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Heterogeneity of human adult and fetal fibrinogen: Detection of derivatives indicative of thrombin proteolysis
398
BIOCHIMICA ET BIOPHYSICAACTA
BBA 36258 H E T E R O G E N E I T Y OF HUMAN ADULT AND F E T A L F I B R I N O G E N : DETECTION OF DERIVATIVES INDICATIVE OF THROMBIN PROTEOLYSIS
DONALD A. MILLS AND SIMON KARPATK1N Department of Medicine, New York University Medical Center, 55 ° First Avenue, New York, N . Y . zoor6 ( U . S . A . ) (Received July 3rd, 1972)
SUMMARY
Low solubility, bulk and high solubility fibrinogen fractions from human adult and cord plasmas were compared. Plasma was collected and fractionated in the presence of e-aminocaproic acid, and plasmin degradation products were not detected. Corresponding adult and cord fibrinogen fractions were similar on sodium dodecyl sulfate gel electrophoresis, in percentage clottability and in hexose content. However, the adult low solubility and bulk fractions clotted more rapidly with thrombin than the corresponding cord fractions. In addition, adult fibrinogen contained a greater proportion than cord fibrinogen of the low solubility fraction (mean 39% as compared to 32°,'o, P < o.oi). Low solubility fractions characteristically had the shortest thrombin clotting times and formed coagula when frozen and thawed. As determined by sodium dodecyl sulfate gel electrophoresis, the coagula contained about equal amounts of a(A)-chain and a-chain, the latter resulting presumably from liberation of fibrinopeptide A by thrombin.
INTRODUCTION
Our previous study 1 has shown that several different fibrinogen components in normal adult plasma can be distinguished by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Reports2, s that fibrinogen of the human fetus differs from that of the adult have raised the possibility that one or more components of adult fibrinogen may actually be fetal fibrinogen persisting into adult life. Therefore, in the present work we have fractionated and compared adult and cord fibrinogens. It has been suggested 4 that prolonged thrombin clotting times of cord plasmas may be due to fibrinogen degradation products rather than to a special characteristic of cord blood fibrinogen. Our experience with adult fibrinogenl, 5 has shown that sodium dodecyl sulfate gel electrophoresis is quite sensitive in detecting alteration of fibrinogen caused by proteolysis. Biochim. ]Biophys. -dcta, 285 (I972) 398-403
HETEROGENEITY OF FIBRONOGEN
399
METHODS AND MATERIALS
9 vol. of blood were mixed with I vol. of ACD-A anticoagulant containing o. I M e-aminocaproic acid, an inhibitor of fibrinolysis. Placental (cord) blood samples were obtained immediately after delivery and with the umbilical cord clamped, by venepuncture at the juncture of placenta and cord. Blood was cooled in ice water and centrifuged at 4 °C. Plasma was stored frozen prior to fractionation. Pairs of adult and cord plasmas were fractionated and analyzed together. To each plasma was added (dry) e-aminocaproic acid to 0.2 M and glycine to 2.1 M. After 2 h at o °C the precipitated fibrinogen was spun down and dissolved in one-third plasma volume of 0.05 M citrate-o.2 M e-aminocaproic acid, pH 7.4. After a second precipitation with glycine the fibrinogen concentration was adjusted to 3 mg/ml in the citratee-aminoeaproic acid buffer. Low solubility fibrinogen was precipitated by addition of 2 vol. of 0.75 M glycine-3% ethanol at o °C and centrifuged after 2 h. The bulk fibrinogen fraction was precipitated from the supernate by addition of ethanol to 8% at o °C. Similarly, high solubility fibrinogen was precipitated by addition of ethanol to 20% at --5 °C. Fibrinogen concentration and clottability were determined using absorbance at 280 nm ~. Clotting time of plasma (0.2 ml) was determined by addition of 0.2 ml bovine thrombin (Parke-Davis), 4 units/ml in buffer (o.i M e-aminocaproic acid0.075 M NaCl-o.oI 5 M sodium phosphate, pH 6.4) at room temperature. Fibrinogen fraction clotting time was determined using 0.2 ml of the fraction (2 mg/ml in citratee-aminocaproic acid buffer) and o.2 ml of thrombin (2o units/ml). For sodium dodecyl sulfate gel electrophoresis clots were prepared with purified bovine thrombin, in the presence of p-hydroxymercuriphenylsulfonic acid as previously described 1. Hexose was assayed by the phenol-sulfuric acid method 7. RESULTS AND DISCUSSION
Fractionation of adult and cord fibrinogen Individual fibrinogen preparations from 12 adult and 12 cord plasmas were fractionally precipitated into low solubility, bulk, and high solubility fractions. Protein distribution and some properties of the fractions are summarized in Table I. With both adult and cord samples the low solubility fractions were similar in percentage clottability and hexose content to bulk fractions, but clotted more rapidly. High solubility fractions showed an increased hexose content, decreased percentage clottability, and prolonged clotting times difficult to measure under these conditions. In each adult and cord sample pair the proportion of low solubility fibrinogen (0-2% ethanol fraction) was greater for the adult (mean 39%) than for the cord (mean 32%), with a significance of P < o.oi. In addition, both the low solubility and bulk fractions of adult samples clotted more rapidly than tile corresponding cord sample fractions*. When either bulk or high solubility fractions were frozen and thawed, solutions I n a few e x p e r i m e n t s in which t h r o m b i n c o n c e n t r a t i o n was doubled, adult high solubility fractions also clotted more rapidly t h a n corresponding cord fractions. The significance of these results is n o t k n o w n because of scarcity of material for f u r t h e r testing.
Biochim. Biophys. Acta, 285 (1972) 398-403
400
D. A. MILLS, S. KARPATKIN
TABLE I COMPARISON OF ADULT AND CORD FIBRINOGEN FRACTIONS FROM 12 PLASMA SAMPLE PAIRS Glycine p r e c i p i t a t e s f r o m p l a s m a s were dissolved a n d r e p r e c i p i t a t e d into low solubility, b u l k a n d h i g h solubility fibrinogen f r a c t i o n s a t 0-2%o, 2-8%o a n d 8 - 2 0 % ethanol, respectively. M e a n v a l u e s ± S.D. for t h e m e a s u r e m e n t s are given. Significant differences b e t w e e n c o r r e s p o n d i n g a d u l t a n d cord f r a c t i o n s are n o t e d : ~P < o.ot, * ' P < 0.025 b y t h e S t u d e n t t-test.
Fibrinogen fraction
% of total
Clotting time (s)
% Clottability
% Hexose
L o w solubility
39 32 53 60 8 8
(i5)* (~:7)* (±5)* (±6)* (±2)
14 ( ± 4)** 19 ( ± 4)** 51 (2:19)* 92 (--30)* >ioo
94 ( ± 2 ) 95 ( ± 2 ) 96 ( ± 2 ) 97 (x:2) 89*
(-_E2)
>IOO
90~"
1. 4 1. 5 1. 4 1. 4 1.8 1.9
Adult Cord Bulk Adult Cord H i g h solubility A d u l t Cord
(_~o.i) (+o.i) (io.I) (±o.i) (±o.i) (±o.I)
* Single d e t e r m i n a t i o n on pooled sample.
remained relatively clear. In contrast, when low solubility fractions were frozen and thawed, coagula formed which could be removed with a glass rod and blotted to give a weak fibrin-like net. In a single experiment the coagulum from pooled adult plasma amounted to 7%0 of the total fibrinogen (clottable protein) compared to 4% for the corresponding pooled cord plasma fraction.
Comparison of adult and cord fibrinogen fractions on sodium dodecyl sulfate gel electrophoresis In Fig. I the low solubility-fraction coagula from pooled adult and pooled cord plasmas are compared with bulk fractions on sodium dodecyl sulfate gel electrophoresis, after reduction of disulfide bonds. Fig. IA shows adult bulk fibrinogen which has major and minor a(A)-polypeptide chains along with fl(B)- and v-chains. After clotting with thrombin (release of A and B fibrinopeptides) the major and minor a-chains and E-chain migrate closer to the v-chain (Fig. IB). Fig. IC is a mixture of equal amounts of bulk fraction fibrinogen and fibrin, in which a(A)-chain and a-chain major bands are distinguishable. The/5(B)- and E-chains form a broad band which is not well resolved. Fig. ID shows the coagulum from the adult low solubility fraction. The upper region contains two major bands of about equal intensity which in additional mixing experiments (not shown) were indistinguishable from the a(A)and a-chains. The mobility of the central band (molecular weight 59 ooo) corresponded to that of the /~(B)-chain, i.e. with the B-fibrinopeptide intact. The same results were found using cord fibrinogen fractions in place of adult fractions (Figs I E - I H ) . It should be noted that patterns of (whole) low solubility fractions (not shown) were not distinguishable from bulk fractions, presumably due to masking of small amounts of a-chain by excess a(A)-chain. A high-molecular-weight component was present in both adult and cord coagula (most easily seen in Fig. IH) and in trace amounts in the other fractions. This was found on gel to be indistinguishable from the v-chain dimer formed by activated Factor X l I I (fibrin stabilizing factor)S, 9. The high solubility fraction of adult fibrinogen is characterized by a decreased content of (intact) a(A)-chains s, as shown in Fig. 2A. The same was found for cord Biochim. Biophys. -4cta~ 285 (1972) 398-403
HETEROGENEITY OF FIBRINOGEN
T
401
....
and
(8),
Fig. I. Sodium dodecyl sulfate gel electrophoresis patterns of adult and cord low solubility coagula and bulk fibrinogen fractions after reduction of disulfide bonds (9% gels). A, adult bulk fraction fibrinogen; B, fibrin obtained by clotting adult bulk fibrinogen; C, mixture of adult bulk fibrinogen and fibrin; D, coagulum from adult low solubility fibrinogen (approx. 3° f,g protein electrophorized at 7 m.~ per gel for 18 h); E - H , as A-D except cord fractions (approx. 20/*g protein) rather than adult fractions. Arrow indicates the mobility of the v-chain dimer.
high solubility fraction (Fig. 2B). A mixture of adult and cord bulk fibrinogens (Fig. 2C) is shown for comparison. Fig. 2D is a mixture of adult and cord fibrins derived from the high solubility fractions. In additional mixing experiments with both reduced and non-reduced 1 samples, qualitative differences between corresponding adult and cord fibrinogen fractions were not demonstrable by sodium dodecyl sulfate gel electrophoresis. The same was true for whole plasma clots. As in Figs I and 2, slight quantitative variation was observed but this was common to both adult and cord fractions when comparing several individual samples. These studies show that the heterogeneity of human adult fibrinogen demonstrable by sodium dodecyl sulfate gel electrophoresis 1,s is not due to persistence of fetal fibrinogen. It has been reported2, a that human cord fibrinogen has a higher phosphorus content, is eluted in a higher salt concentration from DEAE-cellulose, and has a prolonged thrombin clotting time relative to adult fibrinogen. Differences in tryptic peptide maps have also been noted. Recently, fibrinogen preparations from adult and fetal sheep have been compared ~° and the authors have discussed the possible affects of thrombin activation and fibrinogenolysis in vitro on the fibrinogen clotting times. In the present study, samples were collected and fractionated in the presence of e-aminocaproic acid, and plasmin digest products detectable by sodium dodecyl sulfate eleetrophoresisl, s were not found in either adult or cord fractions. However, Biochim. Biophys. ~4cta, 285 (1972) 398 403
402
D . A . MILLS, S. KARPATKIN
A
B
C
O
Fig. 2. S o d i u m dodecyl sulfate gel electrophoresis p a t t e r n s of a d u l t a n d cord bulk a n d h i g h solubility fibrinogen f r a c t i o n s a f t e r r e d u c t i o n of disulfide b o n d s (approx. 45 t~g p r o t e i n electrophorized a t 7 m A per gel for I8 h). A, a d u l t high solubility fibrinogen; B, cord h i g h solubility fibrinogen; C, m i x t u r e of a d u l t a n d cord b u l k fibrinogens ; D, m i x t u r e of a d u l t a n d cord fibrins o b t a i n e d b y clotting h i g h solubility fibrinogens. B a n d d e s i g n a t i o n s are as Fig. i.
the coagula from low solubility fractions do appear to represent derivatives of limited thrombin action, containing about equal amounts of a(A)-chains and a-chains by the criterion of sodium dodecyl sulfate gel electrophoresis. The presence of a-chains would indicate removal of fibrinopeptide A by the limited action of thrombin*. The more extensive action of thrombin (removal of fibrinopeptide B to form fl-chains) was not detected**. A fibrinogen derivative deficient in fibrinopeptide A but not B (cryoprofibrin) was first characterized from plasma of endotoxin-treated rabbits 11. The material seemed to be closely related to a previously described heparin-precipitable fraction 12-14, and it is noteworthy that levels of heparin-precipitable fraction were found to be very low in plasma of newborn infants relative to adult human levels 14. It m a y be doubted that appreciable amounts of thrombin-activated derivatives are present normally in vivo, and such materials detected in the present work could have arisen under the conditions of isolation. In either case, if a greater amount of thrombin-activated derivatives were present in the adult samples, this could explain the differences between adult and cord fibrinogen fractions in precipitability and clotting time (Table I). However, structural differences not detectable by sodium dodecyl sulfate gel electrophoresis could also explain these findings. It should be * A similar a l t e r a t i o n of a ( A ) - c h a i n s b y a p l a s m a e n z y m e o t h e r t h a n t h r o m b i n h a s n o t been r e p o r t e d and, specifically, p l a s m i n a c t i o n m a y be ruled outl, 5. *" T h e presence (Fig. I) of m a t e r i a l i n d i s t i n g u i s h a b l e f r o m y - c h a i n d i m e r p r o d u c e d b y Factor X [ 11 indirectly indicates t h r o m b i n action, since F a c t o r X I I [ z y m o g e n is a c t i v a t e d b y t h r o m b i n proteolysis 9.
Biochim. Biophys..4cta, 285 (1972) 398-403
403
HETEROGENEITY OF FIBRINOGEN
n o t e d t h a t t h e a b i l i t y o f s o d i u m d o d e c y l s u l f a t e gel e l e c t r o p h o r e s i s t o d e t e c t t h r o m b i n - a c t i v a t e d d e r i v a t i v e s m a y b e o f use in t h e c h a r a c t e r i z a t i o n o f t h o s e f i b r i n o g e n related materials
postulated
to
be indicative of intravascular
coagulation
(e.g.
r e f s 15, 16). ACKNOWLEDGEMENTS This work was supported by a grant from the New York Heart Association.
REFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16
D. Mills and S. Karpatkin, Biochim. Biophys. Acta, 251 (1971) 121. I. Witt, H. Mr'filer and W. Kt~nzer, Thromb. Diath. Haemorrh., 22 (1969) ioi. I. Witt and H. Mailer, Biochim. Biophys. Acta, 221 (197 o) 402. A. von Felten and P. VV. Straub, Thromb. Diath. Haemorrh.. 22 (I969) 273. D. A. Mills, Biochim. Biophys. Acta, 263 (1972) 619. D. A. Mills, R. Coyne, B. Pollara and R. W. Von Korff, Biochim. Biophys. Acta, 86 (1964) 527 . M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith, Anal. Chem., 28 (1956) 35 ° • D. Mills and S. Karpatkin, Biochem. Biophys. Res. Commun., 4 ° (197 o) 206. M. L. Schwartz, S. V. Pizzo, R. L. Hill and P. A. McKee, J. Biol. Chem., 246 (1971) 5851. W. Loly, L. G. Israels, A. J. Bishop and E. D. Israels, Thromb. Diath. Haemorrh., 26 (1971) 526. J. R. Shainoff and 1. H. Page, Circ. Res., 8 (196o) lOl 3 . L. Thomas, R. T. Smith and R. Von Korff, Proc. Soc. Exp. Biol. Med., 86 (1954) 813. R. T. Smith and R. W. Von Korff, J. Clin. Invest., 36 (1957) 596. R. T. Smith, J. Clin. Invest., 36 (1957) 605. P. A. McKee, J. M. Kalbfleisch and R. M. Bird, J. Lab. Clin. Med., 61 (1963) 203. S. Niewiarowski and V. Gurewich, J. Lab. Clin. Med., 77 (1971) 665.
Biochim. Biophys. Acta, 285 (1972) 398-4o3
BIOCHIMICA ET BIOPHYSICAACTA
BBA 36258 H E T E R O G E N E I T Y OF HUMAN ADULT AND F E T A L F I B R I N O G E N : DETECTION OF DERIVATIVES INDICATIVE OF THROMBIN PROTEOLYSIS
DONALD A. MILLS AND SIMON KARPATK1N Department of Medicine, New York University Medical Center, 55 ° First Avenue, New York, N . Y . zoor6 ( U . S . A . ) (Received July 3rd, 1972)
SUMMARY
Low solubility, bulk and high solubility fibrinogen fractions from human adult and cord plasmas were compared. Plasma was collected and fractionated in the presence of e-aminocaproic acid, and plasmin degradation products were not detected. Corresponding adult and cord fibrinogen fractions were similar on sodium dodecyl sulfate gel electrophoresis, in percentage clottability and in hexose content. However, the adult low solubility and bulk fractions clotted more rapidly with thrombin than the corresponding cord fractions. In addition, adult fibrinogen contained a greater proportion than cord fibrinogen of the low solubility fraction (mean 39% as compared to 32°,'o, P < o.oi). Low solubility fractions characteristically had the shortest thrombin clotting times and formed coagula when frozen and thawed. As determined by sodium dodecyl sulfate gel electrophoresis, the coagula contained about equal amounts of a(A)-chain and a-chain, the latter resulting presumably from liberation of fibrinopeptide A by thrombin.
INTRODUCTION
Our previous study 1 has shown that several different fibrinogen components in normal adult plasma can be distinguished by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Reports2, s that fibrinogen of the human fetus differs from that of the adult have raised the possibility that one or more components of adult fibrinogen may actually be fetal fibrinogen persisting into adult life. Therefore, in the present work we have fractionated and compared adult and cord fibrinogens. It has been suggested 4 that prolonged thrombin clotting times of cord plasmas may be due to fibrinogen degradation products rather than to a special characteristic of cord blood fibrinogen. Our experience with adult fibrinogenl, 5 has shown that sodium dodecyl sulfate gel electrophoresis is quite sensitive in detecting alteration of fibrinogen caused by proteolysis. Biochim. ]Biophys. -dcta, 285 (I972) 398-403
HETEROGENEITY OF FIBRONOGEN
399
METHODS AND MATERIALS
9 vol. of blood were mixed with I vol. of ACD-A anticoagulant containing o. I M e-aminocaproic acid, an inhibitor of fibrinolysis. Placental (cord) blood samples were obtained immediately after delivery and with the umbilical cord clamped, by venepuncture at the juncture of placenta and cord. Blood was cooled in ice water and centrifuged at 4 °C. Plasma was stored frozen prior to fractionation. Pairs of adult and cord plasmas were fractionated and analyzed together. To each plasma was added (dry) e-aminocaproic acid to 0.2 M and glycine to 2.1 M. After 2 h at o °C the precipitated fibrinogen was spun down and dissolved in one-third plasma volume of 0.05 M citrate-o.2 M e-aminocaproic acid, pH 7.4. After a second precipitation with glycine the fibrinogen concentration was adjusted to 3 mg/ml in the citratee-aminoeaproic acid buffer. Low solubility fibrinogen was precipitated by addition of 2 vol. of 0.75 M glycine-3% ethanol at o °C and centrifuged after 2 h. The bulk fibrinogen fraction was precipitated from the supernate by addition of ethanol to 8% at o °C. Similarly, high solubility fibrinogen was precipitated by addition of ethanol to 20% at --5 °C. Fibrinogen concentration and clottability were determined using absorbance at 280 nm ~. Clotting time of plasma (0.2 ml) was determined by addition of 0.2 ml bovine thrombin (Parke-Davis), 4 units/ml in buffer (o.i M e-aminocaproic acid0.075 M NaCl-o.oI 5 M sodium phosphate, pH 6.4) at room temperature. Fibrinogen fraction clotting time was determined using 0.2 ml of the fraction (2 mg/ml in citratee-aminocaproic acid buffer) and o.2 ml of thrombin (2o units/ml). For sodium dodecyl sulfate gel electrophoresis clots were prepared with purified bovine thrombin, in the presence of p-hydroxymercuriphenylsulfonic acid as previously described 1. Hexose was assayed by the phenol-sulfuric acid method 7. RESULTS AND DISCUSSION
Fractionation of adult and cord fibrinogen Individual fibrinogen preparations from 12 adult and 12 cord plasmas were fractionally precipitated into low solubility, bulk, and high solubility fractions. Protein distribution and some properties of the fractions are summarized in Table I. With both adult and cord samples the low solubility fractions were similar in percentage clottability and hexose content to bulk fractions, but clotted more rapidly. High solubility fractions showed an increased hexose content, decreased percentage clottability, and prolonged clotting times difficult to measure under these conditions. In each adult and cord sample pair the proportion of low solubility fibrinogen (0-2% ethanol fraction) was greater for the adult (mean 39%) than for the cord (mean 32%), with a significance of P < o.oi. In addition, both the low solubility and bulk fractions of adult samples clotted more rapidly than tile corresponding cord sample fractions*. When either bulk or high solubility fractions were frozen and thawed, solutions I n a few e x p e r i m e n t s in which t h r o m b i n c o n c e n t r a t i o n was doubled, adult high solubility fractions also clotted more rapidly t h a n corresponding cord fractions. The significance of these results is n o t k n o w n because of scarcity of material for f u r t h e r testing.
Biochim. Biophys. Acta, 285 (1972) 398-403
400
D. A. MILLS, S. KARPATKIN
TABLE I COMPARISON OF ADULT AND CORD FIBRINOGEN FRACTIONS FROM 12 PLASMA SAMPLE PAIRS Glycine p r e c i p i t a t e s f r o m p l a s m a s were dissolved a n d r e p r e c i p i t a t e d into low solubility, b u l k a n d h i g h solubility fibrinogen f r a c t i o n s a t 0-2%o, 2-8%o a n d 8 - 2 0 % ethanol, respectively. M e a n v a l u e s ± S.D. for t h e m e a s u r e m e n t s are given. Significant differences b e t w e e n c o r r e s p o n d i n g a d u l t a n d cord f r a c t i o n s are n o t e d : ~P < o.ot, * ' P < 0.025 b y t h e S t u d e n t t-test.
Fibrinogen fraction
% of total
Clotting time (s)
% Clottability
% Hexose
L o w solubility
39 32 53 60 8 8
(i5)* (~:7)* (±5)* (±6)* (±2)
14 ( ± 4)** 19 ( ± 4)** 51 (2:19)* 92 (--30)* >ioo
94 ( ± 2 ) 95 ( ± 2 ) 96 ( ± 2 ) 97 (x:2) 89*
(-_E2)
>IOO
90~"
1. 4 1. 5 1. 4 1. 4 1.8 1.9
Adult Cord Bulk Adult Cord H i g h solubility A d u l t Cord
(_~o.i) (+o.i) (io.I) (±o.i) (±o.i) (±o.I)
* Single d e t e r m i n a t i o n on pooled sample.
remained relatively clear. In contrast, when low solubility fractions were frozen and thawed, coagula formed which could be removed with a glass rod and blotted to give a weak fibrin-like net. In a single experiment the coagulum from pooled adult plasma amounted to 7%0 of the total fibrinogen (clottable protein) compared to 4% for the corresponding pooled cord plasma fraction.
Comparison of adult and cord fibrinogen fractions on sodium dodecyl sulfate gel electrophoresis In Fig. I the low solubility-fraction coagula from pooled adult and pooled cord plasmas are compared with bulk fractions on sodium dodecyl sulfate gel electrophoresis, after reduction of disulfide bonds. Fig. IA shows adult bulk fibrinogen which has major and minor a(A)-polypeptide chains along with fl(B)- and v-chains. After clotting with thrombin (release of A and B fibrinopeptides) the major and minor a-chains and E-chain migrate closer to the v-chain (Fig. IB). Fig. IC is a mixture of equal amounts of bulk fraction fibrinogen and fibrin, in which a(A)-chain and a-chain major bands are distinguishable. The/5(B)- and E-chains form a broad band which is not well resolved. Fig. ID shows the coagulum from the adult low solubility fraction. The upper region contains two major bands of about equal intensity which in additional mixing experiments (not shown) were indistinguishable from the a(A)and a-chains. The mobility of the central band (molecular weight 59 ooo) corresponded to that of the /~(B)-chain, i.e. with the B-fibrinopeptide intact. The same results were found using cord fibrinogen fractions in place of adult fractions (Figs I E - I H ) . It should be noted that patterns of (whole) low solubility fractions (not shown) were not distinguishable from bulk fractions, presumably due to masking of small amounts of a-chain by excess a(A)-chain. A high-molecular-weight component was present in both adult and cord coagula (most easily seen in Fig. IH) and in trace amounts in the other fractions. This was found on gel to be indistinguishable from the v-chain dimer formed by activated Factor X l I I (fibrin stabilizing factor)S, 9. The high solubility fraction of adult fibrinogen is characterized by a decreased content of (intact) a(A)-chains s, as shown in Fig. 2A. The same was found for cord Biochim. Biophys. -4cta~ 285 (1972) 398-403
HETEROGENEITY OF FIBRINOGEN
T
401
....
and
(8),
Fig. I. Sodium dodecyl sulfate gel electrophoresis patterns of adult and cord low solubility coagula and bulk fibrinogen fractions after reduction of disulfide bonds (9% gels). A, adult bulk fraction fibrinogen; B, fibrin obtained by clotting adult bulk fibrinogen; C, mixture of adult bulk fibrinogen and fibrin; D, coagulum from adult low solubility fibrinogen (approx. 3° f,g protein electrophorized at 7 m.~ per gel for 18 h); E - H , as A-D except cord fractions (approx. 20/*g protein) rather than adult fractions. Arrow indicates the mobility of the v-chain dimer.
high solubility fraction (Fig. 2B). A mixture of adult and cord bulk fibrinogens (Fig. 2C) is shown for comparison. Fig. 2D is a mixture of adult and cord fibrins derived from the high solubility fractions. In additional mixing experiments with both reduced and non-reduced 1 samples, qualitative differences between corresponding adult and cord fibrinogen fractions were not demonstrable by sodium dodecyl sulfate gel electrophoresis. The same was true for whole plasma clots. As in Figs I and 2, slight quantitative variation was observed but this was common to both adult and cord fractions when comparing several individual samples. These studies show that the heterogeneity of human adult fibrinogen demonstrable by sodium dodecyl sulfate gel electrophoresis 1,s is not due to persistence of fetal fibrinogen. It has been reported2, a that human cord fibrinogen has a higher phosphorus content, is eluted in a higher salt concentration from DEAE-cellulose, and has a prolonged thrombin clotting time relative to adult fibrinogen. Differences in tryptic peptide maps have also been noted. Recently, fibrinogen preparations from adult and fetal sheep have been compared ~° and the authors have discussed the possible affects of thrombin activation and fibrinogenolysis in vitro on the fibrinogen clotting times. In the present study, samples were collected and fractionated in the presence of e-aminocaproic acid, and plasmin digest products detectable by sodium dodecyl sulfate eleetrophoresisl, s were not found in either adult or cord fractions. However, Biochim. Biophys. ~4cta, 285 (1972) 398 403
402
D . A . MILLS, S. KARPATKIN
A
B
C
O
Fig. 2. S o d i u m dodecyl sulfate gel electrophoresis p a t t e r n s of a d u l t a n d cord bulk a n d h i g h solubility fibrinogen f r a c t i o n s a f t e r r e d u c t i o n of disulfide b o n d s (approx. 45 t~g p r o t e i n electrophorized a t 7 m A per gel for I8 h). A, a d u l t high solubility fibrinogen; B, cord h i g h solubility fibrinogen; C, m i x t u r e of a d u l t a n d cord b u l k fibrinogens ; D, m i x t u r e of a d u l t a n d cord fibrins o b t a i n e d b y clotting h i g h solubility fibrinogens. B a n d d e s i g n a t i o n s are as Fig. i.
the coagula from low solubility fractions do appear to represent derivatives of limited thrombin action, containing about equal amounts of a(A)-chains and a-chains by the criterion of sodium dodecyl sulfate gel electrophoresis. The presence of a-chains would indicate removal of fibrinopeptide A by the limited action of thrombin*. The more extensive action of thrombin (removal of fibrinopeptide B to form fl-chains) was not detected**. A fibrinogen derivative deficient in fibrinopeptide A but not B (cryoprofibrin) was first characterized from plasma of endotoxin-treated rabbits 11. The material seemed to be closely related to a previously described heparin-precipitable fraction 12-14, and it is noteworthy that levels of heparin-precipitable fraction were found to be very low in plasma of newborn infants relative to adult human levels 14. It m a y be doubted that appreciable amounts of thrombin-activated derivatives are present normally in vivo, and such materials detected in the present work could have arisen under the conditions of isolation. In either case, if a greater amount of thrombin-activated derivatives were present in the adult samples, this could explain the differences between adult and cord fibrinogen fractions in precipitability and clotting time (Table I). However, structural differences not detectable by sodium dodecyl sulfate gel electrophoresis could also explain these findings. It should be * A similar a l t e r a t i o n of a ( A ) - c h a i n s b y a p l a s m a e n z y m e o t h e r t h a n t h r o m b i n h a s n o t been r e p o r t e d and, specifically, p l a s m i n a c t i o n m a y be ruled outl, 5. *" T h e presence (Fig. I) of m a t e r i a l i n d i s t i n g u i s h a b l e f r o m y - c h a i n d i m e r p r o d u c e d b y Factor X [ 11 indirectly indicates t h r o m b i n action, since F a c t o r X I I [ z y m o g e n is a c t i v a t e d b y t h r o m b i n proteolysis 9.
Biochim. Biophys..4cta, 285 (1972) 398-403
403
HETEROGENEITY OF FIBRINOGEN
n o t e d t h a t t h e a b i l i t y o f s o d i u m d o d e c y l s u l f a t e gel e l e c t r o p h o r e s i s t o d e t e c t t h r o m b i n - a c t i v a t e d d e r i v a t i v e s m a y b e o f use in t h e c h a r a c t e r i z a t i o n o f t h o s e f i b r i n o g e n related materials
postulated
to
be indicative of intravascular
coagulation
(e.g.
r e f s 15, 16). ACKNOWLEDGEMENTS This work was supported by a grant from the New York Heart Association.
REFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16
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