- Email: [email protected]
Fibrinogen
The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746 www.elsevier.com/locate/ijbcb
Molecules in focus
Fibrinogen Sarah Herrick b,*, Olivier Blanc-Brude b, Andrew Gray a, Georey Laurent b a
Discovery Biology, P®zerCentral Research, Sandwich, Kent, UK Centre for Cardiopulmonary Biochemistry and Respiratory Medicine, The Rayne Institute, 5 University Street, University College London Medical School, London WC1E 6JJ, UK
b
Received 16 March 1999; received in revised form 16 March 1999; accepted 9 April 1999
Abstract Fibrinogen is a blood-borne glycoprotein comprised of three pairs of nonidentical polypeptide chains. Following vascular injury, ®brinogen is cleaved by thrombin to form ®brin which is the most abundant component of blood clots. As well as controlling blood loss at sites of tissue damage, other properties of ®brinogen have recently been discovered. For example, various cleavage products of ®brinogen and ®brin, released during coagulation and ®brinolysis, respectively, regulate cell adhesion and spreading, display vasoconstrictor and chemotactic activities, and are mitogens for several cell types including ®broblasts, endothelial and smooth muscle cells. Current research aims to de®ne the bioactive ®brinogen molecule moieties and cellular receptors involved in these processes. Future studies may provide us with new opportunities to develop agents which are useful in promoting tissue repair or conversely in inhibiting ®brosis in in¯ammatory and ®broproliferative diseases where endothelial cell damage or chronic leakage of blood proteins is a feature. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Fibrinogen; Fibrin degradation products; Integrins; Haemostasis
1. Introduction Human ®brinogen is a circulating 340 kDa glycoprotein, primarily synthesised by hepatocytes. It is comprised of two symmetric half molecules, each consisting of one set of three dierent polypeptide chains termed Aa, Bb and g (Fig. 1). The molecule is highly heterogenous due to alterna-
tive splicing, extensive post-translational modi®cation and proteolytic degradation. Furthermore, there is polymorphic variation in the Aa and Bb chains, leading to the suggestion that each individual may carry over one million nonidentical ®brinogen molecules in their blood [1].
2. Structure * Corresponding author. Tel.: +44-171-209-6016; fax: +44171-209-6973. E-mail address: [email protected] (S. Herrick)
Each of the three polypeptide chains of the ®brinogen molecule is encoded by a separate
1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 3 2 - 1
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S. Herrick et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746
Fig. 1. Schematic diagram of ®brinogen (adapted from Ref. [16]). There are three sets of nonidentical chains which are bound by disulphide bonds at the amino-terminal end. Removal of ®brinopeptides A and B at this end via the action of thrombin, leads to cross-linking and the formation of the ®brin clot.
gene located on chromosome four. The Aa (6 exons) and g (10 exons) ®brinogen chain genes are orientated in tandem and are transcribed in the opposite direction to the Bb (8 exons) ®brinogen chain gene. Alternative splicing may occur in the Aa and g ®brinogen chain genes leading to the production of peptide variants. The predominant Aa chain of circulating ®brinogen contains around 610 amino acid residues (70 kDa), the Bb chain of ®brinogen consists of 461 amino acids (56 kDa). The g chain is heterogenous with respect to both charge and size, however, the most abundant form, denoted as g- or gA, consists of 411 residues (48 kDa). The ®brinogen molecule has three distinct domains (see Fig. 1); two terminal D domains (67 kDa), each linked to a single central E domain (33 kDa) by a triple-stranded array of the polypeptide chains believed to exist in the form of a helical coiled coils. The three constitutive chains and the two halves of the ®brinogen molecule are held together by a series of 29 disul-
phide bonds with all 58 cysteine residues of ®brinogen participating in these interactions. There is extensive post-translational modi®cation of the protein including phosphorylation, sulphation, glycosylation and hydroxylation [1].
3. Synthesis and degradation Fibrinogen is synthesised by hepatocytes and circulates as a component of blood at a concentration of approximately 9 mM with a half-life of around 100 h. During episodes of in¯ammation, the synthesis of ®brinogen is drastically enhanced, an eect thought to be mediated by interleukin-6 (IL-6), glucocorticoids and oncostatin M. Interleukin-6 and glucocorticoid response elements have been identi®ed in all three ®brinogen chain genes, whereas the oncostatin M response elements have not been mapped.
S. Herrick et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746
Furthermore, the Bb and Aa chain gene promoters contain a hepatic nuclear factor-1 (HNF-1) binding site which appears to contribute to the liver speci®c expression of these genes. Liver speci®c expression of the g chain gene is mediated by an upstream stimulatory factor (USF) in conjunction with other upstream elements such as TATA- and CAAT-like sequences. Interestingly, recent work suggests that extra-hepatic epithelial cells, in particular lung alveolar epithelium, are also able to synthesise and secrete ®brinogen locally in response to proin¯ammatory mediators [2]. At sites of tissue injury, ®brinogen is converted to ®brin by a-thrombin with the release of ®brinopeptides A and B from the amino-terminal ends of the Aa and Bb chains respectively (Fig. 1). The ®brin monomers polymerise spontaneously and undergo intermolecular crosslinking to form a stable ®brin clot in the presence of transglutaminase (factor XIIIa) [3]. Subsequently, the ®brin matrix is degraded into an number of fragments, including X, Y, D and E, primarily through the action of plasmin.
743
Fig. 2. Key functions of ®brin(ogen) and its cleavage products. Fibrinogen and its cleavage products have well described roles in haemostasis where ®brin forms a clot limiting blood loss and providing a key substrate of the provisional matrix which is vital for normal repair. It is also recognised that ®brin(ogen) and its breakdown products are capable of promoting other vital events in tissue repair including blood vessel tone (vasoconstriction), angiogenesis, directed cell migration and proliferation of various cells including ®broblasts, smooth muscle cells and lymphocytes. The later data suggest it may in¯uence the local immune response.
However, other proteases, such as neutrophil elastase, mast cell tryptase, matrix metalloproteases and cathepsins D and G, are also able to degrade ®brin to produce an additional array of cleavage products.
Table 1 Integrin and nonintegrin receptors which bind to known recognition sites on human ®brin(ogen) or its cleavage products and regulate cell function Integrin receptor
Cell types
Recognition sites
Cellular function
aIIbb3 (GPIIb/IIIa)
platelets
avb3
adhesion aggregation and clot retraction adhesion and spreading
a5b1 amb2 (CD11b/CD18) axb2 (CD11c/CD18)
endothelial cells, melanoma cells, ®broblasts endothelial cells monocytes, neutrophils neutrophils B lymphocytes
g400±411, Aa572±574, Aa95±97 Aa572±574 Aa572±574 g190±202, g377±395 Aa17±19
adhesion phagocytosis and adhesion adhesion
Nonintegrin receptor
Cell types
Recognition sites
Cellular function
130 kD receptor
endothelial cells
Bb15±42
CD-44 surface receptor
endothelial cells, ®broblasts, tumour cells ®broblasts hemopoietic cells lymphoid cell lines endothelial cells, ®broblasts, mesothelial cells
±
von-Willebrand factor release adhesion and migration proliferation proliferation proliferation adhesion
Calreticulin 92±94 kD receptor (MFR) ICAM-1
Bb chain ± g117±133 g117±133
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S. Herrick et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746
4. Biological function Following vascular injury, there are two principal mechanisms by which ®brinogen participates in controlling blood loss. It acts as an adhesive protein essential for platelet aggregation as well as forming an insoluble ®brin clot in the ®nal stage of the blood coagulation cascade. It is therefore interesting that ®brinogen-de®cient mice are able to control bleeding following mild trauma [4]. This has led to the assumption that platelets can bind to alternative matrix molecules to establish a platelet-rich clot to prevent bleeding. However, this level of protection is clearly insucient following major vascular injury, resulting in uncontrolled bleeding as observed in individuals with congenital ®brinogen de®ciency. Although clearly important in haemostasis, the ®brin clot also provides a scaold for cell adhesion, spreading, migration and proliferation. Structural changes that accompany the conversion of ®brinogen to ®brin expose new receptor binding sites providing a basis for ®brin speci®c cell responses. Furthermore, recent studies have suggested that ®brin(ogen) cleavage products released during coagulation and subsequent ®brinolysis, may be bioactive and exert direct eects on ®broblasts and other cell types associated with the wound healing process (Fig. 2). Human ®brin(ogen) contains three potential integrin binding sites, two RGD sequences within the Aa chain (RGDS a572±575 and RGDF a95±98) and a non-RGD sequence in the g chain (carboxy terminal g400±411), but can also interact with cells through nonintegrin receptors (Table 1). For example, ®brinogen mediates the adhesion and transendothelial migration of leucocytes by acting as a molecular bridge between the cell types through ICAM-1 dependent pathways [5]. In addition, ®brin(ogen) binds to other extracellular matrix molecules and can act as a reservoir for growth factors, proteases and protease inhibitors. In particular, ®brin binds plasminogen and its activator (tissue-type plasminogen activator) to form a ternary complex which enhances the conversion of plasminogen into the ®brinolytic enzyme, plasmin and protects them from inactivation by their inhibitors.
4.1. Mitogenic eects of ®brin(ogen) and its cleavage products Fibrin(ogen) cleavage products are mitogens for a variety of cell types, including endothelial cells, ®broblasts, mesothelial cells, smooth muscle cells and lymphocytes. Fibroblast and endothelial cell proliferation on ®brin in vitro is enhanced by ®brinopeptide B cleavage and exposure of the amino-terminus of the ®brin Bb chain, suggesting that speci®c structural features of the provisional ®brin matrix formed at sites of injury may modulate the proliferative response of these cells [6]. In contrast, intact ®brinogen is mitogenic for both lymphoid cell lines and human hemopoietic progenitors, an eect mediated by two possible nonintegrin binding sites, a 92±94 kDa mitogenic ®brinogen receptor (MFR) and ICAM-1. The MFR is down-regulated in plasma and serum which may explain why normal cells do not proliferate in the peripheral circulation [7]. The cleavage products of ®brin(ogen) can also regulate cell proliferation. Fibrinopeptides A and B, as well as partially degraded ®brinogen products released following cleavage by thrombin, are mitogens for ®broblasts in vitro and therefore may provide an early stimulus for proliferation during the initial coagulation stage of tissue repair [8]. In addition, the ®brin degradation product, fragment E, stimulates vascular proliferation, including angiogenesis, and is thought to play a role in pathological lesion development such as atherosclerotic plaque growth, as well as, wound healing and breast cancer [9]. 4.2. Vasoactive eects of ®brin(ogen) and its cleavage products Peptides derived from proteolytic cleavage of ®brin(ogen) have been shown to have numerous vasoactive eects, including vasoconstriction, vasodilation, and increased vascular permeability [10]. However, some of these eects may be indirect via the release of known vasoactive mediators, such as histamine, bradykinin and products of the arachidonic acid cascade. For example, intact ®brinogen can bind directly to ICAM-1 on saphenous vein endothelium to in-
S. Herrick et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746
itiate signalling pathways leading to the synthesis of vasoactive mediators [11]. Furthermore, ®brin and its cleavage products cause ICAM-1 upregulation on endothelial cells thus providing a link between ®brin deposition and adhesion molecule expression which may lead subsequently to leucocyte accumulation and transendothelial extravasation [12]. 4.3. Migratory eects of ®brin(ogen) and its cleavage products A variety of cell types speci®cally adhere to and spread on a ®brin(ogen) matrix including endothelial cells, ®broblasts, macrophages, smooth muscle cells, keratinocytes and tumour cells. During tissue repair, the provisional ®brin matrix provides a critical scaold for the migration of a variety of cells into the wound site subsequently resulting in re-epithelialisation, vascularisation and the deposition of collagen [13]. Fibrin(ogen) cleavage products have been shown to act as chemoattractants for neutrophils, ®broblasts and monocytes. Fibrinopeptide B (Bb1±14), released from ®brinogen by thrombin, and plasmic-derived ®brinogen peptide (Bb1±42) stimulate the migration of neutrophils and ®broblasts with an activity comparable to that of leukotriene B4 and platelet-derived growth factor (PDGF) respectively. In addition to ®brinopeptide B, ®brin degradation products, fragments D and E, are chemoattractants for monocytes. The presence of several independent chemotactic domains in the amino-terminal region of the ®brinogen Bb chain indicates that proteolysis of ®brinogen by thrombin, neutrophil elastase or plasmin releases potent chemotactic fragments, capable of mobilising leucocytes and ®broblasts to the site of injury and thus potentiating the in¯ammatory response [14]. 5. Therapeutic potential Thrombus formation is a major complication and cause of mortality and morbidity following vascular injury. A number of approaches are already used to prevent thrombus formation, but
745
the existing agents can induce allergies and gastric bleeding (aspirin), need to be administered parenterally (heparin), or can cause severe drug reactions (warfarin). Alternative forms of therapy are therefore being sought. It is well recognised that ®brinogen plays a central role in thrombus formation, causing platelet aggregation and facilitating their activation. Since aIIbb3 (GPIIb/IIIa) was identi®ed as the platelet's principal receptor for ®brinogen, a range of peptide antagonists has been designed around the RGD sequence of ®brinogen to block its interaction with platelets. Recent studies have resulted in the synthesis of orally active nonpeptide antagonists which inactivate the receptor. GPIIb/IIIa inhibitors are signi®cantly more eective than heparin in controlling platelet-rich arterial thrombosis, but are of little use in venous thrombosis, where ®brin is the key mediator of thrombus formation. One potential application emanating from recent developments is the use of agents directed against ®brin(ogen)±cell interactions to control tissue repair and ®brosis. By controlling ®brinogen degradation, the distribution of its fragments and interactions between these fragments and their receptors, the rate and quality of the repair process may be modi®ed. Promotion of ®broblast migration and angiogenesis by ®brin cleavage products may be useful in some settings such as nonhealing wounds. Alternatively, the inhibition of these processes may be helpful in ®broproliferative conditions. Clinical applications aim to stimulate healing through the administration of `®brin sealants' and may be used in a variety of surgical procedures as haemostatic and delivery agents, tissue adhesives, barriers to ¯uid leakage, and packing materials [15]. An improved understanding of the structure and function of ®brinogen and its cleavage products as well as their speci®c receptors will reveal novel drug targets to modulate cell function during tissue repair and ®broproliferative disorders.
References [1] A.H. Henschen-Edman, Human ®brinogen occurs as over 1 million non-identical molecules, in: M.Z. Atassi,
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[2]
[3]
[4]
[5]
[6] [7]
[8]
S. Herrick et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746 E. Appella (Eds.), Methods in Protein Structure Analysis, Plenum Press, New York, 1995, pp. 435±443. G. Guadiz, L.A. Sporn, R.A. Goss, S.O. Lawrence, V.J. Marder, P.J. Simpson-Haidaris, Polarized secretion of ®brinogen by lung epithelial cells, Am. J. Respir. Cell Mol. Biol. 17 (1997) 60±69. M.W. Mosesson, Fibrinogen and ®brin polymerisation: appraisal of the binding events that accompany ®brin generation and ®brin clot assembly, Blood Coagul. Fibrinol. 8 (1997) 257±267. T.T. Suh, K. HolmbaÈck, N.J. Jensen, C.C. Daugherty, K. Small, D.I. Simon, S.S. Potter, J.L. Degen, Resolution of spontaneous bleeding events but failure of pregnancy in ®brinogen-de®cient mice, Genes Dev. 9 (1995) 2020±2033. L.R. Languino, A. Duperray, K.J. Joganic, M. Fornaro, G.B. Thornton, D.C. Alteri, Regulation of leucocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-®brinogen recognition, Proc. Natl. Acad. Sci. USA 28 (1995) 1505±1509. L.A. Sporn, A.A. Bunce, C.W. Francis, Cell proliferation on ®brin: modulation by ®brinopeptide cleavage, Blood 86 (1995) 1802±1810. A. Heron, J.P. LeÂvesque, A. Hatzfeld, S. Kisselev, M.N. Monier, P. Krief, J. Hatzfeld, Mitogenic eect of ®brinogen on hematopoietic cells: involvement of two distict speci®c receptors, MFR and ICAM-1, Biochem. Biophys. Res. Commun. 246 (1998) 231±237. A.J. Gray, P.W. Parks, T.J. Broekelmann, G.J. Laurent, J.T. Reeves, K.R. Stenmark, R.P. Mecham, The mito-
[9]
[10]
[11] [12] [13]
[14]
[15] [16]
genic eects of the Bb chain of ®brinogen are mediated through cell surface calreticulin, J. Biol. Chem. 270 (1995) 26602±26606. W.D. Thompson, E.B. Smith, C.M. Stirk, J. Wang, Fibrin degradation products in growth stimulatory extracts of pathological lesions, Blood Coagul. Fibrinol. 4 (1993) 113±115. C.W. Francis, L.A. Bunce, L.A. Sporn, Endothelial cell responses to ®brin mediated by ®brinopeptide B cleavage and amino terminus of the b chain, Blood Cells 19 (1993) 291±307. R.C.J. Hicks, J. Golledge, R. Mir-Hasseine, J.T. Powell, Vasoactive eects of ®brinogen on saphenous vein, Nature 379 (1996) 818±820. J. Qi, D.L. Kreutzer, T.H. Piela-Smith, Fibrin induction of ICAM-1 expression in human vascular endothelial cells, J. Immunol. 158 (1997) 1880±1886. D. Greiling, R.A.F. Clark, Fibronectin provides a conduit for ®broblast transmigration from a collagenous stroma into ®brin clot provisional matrix, J. Cell Sci. 110 (1997) 861±870. W.F. Skogen, R.M. Senior, G.L. Grin, G.N. Wilner, Fibrinogen-derived peptide B beta 1±42 is a multidomained neutrophil chemoattractant, Blood 71 (1988) 1475±1479. D.H. Sierra, Fibrin sealant adhesive systems: a review of their chemistry, material properties and clinical applications, J. Biomater. Appl. 7 (1993) 309±352. M.W. Mosesson, Fibrin polymerization and its regulatory role in hemostasis, J. Lab. Clin. Med. 116 (1990) 8± 17.
Molecules in focus
Fibrinogen Sarah Herrick b,*, Olivier Blanc-Brude b, Andrew Gray a, Georey Laurent b a
Discovery Biology, P®zerCentral Research, Sandwich, Kent, UK Centre for Cardiopulmonary Biochemistry and Respiratory Medicine, The Rayne Institute, 5 University Street, University College London Medical School, London WC1E 6JJ, UK
b
Received 16 March 1999; received in revised form 16 March 1999; accepted 9 April 1999
Abstract Fibrinogen is a blood-borne glycoprotein comprised of three pairs of nonidentical polypeptide chains. Following vascular injury, ®brinogen is cleaved by thrombin to form ®brin which is the most abundant component of blood clots. As well as controlling blood loss at sites of tissue damage, other properties of ®brinogen have recently been discovered. For example, various cleavage products of ®brinogen and ®brin, released during coagulation and ®brinolysis, respectively, regulate cell adhesion and spreading, display vasoconstrictor and chemotactic activities, and are mitogens for several cell types including ®broblasts, endothelial and smooth muscle cells. Current research aims to de®ne the bioactive ®brinogen molecule moieties and cellular receptors involved in these processes. Future studies may provide us with new opportunities to develop agents which are useful in promoting tissue repair or conversely in inhibiting ®brosis in in¯ammatory and ®broproliferative diseases where endothelial cell damage or chronic leakage of blood proteins is a feature. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Fibrinogen; Fibrin degradation products; Integrins; Haemostasis
1. Introduction Human ®brinogen is a circulating 340 kDa glycoprotein, primarily synthesised by hepatocytes. It is comprised of two symmetric half molecules, each consisting of one set of three dierent polypeptide chains termed Aa, Bb and g (Fig. 1). The molecule is highly heterogenous due to alterna-
tive splicing, extensive post-translational modi®cation and proteolytic degradation. Furthermore, there is polymorphic variation in the Aa and Bb chains, leading to the suggestion that each individual may carry over one million nonidentical ®brinogen molecules in their blood [1].
2. Structure * Corresponding author. Tel.: +44-171-209-6016; fax: +44171-209-6973. E-mail address: [email protected] (S. Herrick)
Each of the three polypeptide chains of the ®brinogen molecule is encoded by a separate
1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 3 2 - 1
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S. Herrick et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746
Fig. 1. Schematic diagram of ®brinogen (adapted from Ref. [16]). There are three sets of nonidentical chains which are bound by disulphide bonds at the amino-terminal end. Removal of ®brinopeptides A and B at this end via the action of thrombin, leads to cross-linking and the formation of the ®brin clot.
gene located on chromosome four. The Aa (6 exons) and g (10 exons) ®brinogen chain genes are orientated in tandem and are transcribed in the opposite direction to the Bb (8 exons) ®brinogen chain gene. Alternative splicing may occur in the Aa and g ®brinogen chain genes leading to the production of peptide variants. The predominant Aa chain of circulating ®brinogen contains around 610 amino acid residues (70 kDa), the Bb chain of ®brinogen consists of 461 amino acids (56 kDa). The g chain is heterogenous with respect to both charge and size, however, the most abundant form, denoted as g- or gA, consists of 411 residues (48 kDa). The ®brinogen molecule has three distinct domains (see Fig. 1); two terminal D domains (67 kDa), each linked to a single central E domain (33 kDa) by a triple-stranded array of the polypeptide chains believed to exist in the form of a helical coiled coils. The three constitutive chains and the two halves of the ®brinogen molecule are held together by a series of 29 disul-
phide bonds with all 58 cysteine residues of ®brinogen participating in these interactions. There is extensive post-translational modi®cation of the protein including phosphorylation, sulphation, glycosylation and hydroxylation [1].
3. Synthesis and degradation Fibrinogen is synthesised by hepatocytes and circulates as a component of blood at a concentration of approximately 9 mM with a half-life of around 100 h. During episodes of in¯ammation, the synthesis of ®brinogen is drastically enhanced, an eect thought to be mediated by interleukin-6 (IL-6), glucocorticoids and oncostatin M. Interleukin-6 and glucocorticoid response elements have been identi®ed in all three ®brinogen chain genes, whereas the oncostatin M response elements have not been mapped.
S. Herrick et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746
Furthermore, the Bb and Aa chain gene promoters contain a hepatic nuclear factor-1 (HNF-1) binding site which appears to contribute to the liver speci®c expression of these genes. Liver speci®c expression of the g chain gene is mediated by an upstream stimulatory factor (USF) in conjunction with other upstream elements such as TATA- and CAAT-like sequences. Interestingly, recent work suggests that extra-hepatic epithelial cells, in particular lung alveolar epithelium, are also able to synthesise and secrete ®brinogen locally in response to proin¯ammatory mediators [2]. At sites of tissue injury, ®brinogen is converted to ®brin by a-thrombin with the release of ®brinopeptides A and B from the amino-terminal ends of the Aa and Bb chains respectively (Fig. 1). The ®brin monomers polymerise spontaneously and undergo intermolecular crosslinking to form a stable ®brin clot in the presence of transglutaminase (factor XIIIa) [3]. Subsequently, the ®brin matrix is degraded into an number of fragments, including X, Y, D and E, primarily through the action of plasmin.
743
Fig. 2. Key functions of ®brin(ogen) and its cleavage products. Fibrinogen and its cleavage products have well described roles in haemostasis where ®brin forms a clot limiting blood loss and providing a key substrate of the provisional matrix which is vital for normal repair. It is also recognised that ®brin(ogen) and its breakdown products are capable of promoting other vital events in tissue repair including blood vessel tone (vasoconstriction), angiogenesis, directed cell migration and proliferation of various cells including ®broblasts, smooth muscle cells and lymphocytes. The later data suggest it may in¯uence the local immune response.
However, other proteases, such as neutrophil elastase, mast cell tryptase, matrix metalloproteases and cathepsins D and G, are also able to degrade ®brin to produce an additional array of cleavage products.
Table 1 Integrin and nonintegrin receptors which bind to known recognition sites on human ®brin(ogen) or its cleavage products and regulate cell function Integrin receptor
Cell types
Recognition sites
Cellular function
aIIbb3 (GPIIb/IIIa)
platelets
avb3
adhesion aggregation and clot retraction adhesion and spreading
a5b1 amb2 (CD11b/CD18) axb2 (CD11c/CD18)
endothelial cells, melanoma cells, ®broblasts endothelial cells monocytes, neutrophils neutrophils B lymphocytes
g400±411, Aa572±574, Aa95±97 Aa572±574 Aa572±574 g190±202, g377±395 Aa17±19
adhesion phagocytosis and adhesion adhesion
Nonintegrin receptor
Cell types
Recognition sites
Cellular function
130 kD receptor
endothelial cells
Bb15±42
CD-44 surface receptor
endothelial cells, ®broblasts, tumour cells ®broblasts hemopoietic cells lymphoid cell lines endothelial cells, ®broblasts, mesothelial cells
±
von-Willebrand factor release adhesion and migration proliferation proliferation proliferation adhesion
Calreticulin 92±94 kD receptor (MFR) ICAM-1
Bb chain ± g117±133 g117±133
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S. Herrick et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746
4. Biological function Following vascular injury, there are two principal mechanisms by which ®brinogen participates in controlling blood loss. It acts as an adhesive protein essential for platelet aggregation as well as forming an insoluble ®brin clot in the ®nal stage of the blood coagulation cascade. It is therefore interesting that ®brinogen-de®cient mice are able to control bleeding following mild trauma [4]. This has led to the assumption that platelets can bind to alternative matrix molecules to establish a platelet-rich clot to prevent bleeding. However, this level of protection is clearly insucient following major vascular injury, resulting in uncontrolled bleeding as observed in individuals with congenital ®brinogen de®ciency. Although clearly important in haemostasis, the ®brin clot also provides a scaold for cell adhesion, spreading, migration and proliferation. Structural changes that accompany the conversion of ®brinogen to ®brin expose new receptor binding sites providing a basis for ®brin speci®c cell responses. Furthermore, recent studies have suggested that ®brin(ogen) cleavage products released during coagulation and subsequent ®brinolysis, may be bioactive and exert direct eects on ®broblasts and other cell types associated with the wound healing process (Fig. 2). Human ®brin(ogen) contains three potential integrin binding sites, two RGD sequences within the Aa chain (RGDS a572±575 and RGDF a95±98) and a non-RGD sequence in the g chain (carboxy terminal g400±411), but can also interact with cells through nonintegrin receptors (Table 1). For example, ®brinogen mediates the adhesion and transendothelial migration of leucocytes by acting as a molecular bridge between the cell types through ICAM-1 dependent pathways [5]. In addition, ®brin(ogen) binds to other extracellular matrix molecules and can act as a reservoir for growth factors, proteases and protease inhibitors. In particular, ®brin binds plasminogen and its activator (tissue-type plasminogen activator) to form a ternary complex which enhances the conversion of plasminogen into the ®brinolytic enzyme, plasmin and protects them from inactivation by their inhibitors.
4.1. Mitogenic eects of ®brin(ogen) and its cleavage products Fibrin(ogen) cleavage products are mitogens for a variety of cell types, including endothelial cells, ®broblasts, mesothelial cells, smooth muscle cells and lymphocytes. Fibroblast and endothelial cell proliferation on ®brin in vitro is enhanced by ®brinopeptide B cleavage and exposure of the amino-terminus of the ®brin Bb chain, suggesting that speci®c structural features of the provisional ®brin matrix formed at sites of injury may modulate the proliferative response of these cells [6]. In contrast, intact ®brinogen is mitogenic for both lymphoid cell lines and human hemopoietic progenitors, an eect mediated by two possible nonintegrin binding sites, a 92±94 kDa mitogenic ®brinogen receptor (MFR) and ICAM-1. The MFR is down-regulated in plasma and serum which may explain why normal cells do not proliferate in the peripheral circulation [7]. The cleavage products of ®brin(ogen) can also regulate cell proliferation. Fibrinopeptides A and B, as well as partially degraded ®brinogen products released following cleavage by thrombin, are mitogens for ®broblasts in vitro and therefore may provide an early stimulus for proliferation during the initial coagulation stage of tissue repair [8]. In addition, the ®brin degradation product, fragment E, stimulates vascular proliferation, including angiogenesis, and is thought to play a role in pathological lesion development such as atherosclerotic plaque growth, as well as, wound healing and breast cancer [9]. 4.2. Vasoactive eects of ®brin(ogen) and its cleavage products Peptides derived from proteolytic cleavage of ®brin(ogen) have been shown to have numerous vasoactive eects, including vasoconstriction, vasodilation, and increased vascular permeability [10]. However, some of these eects may be indirect via the release of known vasoactive mediators, such as histamine, bradykinin and products of the arachidonic acid cascade. For example, intact ®brinogen can bind directly to ICAM-1 on saphenous vein endothelium to in-
S. Herrick et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 741±746
itiate signalling pathways leading to the synthesis of vasoactive mediators [11]. Furthermore, ®brin and its cleavage products cause ICAM-1 upregulation on endothelial cells thus providing a link between ®brin deposition and adhesion molecule expression which may lead subsequently to leucocyte accumulation and transendothelial extravasation [12]. 4.3. Migratory eects of ®brin(ogen) and its cleavage products A variety of cell types speci®cally adhere to and spread on a ®brin(ogen) matrix including endothelial cells, ®broblasts, macrophages, smooth muscle cells, keratinocytes and tumour cells. During tissue repair, the provisional ®brin matrix provides a critical scaold for the migration of a variety of cells into the wound site subsequently resulting in re-epithelialisation, vascularisation and the deposition of collagen [13]. Fibrin(ogen) cleavage products have been shown to act as chemoattractants for neutrophils, ®broblasts and monocytes. Fibrinopeptide B (Bb1±14), released from ®brinogen by thrombin, and plasmic-derived ®brinogen peptide (Bb1±42) stimulate the migration of neutrophils and ®broblasts with an activity comparable to that of leukotriene B4 and platelet-derived growth factor (PDGF) respectively. In addition to ®brinopeptide B, ®brin degradation products, fragments D and E, are chemoattractants for monocytes. The presence of several independent chemotactic domains in the amino-terminal region of the ®brinogen Bb chain indicates that proteolysis of ®brinogen by thrombin, neutrophil elastase or plasmin releases potent chemotactic fragments, capable of mobilising leucocytes and ®broblasts to the site of injury and thus potentiating the in¯ammatory response [14]. 5. Therapeutic potential Thrombus formation is a major complication and cause of mortality and morbidity following vascular injury. A number of approaches are already used to prevent thrombus formation, but
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the existing agents can induce allergies and gastric bleeding (aspirin), need to be administered parenterally (heparin), or can cause severe drug reactions (warfarin). Alternative forms of therapy are therefore being sought. It is well recognised that ®brinogen plays a central role in thrombus formation, causing platelet aggregation and facilitating their activation. Since aIIbb3 (GPIIb/IIIa) was identi®ed as the platelet's principal receptor for ®brinogen, a range of peptide antagonists has been designed around the RGD sequence of ®brinogen to block its interaction with platelets. Recent studies have resulted in the synthesis of orally active nonpeptide antagonists which inactivate the receptor. GPIIb/IIIa inhibitors are signi®cantly more eective than heparin in controlling platelet-rich arterial thrombosis, but are of little use in venous thrombosis, where ®brin is the key mediator of thrombus formation. One potential application emanating from recent developments is the use of agents directed against ®brin(ogen)±cell interactions to control tissue repair and ®brosis. By controlling ®brinogen degradation, the distribution of its fragments and interactions between these fragments and their receptors, the rate and quality of the repair process may be modi®ed. Promotion of ®broblast migration and angiogenesis by ®brin cleavage products may be useful in some settings such as nonhealing wounds. Alternatively, the inhibition of these processes may be helpful in ®broproliferative conditions. Clinical applications aim to stimulate healing through the administration of `®brin sealants' and may be used in a variety of surgical procedures as haemostatic and delivery agents, tissue adhesives, barriers to ¯uid leakage, and packing materials [15]. An improved understanding of the structure and function of ®brinogen and its cleavage products as well as their speci®c receptors will reveal novel drug targets to modulate cell function during tissue repair and ®broproliferative disorders.
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