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Prothrombin activation by encephalitogenic brain protein
EXPERIMENTAL
AND
Prothrombin
MOLECULAR
Activation K. D. MILLER,
Division
5, 575479
PATHOLOGY
by
(1966)
Encephalitogenic
A. W. PHELAN,
Brain
Protein1
AND D. N. COLLINS
of Laboratories and Research, New York State Department of Health,’ Department of Pathology, Albany Medical College, Albany, New York” Received
August
II,
and
1966
Experimental allergic encephalomyelitis (EAE) in guinea pigs, produced by inoculation of products of brain or spinal cord along with Freund’s complete adjuvant, was recently attributed, amqng other fractions, to a lipid-free brain protein (Kies and Alvord, 1959a; Roboz and Henderson, 1959; Kies et al., 1960; Caspary and Field, 196.5; Honegger, 1965). This protein is strongly basic (Caspary and Field, 1965; Honegger, 1965) and as such is a potential participant in the so-called nonenzymic phase of prothrombin activation (Miller, 1960; Miller et al., 1961). The present work describes the prothrombin-activating activity of encephalitogenic brain protein preparations. MATERIALS
AND METHODS
Procedures for preparation of the EAE protein followed largely those of Kies ( 1965a). In a typical preparation, 25 guinea pig brains were homogenizedwith cold (4’C) saline in a glasshomogenizer with Teflon pestle, and washed three times with cold saline, with centrifugation of the sediment each time at 6500 X g. The residue was ground with cold (--1O’C) acetone until nearly dry, extracted three times with 20 volumes of cold (4’C) chloroform:methanol (2: l), then three times with acetone to remove residual chloroform. The residue (5.13 gm) was stirred 1 hour at 4’C with 110 ml of water and centrifuged at 6500 x: g for 30 minutes. The wet, insoluble residue was then stirred for 4 days in 235 ml of 0.01 N HCl at 4°C. Centrifugation (6500 X y; 30 minutes) gave 240 ml of extract of pH 1.5. Dry ammonium sulfate (85.5 gm) was added slowly with continuous stirring at 3°C and the precipitate formed in 1 hour was recovered by centrifugation at 6500 X g. The precipitate was dissolved in 50 ml of distilled water (4°C). The pH was adjusted to 7.0, and the solution was dialyzed against six changesof distilled water, each water volume 180 times the volume of the extract. Lyophilization of the supernatant yielded 309 mg of dry material. Most investigations of EAE and prothrombin-activating activities were conducted with this type of preparation. Further purification, such as by Sephadex G-100 (Caspary and Field, 1965) and G-150 chromatography, was achieved but quantities were insufficient for adequate testing. The secondof two peaks recovered in G-150 chroma1 Supported in part by grants-in-aid from the National Health (HE-09902) and the American National Red Cross 2 Thrombosis and Vascular Disease Research Laboratory. 3 Affiliated with the Institute of Preventive Medicine.
Heart Institute, (K-29915).
National
Institutes
of
576
K.
D.
MILLER,
A.
W.
PHELAN,
AND
D.
N.
COLLINS
tography (0.1 M acetate buffer, pH 5.0) of a human brain preparation crystallized on concentration by vacuum dialysis was also insufficient in quantity for study. Horse prothrombin was prepared according to Miller and McGarrahan (to be published). Synthetic poly-L-lysine hydrobromide (lot PLH 6101) was obtained from Schwartz BioResearch, Inc., Orangeburg, New York. Encephalitogenic activity (EAE) was determined on weighed samples of the dry brain fractions homogenized in Freund’s adjuvant containing 5 mg of BCG (Freund et al., 1947) per milliliter. Portions of the suspensions (0.1 ml), each containing 0.5 mg of BCG and varying amounts of encephalitogen, were injected once intracutaneously over the sternum of SOO- to 800-gm male guinea pigs. Controls included injections of adjuvant without encephalitogen. Occurrence of EAE was determined by the development of physical signs and microscopic pathology. The animals were observed daily for alterations in general physical condition, weight loss, altered righting reflex, and paralysis. As paralytic signs developed, animals were sacrificed and the brain and spinal cord fixed in Bouin’s fluid. Twenty-eight days post-injection the survivors were also sacrificed. The CNS samples from all animals were sectioned and examined for microscopic evidence of encephalomyelitis. The prothrombin activating effect of the encephalitogenic preparations was studied at varying pH, temperature, and NaCl, CaCl>, and glycerol concentrations, Optimum reaction conditions were compared to those of polylysine as well as other synthetic and naturally occurring activators (Miller, 1960; Miller et al., 1961). The reaction tubes contained 1725 Iowa units of horse prothrombin per milliliter ( 1.6 mg/ml) and l-3 mg/ml encephalitogen. After determination of optimum conditions of pH, temperature, etc., the encephalitogenic protein was varied to determine the optimum ratio to prothrombin for activation and also the smallest amount necessary for initiation of activation ( [Ali; Miller and LeGere, 1963). Controls included mixtures of prothrombin in the optimum solvent but without encephalitogen. The polylysine effect on prothrombin activation serves as a reference reaction. The prothrombin activating effect of the encephalitogen was also determined after heating a 1s solution at 100°C for 15 minutes. Prothrombin and thrombin were determined according to Ware and Seegers ( 1949). The reference standard was the prothrombin (300 Iowa units/ml) contained in a pool of oxalated bovine plasma (1 part 1.34% sodium oxalate to 9 parts blood). RESULTS Injection of two guinea pig encephalitogenic protein preparations into guinea pigs produced symptoms and microscopic lesions as indicated in Table I. Because of the large amounts of material needed for studies of prothrombin activation, a titration to determine the EDhO, the smallest amount causing the average disease in 50% of a larger number of animals (Alvord, 1959), was not determined. Horse prothrombin was activated with the encephalitogenic protein preparations. Optimal conditions were at pH 7.5, 18”C, 1.0 M glycerol and 0.05 ionic strength. Calcium chloride had no effect on the reaction, and activation progressed as well in EDTA as in Tris buffer. Subsequent reactions were carried out on solutions of prothrombin and the encephalitogenic materials in 0.05 M Tris buffer, pH 7.5, containing 1.0 M glycerol, at 18°C.
EXPERIMENTAL
ALLERGIC
ENCEPHALOMYELITIS
577
The optimum weight ratio of one encephalitogen preparation to prothrombin was 5: 1, for a second preparation, 2 : 1. The [Ai] for this second preparation, the smallest amount necessary for any activation of prothrombin in 24 hours, was 0.77 mg activator per milligram of horse prothrombin. Extraction of the prothrombin with chloroform:methanol (2: 1) did not affect these values. The relative rates of activation at optimum ratios of the encephalitogen and polylysine to prothrombin are depicted in Fig. 1. Heating the encephalitogen to 1OO’C d.id not affect its prothrombin activating properties. TABLE GUINEA
PIG ENCEPHALOMYELITIS
and
No. animals injected
Preparation amount
1
FROM
HOMOLWOUS
BRAIN
Incidence of physical sign9
PROTEIN
Incidence of microscopic lesionsf)
I
(2.5 mg)
5
3!5
s/s
I II None
(1.25 mg) (0.50 mg) (BCG-adjuvant
5 5
3/5 3/5
4/5 415
O/IS
O/IS
15
only)
a Physical signs = altered righting reflexes, paralysis, general inactivity with weight loss. b Microscopic lesions = meningoencephalomyelitis in any of the sections taken at l-cm levels the brain and spinal cord.
TIME
of
(HOURS)
FIG. 1. Horse prothrombin activation with polylysine and EAE and 1.044 glycerol. o-o 0.8 mg polylysine + 2.0 mg prothrombin; -+ 1.6 mg prothrombin.
protein x-x
in 0.0544 Tris, pH 7.5, 3.5 mg EAE protein
DISCUSSION No immediate significance is attached to prothrombin activation in the genesisof EAE. A wealth of studies suggestsimmunologic mechanismsare involved in the etiology of the disease(Kies and Alvord, 1959b). There is, however, the possibility that encephalitogen-antibody complexes,as combinations of two basic proteins, might provid.e sufficient concentrations of basic groupings to influence local thrombin formations. The effects of thrombin on the accumulation of eosinophils at skin windows were recently studied by Riddle and Barnhart (1965), but little is known of reac-
578
K.
D. MILLER,
A. W.
PHELAN,
AND
D. N.
COLLINS
tions to thrombin in the CNS. Copley et al. (1966) suggest fibrinopeptides may enhance capillary permeability. The low temperature (18OC) and 1.0 M glycerol optima for prothrombin activation in the presence of the encephalitogen, as well as its independence from lipid and calcium ion effects, are characteristic of a variety of synthetic, so-called “nonenzymic” prothrombin activators (Miller, 1960; Miller et al., 1961; Miller, to be published). That the brain protein functions in a similar manner is predictable from its high basicity; analyses indicate the highly purified guinea pig encephalitogen preparations contain 12.8% arginine and 10.70/, lysine (Kies, 1965b). The heat stability of the prothrombin activating property of the brain protein preparation is also characteristic of the encephalitogenic activity (Caspary and Field, 1965). The prothrombin activating characteristic of the encephalitogen is significant in that a brain constituent other than phospholipid displays thromboplastic properties. In the present work the encephalitogen-prothrombin interaction is compared to a reference polylysine-prothrombin reaction. The differing rate of activation at optimum conditions for each substance reacting with the same prothrombin preparation is a common finding among many synthetic and naturally occurring basic macromolecules (Miller and LeGere, 1963 ; Miller, to be published). SUMMARY Preliminary evidence is presented describing the prothrombin-activating property encephalitogenic brain protein preparations. The effect of the encephalitogen activation resembles that of polylysine and other basic polymers.
of heat-stable, on prothrombin
REFERENCES ALVORD, E. C., JR. (1959). Discussion of techniques for quantitation of encephalitogenic activity Encephalomyelitis” (M. W. Kies and E. C. Alvord, Jr., in experimental animals. In “‘Allergic’ eds.), pp. 231-252. Thomas, Springfield, Ill. CASPARY, E. A., and FIELD, E. J. (1965). An encephalitogenic protein of human origin; some chemical and biological properties. Ann. N.Y. Acad. Sci. l.22, 182-198. COPLEY, A. L., HANIG, J. P., LUCHINI, B. W., and ALLEN, R. L., JR. (1966). Capillary permeability enhancing action of fibrinopeptides isolated during fibrin monomer formation. Federation Proc. 25, 446. FREUND, J,, STERN, E. R., and PISANI, T. M. (1947). Isoallergic encephalomyelitis and radiculitis in guinea pigs after one injection of brain and mycobacteria in water-in-oil emulsion. I. Immunol. 57, 179-194. HONEGGER, C. G. (1965). Studies on basic proteins from the central nervous system. Ann. N.Y. Acad. Sci. 122, 199-208. Krxs, M. W. (1965a). Chemical studies on an encephalitogenic protein from guinea pig brain. Ann. N.Y. Acad. Sci. 122, 161-170. KIES, M. W. (1965b). Amino acid analysis of encephalitogenic protein prepared from defatted guinea pig brain. Discussion of An Encephalitogenic Protein of Human Origin; Some Chemical and Biological Properties by E. A. Caspary and E. J. Field. Ann. N.Y. Acad. Sci. 122, 194. Krxs, M. W., and ALVORD, E. C., JR., eds. (1959a). “ ‘Allergic’ Encephalomyelitis.” Thomas, Springfield, Ill. Kms, M. W., and ALVORD, E. C., JR. (1959b). Encephalitogenic activity in guinea pigs of watersoluble protein fractions of nervous tissue. In “ ‘Allergic’ Encephalomyelitis” (M. W. Kies and E. C. Alvord, Jr., eds.), pp. 293-299. Thomas, Springfield, Ill. Kms, M. W., MURPHY, J. B., and ALVORD, E. C., JR. (1960). Fractionation of guinea pig brain protein with an encephalitogenic activity. Federation Proc. 19, 207. MILLER, K. D. (1960). The nonenzymic activation of prothrombin by polylysine. J. Biol. Chem. 235, PC63-64.
EXPERIMENTAL
ALLERGIC
ENCEPHALOMYELITIS
579
MILLER, K. D., to be published. Nonenzymic phase of prothrombin activation. MILLER, K. D., and LEGERE, M. (1963). Some characteristics of nonenzymic prothrombin activation. Federation Proc. 22, 164. MILLER, K. D., and MCGARRAHAN, J. F., to be published. Comparative biochemistry of equine and bovine prothrombins. MILLER, K. D., COPELAND, W. H., and MCGARRAHAN, J. F. (1961). Agents providing nonenzymic prothrombin activation. Proc. Sot. Exptl. Biol. Med. 108, 117-119. R~DLE, J. M., and BARNHART, M. I. (1965). The eosinophil as a source for profibrino!ysin in acute inflammation. Blood 26, 776-794. ROBOZ, E., and HENDERSON, M. (1959). Preparation and properties of water-soluble proteins encephalomyelitic activity. In “ ‘Allergic’ Encephalomyelitis” from bovine cord with “a.Ilergic” (M. W. Kies, E. C. Alvord, Jr., eds.), pp. 281-292. Charles C. Thomas, Springfield, 111. WARE, A. G., and SEEGERS, W. H. (1949). Two-stage procedure for the quantitative determination of prothrombin concentration. Am. /. Clin. Pathol. 19, 471-482.
AND
Prothrombin
MOLECULAR
Activation K. D. MILLER,
Division
5, 575479
PATHOLOGY
by
(1966)
Encephalitogenic
A. W. PHELAN,
Brain
Protein1
AND D. N. COLLINS
of Laboratories and Research, New York State Department of Health,’ Department of Pathology, Albany Medical College, Albany, New York” Received
August
II,
and
1966
Experimental allergic encephalomyelitis (EAE) in guinea pigs, produced by inoculation of products of brain or spinal cord along with Freund’s complete adjuvant, was recently attributed, amqng other fractions, to a lipid-free brain protein (Kies and Alvord, 1959a; Roboz and Henderson, 1959; Kies et al., 1960; Caspary and Field, 196.5; Honegger, 1965). This protein is strongly basic (Caspary and Field, 1965; Honegger, 1965) and as such is a potential participant in the so-called nonenzymic phase of prothrombin activation (Miller, 1960; Miller et al., 1961). The present work describes the prothrombin-activating activity of encephalitogenic brain protein preparations. MATERIALS
AND METHODS
Procedures for preparation of the EAE protein followed largely those of Kies ( 1965a). In a typical preparation, 25 guinea pig brains were homogenizedwith cold (4’C) saline in a glasshomogenizer with Teflon pestle, and washed three times with cold saline, with centrifugation of the sediment each time at 6500 X g. The residue was ground with cold (--1O’C) acetone until nearly dry, extracted three times with 20 volumes of cold (4’C) chloroform:methanol (2: l), then three times with acetone to remove residual chloroform. The residue (5.13 gm) was stirred 1 hour at 4’C with 110 ml of water and centrifuged at 6500 x: g for 30 minutes. The wet, insoluble residue was then stirred for 4 days in 235 ml of 0.01 N HCl at 4°C. Centrifugation (6500 X y; 30 minutes) gave 240 ml of extract of pH 1.5. Dry ammonium sulfate (85.5 gm) was added slowly with continuous stirring at 3°C and the precipitate formed in 1 hour was recovered by centrifugation at 6500 X g. The precipitate was dissolved in 50 ml of distilled water (4°C). The pH was adjusted to 7.0, and the solution was dialyzed against six changesof distilled water, each water volume 180 times the volume of the extract. Lyophilization of the supernatant yielded 309 mg of dry material. Most investigations of EAE and prothrombin-activating activities were conducted with this type of preparation. Further purification, such as by Sephadex G-100 (Caspary and Field, 1965) and G-150 chromatography, was achieved but quantities were insufficient for adequate testing. The secondof two peaks recovered in G-150 chroma1 Supported in part by grants-in-aid from the National Health (HE-09902) and the American National Red Cross 2 Thrombosis and Vascular Disease Research Laboratory. 3 Affiliated with the Institute of Preventive Medicine.
Heart Institute, (K-29915).
National
Institutes
of
576
K.
D.
MILLER,
A.
W.
PHELAN,
AND
D.
N.
COLLINS
tography (0.1 M acetate buffer, pH 5.0) of a human brain preparation crystallized on concentration by vacuum dialysis was also insufficient in quantity for study. Horse prothrombin was prepared according to Miller and McGarrahan (to be published). Synthetic poly-L-lysine hydrobromide (lot PLH 6101) was obtained from Schwartz BioResearch, Inc., Orangeburg, New York. Encephalitogenic activity (EAE) was determined on weighed samples of the dry brain fractions homogenized in Freund’s adjuvant containing 5 mg of BCG (Freund et al., 1947) per milliliter. Portions of the suspensions (0.1 ml), each containing 0.5 mg of BCG and varying amounts of encephalitogen, were injected once intracutaneously over the sternum of SOO- to 800-gm male guinea pigs. Controls included injections of adjuvant without encephalitogen. Occurrence of EAE was determined by the development of physical signs and microscopic pathology. The animals were observed daily for alterations in general physical condition, weight loss, altered righting reflex, and paralysis. As paralytic signs developed, animals were sacrificed and the brain and spinal cord fixed in Bouin’s fluid. Twenty-eight days post-injection the survivors were also sacrificed. The CNS samples from all animals were sectioned and examined for microscopic evidence of encephalomyelitis. The prothrombin activating effect of the encephalitogenic preparations was studied at varying pH, temperature, and NaCl, CaCl>, and glycerol concentrations, Optimum reaction conditions were compared to those of polylysine as well as other synthetic and naturally occurring activators (Miller, 1960; Miller et al., 1961). The reaction tubes contained 1725 Iowa units of horse prothrombin per milliliter ( 1.6 mg/ml) and l-3 mg/ml encephalitogen. After determination of optimum conditions of pH, temperature, etc., the encephalitogenic protein was varied to determine the optimum ratio to prothrombin for activation and also the smallest amount necessary for initiation of activation ( [Ali; Miller and LeGere, 1963). Controls included mixtures of prothrombin in the optimum solvent but without encephalitogen. The polylysine effect on prothrombin activation serves as a reference reaction. The prothrombin activating effect of the encephalitogen was also determined after heating a 1s solution at 100°C for 15 minutes. Prothrombin and thrombin were determined according to Ware and Seegers ( 1949). The reference standard was the prothrombin (300 Iowa units/ml) contained in a pool of oxalated bovine plasma (1 part 1.34% sodium oxalate to 9 parts blood). RESULTS Injection of two guinea pig encephalitogenic protein preparations into guinea pigs produced symptoms and microscopic lesions as indicated in Table I. Because of the large amounts of material needed for studies of prothrombin activation, a titration to determine the EDhO, the smallest amount causing the average disease in 50% of a larger number of animals (Alvord, 1959), was not determined. Horse prothrombin was activated with the encephalitogenic protein preparations. Optimal conditions were at pH 7.5, 18”C, 1.0 M glycerol and 0.05 ionic strength. Calcium chloride had no effect on the reaction, and activation progressed as well in EDTA as in Tris buffer. Subsequent reactions were carried out on solutions of prothrombin and the encephalitogenic materials in 0.05 M Tris buffer, pH 7.5, containing 1.0 M glycerol, at 18°C.
EXPERIMENTAL
ALLERGIC
ENCEPHALOMYELITIS
577
The optimum weight ratio of one encephalitogen preparation to prothrombin was 5: 1, for a second preparation, 2 : 1. The [Ai] for this second preparation, the smallest amount necessary for any activation of prothrombin in 24 hours, was 0.77 mg activator per milligram of horse prothrombin. Extraction of the prothrombin with chloroform:methanol (2: 1) did not affect these values. The relative rates of activation at optimum ratios of the encephalitogen and polylysine to prothrombin are depicted in Fig. 1. Heating the encephalitogen to 1OO’C d.id not affect its prothrombin activating properties. TABLE GUINEA
PIG ENCEPHALOMYELITIS
and
No. animals injected
Preparation amount
1
FROM
HOMOLWOUS
BRAIN
Incidence of physical sign9
PROTEIN
Incidence of microscopic lesionsf)
I
(2.5 mg)
5
3!5
s/s
I II None
(1.25 mg) (0.50 mg) (BCG-adjuvant
5 5
3/5 3/5
4/5 415
O/IS
O/IS
15
only)
a Physical signs = altered righting reflexes, paralysis, general inactivity with weight loss. b Microscopic lesions = meningoencephalomyelitis in any of the sections taken at l-cm levels the brain and spinal cord.
TIME
of
(HOURS)
FIG. 1. Horse prothrombin activation with polylysine and EAE and 1.044 glycerol. o-o 0.8 mg polylysine + 2.0 mg prothrombin; -+ 1.6 mg prothrombin.
protein x-x
in 0.0544 Tris, pH 7.5, 3.5 mg EAE protein
DISCUSSION No immediate significance is attached to prothrombin activation in the genesisof EAE. A wealth of studies suggestsimmunologic mechanismsare involved in the etiology of the disease(Kies and Alvord, 1959b). There is, however, the possibility that encephalitogen-antibody complexes,as combinations of two basic proteins, might provid.e sufficient concentrations of basic groupings to influence local thrombin formations. The effects of thrombin on the accumulation of eosinophils at skin windows were recently studied by Riddle and Barnhart (1965), but little is known of reac-
578
K.
D. MILLER,
A. W.
PHELAN,
AND
D. N.
COLLINS
tions to thrombin in the CNS. Copley et al. (1966) suggest fibrinopeptides may enhance capillary permeability. The low temperature (18OC) and 1.0 M glycerol optima for prothrombin activation in the presence of the encephalitogen, as well as its independence from lipid and calcium ion effects, are characteristic of a variety of synthetic, so-called “nonenzymic” prothrombin activators (Miller, 1960; Miller et al., 1961; Miller, to be published). That the brain protein functions in a similar manner is predictable from its high basicity; analyses indicate the highly purified guinea pig encephalitogen preparations contain 12.8% arginine and 10.70/, lysine (Kies, 1965b). The heat stability of the prothrombin activating property of the brain protein preparation is also characteristic of the encephalitogenic activity (Caspary and Field, 1965). The prothrombin activating characteristic of the encephalitogen is significant in that a brain constituent other than phospholipid displays thromboplastic properties. In the present work the encephalitogen-prothrombin interaction is compared to a reference polylysine-prothrombin reaction. The differing rate of activation at optimum conditions for each substance reacting with the same prothrombin preparation is a common finding among many synthetic and naturally occurring basic macromolecules (Miller and LeGere, 1963 ; Miller, to be published). SUMMARY Preliminary evidence is presented describing the prothrombin-activating property encephalitogenic brain protein preparations. The effect of the encephalitogen activation resembles that of polylysine and other basic polymers.
of heat-stable, on prothrombin
REFERENCES ALVORD, E. C., JR. (1959). Discussion of techniques for quantitation of encephalitogenic activity Encephalomyelitis” (M. W. Kies and E. C. Alvord, Jr., in experimental animals. In “‘Allergic’ eds.), pp. 231-252. Thomas, Springfield, Ill. CASPARY, E. A., and FIELD, E. J. (1965). An encephalitogenic protein of human origin; some chemical and biological properties. Ann. N.Y. Acad. Sci. l.22, 182-198. COPLEY, A. L., HANIG, J. P., LUCHINI, B. W., and ALLEN, R. L., JR. (1966). Capillary permeability enhancing action of fibrinopeptides isolated during fibrin monomer formation. Federation Proc. 25, 446. FREUND, J,, STERN, E. R., and PISANI, T. M. (1947). Isoallergic encephalomyelitis and radiculitis in guinea pigs after one injection of brain and mycobacteria in water-in-oil emulsion. I. Immunol. 57, 179-194. HONEGGER, C. G. (1965). Studies on basic proteins from the central nervous system. Ann. N.Y. Acad. Sci. 122, 199-208. Krxs, M. W. (1965a). Chemical studies on an encephalitogenic protein from guinea pig brain. Ann. N.Y. Acad. Sci. 122, 161-170. KIES, M. W. (1965b). Amino acid analysis of encephalitogenic protein prepared from defatted guinea pig brain. Discussion of An Encephalitogenic Protein of Human Origin; Some Chemical and Biological Properties by E. A. Caspary and E. J. Field. Ann. N.Y. Acad. Sci. 122, 194. Krxs, M. W., and ALVORD, E. C., JR., eds. (1959a). “ ‘Allergic’ Encephalomyelitis.” Thomas, Springfield, Ill. Kms, M. W., and ALVORD, E. C., JR. (1959b). Encephalitogenic activity in guinea pigs of watersoluble protein fractions of nervous tissue. In “ ‘Allergic’ Encephalomyelitis” (M. W. Kies and E. C. Alvord, Jr., eds.), pp. 293-299. Thomas, Springfield, Ill. Kms, M. W., MURPHY, J. B., and ALVORD, E. C., JR. (1960). Fractionation of guinea pig brain protein with an encephalitogenic activity. Federation Proc. 19, 207. MILLER, K. D. (1960). The nonenzymic activation of prothrombin by polylysine. J. Biol. Chem. 235, PC63-64.
EXPERIMENTAL
ALLERGIC
ENCEPHALOMYELITIS
579
MILLER, K. D., to be published. Nonenzymic phase of prothrombin activation. MILLER, K. D., and LEGERE, M. (1963). Some characteristics of nonenzymic prothrombin activation. Federation Proc. 22, 164. MILLER, K. D., and MCGARRAHAN, J. F., to be published. Comparative biochemistry of equine and bovine prothrombins. MILLER, K. D., COPELAND, W. H., and MCGARRAHAN, J. F. (1961). Agents providing nonenzymic prothrombin activation. Proc. Sot. Exptl. Biol. Med. 108, 117-119. R~DLE, J. M., and BARNHART, M. I. (1965). The eosinophil as a source for profibrino!ysin in acute inflammation. Blood 26, 776-794. ROBOZ, E., and HENDERSON, M. (1959). Preparation and properties of water-soluble proteins encephalomyelitic activity. In “ ‘Allergic’ Encephalomyelitis” from bovine cord with “a.Ilergic” (M. W. Kies, E. C. Alvord, Jr., eds.), pp. 281-292. Charles C. Thomas, Springfield, 111. WARE, A. G., and SEEGERS, W. H. (1949). Two-stage procedure for the quantitative determination of prothrombin concentration. Am. /. Clin. Pathol. 19, 471-482.