Binding of Basic Fibroblast Growth Factor to Fibrinogen and Fibrin
1998; Elsevier BV; Volume: 273; Issue: 13 Linguagem: Inglês
10.1074/jbc.273.13.7554
ISSN1083-351X
AutoresAbha Sahni, Tatjana Odrljin, Charles W. Francis,
Tópico(s)Platelet Disorders and Treatments
ResumoFibrin is formed at sites of tissue injury and provides the temporary matrix needed to support the initial endothelial cell responses needed for vessel repair. Basic fibroblast growth factor (bFGF) also acts at sites of injury and stimulates similar vascular cell responses. We have, therefore, investigated whether there are specific interactions between bFGF and fibrinogen and fibrin that could play a role in coordinating these actions. Binding studies were performed using bFGF immobilized on Sepharose beads and soluble125I-labeled fibrinogen and also using Sepharose-immobilized fibrinogen and soluble 125I-bFGF. Both systems demonstrated specific and saturable binding. Scatchard analysis indicated two classes of binding sites for each withKd values of 1.3 and 260 nm using immobilized bFGF; and Kd values of 0.9 and 70 nm using immobilized fibrinogen. After conversion of Sepharose-immobilized fibrinogen to fibrin by treatment with thrombin, bFGF also demonstrated specific and saturable binding with two classes of binding sites having Kd values of 0.13 and 83 nm. Fibrin binding was also investigated by clotting a solution of bFGF and fibrinogen, and two classes of binding sites were demonstrated using this system with Kd values of 0.8 and 261 nm. The maximum molar binding ratios of bFGF to fibrinogen were between 2.0 and 4.0 with the four binding systems. We conclude that bFGF binds specifically and saturably to fibrinogen and fibrin with high affinity, and this may have implications regarding the localization of its effect at sites of tissue injury. Fibrin is formed at sites of tissue injury and provides the temporary matrix needed to support the initial endothelial cell responses needed for vessel repair. Basic fibroblast growth factor (bFGF) also acts at sites of injury and stimulates similar vascular cell responses. We have, therefore, investigated whether there are specific interactions between bFGF and fibrinogen and fibrin that could play a role in coordinating these actions. Binding studies were performed using bFGF immobilized on Sepharose beads and soluble125I-labeled fibrinogen and also using Sepharose-immobilized fibrinogen and soluble 125I-bFGF. Both systems demonstrated specific and saturable binding. Scatchard analysis indicated two classes of binding sites for each withKd values of 1.3 and 260 nm using immobilized bFGF; and Kd values of 0.9 and 70 nm using immobilized fibrinogen. After conversion of Sepharose-immobilized fibrinogen to fibrin by treatment with thrombin, bFGF also demonstrated specific and saturable binding with two classes of binding sites having Kd values of 0.13 and 83 nm. Fibrin binding was also investigated by clotting a solution of bFGF and fibrinogen, and two classes of binding sites were demonstrated using this system with Kd values of 0.8 and 261 nm. The maximum molar binding ratios of bFGF to fibrinogen were between 2.0 and 4.0 with the four binding systems. We conclude that bFGF binds specifically and saturably to fibrinogen and fibrin with high affinity, and this may have implications regarding the localization of its effect at sites of tissue injury. The vascular response to injury requires a coordinated interaction of the hemostatic and inflammatory systems and is regulated by cytokines and growth factors that act locally to regulate cellular proliferation and tissue repair. The hemostatic response results in platelet accumulation at the site of injury, and exposure of blood to tissue factor also leads to the formation of thrombin. Thrombin then cleaves fibrinopeptides from fibrinogen converting it to fibrin, which helps prevent blood loss and also serves as a temporary matrix to support tissue healing and remodeling. The role of fibrin in the cellular response is not passive as a structural matrix only, but rather it plays an active role through specific receptor-mediated interactions with cells of the blood and vessel wall. These result in fibrin-specific responses of endothelial cells including adhesion and spreading (1Bunce L.A. Sporn L.A. Francis C.W. J. Clin. Invest. 1992; 89: 842-850Crossref PubMed Scopus (65) Google Scholar), proliferation (2Sporn L.A. Bunce L.A. Francis C.W. Blood. 1995; 86: 1802-1810Crossref PubMed Google Scholar), protein synthesis (3Kaplan K.L. Mather T. DeMarco L. Solomon S. Arteriosclerosis. 1989; 9: 43-49Crossref PubMed Google Scholar) and secretion (4Ribes J.A. Francis C.W. Wagner D.D. J. Clin. Invest. 1987; 79: 117-123Crossref PubMed Scopus (134) Google Scholar), and angiogenesis (5Chalupowicz D.G. Chowdhury Z.A. Bach T.L. Barsigian C. Martinez J. J. Cell Biol. 1995; 130: 207-215Crossref PubMed Scopus (128) Google Scholar).Cytokines and growth factors are produced in response to injury and also act locally to modulate cell responses to vascular damage. Important among these are members of the fibroblast growth factor family, which includes 13 members exerting a variety of effects on many cells and organ systems (6Bikfalvi A. Klein S. Pintucci G. Rifkin D.B. Endocr. Rev. 1997; 18: 26-45Crossref PubMed Scopus (847) Google Scholar). In particular, bFGF 1The abbreviations used are: bFGF, basic fibroblast growth factor; FGFR, FGF receptor. 1The abbreviations used are: bFGF, basic fibroblast growth factor; FGFR, FGF receptor. increases endothelial cell migration and proliferation and also stimulates angiogenesisin vitro and in vivo (6Bikfalvi A. Klein S. Pintucci G. Rifkin D.B. Endocr. Rev. 1997; 18: 26-45Crossref PubMed Scopus (847) Google Scholar, 7Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1049) Google Scholar). bFGF also regulates the expression of proteolytic mediators of angiogenesis including urokinase-type plasminogen activator and collagenase (8Gualandris A. Presta M. J. Cell. Physiol. 1995; 162: 400-409Crossref PubMed Scopus (42) Google Scholar) and urokinase-type plasminogen activator receptor (9Mignatti P. Mazzieri R. Rifkin D.B. J. Cell Biol. 1991; 113: 1193-1201Crossref PubMed Scopus (157) Google Scholar). The role of bFGF in vessel injury and repair is further supported by evidence that bFGF is released from vessel wall cells after injury (10Villaschi S. Nicosia R. Am. J. Pathol. 1993; 143: 181-190PubMed Google Scholar) and that bFGF mRNA is up-regulated in atherosclerotic arteries (11Hughes S.E. Crossman D. Hall P.A. Cardiovasc. Res. 1993; 27: 1214-1219Crossref PubMed Scopus (99) Google Scholar) and following vessel injury (12Casscells W. Lappi D.A. Olwin B.B. Wai C. Siegman M. Spier E.H. Sasse J. Baird A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7159-7163Crossref PubMed Scopus (127) Google Scholar).The need for fibrin to support endothelial cell spreading, migration, angiogenesis, and the potent stimulation of the same responses by bFGF suggests that these processes may be interrelated. This concept is supported by evidence that fibrin clots are a good matrix to support bFGF-stimulated angiogenesis in vitro (13Deblois C. Cote M.-F. Doillon C.J. Biomaterials. 1994; 15: 665-672Crossref PubMed Scopus (80) Google Scholar, 14Brown, K. J., Maynes, S. F., Bezos, A., Maguire, D. J., Ford, M. D., and Parish, C. R. Lab. Invest., 75, 539–555.Google Scholar). Little information is available, however, regarding specific interactions of bFGF with fibrin. We have, therefore, investigated the association of bFGF with fibrinogen and fibrin, and the results demonstrate high affinity specific and saturable binding.RESULTSBinding of fibrinogen to immobilized bFGF was saturable and specific with nonspecific binding representing less than 20% of the total (Fig. 1 A). Saturation of specific binding occurred at a fibrinogen concentration of 150 nm, and only an increase in nonspecific binding was observed at higher concentrations. In control experiments there was a maximum of 3% binding of 125I-fibrinogen over the same range of concentrations to beads with immobilized anti-bFGF immunoglobulin only or to beads with no protein bound and active sites blocked with ethanolamine. A plot of bound versus bound/free fibrinogen (Fig. 1 B) was nonlinear, suggesting the presence of more than one binding site. This was confirmed by Scatchard analysis, which indicated that binding was best described by a two-site model with apparent Kd values of 1.3 and 260 nm (Table I).Bmax was 6.3 and 35 nm for the high and low affinity sites, respectively, and the maximum molar binding ratio of bFGF to fibrinogen was 4.0.Table IBinding constants for the association of bFGF with fibrinogen and fibrinSystemKd1Kd2Bmax1Bmax2Maximum molar binding ratio of bFGF/fibrin(ogen)nmFibrinogen/bFGF-Sepharose1.3 ± 1.6260 ± 646.3 ± 3.135 ± 2.14.0 ± 0.5bFGF/fibrinogen-Sepharose0.9 ± 0.170 ± 120.3 ± .025.9 ± 0.72.0 ± 0.5bFGF/fibrin-Sepharose0.13 ± 0.183 ± 24.03 ± .015.9 ± 1.42.0 ± 0.9bFGF/fibrin clot0.8 ± 0.7261 ± 700.5 ± 0.360 ± 222.0 ± 0.8 Open table in a new tab To further characterize the protein that bound to bFGF,125I-fibrinogen was passed over a column of immobilized bFGF. Following washing, the specifically bound protein was eluted with 2 mg/ml unlabeled fibrinogen (Fig. 2), and approximately 90% of bound label rapidly eluted in two fractions. SDS-polyacrylamide gel electrophoresis of the eluted protein showed bands consistent with the Aα, Bβ and γ chains of fibrinogen (Fig. 2, inset) establishing that the bound protein was fibrinogen and not a minor contaminant. In control experiments, less than 5% of bound radioactivity was eluted from the column with 2.0 mg/ml ovalbumin, demonstrating specificity of the elution.Figure 2Elution of bound protein from immobilized bFGF. 125I-Fibrinogen (1.0 mg/ml) was passed through a 1-ml column of Sepharose-immobilized bFGF. Following washing, the column was eluted with 2 mg/ml unlabeled fibrinogen, and fractions of 200 μl were collected. Approximately 90% of the bound radioactivity eluted in fractions 5 and 6 (1.0–1.2 ml elution volume). These fractions were pooled, and an aliquot was electrophoresed on a 7% SDS-polyacrylamide gel electrophoresis gel and used to prepare autoradiograms (inset). The polypeptide chain pattern in the eluted pool showed Aα, Bβ, and γ chains of fibrinogen and was similar to that in the starting material.View Large Image Figure ViewerDownload (PPT)The association of bFGF and fibrinogen was also characterized using soluble 125I-radiolabeled bFGF and fibrinogen immobilized on Sepharose beads (Fig. 3). With this system, saturable and specific binding was also observed, and nonspecific binding represented 20% or less of the total. Saturation of specific binding was observed at a bFGF concentration of approximately 75 nm. Scatchard analysis (Fig. 3 B) indicated the presence of two binding sites of different affinities with apparent Kd values of 0.9 and 70 nm and a maximum molar binding ratio of 2.0 (Table I) as compared with 4.0 with radiolabeled fibrinogen binding to Sepharose-immobilized bFGF (Fig. 1 and Table I). Competitive inhibition of the binding was performed to further characterize the specificity and the degree of nonspecific association. 125I-bFGF at a concentration of 0.2 nm was incubated with Sepharose-immobilized fibrinogen and then varying concentrations of unlabeled bFGF was added. The binding of 125I-bFGF to fibrinogen was reduced in a dose-dependent manner with increasing concentrations of unlabeled bFGF (Fig. 3 C), but 28% of the radiolabel remained at 100 nm bFGF, which we interpreted as nonspecific binding.Figure 3A, binding of bFGF to fibrinogen. 125I-bFGF was incubated with fibrinogen immobilized on Sepharose beads, and the amount of bound protein was determined as radioactivity associated with the beads following centrifugation and washing. Nonspecific binding (squares) was determined in the same way in the presence of a 100-fold molar excess of unlabeled bFGF. Specific binding (triangles) was calculated by subtracting the nonspecific from the total bound (diamonds). Each point represents the mean ± S.D. of three different experiments. B, Scatchard plot. The best fit of the data was determined by analysis with the Ligand program and is most consistent with involvement of two distinct binding sites.C, competitive inhibition of binding. Increasing concentrations of unlabeled bFGF were used to competitively inhibit the binding of 125I-bFGF to fibrinogen. Each point represents mean ± S.D. of three different experiments.View Large Image Figure ViewerDownload (PPT)Fibrinogen is converted to fibrin by thrombin, which cleaves fibrinopeptides A and B from the Aα and Bβ chains respectively, forming fibrin monomer, which can then polymerize to form a branching network of fibers. To characterize the association of bFGF with fibrin in the absence of polymerization, we incubated Sepharose-immobilized fibrinogen with thrombin. The antibody-mediated immobilization of fibrinogen to the Sepharose beads prevents or limits association of the resulting fibrin, forming a surface with immobilized fibrin monomer. Binding of bFGF to fibrin (Fig. 4 A) was similar to that seen for fibrinogen using the same system with specific binding approaching saturation between 75 and 100 nm bFGF (Fig. 4 A). Nonspecific binding was low at bFGF concentrations below 15 nm and increased at higher concentrations. In contrast to the binding seen with fibrinogen, the bFGF binding curve at concentrations below 1 nm bFGF suggested the presence of a high affinity binding site of low capacity. This was confirmed by Scatchard analysis (Fig. 4 B), indicating the presence of two binding sites with apparent Kd values of 0.13 and 83 nm (Table I).Figure 4Binding of bFGF to fibrin monomer.Fibrinogen was immobilized on Sepharose beads and then converted to fibrin monomer by incubation with thrombin. 125I-bFGF was incubated with immobilized fibrin, and bound and free ligand were then separated by centrifugation. Nonspecific binding (squares) was measured in the presence of a 100-fold molar excess of unlabeled bFGF, and specific binding (triangles) was determined by subtraction of nonspecific from total binding (diamonds). Each point represents the mean ± S.D. of three different experiments. B, Scatchard plot. The best fit of the data was determined using the Ligand program, and the presence of two distinct binding sites was indicated.View Large Image Figure ViewerDownload (PPT)Characterization of binding to polymerized fibrin presents technical and interpretive problems because of transport of bFGF into the gel is limited, and access to potential binding sites within individual fibrin fibers may also be restricted. We chose, therefore, to add125I-bFGF to a solution of fibrinogen, which was then clotted by the addition of thrombin to avoid problems of transport of bFGF into the gel. Total binding was measured with this clotting system in the absence of an unlabeled competitor, whereas nonspecific binding was measured in the presence of 100-fold molar access of unlabeled bFGF (Fig. 5). Nonspecific binding represented up to 40% of the total (Fig. 5 A). This was higher than that seen with binding to fibrinogen (Figs. 1 and 3) or to fibrin monomer (Fig. 4), possibly reflecting some entrapment of radiolabel within the fibrin gel. A plot of bound versus bound/free125I-bFGF was nonlinear (Fig. 5 B), and Scatchard analysis identified two distinct binding sites with apparentKd values of 0.8 and 261 nm, similar to those for fibrinogen (Table I). The maximum molar binding ratio of bFGF to fibrin was 2.0.Figure 5A, binding of 125I-bFGF to polymerized fibrin. 125I-bFGF was added to a solution of 100 μg/ml fibrinogen and then clotted by the addition of 0.5 units/ml of thrombin. Bound and unbound bFGF were then separated by vacuum filtration, and nonspecific binding (squares) was determined in the presence of 100-fold molar excess of bFGF. Specific binding (triangles) was calculated by subtracting nonspecific from total binding (diamonds). Each point represents the mean ± S.D. of three different experiments. B, Scatchard analysis. The best fit of the data was determined using the Ligand program, and the presence of two distinct binding sites was indicated.View Large Image Figure ViewerDownload (PPT)DISCUSSIONThe results presented demonstrate that bFGF binds specifically and saturably to fibrinogen and fibrin. Two different experimental systems were used to characterize the association with either bFGF or fibrinogen immobilized on Sepharose beads. The results were similar, with both systems identifying high affinity binding sites withKd values of 1.3 and 0.9 nm and lower affinity sites with Kd values of 260 and 70 nm. The association of bFGF with fibrin was also characterized using two systems with either surface-immobilized fibrin or polymerized fibrin. The results of binding to fibrin were similar to those found using fibrinogen with two distinct binding sites. the Kd values for the high and low affinity sites were 0.13 and 0.8 nm and 83 and 261 nm for surface-immobilized and -polymerized fibrin, respectively.The maximum molar binding ratios for bFGF to fibrinogen or fibrin were between 2.0 and 4.0 with the different systems used. Considering that fibrinogen is a dimerically symmetric molecule (19Doolittle R.F. Annu. Rev. Biochem. 1984; 53: 195-229Crossref PubMed Scopus (524) Google Scholar) and that two binding sites with different Kd values were identified, the ratio of 4 bFGF to 1 fibrinogen would be expected and consistent with the presence of two structurally distinct and independent sites on each half-molecule. The expected ratio of 4 was, however, only found using a system in which fibrinogen bound to immobilized bFGF, whereas the ratio was lower using the other three approaches. One potential explanation for the lower binding ratio is that the access of bFGF to potential binding sites was limited. This would be reasonable with polymerized fibrin, as sites in both the D and E domains are involved in the reciprocal binding required for polymerization (20Olexa S.A. Budzynski A.Z. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1374-1378Crossref PubMed Scopus (151) Google Scholar). This could prevent concurrent binding of bFGF to any sites in close proximity or to those otherwise affected by polymerization. A similar explanation could explain reduced binding of bFGF to Sepharose-immobilized fibrinogen or fibrin if the binding site was either close to that recognized by the antibody J88B or altered by antibody binding. An alternative explanation for the lower than expected binding ratio is that one of the binding sites is present on only a minor variant of fibrinogen. There are several such fibrinogen variants including those due to heterogeneity at the carboxyl terminus of the γ chain (21Francis C.W. Marder V.J. Martin S.E. J. Biol. Chem. 1980; 255: 5599-5604Abstract Full Text PDF PubMed Google Scholar, 22Wolfenstein-Todel C. Mosesson M.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5069-5073Crossref PubMed Scopus (105) Google Scholar) or to variations in serine phosphorylation. These sites are known to be important in the molecular interactions and function of fibrinogen and fibrin as the γ chain site is involved both in binding to platelets (23Kloczewiak M. Timmons S. Bednarek M.A. Sakon M. Hawiger J. Biochemistry. 1989; 28: 2915-2923Crossref PubMed Scopus (83) Google Scholar) and in factor XIII cross-linking (24Chen R. Doolittle R.F. Biochemistry. 1971; 10: 4486-4491Crossref Scopus (206) Google Scholar), and phosphorylation of Ser3 of the α chain affects thrombin action (25Hanna L.S. Scheraga H.A. Francis C.W. Marder V.J. Biochemistry. 1984; 23: 4681-4687Crossref PubMed Scopus (52) Google Scholar). This explanation could account for the ratio of 4 found with the system using bFGF immobilized on Sepharose as only those fibrinogen molecules with the high affinity site would bind. By contrast, the epitope recognized by J88B involves a structurally invariant site (17Odrljin T.M. Rybarczyk B.J. Francis C.W. Lawrence S.O. Hamaguchi M. Simpson-Haidaris P.J. Biochim. Biophys. Acta. 1996; 1298: 69-77Crossref PubMed Scopus (24) Google Scholar) so all fibrinogen molecules would be immobilized, whereas only a minority would possess the putative high affinity site. Ongoing studies designed to identify specific sites on fibrinogen and fibrin responsible for bFGF binding will be required to resolve this question.The significance of bFGF binding to fibrinogen and fibrin must be considered in relation to both the tissue distribution of bFGF and to the availability of other sites for binding within the vasculature. bFGF has a wide tissue distribution, and it is synthesized in culture by fibroblasts, endothelial cells, glial cells, and smooth muscle cells (6Bikfalvi A. Klein S. Pintucci G. Rifkin D.B. Endocr. Rev. 1997; 18: 26-45Crossref PubMed Scopus (847) Google Scholar). Vlodavsky et al. (26Vlodavsky I. Fridman R. Sullivan R. Sasse J. Klagsbrun M. J. Cell. Physiol. 1987; 131: 402-408Crossref PubMed Scopus (163) Google Scholar) have shown that endothelial cells synthesize bFGF, which then remains closely associated with the cells and bound to the basement membrane or cell matrix with aKd value of 610 nm. It is inactive as a complex with heparan sulfate or heparin but is stabilized and protected against proteolytic degradation (27Saksela O. Moscatelli D. Sommer A. Rifkin D.B. J. Cell Biol. 1988; 107: 743-751Crossref PubMed Scopus (651) Google Scholar, 28Rosengart T.K. Johnson W.B. Friesel R. Clark R. Maciag T. Biochem. Biophys. Res. Commun. 1988; 152: 432-440Crossref PubMed Scopus (130) Google Scholar). It can be released in active form by proteolytic degradation with plasmin (29Saksela O. Rifkin D.B. J. Cell Biol. 1990; 110: 767-775Crossref PubMed Scopus (433) Google Scholar) or heparitinase and by competition with heparin-like molecules (30Bhaskin P. Doctrow S. Klagsbrun M. Svahn C.M. Folkman J. Vlodavsky I. Biochemistry. 1989; 28: 1737-1743Crossref PubMed Scopus (515) Google Scholar). bFGF is also present normally in plasma at a concentration up to 10 pg/ml (0.6 pm), and elevated levels up to 6 pm can be found in patients after cardiopulmonary bypass (31Medalion B. Merin G. Aingorn H. Miao H.-Q. Nagler A. Elami A. Ishai-Michaeli R. Vlodavsky I. Circulation. 1997; 95: 1853-1862Crossref PubMed Scopus (27) Google Scholar) and chronic liver disease (32Jin-ho K. Tanimizu M. Hyodo I. Kurimoto F. Yamashita T. J. Gastroenterology. 1997; 32: 119-212Crossref PubMed Scopus (52) Google Scholar). The effects of bFGF are mediated through specific receptors, and four distinct genes encoding bFGF cell surface receptor tyrosine kinases have been identified. bFGF binds to FGFR1 and FGFR2 with similar affinities (33Mansukhani A. Dell'Era P. Moscatelli D. Kornbluth S. Hanafusa H. Basilico C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3305-3309Crossref PubMed Scopus (113) Google Scholar, 34Dionne C.A. Crumley G. Bellot F. Kaplan J.M. Searfoss G. Ruta M. Burgess W.H. Jaye M. Schlessinger J. EMBO J. 1990; 9: 2685-2692Crossref PubMed Scopus (544) Google Scholar), and bFGF binds specifically and with higher affinity to intact baby hamster kidney cells with Kdvalues of 20 pm and 2 nm (35Moscatelli D. J. Cell. Physiol. 1987; 131: 123-130Crossref PubMed Scopus (530) Google Scholar).Heparin and heparan sulfate interact with both bFGF and fibrinogen and may play a role in modulating interactions between them. Heparin and heparan sulfate represent low affinity (Kd 470 nm) receptors for bFGF present in abundance on cell surfaces and in the extracellular matrix (36Schlessinger J. Lax J. Lemmon M. Cell. 1995; 83: 357-360Abstract Full Text PDF PubMed Scopus (449) Google Scholar). This binding increases the concentration of bFGF on the cell surface and may thereby promote the interaction of bFGF with high affinity transmembrane-signaling receptors. Heparin is not required for binding of bFGF to specific cell receptors (37Roghani M. Mansukhani A. Dell'Era P. Bellosta P. Basilico C. Rifkin D.B. Moscatelli D. J. Biol. Chem. 1994; 269: 3976-3984Abstract Full Text PDF PubMed Google Scholar), but receptors are activated by receptor dimerization, which is promoted by heparin (38Springer B.A. Pantoliano M.W. Barbera F.A. Gunyuzlu P.L. Thompson L.D. Herblin W.F. Rosenfeld S.A. Book G.W. J. Biol. Chem. 1994; 269: 26879-26884Abstract Full Text PDF PubMed Google Scholar). Also, the affinity of bFGF for FGFR1 is increased approximately 10-fold in the presence of heparan sulfate or heparin (39Pantoliano M.W. Horlick R.A. Springer B.A. VanDyk D.E. Tobery T. Wetmore D.R. Lear J.D. Hahapetian A.T. Bradley J.D. Sisk W.P. Biochemistry. 1994; 33: 10229-10248Crossref PubMed Scopus (229) Google Scholar). Receptor dimerization results in activation of protein tyrosine kinase activity and autophosphorylation and further to activation of the signaling pathway for the initiation of cell proliferation (40Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar). Heparin mediates binding of bFGF to the luminal surface of endothelial cells as indicated by its displacement by incubation of heparin in vitro (31Medalion B. Merin G. Aingorn H. Miao H.-Q. Nagler A. Elami A. Ishai-Michaeli R. Vlodavsky I. Circulation. 1997; 95: 1853-1862Crossref PubMed Scopus (27) Google Scholar) or by heparin infusionin vivo (31Medalion B. Merin G. Aingorn H. Miao H.-Q. Nagler A. Elami A. Ishai-Michaeli R. Vlodavsky I. Circulation. 1997; 95: 1853-1862Crossref PubMed Scopus (27) Google Scholar, 41Thompson R.W. Whalen G.F. Saunders K.B. Hores T. D'more P.A. Growth Factors. 1990; 3: 221-229Crossref PubMed Scopus (27) Google Scholar). Heparin also binds to fibrinogen and fibrin, and it may, therefore, alter their interactions with bFGF. Heparin binds to a site within the fibrinogen D domain (42Mohri H. Iwamatsu A. Ohkubo T. Thromb. Thrombolysis. 1994; 1: 49-54Crossref PubMed Scopus (12) Google Scholar) and with higher affinity (Kd 0.8 μm) to the central E domain (43Odrljin T.M. Shainoff J.R. Lawrence S.O. Simpson-Haidaris P.J. Blood. 1996; 88: 2050-2061Crossref PubMed Google Scholar). Cleavage of fibrinopeptide B during conversion to fibrin exposes a new site at the amino terminus of the β chain including residues 15–42, which represent a higher affinity site (Kd 0.3 μm) that mediates fibrin-endothelial cell interactions (43Odrljin T.M. Shainoff J.R. Lawrence S.O. Simpson-Haidaris P.J. Blood. 1996; 88: 2050-2061Crossref PubMed Google Scholar).The binding of bFGF to fibrinogen has implications regarding the distribution and actions of bFGF within the vasculature. At normal plasma concentrations of fibrinogen (7 μm) and of bFGF (up to 6 pm) nearly all bFGF should be bound to fibrinogen considering the Kd values in the nanomolar range. However, other bFGF binding proteins, α2 macroglobulin (44Dennis P.A. Saksela O. Harpel P. Rifkin D.B. J. Biol. Chem. 1989; 264: 7210-7216Abstract Full Text PDF PubMed Google Scholar) and soluble forms of FGF receptor (45Hanneken A. Ying W. Ling N. Baird A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9170-9174Crossref PubMed Scopus (92) Google Scholar), have also been identified in blood. The binding of bFGF to α2 macroglobulin involves formation of covalent bonds and is slow, requiring up to 4 h to reach completion (44Dennis P.A. Saksela O. Harpel P. Rifkin D.B. J. Biol. Chem. 1989; 264: 7210-7216Abstract Full Text PDF PubMed Google Scholar). Three soluble truncated forms of the high affinity cell receptor FGFR1 have also been identified in plasma as binding proteins for bFGF (45Hanneken A. Ying W. Ling N. Baird A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9170-9174Crossref PubMed Scopus (92) Google Scholar), but neither the plasma concentration nor binding affinities have been described. Further studies will be required to elucidate the distribution of bFGF binding to these plasma proteins and their role in influencing plasma half-life or bFGF activity.The binding of bFGF with fibrinogen and fibrin may have effects locally at sites of vessel injury or disease. Fibrinogen is found in both normal and atherosclerotic arterial walls (46Bini A. Kudryk B.J. Ann. N. Y. Acad. Sci. U. S. A. 1995; 748: 461-470Crossref PubMed Scopus (33) Google Scholar, 47Shekhonin, B. V., Tararak, T. M., Samokhin, G. P., Mitkevich, O. V., Mazurov, A. V., Vinogradov, D. V., Vlasik, T. N., Kalantarov, G. F., and Koteliansky, V. E.Atherosclerosis, 82, 213–226.Google Scholar, 48Bini A. Kudryk G.J. Thromb. Res. 1994; 75: 337-341Abstract Full Text PDF PubMed Scopus (18) Google Scholar) and could, therefore, serve as a binding site for bFGF within the matrix. This may also occur with fibrin that is also present in atherosclerotic vessels (47Shekhonin, B. V., Tararak, T. M., Samokhin, G. P., Mitkevich, O. V., Mazurov, A. V., Vinogradov, D. V., Vlasik, T. N., Kalantarov, G. F., and Koteliansky, V. E.Atherosclerosis, 82, 213–226.Google Scholar) as well as at sites of injury, inflammation, or tumor growth. Binding of bFGF to fibrin could, therefore, localize both molecules to sites where they are needed to support endothelial cell migration, proliferation, and angiogenesis.The binding of bFGF to fibrinogen and fibrin may also have effects on interactions with cell receptors and with signal transduction. Binding of endothelial cells to matrix glycoproteins through integrin receptors alters their sensitivity to growth factor-induced signaling mechanisms (49Klein S. Roghani M. Rifkin D.B. Exper. Suppl. (Basel). 1997; 79: 159-192Crossref PubMed Scopus (80) Google Scholar). Fibrinogen and fibrin can support endothelial cell attachm
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