Binding of Basic Fibroblast Growth Factor to Fibrinogen and Fibrin

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. 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. proliferation L.A. L.A. Francis C.W. and Francis C.W. and and growth factors are in response to injury and also act locally to cell responses to vascular these are of the fibroblast growth factor which a of on cells and systems bFGF fibroblast growth fibroblast growth endothelial cell and proliferation and also stimulates and in bFGF also the of of including and and The role of bFGF in vessel injury and is by that bFGF is from vessel cells injury and that bFGF is in and vessel injury for fibrin to support endothelial cell and the of the responses by bFGF that these may is by that fibrin are a matrix to support in and is regarding specific interactions of bFGF with We have, therefore, investigated the of bFGF with fibrinogen and fibrin, and the results high specific and saturable of fibrinogen to immobilized bFGF was saturable and specific with binding of the of specific binding at a fibrinogen of and an in binding was at there was a maximum of binding of the of to beads with immobilized to beads with and active sites with of fibrinogen was the of binding was by Scatchard which indicated that binding was by a with Kd values of 1.3 and 260 nm was and nm for the high and and the maximum molar binding of bFGF to fibrinogen was for the of bFGF with fibrinogen and molar binding of 0.8 in a the that to was a of immobilized the specifically was with fibrinogen and of in two of the with the and of fibrinogen that the was fibrinogen and not a of was from the with 2.0 of the of from immobilized was through a of Sepharose-immobilized the was with fibrinogen, and of were of the in and These were and an was on a and to The in the and of fibrinogen and was similar to that in the of bFGF and fibrinogen was also using soluble bFGF and fibrinogen immobilized on Sepharose beads this saturable and specific binding was also and binding of the of specific binding was at a bFGF of nm. Scatchard analysis indicated the of two binding sites of with Kd values of 0.9 and 70 nm and a maximum molar binding of 2.0 as with 4.0 with fibrinogen binding to Sepharose-immobilized bFGF and of the binding was performed to the and the of at a of nm was with Sepharose-immobilized fibrinogen and then of bFGF was The binding of to fibrinogen was in a with of bFGF but of the at nm which as binding of bFGF to fibrinogen. was with fibrinogen immobilized on Sepharose and the of was as with the beads and binding was in the in the of a molar of binding was by the from the the of Scatchard The of the was by analysis with the and is with of two binding of binding. of bFGF were to the binding of to fibrinogen. of is to fibrin by thrombin, which cleaves fibrinopeptides and from the and fibrin which then to a of the of bFGF with fibrin in the of Sepharose-immobilized fibrinogen with thrombin. The of fibrinogen to the Sepharose beads of the fibrin, a with immobilized fibrin Binding of bFGF to fibrin was similar to that for fibrinogen using the system with specific binding between and nm bFGF binding was at bFGF nm and at to the binding with fibrinogen, the bFGF binding at nm bFGF the of a high binding site of was by Scatchard analysis the of two binding sites with Kd values of 0.13 and 83 nm of bFGF to fibrin was immobilized on Sepharose beads and then to fibrin by with thrombin. was with immobilized fibrin, and and were then by binding was in the of a molar of and specific binding was by of from binding the of Scatchard The of the was using the and the of two binding sites was of binding to fibrin and of of bFGF the is and to binding sites fibrin may also We therefore, to to a solution of fibrinogen, which was then by the of to of of bFGF the binding was with this clotting system in the of an binding was in the of molar of bFGF binding to of the was that with binding to fibrinogen and to fibrin of the fibrin of was and Scatchard analysis two binding sites with values of 0.8 and 261 similar to for fibrinogen The maximum molar binding of bFGF to fibrin was binding of to was to a solution of fibrinogen and then by the of of thrombin. and bFGF were then by and binding was in the of molar of binding was by from binding the of Scatchard The of the was using the and the of two binding sites was results that bFGF binds specifically and saturably to fibrinogen and systems were to the with bFGF fibrinogen immobilized on Sepharose The results were with systems high binding sites withKd values of 1.3 and 0.9 nm and sites with Kd values of 260 and 70 nm. The of bFGF with fibrin was also using two systems with fibrin The results of binding to fibrin were similar to using fibrinogen with two binding the Kd values for the high and sites were 0.13 and 0.8 nm and 83 and 261 nm for and fibrin, maximum molar binding ratios for bFGF to fibrinogen fibrin were between 2.0 and 4.0 with the systems that fibrinogen is a and that two binding sites with Kd values were the of bFGF to fibrinogen and with the of two and sites on each The of using a system in which fibrinogen to immobilized the was using the for the binding is that the of bFGF to binding sites was with fibrin, as sites in the and are in the binding for could prevent binding of bFGF to sites in to by similar could binding of bFGF to Sepharose-immobilized fibrinogen fibrin the binding site was to that by the by binding. for the binding is that of the binding sites is on a of fibrinogen. are fibrinogen including to at the of the C.W. to in These sites are to in the interactions and of fibrinogen and fibrin as the site is in binding to and in factor and of of the Francis C.W. could for the of with the system using bFGF immobilized on Sepharose as fibrinogen with the high site the by a site Francis C.W. fibrinogen a the high studies to specific sites on fibrinogen and fibrin for bFGF binding to this of bFGF binding to fibrinogen and fibrin in to the tissue of bFGF and to the of sites for binding the bFGF a tissue and it is in by endothelial and cells have that endothelial cells which then with the cells and to the cell matrix with of nm. is as a with but is and in active by with and by with bFGF is also in at a to and to in and The of bFGF are through specific and four bFGF cell have bFGF binds to and with similar and bFGF binds specifically and with to cells with of and nm and with bFGF and fibrinogen and may play a role in interactions between and for bFGF in on cell and in the matrix binding the of bFGF on the cell and may the interaction of bFGF with high is not for binding of bFGF to specific cell but are by which is by the of bFGF for is in the of results in of and and to of the for the of cell proliferation binding of bFGF to the of endothelial cells as indicated by its by of in by also binds to fibrinogen and fibrin, and it therefore, interactions with binds to a site the fibrinogen and with 0.8 to the of conversion to fibrin a site at the of the including which a site that cell interactions binding of bFGF to fibrinogen implications regarding the and of bFGF the of fibrinogen and of bFGF to bFGF to fibrinogen the Kd values in the bFGF binding and soluble of have also in The binding of bFGF to formation of and is to to soluble of the high cell have also in as binding for bFGF but the binding have studies to the of bFGF binding to these and role in bFGF binding of bFGF with fibrinogen and fibrin may have locally at sites of vessel injury is in and and and therefore, as a binding site for bFGF the may also with fibrin that is also in and as as at sites of injury, Binding of bFGF to fibrin therefore, to sites are needed to support endothelial cell and binding of bFGF to fibrinogen and fibrin may also have on interactions with cell and with Binding of endothelial cells to matrix through to growth and fibrin support endothelial cell through of and the formation of a adhesion in the of and the high with the adhesion may between and growth factor The binding of bFGF to fibrinogen and fibrin could between interactions between bFGF with fibrinogen and fibrin in vascular responses may result from the role of bFGF in endothelial cell The binding of bFGF as in this therefore, to its and with fibrinogen and fibrin in and the vascular response to 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. proliferation L.A. L.A. Francis C.W. and Francis C.W. and and growth factors are in response to injury and also act locally to cell responses to vascular these are of the fibroblast growth factor which a of on cells and systems bFGF fibroblast growth fibroblast growth endothelial cell and proliferation and also stimulates and in bFGF also the of of including and and The role of bFGF in vessel injury and is by that bFGF is from vessel cells injury and that bFGF is in and vessel injury The for fibrin to support endothelial cell and the of the responses by bFGF that these may is by that fibrin are a matrix to support in and is regarding specific interactions of bFGF with We have, therefore, investigated the of bFGF with fibrinogen and fibrin, and the results high specific and saturable binding. of fibrinogen to immobilized bFGF was saturable and specific with binding of the of specific binding at a fibrinogen of and an in binding was at there was a maximum of binding of the of to beads with immobilized to beads with and active sites with of fibrinogen was the of binding was by Scatchard which indicated that binding was by a with Kd values of 1.3 and 260 nm was and nm for the high and and the maximum molar binding of bFGF to fibrinogen was for the of bFGF with fibrinogen and molar binding of 0.8 in a the that to was a of immobilized the specifically was with fibrinogen and of in two of the with the and of fibrinogen that the was fibrinogen and not a of was from the with 2.0 of the of bFGF and fibrinogen was also using soluble bFGF and fibrinogen immobilized on Sepharose beads this saturable and specific binding was also and binding of the of specific binding was at a bFGF of nm. Scatchard analysis indicated the of two binding sites of with Kd values of 0.9 and 70 nm and a maximum molar binding of 2.0 as with 4.0 with fibrinogen binding to Sepharose-immobilized bFGF and of the binding was performed to the and the of at a of nm was with Sepharose-immobilized fibrinogen and then of bFGF was The binding of to fibrinogen was in a with of bFGF but of the at nm which as binding of bFGF to fibrinogen. was with fibrinogen immobilized on Sepharose and the of was as with the beads and binding was in the in the of a molar of binding was by the from the the of Scatchard The of the was by analysis with the and is with of two binding of binding. of bFGF were to the binding of to fibrinogen. of is to fibrin by thrombin, which cleaves fibrinopeptides and from the and fibrin which then to a of the of bFGF with fibrin in the of Sepharose-immobilized fibrinogen with thrombin. The of fibrinogen to the Sepharose beads of the fibrin, a with immobilized fibrin Binding of bFGF to fibrin was similar to that for fibrinogen using the system with specific binding between and nm bFGF binding was at bFGF nm and at to the binding with fibrinogen, the bFGF binding at nm bFGF the of a high binding site of was by Scatchard analysis the of two binding sites with Kd values of 0.13 and 83 nm of bFGF to fibrin was immobilized on Sepharose beads and then to fibrin by with thrombin. was with immobilized fibrin, and and were then by binding was in the of a molar of and specific binding was by of from binding the of Scatchard The of the was using the and the of two binding sites was of binding to fibrin and of of bFGF the is and to binding sites fibrin may also We therefore, to to a solution of fibrinogen, which was then by the of to of of bFGF the binding was with this clotting system in the of an binding was in the of molar of bFGF binding to of the was that with binding to fibrinogen and to fibrin of the fibrin of was and Scatchard analysis two binding sites with values of 0.8 and 261 similar to for fibrinogen The maximum molar binding of bFGF to fibrin was binding of to was to a solution of fibrinogen and then by the of of thrombin. and bFGF were then by and binding was in the of molar of binding was by from binding the of Scatchard The of the was using the and the of two binding sites was Binding of fibrinogen to immobilized bFGF was saturable and specific with binding of the of specific binding at a fibrinogen of and an in binding was at there was a maximum of binding of the of to beads with immobilized to beads with and active sites with of fibrinogen was the of binding was by Scatchard which indicated that binding was by a with Kd values of 1.3 and 260 nm was and nm for the high and and the maximum molar binding of bFGF to fibrinogen was the that to was a of immobilized the specifically was with fibrinogen and of in two of the with the and of fibrinogen that the was fibrinogen and not a of was from the with 2.0 of the The of bFGF and fibrinogen was also using soluble bFGF and fibrinogen immobilized on Sepharose beads this saturable and specific binding was also and binding of the of specific binding was at a bFGF of nm. Scatchard analysis indicated the of two binding sites of with Kd values of 0.9 and 70 nm and a maximum molar binding of 2.0 as with 4.0 with fibrinogen binding to Sepharose-immobilized bFGF and of the binding was performed to the and the of at a of nm was with Sepharose-immobilized fibrinogen and then of bFGF was The binding of to fibrinogen was in a with of bFGF but of the at nm which as binding. is to fibrin by thrombin, which cleaves fibrinopeptides and from the and fibrin which then to a of the of bFGF with fibrin in the of Sepharose-immobilized fibrinogen with thrombin. The of fibrinogen to the Sepharose beads of the fibrin, a with immobilized fibrin Binding of bFGF to fibrin was similar to that for fibrinogen using the system with specific binding between and nm bFGF binding was at bFGF nm and at to the binding with fibrinogen, the bFGF binding at nm bFGF the of a high binding site of was by Scatchard analysis the of two binding sites with Kd values of 0.13 and 83 nm of binding to fibrin and of of bFGF the is and to binding sites fibrin may also We therefore, to to a solution of fibrinogen, which was then by the of to of of bFGF the binding was with this clotting system in the of an binding was in the of molar of bFGF binding to of the was that with binding to fibrinogen and to fibrin of the fibrin of was and Scatchard analysis two binding sites with values of 0.8 and 261 similar to for fibrinogen The maximum molar binding of bFGF to fibrin was results that bFGF binds specifically and saturably to fibrinogen and systems were to the with bFGF fibrinogen immobilized on Sepharose The results were with systems high binding sites withKd values of 1.3 and 0.9 nm and sites with Kd values of 260 and 70 nm. The of bFGF with fibrin was also using two systems with fibrin The results of binding to fibrin were similar to using fibrinogen with two binding the Kd values for the high and sites were 0.13 and 0.8 nm and 83 and 261 nm for and fibrin, maximum molar binding ratios for bFGF to fibrinogen fibrin were between 2.0 and 4.0 with the systems that fibrinogen is a and that two binding sites with Kd values were the of bFGF to fibrinogen and with the of two and sites on each The of using a system in which fibrinogen to immobilized the was using the for the binding is that the of bFGF to binding sites was with fibrin, as sites in the and are in the binding for could prevent binding of bFGF to sites in to by similar could binding of bFGF to Sepharose-immobilized fibrinogen fibrin the binding site was to that by the by binding. for the binding is that of the binding sites is on a of fibrinogen. are fibrinogen including to at the of the C.W. to in These sites are to in the interactions and of fibrinogen and fibrin as the site is in binding to and in factor and of of the Francis C.W. could for the of with the system using bFGF immobilized on Sepharose as fibrinogen with the high site the by a site Francis C.W. fibrinogen a the high studies to specific sites on fibrinogen and fibrin for bFGF binding to this of bFGF binding to fibrinogen and fibrin in to the tissue of bFGF and to the of sites for binding the bFGF a tissue and it is in by endothelial and cells have that endothelial cells which then with the cells and to the cell matrix with of nm. is as a with but is and in active by with and by with bFGF is also in at a to and to in and The of bFGF are through specific and four bFGF cell have bFGF binds to and with similar and bFGF binds specifically and with to cells with of and nm and with bFGF and fibrinogen and may play a role in interactions between and for bFGF in on cell and in the matrix binding the of bFGF on the cell and may the interaction of bFGF with high is not for binding of bFGF to specific cell but are by which is by the of bFGF for is in the of results in of and and to of the for the of cell proliferation binding of bFGF to the of endothelial cells as indicated by its by of in by also binds to fibrinogen and fibrin, and it therefore, interactions with binds to a site the fibrinogen and with 0.8 to the of conversion to fibrin a site at the of the including which a site that cell interactions binding of bFGF to fibrinogen implications regarding the and of bFGF the of fibrinogen and of bFGF to bFGF to fibrinogen the Kd values in the bFGF binding and soluble of have also in The binding of bFGF to formation of and is to to soluble of the high cell have also in as binding for bFGF but the binding have studies to the of bFGF binding to these and role in bFGF binding of bFGF with fibrinogen and fibrin may have locally at sites of vessel injury is in and and and therefore, as a binding site for bFGF the may also with fibrin that is also in and as as at sites of injury, Binding of bFGF to fibrin therefore, to sites are needed to support endothelial cell and binding of bFGF to fibrinogen and fibrin may also have on interactions with cell and with Binding of endothelial cells to matrix through to growth and fibrin support endothelial cell through of and the formation of a adhesion in the of and the high with the adhesion may between and growth factor The binding of bFGF to fibrinogen and fibrin could between interactions between bFGF with fibrinogen and fibrin in vascular responses may result from the role of bFGF in endothelial cell The binding of bFGF as in this therefore, to its and with fibrinogen and fibrin in and the vascular response to injury. The results that bFGF binds specifically and saturably to fibrinogen and systems were to the with bFGF fibrinogen immobilized on Sepharose The results were with systems high binding sites withKd values of 1.3 and 0.9 nm and sites with Kd values of 260 and 70 nm. The of bFGF with fibrin was also using two systems with fibrin The results of binding to fibrin were similar to using fibrinogen with two binding the Kd values for the high and sites were 0.13 and 0.8 nm and 83 and 261 nm for and fibrin, The maximum molar binding ratios for bFGF to fibrinogen fibrin were between 2.0 and 4.0 with the systems that fibrinogen is a and that two binding sites with Kd values were the of bFGF to fibrinogen and with the of two and sites on each The of using a system in which fibrinogen to immobilized the was using the for the binding is that the of bFGF to binding sites was with fibrin, as sites in the and are in the binding for could prevent binding of bFGF to sites in to by similar could binding of bFGF to Sepharose-immobilized fibrinogen fibrin the binding site was to that by the by binding. for the binding is that of the binding sites is on a of fibrinogen. are fibrinogen including to at the of the C.W. to in These sites are to in the interactions and of fibrinogen and fibrin as the site is in binding to and in factor and of of the Francis C.W. could for the of with the system using bFGF immobilized on Sepharose as fibrinogen with the high site the by a site Francis C.W. fibrinogen a the high studies to specific sites on fibrinogen and fibrin for bFGF binding to this The of bFGF binding to fibrinogen and fibrin in to the tissue of bFGF and to the of sites for binding the bFGF a tissue and it is in by endothelial and cells have that endothelial cells which then with the cells and to the cell matrix with of nm. is as a with but is and in active by with and by with bFGF is also in at a to and to in and The of bFGF are through specific and four bFGF cell have bFGF binds to and with similar and bFGF binds specifically and with to cells with of and nm and with bFGF and fibrinogen and may play a role in interactions between and for bFGF in on cell and in the matrix binding the of bFGF on the cell and may the interaction of bFGF with high is not for binding of bFGF to specific cell but are by which is by the of bFGF for is in the of results in of and and to of the for the of cell proliferation binding of bFGF to the of endothelial cells as indicated by its by of in by also binds to fibrinogen and fibrin, and it therefore, interactions with binds to a site the fibrinogen and with 0.8 to the of conversion to fibrin a site at the of the including which a site that cell interactions The binding of bFGF to fibrinogen implications regarding the and of bFGF the of fibrinogen and of bFGF to bFGF to fibrinogen the Kd values in the bFGF binding and soluble of have also in The binding of bFGF to formation of and is to to soluble of the high cell have also in as binding for bFGF but the binding have studies to the of bFGF binding to these and role in bFGF The binding of bFGF with fibrinogen and fibrin may have locally at sites of vessel injury is in and and and therefore, as a binding site for bFGF the may also with fibrin that is also in and as as at sites of injury, Binding of bFGF to fibrin therefore, to sites are needed to support endothelial cell and The binding of bFGF to fibrinogen and fibrin may also have on interactions with cell and with Binding of endothelial cells to matrix through to growth and fibrin support endothelial cell through of and the formation of a adhesion in the of and the high with the adhesion may between and growth factor The binding of bFGF to fibrinogen and fibrin could between interactions between bFGF with fibrinogen and fibrin in vascular responses may result from the role of bFGF in endothelial cell The binding of bFGF as in this therefore, to its and with fibrinogen and fibrin in and the vascular response to injury.

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