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The peptide β43–47 which increases microvascular permeability is released by plasmic during cleavage of fragment Y of fibrinogen
594
Biochimica et Biophvsica Acta 884 (1986) 594-597 Elsevier
BBA22388
The peptide fl43-47 which increases microvascular permeability is released by plasmin during cleavage of fragment Y of fibdnogen Czeslaw S. Cierniewski, Jacek Poniatowski and Jolanta Urbanczyk Department of Biophysics, Institute of Physiology and Biochemisto,, Medical School of Lodz, Lodz (Poland) (Received 4 April 1986) (Revised manuscript received 27 August 1986)
Key words: Synthetic peptide; Plasmin release; Microvascular permeability; Fibrinogen cleavage
A synthetic pentapeptide corresponding to sequence 43-47 of human fibrinogen Bfl chain elicited, in rabbits, antibodies that during immunoblotting recognized intact fibrinogen, fragments X and Y as well as the Bfl chain. Since fragment Y is the last peptide product which reacts with anti-~43-47 antibodies, splitting of fragment Y into fragment D and fragment E must be accompanied by plasmin cleavage of the peptide bond flLys-47-Aia-48.
Introduction The sequence of structural changes in human fibrinogen during digestion by plasmin was established a long time ago based on the molecular weight of the products, their carbohydrate content and the relative amounts of each product present during digestion [1]. Accordingly, the major products of fibrinogen degradation have been isolated and ~heir amino acid sequence determined [2-5]. In addition to the large molecular weight intermediate products (fragments X and Y) and terminal products (fragments D and E), a variety of low molecular weight fragments are released from fibrinogen by plasmin. These low molecular weight split products of fibrin(ogen) have various biological properties [6,7]. Among them, a pentapeptide, Ala-Arg-Pro-Ala-Lys, is known to increase microvascular permeability [8] and is supposed to be involved in the establishment of pulmonary edema in conditions with fibrin accumulation in the lungs Correspondence: Department of Biophysics, Institute of Physiology and Biochemistry, Medical School of Lodz, 3 Lindley St., Lodz 90-131, Poland.
[9]. This peptide also induced dilation of bovine mesenteric arteries and caused a release of prostacyclin as well as an increase in cyclic AMP in blood vessels [10]. The aim of this report was to demonstrate at which step of plasmin degradation this physiologically active peptide is released from fibrin(ogen) to the environment. For this purpose pentapeptide Ala-Arg-Pro-Ala-Lys and its analog Tyr-Ala-ProAla-Lys were synthesized and used to produce specific antibodies. The latter were next employed in radioimmunoassay analysis and immunoblotting experiments in order to localize this peptide segment in fibrinogen and its plasmic fragments. Experimental procedures
Preparativeprocedures Human fibrinogen was isolated from normal human citrated plasma by precipitation with ammonium sulfate (0.85 M) and glycine (2.1 M) followed by gel filtration on Sepharose CL-6B and another ammonium sulfate (0.65 M) precipitation as described previously [11]. Fibrinogen (500 mg), suspended in 0.15 M Tris-
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
595 HC1 buffer (pH 7.8) containing 5 mM calcium chloride and 0.02% sodium azide, was digested with human plasmin (KABI) to obtain stage 2 or stage 3 digests. A stage 2 digest of fibrinogen contains mainly fragment X but also fragments Y, D, and E. A stage 3 digest is characterized by the presence of fragments D and E only [12]. Digestion was stopped by an addition of trasylol (aprotinin, 10000 KIU/ml; Mobay Chemical Corp., New York, NY) in the ratio of 250 KIU/1 CTA unit of plasmin. Fragment D1 was obtained from a plasmic digest of human fibrinogen (100 rag) by column (2.5 × 130 cm) gel filtration on Sephadex G-100 (Pharmacia, Piscataway, N J) in 10% acetic acid. Elution rate was 15 ml/h and 3 ml fractions were collected. The peak containing fragment D1 was pooled and freeze-dried. Alternatively, non-denatured fragment D1 was obtained from a plasmic digest of fibrinogen by preparative electrophoresis on a Pevikon block [13]. Purified fragment D1 was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained remnants of a ( M r 12000), fl (M r 42000) and "r (Mr 39 000) chains. Fragment E1 was isolated from plasmic digest stage 3 of fibrinogen by gel filtration on Sepharose CL-6B [19]. The pentapeptide Ala-Arg-Pro-Ala-Lys corresponding to fl43-47 sequence of human fibrinogen and its analog Tyr-Ala-Arg-Pro-Ala-Lys were synthesized by a classical solution method using reagents as described previously [15]. The purity of peptides was checked by amino acid composition analysis.
Analytic procedures Protein concentration was determined either by the microbiuret method [16] or by spectrophotometry at 280 nm using an absorption coefficient at 1 mg/ml of 1.5, 1.6, 2.0 and 1.35 for fibrinogen, fragment D1, fragment E1 and IgG, respectively. Amino acid composition was determined on a Beckman model 121 M analyzer from 24 h hydrolysates in 6.0 M HC1 at 100" C in vacuo [17]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to Laemmli [18]. Completed gels were transferred electrophoretically (6 V/era for 15 h at 4"C) onto
nitrocellulose paper for staining with a specific antibody [19]. The buffer contained 25 mM Tris, 192 mM glycine (pH 8.2), 20% (v/v) methanol. For antibody staining, completed transfers were incubated at 20°C on a rocker (Ames aliquot mixer, Miles Laboratories, Elkhart, IN) as follows: 5 rain in 0.15 M NaC1, 0.01 M Tris-glycine (pH 7.5), which was the buffer used throughout; 30 min in 1% bovine serum albumin in the buffer; 1-2 h in bovine serum albumin containing antiserum; 20 min in several changes of the buffer containing 0.1% Triton X-100 to remove excess first antibody; 1 h in 30 #1 peroxidase-conjugated goat anti-rabbit IgG (Cappel Laboratories, Chester, PA); 20 min in several changes of the buffer to remove second antibody; 5 min in substrate solution (10 mg 4-chloro-l-naphthol dissolved in 3 ml methanol and added to 47 ml of the buffer containing 50 #I 30% H202). All volumes were 20-25 ml, the minimum to ensure uniform wetting of paper. Sensitivity was of the order of 25 ng using the peroxidase conjugate.
Immunologicalprocedures The pentapeptide fl43-47 was conjugated with bovine serum albumin and thyroglobulin by the glutaraldehyde technique [20]. Antisera specific to fl43-47 were produced in three rabbits. After sonication of the aggregated carrier proteins with the pentapeptide, aliquots containing 200 #g of fl43-47 were emulsified with complete Freund's adjuvant and injected through 30-40 intradermal sites in each rabbit. During the first three immunizations the pentapeptide immobilized on bovine serum albumin was injected; the next injections were made using the peptide coupled to thyroglobulin. Rabbits were bled 7 days after each injection. Antibody titers were determined by radioimmunoassay [21] using radioiodinated Tyr-Ala-ArgPro-Ala-Lys and fibrinogen and the highest titer antisera from each rabbit were selected. In the experimental procedure, 0.1 ml of the radioiodinated peptide (1-10 -x° mol/1) or fibrinogen (5-10 -1° mol/l) was mixed with 0.1 ml of the diluted antiserum. The samples were incubated overnight at 4°C. Then 0.1 ml of the goat antirabbit IgG antiserum was added to the mixture. After 3 h of incubation at 4oC tubes had been centrifuged for 10 min at 2000 × g. The super-
596
natant was decanted and the precipitate was washed twice with 0.5 ml 0.14 M NaC1 buffered with 0.01 M sodium phosphate (pH 7.1). All dilutions were made in 0.1 M borate buffer (pH 8.3), containing 2% of normal rabbit serum and heparin at a concentration of 200 units per 1 ml. Results and Discussion
Several low molecular weight peptides released during degradation of fibrin show a potent biological activity [11,12]. Since there is continuous intravascular fibrin deposition and resolution, these peptides may be liberated locally in large amounts and contribute significantly to circulation hemodynamics. Among these physiologically active peptides, fl43-47 is well characterized [22]. The synthetic fl43-47 peptide exhibited full potency and structural requirements for its ability
to increase microvascular permeability are established [22]. In this study we attempted to determine at what stage the fl43-47 peptide is released from the fibrinogen molecule during plasmin cleavage. For this purpose fibrinogen digest stage 2 and purified fragment D and fragment E were separated by polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose sheets. Immunostaining of the sheets (Western blotting) with anti-f143-47 antibody and peroxidase-conjugated second antibody allowed us to detect which intermediate products of fibrinogen catabolism still contained the fl43-47 sequence (Fig. 1). Preimmune serum did not react with fibrinogen in this system (not shown). Besides intact fibrinogen, anti-f143-47 antibody recognized species of fragment X and fragment Y. No detectable fl43-47 sequence was observed in
Fg ........ FgX {.~i~i-~ ~-~ti Fg¥ . . . . . . . . . . .
l
i
B
1
2
3
4
Fig. 1. Western blot detection of fl43-47 sequence in intact fibrinogen (Fg) (lane 1), stage 2 plasmin digest of fibrinogen (lane 2), isolated fragment D1 (lane 3) and E1 (lane 4). Gels presented in panel A were stained for protein with Coomassie Brilliant Blue. Gels in panel B were electrophoretically blotted and stained with anti-f143-47 antibodies and peroxidaseconjugated second antibody by the Western blot procedure of Towbin et al. [19].
1
2
Fig. 2. Binding of anti-f143-47 antibody to the Bfl chain of human fibrinogen. Samples of reduced fibrinogen were run in 7% sodium dodecyl sulfate-polyacrylamide gels and stained with Coomassie Brilliant blue (lane 1) or electrophoretically transferred to the nitrate cellulose and stained with anti-/~43-47 antibodies followed by peroxidase-conjugated second antibody (lane 2) [19].
597
References
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a
IL
:
\ l
2(
lo 2
\. ~ ,
m3
lo 4
1o s
ANTI/~43.47 ANTISERUM DILUTION
Fig. 3. Binding of anti-f143-47 antibodies to radioiodinated human fibrinogen (125I-Fg) evaluated by radioimmunoassay. The reaction of anti-f143-47 antiserum (A) and the lack of binding in the case of preimmune rabbit serum (~,) to fibrinogen are demonstrated.
fragment E. Essentially there was only immunostaining of the fl chain with anti-f143-47 antibody when reduced fibrinogen was analyzed (Fig. 2). However, prolonged exposure of the sheets loaded with antibody to the fl43-47 and peroxidase-conjugated second antibody to the substrate resulted in appearance of a weak band in the position of the y chain as well. The antisera raised in three rabbits against fl43-47 precipitated also radioiodinated fibrinogen which was not exposed to sodium dodecyl sulfate (Fig. 3). These studies confirm our previous observations that sequence 43-53 of the fl chain is exposed on the intact fibrinogen molecule [23]. The presence of the fl43-47 segment in fragment Y and its absence from fragment E1 indicates that this segment is released at the stage when fragment Y is cleaved by plasmin to fragment D and fragment E.
1 Pizzo, S.V., Schwartz, M.L., Hill, R.L. and McKee, P.A. (1972) J. Biol. Chem. 247, 636-645 2 Lottspeich, F. and Henschen, A. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 935-938 3 Henschen, A. and Lottspeich, F. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1643-1646 4 Watt, K.W.K., Takagi, T. and Doolittle, R.F. (1978) Proc. Nail. Acad. Sci. USA 75, 1731-1735 5 Doolitfle, R.F., Watt, K.W.K., Cottrell, B.A., Strong, D.D. and Riley, M. (1979) Nature 280, 464-468 6 Buluk, K., Malofiejew, M. and Czokalo, M. (1966) Bull. Acad. Pol. Sci. (Biol.) 14, 193-197 7 Malofiejew, M. and Buluk, K. (1968) Pol. Tyg. Lek. 17, 619-621 8 Belew, M., Gerdin, B., Porath, J. and Saldeen, T. (1978) Thromb. Res. 13, 983-994 9 Saldeen, T. (1979) in The microembolism syndrome (Saldeen, T., ed.), pp. 7-14, Almquist & Wiksell International, Stockholm 10 Anderson, R.G.G., Saldeen, K. and Saldeen, T. (1983) Thromb. Res. 30, 213-218 11 Pandya, B.V. and Budzynski, A.Z. (1984) Biochemistry 231 460-470 12 Marder, V.J., Shulman, M.R. and Caroll, W.R. (1967) Trans. Assoc. Am. Physicians 53, 156-167 13 Marder, V.J., James, H.J. and Sherry, S. (1969) Thromb. Diath. Haemorrh. 22, 234-239 14 Olexa, S.A., Budzynski, A.Z. and Marder, V.J. (1979) Biochim. Biophys. Acta 570, 39-50 15 Babinska, A., Cierniewsld, C.S., Koziolkiweicz, W. and Janecka, A. (1985) Int. J. Peptide Protein Res. 24, 69-75 16 Itzhaki, R.F. and Gill, D.M. (1969) Anal. Biochem. 9, 401-410 17 Spackman, D.H., Stern, W.H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206 18 Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685 19 Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354 20 Avrameas, S. and Trnyck, T. (1969) Immunochemistry 6, 53-66 21 Ciemiewski, C.S., Janiak, A., Nowak, P. and Augustyniak, W. (1982) Thromb. Haemostasis 48, 33-37 22 Gerdin, B., Lindberg, G., Ragnarsson, U., Saldeen, T. and Wallin, R. (1983) Biochim. Biophys. Acta 757, 366-370 23 Cierniewski, C.S. and Edgington, T.S. (1979) Thromb. Res. 14, 747-764
Biochimica et Biophvsica Acta 884 (1986) 594-597 Elsevier
BBA22388
The peptide fl43-47 which increases microvascular permeability is released by plasmin during cleavage of fragment Y of fibdnogen Czeslaw S. Cierniewski, Jacek Poniatowski and Jolanta Urbanczyk Department of Biophysics, Institute of Physiology and Biochemisto,, Medical School of Lodz, Lodz (Poland) (Received 4 April 1986) (Revised manuscript received 27 August 1986)
Key words: Synthetic peptide; Plasmin release; Microvascular permeability; Fibrinogen cleavage
A synthetic pentapeptide corresponding to sequence 43-47 of human fibrinogen Bfl chain elicited, in rabbits, antibodies that during immunoblotting recognized intact fibrinogen, fragments X and Y as well as the Bfl chain. Since fragment Y is the last peptide product which reacts with anti-~43-47 antibodies, splitting of fragment Y into fragment D and fragment E must be accompanied by plasmin cleavage of the peptide bond flLys-47-Aia-48.
Introduction The sequence of structural changes in human fibrinogen during digestion by plasmin was established a long time ago based on the molecular weight of the products, their carbohydrate content and the relative amounts of each product present during digestion [1]. Accordingly, the major products of fibrinogen degradation have been isolated and ~heir amino acid sequence determined [2-5]. In addition to the large molecular weight intermediate products (fragments X and Y) and terminal products (fragments D and E), a variety of low molecular weight fragments are released from fibrinogen by plasmin. These low molecular weight split products of fibrin(ogen) have various biological properties [6,7]. Among them, a pentapeptide, Ala-Arg-Pro-Ala-Lys, is known to increase microvascular permeability [8] and is supposed to be involved in the establishment of pulmonary edema in conditions with fibrin accumulation in the lungs Correspondence: Department of Biophysics, Institute of Physiology and Biochemistry, Medical School of Lodz, 3 Lindley St., Lodz 90-131, Poland.
[9]. This peptide also induced dilation of bovine mesenteric arteries and caused a release of prostacyclin as well as an increase in cyclic AMP in blood vessels [10]. The aim of this report was to demonstrate at which step of plasmin degradation this physiologically active peptide is released from fibrin(ogen) to the environment. For this purpose pentapeptide Ala-Arg-Pro-Ala-Lys and its analog Tyr-Ala-ProAla-Lys were synthesized and used to produce specific antibodies. The latter were next employed in radioimmunoassay analysis and immunoblotting experiments in order to localize this peptide segment in fibrinogen and its plasmic fragments. Experimental procedures
Preparativeprocedures Human fibrinogen was isolated from normal human citrated plasma by precipitation with ammonium sulfate (0.85 M) and glycine (2.1 M) followed by gel filtration on Sepharose CL-6B and another ammonium sulfate (0.65 M) precipitation as described previously [11]. Fibrinogen (500 mg), suspended in 0.15 M Tris-
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
595 HC1 buffer (pH 7.8) containing 5 mM calcium chloride and 0.02% sodium azide, was digested with human plasmin (KABI) to obtain stage 2 or stage 3 digests. A stage 2 digest of fibrinogen contains mainly fragment X but also fragments Y, D, and E. A stage 3 digest is characterized by the presence of fragments D and E only [12]. Digestion was stopped by an addition of trasylol (aprotinin, 10000 KIU/ml; Mobay Chemical Corp., New York, NY) in the ratio of 250 KIU/1 CTA unit of plasmin. Fragment D1 was obtained from a plasmic digest of human fibrinogen (100 rag) by column (2.5 × 130 cm) gel filtration on Sephadex G-100 (Pharmacia, Piscataway, N J) in 10% acetic acid. Elution rate was 15 ml/h and 3 ml fractions were collected. The peak containing fragment D1 was pooled and freeze-dried. Alternatively, non-denatured fragment D1 was obtained from a plasmic digest of fibrinogen by preparative electrophoresis on a Pevikon block [13]. Purified fragment D1 was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained remnants of a ( M r 12000), fl (M r 42000) and "r (Mr 39 000) chains. Fragment E1 was isolated from plasmic digest stage 3 of fibrinogen by gel filtration on Sepharose CL-6B [19]. The pentapeptide Ala-Arg-Pro-Ala-Lys corresponding to fl43-47 sequence of human fibrinogen and its analog Tyr-Ala-Arg-Pro-Ala-Lys were synthesized by a classical solution method using reagents as described previously [15]. The purity of peptides was checked by amino acid composition analysis.
Analytic procedures Protein concentration was determined either by the microbiuret method [16] or by spectrophotometry at 280 nm using an absorption coefficient at 1 mg/ml of 1.5, 1.6, 2.0 and 1.35 for fibrinogen, fragment D1, fragment E1 and IgG, respectively. Amino acid composition was determined on a Beckman model 121 M analyzer from 24 h hydrolysates in 6.0 M HC1 at 100" C in vacuo [17]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to Laemmli [18]. Completed gels were transferred electrophoretically (6 V/era for 15 h at 4"C) onto
nitrocellulose paper for staining with a specific antibody [19]. The buffer contained 25 mM Tris, 192 mM glycine (pH 8.2), 20% (v/v) methanol. For antibody staining, completed transfers were incubated at 20°C on a rocker (Ames aliquot mixer, Miles Laboratories, Elkhart, IN) as follows: 5 rain in 0.15 M NaC1, 0.01 M Tris-glycine (pH 7.5), which was the buffer used throughout; 30 min in 1% bovine serum albumin in the buffer; 1-2 h in bovine serum albumin containing antiserum; 20 min in several changes of the buffer containing 0.1% Triton X-100 to remove excess first antibody; 1 h in 30 #1 peroxidase-conjugated goat anti-rabbit IgG (Cappel Laboratories, Chester, PA); 20 min in several changes of the buffer to remove second antibody; 5 min in substrate solution (10 mg 4-chloro-l-naphthol dissolved in 3 ml methanol and added to 47 ml of the buffer containing 50 #I 30% H202). All volumes were 20-25 ml, the minimum to ensure uniform wetting of paper. Sensitivity was of the order of 25 ng using the peroxidase conjugate.
Immunologicalprocedures The pentapeptide fl43-47 was conjugated with bovine serum albumin and thyroglobulin by the glutaraldehyde technique [20]. Antisera specific to fl43-47 were produced in three rabbits. After sonication of the aggregated carrier proteins with the pentapeptide, aliquots containing 200 #g of fl43-47 were emulsified with complete Freund's adjuvant and injected through 30-40 intradermal sites in each rabbit. During the first three immunizations the pentapeptide immobilized on bovine serum albumin was injected; the next injections were made using the peptide coupled to thyroglobulin. Rabbits were bled 7 days after each injection. Antibody titers were determined by radioimmunoassay [21] using radioiodinated Tyr-Ala-ArgPro-Ala-Lys and fibrinogen and the highest titer antisera from each rabbit were selected. In the experimental procedure, 0.1 ml of the radioiodinated peptide (1-10 -x° mol/1) or fibrinogen (5-10 -1° mol/l) was mixed with 0.1 ml of the diluted antiserum. The samples were incubated overnight at 4°C. Then 0.1 ml of the goat antirabbit IgG antiserum was added to the mixture. After 3 h of incubation at 4oC tubes had been centrifuged for 10 min at 2000 × g. The super-
596
natant was decanted and the precipitate was washed twice with 0.5 ml 0.14 M NaC1 buffered with 0.01 M sodium phosphate (pH 7.1). All dilutions were made in 0.1 M borate buffer (pH 8.3), containing 2% of normal rabbit serum and heparin at a concentration of 200 units per 1 ml. Results and Discussion
Several low molecular weight peptides released during degradation of fibrin show a potent biological activity [11,12]. Since there is continuous intravascular fibrin deposition and resolution, these peptides may be liberated locally in large amounts and contribute significantly to circulation hemodynamics. Among these physiologically active peptides, fl43-47 is well characterized [22]. The synthetic fl43-47 peptide exhibited full potency and structural requirements for its ability
to increase microvascular permeability are established [22]. In this study we attempted to determine at what stage the fl43-47 peptide is released from the fibrinogen molecule during plasmin cleavage. For this purpose fibrinogen digest stage 2 and purified fragment D and fragment E were separated by polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose sheets. Immunostaining of the sheets (Western blotting) with anti-f143-47 antibody and peroxidase-conjugated second antibody allowed us to detect which intermediate products of fibrinogen catabolism still contained the fl43-47 sequence (Fig. 1). Preimmune serum did not react with fibrinogen in this system (not shown). Besides intact fibrinogen, anti-f143-47 antibody recognized species of fragment X and fragment Y. No detectable fl43-47 sequence was observed in
Fg ........ FgX {.~i~i-~ ~-~ti Fg¥ . . . . . . . . . . .
l
i
B
1
2
3
4
Fig. 1. Western blot detection of fl43-47 sequence in intact fibrinogen (Fg) (lane 1), stage 2 plasmin digest of fibrinogen (lane 2), isolated fragment D1 (lane 3) and E1 (lane 4). Gels presented in panel A were stained for protein with Coomassie Brilliant Blue. Gels in panel B were electrophoretically blotted and stained with anti-f143-47 antibodies and peroxidaseconjugated second antibody by the Western blot procedure of Towbin et al. [19].
1
2
Fig. 2. Binding of anti-f143-47 antibody to the Bfl chain of human fibrinogen. Samples of reduced fibrinogen were run in 7% sodium dodecyl sulfate-polyacrylamide gels and stained with Coomassie Brilliant blue (lane 1) or electrophoretically transferred to the nitrate cellulose and stained with anti-/~43-47 antibodies followed by peroxidase-conjugated second antibody (lane 2) [19].
597
References
1oo
a
IL
:
\ l
2(
lo 2
\. ~ ,
m3
lo 4
1o s
ANTI/~43.47 ANTISERUM DILUTION
Fig. 3. Binding of anti-f143-47 antibodies to radioiodinated human fibrinogen (125I-Fg) evaluated by radioimmunoassay. The reaction of anti-f143-47 antiserum (A) and the lack of binding in the case of preimmune rabbit serum (~,) to fibrinogen are demonstrated.
fragment E. Essentially there was only immunostaining of the fl chain with anti-f143-47 antibody when reduced fibrinogen was analyzed (Fig. 2). However, prolonged exposure of the sheets loaded with antibody to the fl43-47 and peroxidase-conjugated second antibody to the substrate resulted in appearance of a weak band in the position of the y chain as well. The antisera raised in three rabbits against fl43-47 precipitated also radioiodinated fibrinogen which was not exposed to sodium dodecyl sulfate (Fig. 3). These studies confirm our previous observations that sequence 43-53 of the fl chain is exposed on the intact fibrinogen molecule [23]. The presence of the fl43-47 segment in fragment Y and its absence from fragment E1 indicates that this segment is released at the stage when fragment Y is cleaved by plasmin to fragment D and fragment E.
1 Pizzo, S.V., Schwartz, M.L., Hill, R.L. and McKee, P.A. (1972) J. Biol. Chem. 247, 636-645 2 Lottspeich, F. and Henschen, A. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 935-938 3 Henschen, A. and Lottspeich, F. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1643-1646 4 Watt, K.W.K., Takagi, T. and Doolittle, R.F. (1978) Proc. Nail. Acad. Sci. USA 75, 1731-1735 5 Doolitfle, R.F., Watt, K.W.K., Cottrell, B.A., Strong, D.D. and Riley, M. (1979) Nature 280, 464-468 6 Buluk, K., Malofiejew, M. and Czokalo, M. (1966) Bull. Acad. Pol. Sci. (Biol.) 14, 193-197 7 Malofiejew, M. and Buluk, K. (1968) Pol. Tyg. Lek. 17, 619-621 8 Belew, M., Gerdin, B., Porath, J. and Saldeen, T. (1978) Thromb. Res. 13, 983-994 9 Saldeen, T. (1979) in The microembolism syndrome (Saldeen, T., ed.), pp. 7-14, Almquist & Wiksell International, Stockholm 10 Anderson, R.G.G., Saldeen, K. and Saldeen, T. (1983) Thromb. Res. 30, 213-218 11 Pandya, B.V. and Budzynski, A.Z. (1984) Biochemistry 231 460-470 12 Marder, V.J., Shulman, M.R. and Caroll, W.R. (1967) Trans. Assoc. Am. Physicians 53, 156-167 13 Marder, V.J., James, H.J. and Sherry, S. (1969) Thromb. Diath. Haemorrh. 22, 234-239 14 Olexa, S.A., Budzynski, A.Z. and Marder, V.J. (1979) Biochim. Biophys. Acta 570, 39-50 15 Babinska, A., Cierniewsld, C.S., Koziolkiweicz, W. and Janecka, A. (1985) Int. J. Peptide Protein Res. 24, 69-75 16 Itzhaki, R.F. and Gill, D.M. (1969) Anal. Biochem. 9, 401-410 17 Spackman, D.H., Stern, W.H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206 18 Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685 19 Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354 20 Avrameas, S. and Trnyck, T. (1969) Immunochemistry 6, 53-66 21 Ciemiewski, C.S., Janiak, A., Nowak, P. and Augustyniak, W. (1982) Thromb. Haemostasis 48, 33-37 22 Gerdin, B., Lindberg, G., Ragnarsson, U., Saldeen, T. and Wallin, R. (1983) Biochim. Biophys. Acta 757, 366-370 23 Cierniewski, C.S. and Edgington, T.S. (1979) Thromb. Res. 14, 747-764