SEARCH

SEARCH BY CITATION

Keywords:

  • cell adhesion;
  • fibrin sealants;
  • fibrin structure;
  • fibrin(ogen);
  • wound healing

Abstract

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

Summary  Fibrinogen and fibrin play an important role in blood clotting, fibrinolysis, cellular and matrix interactions, inflammation, wound healing, angiogenesis, and neoplasia. The contribution of fibrin(ogen) to these processes largely depends not only on the characteristics of the fibrin(ogen) itself, but also on interactions between specific-binding sites on fibrin(ogen), pro-enzymes, clotting factors, enzyme inhibitors, and cell receptors. In this review, the molecular and cellular biology of fibrin(ogen) is reviewed in the context of cutaneous wound repair. The outcome of wound healing depends largely on the fibrin structure, such as the thickness of the fibers, the number of branch points, the porosity, and the permeability. The binding of fibrin(ogen) to hemostasis proteins and platelets as well as to several different cells such as endothelial cells, smooth muscle cells, fibroblasts, leukocytes, and keratinocytes is indispensable during the process of wound repair. High-molecular-weight and low-molecular-weight fibrinogen, two naturally occurring variants of fibrin, are important determinants of angiogenesis and differ in their cell growth stimulation, clotting rate, and fibrin polymerization characteristics. Fibrin sealants have been investigated as matrices to promote wound healing. These sealants may also be an ideal delivery vehicle to deliver extra cells for the treatment of chronic wounds.


Wound healing

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

The first process that takes place as a reaction to a wounding is the prevention of local hemorrhage. This is attained via platelet aggregation and via activation of the hemostasis cascade. The resulting blood clot first contributes to stopping the bleeding and then functions as a provisional matrix for the wound healing that already begins approximately 4 days after injury. New capillaries endow the neostroma with its granular appearance. Leukocytes, fibroblasts, and blood vessels move into the wound space and each contributes to the wound healing process. The macrophages provide a continuing source of cytokines, which are necessary to stimulate fibroplasia and angiogenesis, a process in which new blood vessels are formed from pre-existing ones. During the angiogenesis process, the vascular basement membrane and the fibrin or interstitial matrix are degraded by endothelial cells (ECs), upon which the ECs start to migrate into the matrix and to proliferate by forming new capillary-like tubes [1]. Fibroblasts are important for the production of a new extracellular matrix (ECM), which is necessary to support the additional cell ingrowth [2]. Blood vessels play an important role in sustaining cell metabolism by providing oxygen and nutrients. The integrity of the granulation tissue depends on the presence of biological modifiers (e.g. lipid mediators and growth factors), the activity of target cells, and the environment of the ECM [3]. These processes are regulated by growth factors that may come from the plasma but that can also be released by activated platelets in the wounded areas. Infiltrated peripheral blood monocytes and macrophages are also sources of the synthesis and release of growth factors. In addition, growth factors can be synthesized and secreted by injured and activated parenchymal cells. The newly formed temporary fibrin matrix also promotes granulation tissue formation. Once fibroblasts and ECs express the proper integrin receptors, they invade the fibrin/fibronectin-rich clot in the wound space and start synthesizing a permanent ECM.

Hemostasis in wound healing

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

Platelets are involved in the first step in the process of wound healing [4]. Endothelial injury activates blood platelets and thus initiates the formation of a platelet plug, which is the primary hemostasis process that will stop the bleeding. In parallel, the coagulation cascade is initiated and results in the conversion of soluble fibrinogen to a network of insoluble fibrin fibers. This stabilizes the platelet plug by forming a network that includes platelets via the binding of fibrin to the αIIbβ3 receptors, which are exposed on activated platelets [5]. When platelets are activated, they also secrete a number of growth factors, such as platelet-derived growth factor. These factors stimulate the process of wound healing by the activation of fibroblasts to produce collagen, glycosaminoglycans, and proteoglycans [6,7]. The fibrin matrix not only reduces blood loss, but also is the most important temporary ECM in the wounded area and as such, plays an important role in tissue repair, leukocyte cell adhesion, and EC migration during angiogenesis.

Fibrinogen

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

The fibrinogen molecule is composed of two sets of three different peptide chains: two Aα-chains, two Bβ-chains and two γ-chains, which are interconnected by disulfide bridges. Each fibrinogen chain is encoded by a separate gene and the three genes together form the fibrinogen gene cluster that comprises approximately 50 kb and is located on chromosome 4q22–23. The molecules are elongated 46-nm structures consisting of two outer D-domains, each connected to a central E-domain by a coiled-coil segment. The D- as well as the E-domain contains binding sites that play a role in fibrinogen conversion to fibrin, fibrin assembly, cross-linking, and platelet interactions (thrombin substrate), as well as sites that are available after fibrinopeptide cleavage (thrombin binding site), and sites that become exposed as a consequence of the polymerization process (tPA-dependent plasminogen activation). Although the E-domain contains the site that binds to the active site of thrombin, both D- and E-domain contain other sites that bind thrombin but this does not result in proteolytic cleavage [8,9]

Fibrinogen is an acute-phase protein and either in the early stages of inflammation or on treatment with interleukin-6 (IL-6) and glucocortocoids its synthesis is increased, which is directly related to the enhanced transcription of the three genes [6,7,10–12]. Transcription of the three fibrinogen chains is tightly coordinated and in vitro studies suggest that synthesis of the β-chain is rate limiting and up-regulates the expression of the two other genes [13].

The COOH-terminal regions of the Aα-chains, also called the αC-domains, are formed by residues 221–610 and play a significant role in the modulation of various processes. They are involved in fibrin assembly [14], activation of factor (F) XIII [15], modulation of fibrinolysis [16,17], and cell adhesion via either bound fibronectin or their Aα572–574 Arg-Gly-Asp (RGD) recognition motif [18–20]. A schematic representation of the Aα-chain of fibrinogen is given in Fig. 1.

image

Figure 1. Fibrinogen Aα-chain. The fibrinogen Aα-chain contains a number of sites that interact with cells and protein, which are potentially of interest for wound healing processes. bsl00066bsl00066bsl00066 indicate the sites, where the Aα-chain ends in the low molecular weight (LMW) and LMW’ forms of fibrinogen. αE indicates the part of the Aα-chain that is only present in the αE form of fibrinogen.

Download figure to PowerPoint

In the last step of the coagulation cascade, thrombin cleaves off the fibrinopeptides A and B from the amino-terminal segments of the fibrinogen Aα- and Bβ-chains. Soluble fibrinogen is converted into fibrin monomers, which then form insoluble fibrin polymers and a network of fibrin fibers. This fibrin network is stabilized by FXIIIa which is activated by thrombin during the process of fibrin polymerization, which cross-links the fibrin clot by transglutaminase reactions between two γ-chains or between one γ- and one α-chain [21]. These cross-links provide stability to the fibrin clot and they seem to form a protective barrier preventing the degradation of fibrin by plasmin [22].

Leakage of fibrinogen into the wound

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

The liver is the primary source of plasma fibrinogen, but small amounts of fibrinogen can also be produced by lung epithelium where it may be locally incorporated into the provisional matrix [23]. This process is regulated by cytokines, such as IL-6, IL-1β, and glucocorticoids [24]. Interestingly, treatment of pulmonary cells with IL-1β and the glucocorticocoid dexamethasone resulted in a stimulation of fibrinogen synthesis, whereas this did not occur in alveolar cells. These data suggest that IL-1β may play a role in pulmonary wound repair mechanisms during a local acute phase response [24].

Plasma-derived fibrillar strands within the provisional matrix of cutaneous and vascular wounds have been identified as fibrinogen, which has undergone conformational changes and exposes a fibrin-like epitope. Besides binding to the exogenously produced matrix, the fibrinogen also binds to the cell surface [23]. In addition, growth factors such as fibroblast growth factor-2 (FGF-2) and vascular endothelial cell growth factor (VEGF) bind specifically to fibrinogen (and fibrin), suggesting that fibrinogen is not only important for hemostasis, but also for homeostasis [25]. These data suggest that fibrinogen is assembled into the ECM at sites of tissue damage, where it may contribute to cell type-specific mechanisms of wound repair [25].

Determinants of fibrin structure

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

The fibrin structure can be described by variables such as the thickness of the fibers, the number of branch points, the porosity, and the permeability [26]. The structure of the fibrin matrix affects its biological function. For example, more coarse matrices show a faster fibrinolysis [27] and the pH of the fibrin matrix determines the ingrowth of tubular structures. Opaque matrices at pH 7.0 consist of thick fibers and tube formation proceeds at a faster rate than in transparent matrices at pH 7.8 that consists of thinner fibers [28]. Several conditions may affect the fibrin structure, such as the clotting rate (can be modulated by concentration of thrombin and salt content), the rate of polymerization (determined by FXIII concentration and FXIII activation rate), and the rate of lateral polymerization (affected by fibrinopeptide B release and cross-linking sites on alpha and gamma chains). Chloride ions have been identified as modulators of fibrin polymerization, because these ions control fiber size by inhibiting the growth of thicker, stiffer, and straighter fibers [29,30]. The concentration of thrombin and thus the release-rate of FPA can also have an important impact on the polymerization process. High concentrations (up to 1 U mL−1) induce the formation of thin fibers, whereas low concentrations (0.001 U mL−1) result in thick fibers [31]. Heparins, in particular low molecular weight (LMW)-heparin, also affect the structure of the fibrin clot, as well as the sensitivity of the clot to plasmin-dependent degradation [32] and this affects invasion by tube-forming EC [33].

These alterations in the fibrin structure and function also have implications for wound healing [34]. Especially in diseases where the fibrin structure may be modified, such as in diabetes, obese subjects or in patients with bleeding disorders, disturbances in wound healing are seen [30].

Interactions between fibrin(ogen) and proteins

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

Several hemostasis proteins, such as tissue-type plasminogen activator, plasminogen and FXIII, bind to fibrinogen and fibrin. As hemostasis, in particular the fibrinolytic system, is involved in angiogenesis, these are important interactions in wound healing. The importance of plasminogen in wound healing is clearly illustrated by the impaired skin wound healing and by the marked delay in cutaneous wound repair in plasminogen-deficient mice where the lysis rate of the fibrin matrix is reduced [35]. Binding of FXIII affects the fibrin matrix structure by increasing the tensile strength and stability of the clot [36–38].

The fibrinous matrix of a wound also contains other plasma proteins, such as fibronectin and vitronectin. Fibronectin and vitronectin may act as a bridge molecule between smooth muscle cells and fibrin by binding to the α5β1- or αvβ3-receptor on EC, smooth muscle cells, and other cell types [39]. In addition, fibronectin also binds fibrin exclusively through the αC-domain. This binding site is not accessible in fibrinogen, but becomes exposed in fibrin [40]. Vitronectin directly associates with fibrin, preferentially to the carboxy-terminal γ′-chain of the fibrin(ogen) γA/γ′-chain variant [41].

Interactions between fibrin(ogen) and cells

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

Fibrin(ogen) functions as bridging molecule for many types of cell–cell interactions and provides a critical provisional matrix at sites of injury, inflammation, or infection in which cells can proliferate, organize, and carry out specialized functions. Coated fibrinogen and fibrin matrices have been reported to bind EC, smooth muscle cells, keratinocytes, fibroblasts, and leukocytes. These cells can bind directly to fibrin(ogen) via cell surface integrin receptors and non-integrin (e.g. VE-Cadherin, I-CAM-1, P-selectin, and GPIba) receptors [42–44]. Integrins, transmembrane cell adhesion molecules that consist of an α- and β-subunit, have been demonstrated to bind fibrinogen or fibrin, and are αMβ2 on leukocytes [45], αIIbβ3 on platelets [46] and αvβ3, αvβ5 and α5β1 on EC [25,47–49] and fibroblasts [50–52].

Clot retraction by nucleated cells is very important for proper wound healing [53]. Binding of α5β1-integrins to fibrinogen in the clot promotes the retraction of the clot and changes the shape of the cell [54]. The contribution of the αvβ3-integrin in clot retraction during vascular healing has been demonstrated in many studies [55] as well as the involvement of the αIIbβ3- and αMβ2-integrins [56]. In the following part of the review, we will discuss interactions with fibrin(ogen) by cell type.

Fibrin and inflammatory cells

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

Soon after the formation of a fibrin clot, phagocytes invade it. Infiltration by granulocytes and monocytes into injured tissues is regulated not only by chemo-attractant factors, such as fibrinopeptides cleaved from fibrinogen by thrombin [57], fibrin degradation products produced by plasmin degradation of fibrin [58], but also fragments of collagen [59], elastin [60], fibronectin [61], enzymatically active thrombin [62], and transforming growth factor (TGF)-β [63]. Leukocytes at the wound site eliminate contaminating bacteria via phagocytosis, in which the generation of toxic oxygen radicals and enzymatic digestion plays a significant role [64,65]. The circulating monocytes attach to the endothelium of blood vessels at the site of injury and migrate through the vessel wall into the ECM [66]. Fibrin(ogen) modulates the activity of monocytes and macrophages and therefore plays an important role in the transition rate between wound inflammation and tissue repair [67]. Remarkably, keratinocytes do not interact with fibrinogen because they miss the αVβ3 receptor and this is considered to be the reason why migrating epidermis dissects the fibrin eschar from wounds [68].

Fibrin and endothelial cells

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

The infiltration of leukocytes into the clot is followed by the invasion of EC. These EC are recruited from tissues adjacent to the wound [69] and bind fibrinogen through different cell adhesion receptors. Interactions of EC with fibrin(ogen) are implicated in wound healing and angiogenesis. Once the ECs have infiltrated the provisional matrix, they realign to become new vascular structures (angiogenesis). During this process, they, together with different cell types, degrade fibrin by the induction of plasmin activities, metalloproteinases, and the generation of free radicals [70,71].

Integrins play an important role in wound healing and angiogenesis by facilitating the binding of EC to ECM proteins, such as fibrin(ogen) [72]. The α-chain of fibrinogen contains RGD sequences at positions Aα95–97 and Aα572–574 [73], but several studies have shown that the Aα95–97 RGD motif does not play a significant role in EC adhesion [18,74]. The RGD-sequence at position 572–574 of the Aα-chain of fibrinogen binds the αvβ3-integrin in humans [75], but the sequence is not conserved in other species. However, the lack of conservation is not associated with differences in cellular interaction or deficient wound healing, suggesting that the fibrinogen can bind to other RGD-motifs. For example, in bovines, the RGD-sequence at position 252–254 of the Aα-chain, which is homologous to an RGG-motif in human Aα-fibrinogen, compensates for the lack of Aα572–574 in the bovine fibrinogen Aα-chain [75]. Furthermore, RGD-independent interactions also occur. Fibrinogen has additional binding sites for αvβ3-integrin in the γ-chain [76] and recombinant fibrinogen missing the RGD-sequences still preserves its interactions with cells [75].

The congenital dysfibrinogenemia fibrinogenNieuwegein has a truncated Aα-chain and lacks the binding site for αvβ3-integrin as well as the cross-linking site for transglutaminase. Although EC adhesion is not affected, tube formation in an in vitro angiogenesis model is strongly reduced, indicating that the RGD-sequence at position 572–574 is involved in angiogenesis [77]. These observations are supported by the fact that the 572–574 RGD sequence is also required for the interaction of fibrinogen with α5β1-integrin [49,78], which plays an important role in cell adhesion and angiogenesis [79]. However, these observations may be influenced by the albumin molecule that is bound to the free sulfhydryl-group of the truncated Aα-chain of the fibrinogenNieuwegein molecule, which may affect the fibrin structure, and endothelial invasion, and tube formation in the fibrin matrix.

The adhesive potential of EC to fibrin(ogen) is also influenced by the cross-linking (by transglutaminase) and the coagulation (by thrombin) of fibrinogen [80]. Transglutaminase-mediated oligomerization of the αC-domains of fibrinogen promotes integrin clustering and thereby increases cell adhesion and spreading, which stimulates fibrinogen to bind αvβ3-, αvβ5- and α5β1-integrins on EC. The oligomerization also promoted integrin-dependent cell signaling via focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), which results in an increased cell adhesion and cell migration [81].

Fibrin can also stabilize the expression of αvβ3-integrin on cultured human microvascular EC and therefore promote migration of these cells on provisional matrix proteins [82]. ECs interact with fibrin via a number of receptors, such as ICAM-1, VE-Cadherin, CD-44, and integrins. It has been observed that ICAM-1 binds the 117–133 sequence on the γ-chain of fibrinogen [83]. The β15–42 sequence on fibrin plays an important role during the process of neovascularization [84]. It has been demonstrated that the first extracellular domain of VE-cadherin (cadherin 5) binds to this sequence [85]. The 572–574 RGD sequence on the Aα-chain of fibrin binds integrin αvβ3 and α5β1, and plays a significant role during angiogenesis [75,77]. However, the 572–574 RGD sequence is not required for clot retraction by human EC [86].

Although fibrinogen is not necessarily involved in angiogenesis in tumor mouse models, fibrinogen is a critical determinant of the metastatic potential of circulating tumors. The reduction in lung metastasis in fibrinogen-deficient mice could not be explained by a reduction in tumor growth. Studies have demonstrated that fibrin(ogen) supports the adhesion and survival of the tumor cells in the lung [87,88].

Fibrin and Angiogenesis

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

Angiogenesis is a complex process, in which ECs are stimulated by growth factors, such as VEGF or FGF-2, to proliferate and migrate into the ECM to form new capillaries. The invasion of the ECM by EC is controlled by proteolytic enzymes of the plasminogen activator/plasmin system and the matrix metalloproteinases. The cells proliferate and elongate and vessel stabilization is finally achieved by interaction with pericytes and the reconstitution of the basement membrane [89,90].

It has been reported that, in addition to growth factors such as FGF-2 and VEGF, fibrin has a direct effect on angiogenesis. Fibrin-containing chambers, which were implanted subcutaneously in guinea pigs, induced an angiogenic response within 4 days. Vessels were formed and entered the chambers through surface pores [91,92]. The fibrin matrix also appears as an excellent substrate for the invasion of EC and subsequent formation of new capillary-like structures [93,94].

In addition to the supportive role of fibrin toward endothelial adhesion and angiogenesis, it has recently become clear that the degradation products of fibrinogen can also act as angiogenesis inhibitors. Fibrinogen degradation product E contains a stabilized β-band sequence, recognized as alphastatin, that inhibits angiogenesis in vitro and in vivo [95,96].

Fibrin and fibroblasts

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

During the wound-healing process, fibroblasts migrate into the wound bed, and this process starts about 5 days postinjury. Growth factors, in particular PDGF and TGF-β, together with fibrin and fibronectin stimulate fibroblasts to proliferate, express appropriate integrin receptors, and migrate into the wound [97,98]. In addition, the characteristics of the fibrin network in the wound bed affect the proliferation and migration of the fibroblasts [99]. Patients with disturbed wound healing, such as obese or diabetic patients, may experience improved wound healing when specific fibrin sealant preparations are administered to the wound. Once the fibroblasts enter the wound, they replace the fibrin by generating type I collagen as well as other ECM proteins. At day 7, abundant ECM molecules have accumulated and fibroblasts switch to the myofibroblast phenotype, complete with actin bundles in the cytoplasmic area close to the plasma membrane. These myofibroblasts extend pseudopodia as soon as they reach the ECM and retract the pseudopodia once they attach to the ECM molecules, such as fibronectin and collagen. When these processes have finished, the fibroblasts go into apoptosis [100].

Fibrinogen heterogeneity

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

Fibrinogen has been described as a heterogeneous molecule. Heterogeneity occurs at the level of splicing variants, phosphorylation or glycosylation, but a number of genetic polymorphisms also result in heterogeneity [101]. These heterogeneity factors can all affect fibrin matrix characteristics and may thus explain part of the difference that is observed between individuals in wound healing. Some common variants that occur in all individuals may affect wound healing and will be discussed.

Fibrinogen is synthesized as high molecular weight (HMW, MW 340 kDa) fibrinogen and partially degraded in the circulation to low molecular weight (LMW, MW 305 kDa) and low molecular weight (LMW′, MW 270 kDa) fibrinogen forms, with one or two Aα-chains partially degraded at the C-terminus, respectively [102–104]. These three naturally occurring forms of fibrinogen differ in their cell growth stimulation (angiogenesis), clotting rate, and fibrin polymerization characteristics. The C-terminal region of the Aα-chain contains binding sites for cells and sites that are involved in lateral growth of the fibrin chains, which are responsible for the clot structure characteristics. In addition, the heterogeneity in naturally occurring fibrinogen variants, HMW- and LMW-fibrinogen influence the angiogenic capacity of blood vessels in fibrin matrices and may have an impact on the use of fibrin sealants. HMW-fibrinogen favors cell growth and leads to an increased cell and vessel ingrowth, compared with unfractioned and LMW-fibrinogen (E.L. Kaijzel, P. Koolwijk, M.G.M. van Erck, U.W.M. van Hinsbergh, M.P.M. de Maat, unpublished data).

Another form of fibrinogen is γ′-variant, a splice variant with an altered C-terminal sequence in its γ-chain. The FXIII-binding sites and the high-affinity non-substrate thrombin-binding site are located on the γ′-chain [37]. In this variant γ′-chain, the four C-terminal amino acids are replaced by 20 unique amino acids, thus making it larger than the γ-chain (51.5 and 49.5 kDa, respectively). This form is more resistant to lysis, mainly because the fibrin cross-linking is increased. The γ′-fibrinogen variant lacks the binding site for the platelet integrin αIIbβ3, but new binding sites for FXIII B-domain and thrombin are present [56,105,106]. Individuals with higher γ′-fibrinogen levels will have thicker fibers because the fibrinopeptide B release is delayed, which is associated with the delayed lateral aggregation of protofibrils [37,105].

Another variant form of fibrinogen is the Fib420-form (also called AαE), where the C-terminus of the Aα-chain has been extended (differential splicing of an extra exon), resulting in a nodular structure at the C-terminus. This form of fibrinogen has a molecular weight of 420 kDa, while HMW fibrinogen has a molecular weight of 340 kDa. It has been suggested that the Aα-chain of this form of fibrinogen is more resistant to lysis [107,108] and it may be expected that the α-γ cross-linking by FXIII will be less efficient because of steric hindrance by the extension of the α-chain.

In addition to these variants that occur in all individuals, two coding polymorphisms in the fibrinogen cluster have also been reported and that introduce an amino acid change in the mature fibrinogen protein. The first one is located in the Aα-gene and results in a substitution of threonine by alanine at residue 312 of the Aα-chain [109]. This polymorphism is located in an area of the molecule that contains FXIII cross-linking sites. Indeed, Ala312 is associated with larger average fibrin fiber diameters, as Thr312 increased FXIII-dependent α-chain cross-linking and stiffness of the clot, [38]. The second polymorphism is the arginine to lysine substitution at codon 448 [109]. This polymorphism is located in the C-terminal domain of the Bβ-chain, where it could have an effect on the configuration of this domain. It has been suggested in one study that the Lys448 allele is associated with lower clot permeability and a tighter and finer structure than the Arg448 allele [110], but another study could not confirm this [111]. A direct effect of these polymorphisms on wound healing has not yet been described.

Fibrin sealants

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

Fibrin sealant products consist of purified fibrinogen and thrombin. When these two components are mixed, a fibrin clot is formed and this clot is used as a sealant to achieve hemostasis and tissue sealing in several surgical procedures, especially when other procedures for wound closure cannot be used [112]. The fibrin sealant matrix can also be used to promote wound healing. Promising results were reported when fibroblasts or keratinocytes are added to the fibrin solution, thus using the fibrin sealant as a vehicle to deliver extra fibroblasts to the wound to enhance the healing process [99,113]. The results of the different fibrin sealant preparations are not consistent, and this may be explained by the quality of the fibrinogen and variation in the additional factors that are present in the fibrinogen solution. This could be not only fibronectin, but also coagulation, fibrinolysis (FXIII, plasminogen, plasminogen activator, plasminogen activator inhibitor, and thrombin), and growth factors (transforming growth factor-β, FGF-2, and VEGF) are present [114]. For example, sealants containing FXIII promote an increased tensile strength and stability of the clot, and fibronectin binds to the alphaC-domain of fibrin [40].

Conclusions

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References

Fibrinogen is converted to fibrin, which forms a cohesive network, and provides a temporary support for wound healing. The structural composition of fibrin and the binding of fibrin to cells and proteins highly determine the wound healing process. Beyond behaving as a provisional matrix, fibrin actively recruits cells to trigger fibrin-mediated responses, such as cell adhesion, migration, proliferation, and tubule formation. These events are also regulated by the naturally occurring fibrinogen variants HMW- and LMW-fibrinogen. HMW-fibrinogen promotes cell growth and vessel formation, which may be beneficial during wound repair.

References

  1. Top of page
  2. Abstract
  3. Wound healing
  4. Hemostasis in wound healing
  5. Fibrinogen
  6. Leakage of fibrinogen into the wound
  7. Determinants of fibrin structure
  8. Interactions between fibrin(ogen) and proteins
  9. Interactions between fibrin(ogen) and cells
  10. Fibrin and inflammatory cells
  11. Fibrin and endothelial cells
  12. Fibrin and Angiogenesis
  13. Fibrin and fibroblasts
  14. Fibrinogen heterogeneity
  15. Fibrin sealants
  16. Conclusions
  17. Acknowledgements
  18. References
  • 1
    Staton CA, Brown NJ, Lewis CE. The role of fibrinogen and related fragments in tumour angiogenesis and metastasis. Expert Opin Biol Ther 2003; 3: 110520.
  • 2
    Zavan B, Brun P, Vindigni V, Amadori A, Habeler W, Pontisso P et al. Extracellular matrix-enriched polymeric scaffolds as a substrate for hepatocyte cultures: in vitro and in vivo studies. Biomaterials 2005; 26: 703845.
  • 3
    Juliano RL, Haskill S. Signal transduction from the extracellular matrix. J Cell Biol 1993; 120: 57785.
  • 4
    Baum CL, Arpey CJ. Normal cutaneous wound healing: clinical correlation with cellular and molecular events. Dermatol Surg 2005; 31: 67486.
  • 5
    Fang J, Hodivala-Dilke K, Johnson BD, Du LM, Hynes RO, White GC, Wilcox DA. Therapeutic expression of the platelet-specific integrin, alphaIIbbeta3, in a murine model for Glanzmann thrombasthenia. Blood 2005; 106: 26719.
  • 6
    Bauer EA, Cooper TW, Huang JS, Altman J, Deuel TF. Stimulation of in vitro human skin collagenase expression by platelet-derived growth factor. Proc Natl Acad Sci USA 1985; 82: 41326.
  • 7
    Brissett AE, Hom DB. The effects of tissue sealants, platelet gels, and growth factors on wound healing. Curr Opin Otolaryngol Head Neck Surg 2003; 11: 24550.
  • 8
    Pechik I, Madrazo J, Mosesson MW, Hernandez I, Gilliland GL, Medved L. Crystal structure of the complex between thrombin and the central ‘E’ region of fibrin. Proc Natl Acad Sci USA 2004; 101: 271823.
  • 9
    Liu CY, Nossel HL, Kaplan KL. The binding of thrombin by fibrin. J Biol Chem 1979; 254: 104215.
  • 10
    Crabtree GR, Kant JA. Coordinate accumulation of the mRNAs for the alpha, beta, and gamma chains of rat fibrinogen following defibrination. J Biol Chem 1982; 257: 72779.
  • 11
    Grieninger G, Hertzberg KM, Pindyck J. Fibrinogen synthesis in serum-free hepatocyte cultures: stimulation by glucocorticoids. Proc Natl Acad Sci USA 1978; 75: 550610.
  • 12
    Otto JM, Grenett HE, Fuller GM. The coordinated regulation of fibrinogen gene transcription by hepatocyte-stimulating factor and dexamethasone. J Cell Biol 1987; 105: 106772.
  • 13
    Roy S, Overton O, Redman C. Overexpression of any fibrinogen chain by Hep G2 cells specifically elevates the expression of the other two chains. J Biol Chem 1994; 269: 6915.
  • 14
    Cierniewski CS, Budzynski AZ. Involvement of the alpha chain in fibrin clot formation. Effect of monoclonal antibodies. Biochemistry 1992; 31: 424853.
  • 15
    Credo RB, Curtis CG, Lorand L. Alpha-chain domain of fibrinogen controls generation of fibrinoligase (coagulation factor XIIIa). Calcium ion regulatory aspects. Biochemistry 1981; 20: 37708.
  • 16
    Medved L, Tsurupa G, Yakovlev S. Conformational changes upon conversion of fibrinogen into fibrin. The mechanisms of exposure of cryptic sites. Ann N Y Acad Sci 2001; 936: 185204.
  • 17
    Tsurupa G, Medved L. Fibrinogen alpha C domains contain cryptic plasminogen and tPA binding sites. Ann N Y Acad Sci 2001; 936: 32830.
  • 18
    Cheresh DA, Berliner SA, Vicente V, Ruggeri ZM. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell 1989; 58: 94553.
  • 19
    Corbett SA, Lee L, Wilson CL, Schwarzbauer JE. Covalent cross-linking of fibronectin to fibrin is required for maximal cell adhesion to a fibronectin-fibrin matrix. J Biol Chem 1997; 272: 249995005.
  • 20
    Corbett SA, Schwarzbauer JE. Fibronectin-fibrin cross-linking: a regulator of cell behavior. Trends Cardiovasc Med 1998; 8: 35762.
  • 21
    Weisel JW, Francis CW, Nagaswami C, Marder VJ. Determination of the topology of factor XIIIa-induced fibrin gamma-chain cross-links by electron microscopy of ligated fragments. J Biol Chem 1993; 268: 2661824.
  • 22
    Scott EM, Ariens RA, Grant PJ. Genetic and environmental determinants of fibrin structure and function: relevance to clinical disease. Arterioscler Thromb Vasc Biol 2004; 24: 155866.
  • 23
    Guadiz G, Sporn LA, Simpson-Haidaris PJ. Thrombin cleavage-independent deposition of fibrinogen in extracellular matrices. Blood 1997; 90: 264453.
  • 24
    Nguyen MD, Simpson-Haidaris PJ. Cell type-specific regulation of fibrinogen expression in lung epithelial cells by dexamethasone and interleukin-1beta. Am J Respir Cell Mol Biol 2000; 22: 20917.
  • 25
    Rybarczyk BJ, Lawrence SO, Simpson-Haidaris PJ. Matrix-fibrinogen enhances wound closure by increasing both cell proliferation and migration. Blood 2003; 102: 403543.
  • 26
    Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Ann NY Acad Sci 2001; 936: 1130.
  • 27
    Collet JP, Park D, Lesty C, Soria J, Soria C, Montalescot G, Weisel JW. Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy. Arterioscler Thromb Vasc Biol 2000; 20: 135461.
  • 28
    Collen A, Koolwijk P, Kroon M, Van Hinsbergh VW. Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells. Angiogenesis 1998; 2: 15365.
  • 29
    Di Stasio E, Nagaswami C, Weisel JW, Di Cera E. Cl- regulates the structure of the fibrin clot. Biophys J 1998; 75: 19739.
  • 30
    Standeven KF, Ariens RA, Grant PJ. The molecular physiology and pathology of fibrin structure/function. Blood Rev 2005; 19: 27588.
  • 31
    Carr Jr ME, Hermans J. Size and density of fibrin fibers from turbidity. Macromolecules 1978; 11: 4650.
  • 32
    Parise P, Morini M, Agnelli G, Ascani A, Nenci GG. Effects of low molecular weight heparins on fibrin polymerization and clot sensitivity to t-PA-induced lysis. Blood Coagul Fibrinolysis 1993; 4: 7217.
  • 33
    Collen A, Smorenburg SM, Peters E, Lupu F, Koolwijk P, Van Noorden C, Van Hinsbergh VW. Unfractionated and low molecular weight heparin affect fibrin structure and angiogenesis in vitro. Cancer Res 2000; 60: 6196200.
  • 34
    Weisel JW. Fibrinogen and fibrin. Adv Protein Chem 2005; 70: 24799.
  • 35
    Romer J, Bugge TH, Pyke C, Lund LR, Flick MJ, Degen JL, Dano K. Impaired wound healing in mice with a disrupted plasminogen gene. Nat Med 1996; 2: 28792.
  • 36
    Ariens RA, Lai TS, Weisel JW, Greenberg CS, Grant PJ. Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood 2002; 100: 74354.
  • 37
    Cooper AV, Standeven KF, Ariens RA. Fibrinogen gamma-chain splice variant gamma’ alters fibrin formation and structure. Blood 2003; 102: 53540.
  • 38
    Standeven KF, Grant PJ, Carter AM, Scheiner T, Weisel JW, Ariens RA. Functional analysis of the fibrinogen Aalpha Thr312Ala polymorphism: effects on fibrin structure and function. Circulation 2003; 107: 232630.
  • 39
    Ikari Y, Yee KO, Schwartz SM. Role of alpha5beta1 and alphavbeta3 integrins on smooth muscle cell spreading and migration in fibrin gels. Thromb Haemost 2000; 84: 7015.
  • 40
    Makogonenko E, Tsurupa G, Ingham K, Medved L. Interaction of fibrin(ogen) with fibronectin: further characterization and localization of the fibronectin-binding site. Biochemistry 2002; 41: 790713.
  • 41
    Podor TJ, Peterson CB, Lawrence DA, Stefansson S, Shaughnessy SG, Foulon DM, Butcher M, Weitz JI. Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin. J Biol Chem 2000; 275: 1978894.
  • 42
    Bennett JS. Platelet-fibrinogen interactions. Ann N Y Acad Sci 2001; 936: 34054.
  • 43
    Martinez J, Ferber A, Bach TL, Yaen CH. Interaction of fibrin with VE-cadherin. Ann N Y Acad Sci 2001; 936: 386405.
  • 44
    Ugarova TP, Yakubenko VP. Recognition of fibrinogen by leukocyte integrins. Ann N Y Acad Sci 2001; 936: 36885.
  • 45
    Yakovlev S, Zhang L, Ugarova T, Medved L. Interaction of fibrin(ogen) with leukocyte receptor alpha M beta 2 (Mac-1): further characterization and identification of a novel binding region within the central domain of the fibrinogen gamma-module. Biochemistry 2005; 44: 61726.
  • 46
    Podolnikova NP, Yakubenko VP, Volkov GL, Plow EF, Ugarova TP. Identification of a novel binding site for platelet integrins alpha IIb beta 3 (GPIIbIIIa) and alpha 5 beta 1 in the gamma C-domain of fibrinogen. J Biol Chem 2003; 278: 322518.
  • 47
    Nisato RE, Tille JC, Jonczyk A, Goodman SL, Pepper MS. alphav beta 3 and alphav beta 5 integrin antagonists inhibit angiogenesis in vitro. Angiogenesis 2003; 6: 10519.
  • 48
    Sahni A, Francis CW. Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires alphavbeta3. Blood 2004; 104: 363541.
  • 49
    Suehiro K, Gailit J, Plow EF. Fibrinogen is a ligand for integrin alpha5beta1 on endothelial cells. J Biol Chem 1997; 272: 53606.
  • 50
    Farrell DH, Al Mondhiry HA. Human fibroblast adhesion to fibrinogen. Biochemistry 1997; 36: 11238.
  • 51
    Gailit J, Clark RA. Studies in vitro on the role of alpha v and beta 1 integrins in the adhesion of human dermal fibroblasts to provisional matrix proteins fibronectin, vitronectin, and fibrinogen. J Invest Dermatol 1996; 106: 1028.
  • 52
    Gailit J, Clarke C, Newman D, Tonnesen MG, Mosesson MW, Clark RA. Human fibroblasts bind directly to fibrinogen at RGD sites through integrin alpha(v)beta3. Exp Cell Res 1997; 232: 11826.
  • 53
    Bennett JS, Kolodziej MA. Disorders of platelet function. Dis Mon 1992; 38: 577631.
  • 54
    Corbett SA, Schwarzbauer JE. Requirements for alpha(5)beta(1) integrin-mediated retraction of fibronectin-fibrin matrices. J Biol Chem 1999; 274: 209438.
  • 55
    Sajid M, Stouffer GA. The role of alpha(v)beta3 integrins in vascular healing. Thromb Haemost 2002; 87: 18793.
  • 56
    Farrell DH. Pathophysiologic roles of the fibrinogen gamma chain. Curr Opin Hematol 2004; 11: 1515.
  • 57
    Richardson DL, Pepper DS, Kay AB. Chemotaxis for human monocytes by fibrinogen-derived peptides. Br J Haematol 1976; 32: 50713.
  • 58
    Gross TJ, Leavell KJ, Peterson MW. CD11b/CD18 mediates the neutrophil chemotactic activity of fibrin degradation product D domain. Thromb Haemost 1997; 77: 894900.
  • 59
    Postlethwaite AE, Kang AH. Collagen-and collagen peptide-induced chemotaxis of human blood monocytes. J Exp Med 1976; 143: 1299307.
  • 60
    Senior RM, Griffin GL, Mecham RP. Chemotactic activity of elastin-derived peptides. J Clin Invest 1980; 66: 85962.
  • 61
    Clark RA, Wikner NE, Doherty DE, Norris DA. Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment. J Biol Chem 1988; 263: 1211523.
  • 62
    Bar-Shavit R, Benezra M, Eldor A, Hy-Am E, Fenton JW, Wilner GD, Vlodavsky I. Thrombin immobilized to extracellular matrix is a potent mitogen for vascular smooth muscle cells: nonenzymatic mode of action. Cell Regul 1990; 1: 45363.
  • 63
    Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, Sporn MB. Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci USA 1987; 84: 578892.
  • 64
    Segal AW. How neutrophils kill microbes. Annu Rev Immunol 2005; 23: 197223.
  • 65
    Taylor JV, Gordon LE, Hall H, Heinzelmann M, Polk Jr HC. Differences between bacterial species shown by simultaneous assessment of neutrophil phagocytosis and generation of reactive oxygen intermediates in trauma patients. Arch Surg 1999; 134: 12227.
  • 66
    Doherty DE, Haslett C, Tonnesen MG, Henson PM. Human monocyte adherence: a primary effect of chemotactic factors on the monocyte to stimulate adherence to human endothelium. J Immunol 1987; 138: 176271.
  • 67
    Flick MJ, Du X, Degen JL. Fibrin(ogen)-alpha M beta 2 interactions regulate leukocyte function and innate immunity in vivo. Exp Biol Med (Maywood) 2004; 229: 110510.
  • 68
    Kubo M, Van de Water L, Plantefaber LC, Mosesson MW, Simon M, Tonnesen MG, Taichman L, Clark RA. Fibrinogen and fibrin are anti-adhesive for keratinocytes: a mechanism for fibrin eschar slough during wound repair. J Invest Dermatol 2001; 117: 136981.
  • 69
    Gorodetsky R, Clark RA, An J, Gailit J, Levdansky L, Vexler A, Berman E, Marx G. Fibrin microbeads (FMB) as biodegradable carriers for culturing cells and for accelerating wound healing. J Invest Dermatol 1999; 112: 86672.
  • 70
    Collen A, Hanemaaijer R, Lupu F, Quax PH, Van Lent N, Grimbergen J, Peters E, Koolwijk P, Van Hinsberg VW. Membrane-type matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix. Blood 2003; 101: 18107.
  • 71
    Marx G. Immunological monitoring of Fenton fragmentation of fibrinogen. Free Radic Res Commun 1991; 12–13: 51720.
  • 72
    Ruoslahti E. RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 1996; 12: 697715.
  • 73
    Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987; 238: 4917.
  • 74
    Farrell DH, Thiagarajan P. Binding of recombinant fibrinogen mutants to platelets. J Biol Chem 1994; 269: 22631.
  • 75
    Thiagarajan P, Rippon AJ, Farrell DH. Alternative adhesion sites in human fibrinogen for vascular endothelial cells. Biochemistry 1996; 35: 416975.
  • 76
    Yokoyama K, Zhang XP, Medved L, Takada Y. Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains. Biochemistry 1999; 38: 58727.
  • 77
    Collen A, Maas A, Kooistra T, Lupu F, Grimbergen J, Haas FJ, Biesma DH, Koolwijk P, Koopman J, Van Hinsbergh VW. Aberrant fibrin formation and cross-linking of fibrinogen Nieuwegein, a variant with a shortened Aalpha-chain, alters endothelial capillary tube formation. Blood 2001; 97: 97380.
  • 78
    Suehiro K, Mizuguchi J, Nishiyama K, Iwanaga S, Farrell DH, Ohtaki S. Fibrinogen binds to integrin alpha(5)beta(1) via the carboxyl-terminal RGD site of the Aalpha-chain. J Biochem (Tokyo) 2000; 128: 70510.
  • 79
    Francis SE, Goh KL, Hodivala-Dilke K, Bader BL, Stark M, Davidson D, Hynes RO. Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies. Arterioscler Thromb Vasc Biol 2002; 22: 92733.
  • 80
    Dallabrida SM, Falls LA, Farrell DH. Factor XIIIa supports microvascular endothelial cell adhesion and inhibits capillary tube formation in fibrin. Blood 2000; 95: 258692.
  • 81
    Belkin AM, Tsurupa G, Zemskov E, Veklich Y, Weisel JW, Medved L. Transglutaminase-mediated oligomerization of the fibrin(ogen) alphaC domains promotes integrin-dependent cell adhesion and signaling. Blood 2005; 105: 35618.
  • 82
    Feng X, Clark RA, Galanakis D, Tonnesen MG. Fibrin and collagen differentially regulate human dermal microvascular endothelial cell integrins: stabilization of alphav/beta3 mRNA by fibrin1. J Invest Dermatol 1999; 113: 9139.
  • 83
    Altieri DC, Duperray A, Plescia J, Thornton GB, Languino LR. Structural recognition of a novel fibrinogen gamma chain sequence (117–133) by intercellular adhesion molecule-1 mediates leukocyte–endothelium interaction. J Biol Chem 1995; 270: 6969.
  • 84
    Chalupowicz DG, Chowdhury ZA, Bach TL, Barsigian C, Martinez J. Fibrin II induces endothelial cell capillary tube formation. J Cell Biol 1995; 130: 20715.
  • 85
    Bach TL, Barsigian C, Chalupowicz DG, Busler D, Yaen CH, Grant DS, Martinez J. VE-Cadherin mediates endothelial cell capillary tube formation in fibrin and collagen gels. Exp Cell Res 1998; 238: 32434.
  • 86
    Smith RA, Mosesson MW, Rooney MM, Lord ST, Daniels AU, Gartner TK. The role of putative fibrinogen Aalpha-, Bbeta-, and GammaA-chain integrin binding sites in endothelial cell-mediated clot retraction. J Biol Chem 1997; 272: 220805.
  • 87
    Palumbo JS, Kombrinck KW, Drew AF, Grimes TS, Kiser JH, Degen JL, Bugge TH. Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells. Blood 2000; 96: 33029.
  • 88
    Criscuoli ML, Nguyen M, Eliceiri BP. Tumor metastasis but not tumor growth is dependent on Src-mediated vascular permeability. Blood 2005; 105: 150814.
  • 89
    Carmeliet P. Angiogenesis in health and disease. Nat Med 2003; 9: 65360.
  • 90
    Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med 2003; 3: 64351.
  • 91
    Dvorak HF, Harvey VS, Estrella P, Brown LF, McDonagh J, Dvorak AM. Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Lab Invest 1987; 57: 67386.
  • 92
    Liu HM, Wang DL, Liu CY. Interactions between fibrin, collagen and endothelial cells in angiogenesis. Adv Exp Med Biol 1990; 281: 31931.
  • 93
    Koolwijk P, Van Erck MG, De Vree WJ, Vermeer MA, Weich HA, Hanemaaijer R, Van Hinsbergh VW. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol 1996; 132: 117788.
  • 94
    Pepper MS, Belin D, Montesano R, Orci L, Vassalli JD. Transforming growth factor-beta 1 modulates basic fibroblast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro. J Cell Biol 1990; 111: 74355.
  • 95
    Brown NJ, Staton CA, Rodgers GR, Corke KP, Underwood JC, Lewis CE. Fibrinogen E fragment selectively disrupts the vasculature and inhibits the growth of tumours in a syngeneic murine model. Br J Cancer 2002; 86: 18136.
  • 96
    Staton CA, Brown NJ, Rodgers GR, Corke KP, Tazzyman S, Underwood JC, Lewis CE. Alphastatin, a 24-amino acid fragment of human fibrinogen, is a potent new inhibitor of activated endothelial cells in vitro and in vivo. Blood 2004; 103: 6016.
  • 97
    Becker JC, Domschke W, Pohle T. Biological in vitro effects of fibrin glue: fibroblast proliferation, expression and binding of growth factors. Scand J Gastroenterol 2004; 39: 92732.
  • 98
    Kilarski WW, Jura N, Gerwins P. An ex vivo model for functional studies of myofibroblasts. Lab Invest 2005; 85: 64354.
  • 99
    Cox S, Cole M, Tawil B. Behavior of human dermal fibroblasts in three-dimensional fibrin clots: dependence on fibrinogen and thrombin concentration. Tissue Eng 2004; 10: 94254.
  • 100
    Clark RA. Regulation of fibroplasia in cutaneous wound repair. Am J Med Sci 1993; 306: 428.
  • 101
    De Maat MPM, Verschuur M. Fibrinogen heterogeneity: inherited and non-inherited. Curr Opin Hematol 2005; 12: 37783.
  • 102
    Holm B, Nilsen DW, Kierulf P, Godal HC. Purification and characterization of 3 fibrinogens with different molecular weights obtained from normal human plasma. Thromb Res 1985; 37: 16576.
  • 103
    Holm B, Brosstad F, Kierulf P, Godal HC. Polymerization properties of two normally circulating fibrinogens, HMW and LMW. Evidence that the COOH-terminal end of the a-chain is of importance for fibrin polymerization. Thromb Res 1985; 39: 595606.
  • 104
    Holm B, Nilsen DW, Godal HC. Evidence that low molecular fibrinogen (LMW) is formed in man by degradation of high molecular weight fibrinogen (HMW). Thromb Res 1986; 41: 87984.
  • 105
    Collet JP, Nagaswami C, Farrell DH, Montalescot G, Weisel JW. Influence of gamma’ fibrinogen splice variant on fibrin physical properties and fibrinolysis rate. Arterioscler Thromb Vasc Biol 2004; 24: 3826.
  • 106
    Mosesson MW. Fibrinogen gamma chain functions. J Thromb Haemost 2003; 1: 2318.
  • 107
    Fu Y, Grieninger G. Fib420: a normal human variant of fibrinogen with two extended alpha chains. Proc Natl Acad Sci USA 1994; 91: 26258.
  • 108
    Grieninger G. Contribution of the alpha EC domain to the structure and function of fibrinogen-420. Ann N Y Acad Sci 2001; 936: 4464.
  • 109
    Baumann RE, Henschen AH. Human fibrinogen polymorphic site analysis by restriction endonuclease digestion and allele-specific polymerase chain reaction amplification: identification of polymorphisms at positions A alpha 312 and B beta 448. Blood 1993; 82: 211724.
  • 110
    Lim BC, Ariens RA, Carter AM, Weisel JW, Grant PJ. Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk. Lancet 2003; 361: 142431.
  • 111
    Maghzal GJ, Brennan SO, George PM. Fibrinogen B beta polymorphisms do not directly contribute to an altered in vitro clot structure in humans. Thromb Haemost 2003; 90: 10218.
  • 112
    Rosso F, Marino G, Giordano A, Barbarisi M, Parmeggiani D, Barbarisi A. Smart materials as scaffolds for tissue engineering. J Cell Physiol 2005; 203: 46570.
  • 113
    Horch RE, Bannasch H, Stark GB. Transplantation of cultured autologous keratinocytes in fibrin sealant biomatrix to resurface chronic wounds. Transplant Proc 2001; 33: 6424.
  • 114
    Clark RA. Fibrin glue for wound repair: facts and fancy. Thromb Haemost 2003; 90: 10036.