioactivity plasma dilution as the percentage of total radioactivity (T). Dose response curves were drawn by plotting the values of B/T (after logit transformation) against the logs of plasma dilutions. A regression equation was used to calculate the slopes of reference (pooled human) and test (human and animal) plasmas. Parallelism of the dose-response curves was assessed by means of a slope distribution test exploring the hypothesis of identity of slopes of animal and pooled human plasma (6). The maximum bound radioactivity(MBR) was also used to compare the antigenic reactivity of human and animal plasmas. RESULTS Human plasma - The IRMA dose-response curve of pooled human plasma had a sigmoid shape with both human and rabbit Ab, being characterized by and ascending linear part and a plateau (Fig. l-2). The slopes of the regression lines of human plasmas from 10 healthy subjects ranged between 0. 57 and 0.69 with human Ab, whereas the rabbit Ab IRMA resulted in lower values (range : 0.48-o. 54). The values of MBR showed similar differences between human and rabbit Abs (Table ). - Striking differences were found when plasmas from monkeys of Primates three different families were tested in the IRMA (Fig. 1). With both human and rabbit Ab, plasmas from gorilla and chimpanzee (Pongidae) gave results very similar to human plasma in term of slopes, MBR and parallelism of the dose-response curves. Samples from moor and patas monkeys (Cercopithecidae) did not significantly differ from human plasma with the human Ab IRMA but resulted in a marked decrease of MBR employing the rabbit Ab, though the slopes were significantly similar to those of human plasma (Table). Finally, plasma from a monkey of the Cebidae family (brown capuchin) gave low values of slope and MBR as well as non-parallel curves with rabbit Ab, whereas these measurements were not markedly different from those of human plasma using human Ab. Other mammalian orders - When plasma from 21 mammals belonging to six different orders were tested in the IRMA, values of slopes and MBR were generally lower than those of human plasma (Table). The differences were more marked with rabbit than human Ab IRMA. This was particularly evident when parallelism of the dose-response curves was considered. Only Suidae (pig ‘and wild boar) gave significantly parallel lines with the rabbit Ab, whereas the majority of mammalian plasmas gave parallel lines with human Ab only (Fig. 2). MBR values, however, were much lower than in human and other primate plasmas(Table).
FACTOR VIII
476
Human
.
.
IN ANIMAL SPECIES
Vo1.17,No.3/4
Antibody
.
.
.
_
.
.
.
.
.
.
-
60, c)
so.
3
40.
E d
30.
s
20.
z
10.
g
5.
is m
2.
Rabbit
Antibody Chimpuaa
BmwnCupuchin
B
r
1, 7,
409eo
1
10240
r
1
.
RECIPROCAL
I
I
640
2560 OF
I
160
PLASMA FIG.
.
.
1
40
.
1
10
DILUTION
1
IRMA dose-response curves of pooled human plasma ( 0 1, Chimpanzee ( ??) Moor Monkey ( A ) and Brown Capuchin ( 0) plasma using human antibody (upper graph) or rabbit antibody (lower graph). Reciprocal of plasma dilutions are reported logarithmically on the horizontal axis and logit of percentage bound radioactivity on the vertical axis.
Vo1.17,No.3/4
FACTOR
Human
VIII IN ANIMAL
SPECIES
477
Antibody
60, 50. 40.
M8n
30.
Pig
20. OX
10. 5.
--+.t
1c r8ne
2. 1, ,
.
,
60, 60,
Rabbit
Ant ibody
40, 30. 20.
~--Y-A
40660 - 10240 - 2&o RECIPROCAL
. 640 OF
Crme
Ii0
PLASMA
-
40
.
,b
’
DILUTION
FIG. 2 IRMA dose-response curves of pooled human plasma ( 0 ), Pig ( I 1, Ox ( A ) and Demoiselle Crane ( 0) plasma using human antibody (upper graph) or rabbit antibody (lower graph). Reciprocal of plasma dilutions are reported logarithmically on the horizontal axis and logit of percentage bound radioactivity on the vertical axis.
FACTOR VIII IN ANIMAL
478
TABLE -
Slopes and maximum bamd radioactivity
SPECIES
in plasma of different animal species
Source of plasma Order
Human Ab Family
Primates
Vo1.17,No.3/4
Species
MBR%
Rabbit
Slope 69
Ab
MBRW
Slope
49-53
0.48-Q. 5
Hominoidea
Man (Homo sapiens)
56-64
0.57-o.
Pongidae
Cbimpansae
60
0.59+++
47
0.57*
61
0.61++t
41
0.51+t+
51
0.55+++
32
0.40++
57
0.57+++
35
0.40++-
Brown caplchin (Cebus anella)
47
0.52++
12
0.23
Dog (Cams familiaris)
40
0.48+
23
0.36
coyote
(C.lawlq)
35
0.46+
21
0.35
(Pan troKlodytes) Gorilla (Gailla Cercopithecedae
xorilla)
Moor mon,ey (Macaca
maurus)
Patas monkey (Ewhrocebur Cebidae
Carnivaa
Canidae
Felidae
Perissodactyla
vatas)
Cat (Felis catus)
42
0.42+
9
0.25
Lynx (F. lynx)
38
0.42+
13
0.27
Jaguar (Panthera onca)
34
0.44+
10
0.17
Umidae
Bear (Tremarcta
13
0.31
13
0. 16
Equidae
Hme
33
0.41+
10
0.35
30
0.42+
7
0.35
0.50+
17
0.31
ccnatus)
(Eouus caballus)
Zebra (E. sebm) Tapiridae &tiodpCtyla
Suidae
Camelidae
Cervidae Bovidae
Caviidae
Taf
(Taniristemesth) 32
Pig (Sus so&a)
36
0.52*
22
0.37+
Wild boar (Sus scrofal,
33
0. Sl++
23
0.41+
Camel (Camelius bactrianur)
10
0.42+
9
0.27
Guanaco (Ipma xuanacoe)
18
0.42+
9
0.25
Red deer (Cervuaeleuhar)
27
0.41+
13
0.34
Siha deer (C. ninpon)
28
0.46+
12
0.33
Ox (Bee taurug)
16
0.33
Sheep (Ovis pries )
26
Kudu (Tranclcuhas sQweiceroQ Guinea pig (Cavia
9
0.29
0.44+
11
0.31
17
0.31
12
0.32
24
0.33
9
0.14
Rabbit (Oryctolaxus cuniculus)
26
0.37
0
0
Wallaby arisce)
24
0.’44+
6
0.12
PorceDus) bgomm@=
Leporidae
klarsupialia
Asterisk
(Protemnodon
indicate the degree of parallelism of the doss-reqonse
curves assessed by means of slop
dishibution
tes& exploring the hypothesis of identity of sloPes of test and Pooled human plasma. Tha values for human plasma are the ranges observed in plasmas dram 10 healthy subjectx of bah sexes. ++t P
4
++
P
< 0.01
0.001
+
P
< 0.05
Vo1.17,No.3/4
FACTOR VIII
IN ANII?AL SPECIES
479
Bird - Only one avian plasma (demoiselle crane) could be tested. With rabbit kb,the bound radioactivity was not different from that of the blank samples, whereas human Ab IRMA reached a MBR of 8 per cent ( Fig. 2 1. DISCUSSION Immunoradiometric assays are useful tools for studying quantitative and qualitative abnormalities of the F VI II protein in humans (2,3). No specific attempt was made in this study to quantitate F VI I I in. animal plasma by IRMA., However, inspection of the dose-response curves clearly demonstrates that in all the tested mammals the plasma concentration of FVIII is much higher than in human plasma.IRMA with human and rabbit Ab was employed to provide informations on the antigenic structure of animal FVIII and to allow a qualitative comparison with the human protein. In general, the human Ab had a wider spectrum of reactivity with F VIII of various animal species than rabbit Ab. This may be explained by the fact that when’ normal rabbits, whose plasma already contains F VI II ,. are immunized with human .F VI !I , the antibody response is restricted to the specific sites of the human molecule : there is therefore a limited degree of cross-reac tivity with antigenic sites shared by other species. In patients with severe VWD, lacking F VI 1 I in their plasma, multiple antigenic stimulations with a totally foreign protein introduced by replacement therapy are more likely to generate antibodies with broader specificity. Thus, rabbit Ab IRMA was more useful than the method employing human Ab to assess the varied antigenie reactivity of animal F VI I I , a 6 well as any difference or similarity with the human protein. In this regard, the study of non-human primate plasma was of particular interest. The chimpanzee and gorilla, which are most closely related to man in the phylogenetic classification, gave dose-response curves barely distinguishable from human plasma in terms of steepness of the ascending part of MBR. Old World primates, such as moor and the dose-response curve and patas monkeys (Cercopithecidae), showed a consistent decrease of MBR with rabbit Ab, and, to a lesser extent, also with human Ab. Since the decrease of antibody binding capacity could not be improved by increasing the incubation time in the two successive reaction phases of the assay , this difference cannot be accounted for by lack of equilibrium attainement but rather by a lower number of antigenic sites shared with human FVIII . Fitily, plasma from the brown capuchin, the only studiea rJew World monkey (Cebidae), showed more striking differences of the dose response-curve (lack of parallelism, low MBR), suggesting that the antigenic properties of F VI I I diverge markedly from those of human plasma. The anomalies of the IRMA dose response curves of thecebidae and Cercopithecidae are similar to those reported by Peake and Bloom (2) and Ardaillou et al (3) in human “variant” VWD, which are thought to be related to a deficiency of high-molecul ar weight oligomers of the FVIII multimeric series. Itwouldbe of interest to see if the similar antigenic behaviour of these monkey plasmas in the IRMA system also reflects the degree of polymerization of the F VI I I protein Human Ab showed some degree of cross-rectivity with all mammalian and, to a lesser extent, with plasma of the only studied bird specimens,
480
FACTOR VIII
Vo1.17,No.3/4
IN ANIMAL SPECIES
(demoiselle crane). The rabbit Ab did not give any cross-class antigenic reac tivity with demoiselle crane plasma and, as expected, with rabbit plasma. _ The most primitive mammal examined, the red-necked wallaby, was also poorly reactive.There is no simple zoological explanation for the degree of antigenic similarity or difference found among F VI I I of the remaining mammalian orders using either human or rabbit Abs. On the whole, the IRMA slope values suggest that the antigenic properties of F VI I I from various mammalian plasmas were reasonally similar, particularly when the human Ab was employed. This is in contrast with the finding that the platelet F VI I I reactions involved in aggregation with or without ristocetin showed a great deal of species specificity in mammals (7). It is likely that subtle differences in F VI I I structure and composition existing among species are sufficient to modify profaandy the functional properties of the protein, whereas the antigenic sites are probably less affected. REFERENCES 1.
RUGGERI, Z. M., CIAVARELLA, N., MANNUCCI,P.M., MOLINARI, A., DAMMACCO, F., LAVERGNE, J. M., MEYER, D. Familial incidence of precipitating antibodies in von Willebrand’s disease. A study of four cases. J. Lab. Clin.Med. 94, 60-75, 1979.
2.
assay PEAKE, I. R. , B,LOOM, A. L. The use of an immunoradiometric for Factor VIII related antigen in the study of atypical von Willebrand’s disease. Thromb. Res. 10, 27-37,1977.
3.
ARDAILLOU, N. , GIRMA, J. P. , MEYER, D , LAVERGNE, J. M. , disease. SHOA’I, I. , LARRIEU, M. J., Variants oi von Willebrand’s Demonstration of decreased antigenic reactivity by immunoradiometric assay. Thromb. Res. 12, 817-830,1978.
4.
HAWKEY, C. M. in Comparative Medical Books, London, 1975.
5.
RUGGERI, Z. M. , MANNUCCI, P.M., JEFFCOATE, S. L., INGRAM, Immunoradiometric assay of Factor VIII related antigen with G. I. C., observations in 32 patients with von Willebrand’s disease. Br. J. Haemats. 33, 221-232,1976.
6.
BALAAM, L. N., in Fundamentals London, 19 72.
7.
BRINKHOUS, K. M., THOMAS, B. D., IBRAHIM, S. A. , READ, M. S., Plasma levels of platelet aggregating factor/van Willebrand factor in various species. Thromb. Res. 11, 345-355,1977.
Mammalian
Haematology,
of biometry,
George
Heinneman
Allen & Unwin,