Fibrinolysis Regulation: A Promising Approach to Promote Osteogenesis

Abstract

Soon after bone fracture, the initiation of the coagulation cascade results in the formation of a blood clot, which acts as a natural material to facilitate cell migration and osteogenic differentiation at the fracture site. The existence of hematoma is important in early stage of bone healing, but the persistence of hematoma is considered harmful for bone regeneration. Fibrinolysis is recently regarded as a period of critical transition in angiogenic–osteogenic coupling, it thereby is vital for the complete healing of the bone. Moreover, the enhanced fibrinolysis is proposed to boost bone regeneration through promoting the formation of blood vessels, and fibrinolysis system as well as the products of fibrinolysis also play crucial roles in the bone healing process. Therefore, the purpose of this review is to elucidate the fibrinolysis-derived effects on osteogenesis and summarize the potential approaches—improving bone healing by regulating fibrinolysis, with the purpose to further understand the integral roles of fibrinolysis in bone regeneration and to provide theoretical knowledge for potential fibrinolysis-related osteogenesis strategies.

Impact statement

Fibrinolysis emerging as a new and viable therapeutic intervention to be contained within osteogenesis strategies, however to now, there have been no review articles which collates the information between fibrinolysis and osteogenesis. This review, therefore, focusses on the effects that fibrinolysis exerts on bone healing, with a purpose to provide theoretical reference to develop new strategies to modulate fibrinolysis to accelerate fibrinolysis thus enhancing bone healing.

Introduction

Large bone defects caused by trauma, tumor, or bone-related diseases result in significant social issues and remain as major treatment challenges in reconstructive surgery and orthopedic surgery.¹ Bone has an excellent intrinsic capability of self-healing within a small defect, whereas large segmental bone defects fail to undergo spontaneous healing.^2–4 Delayed or incomplete healing of common fractures leads to serious compromise of overall health status, calling the need for advanced therapeutic approaches.

Bone injury is often accompanied by the damage of blood vessels, thus resulting in the formation of hematoma followed by a coagulation cascade situated between the broken fragments. The hematoma (fibrin network) formation is the first stage of bone regeneration, which has long been considered to stimulate the local inflammation and facilitate the recruitment of mesenchymal progenitor cells in the bone defect area.⁵ The fibrin network will then be degraded by plasmin. The timely fibrinolysis is crucial for bone regeneration, as the persistent fibrin in the cartilage callus (during bone fracture repair) results in the failure of new blood vessel invasion into the tissue of the callus, thus causing delayed bone healing; conversely, the removal of fibrin in the fracture sites can lead to normal bone regeneration.^6,7 Similarly, in a rat model, the inhibition of the dissolution of fibrin (fibrinolysis) by applying aprotinin and ɛ-amino-n-caproic acid (ɛ-ACA) is detrimental to normal wound healing.⁸ Those findings indicated that fibrinolysis may be a potential regulatory target to facilitate bone regeneration.

Additionally, amounts of evidence has shown that the intrinsic conformation of the blood clots, which facilitates fibrinolysis, results in enhanced bone healing, compared with those blood clots resistant to fibrinolysis.^9–14 Moreover, the components of fibrinolysis system, such as plasminogen (PLG) and plasmin, have crucial influence on bone metabolism and osteogenesis-associated cells.^15,16 Furthermore, the products of fibrinolysis, for example, fragment D can affect the recruitment of inflammatory cell and the release of inflammatory factors in the early bone healing stage.¹⁷ Therefore, in this review, we draw attention to the influence of fibrinolysis on osteogenesis, with purpose to support the optimization of fibrinolysis-targeting strategies to enhance bone healing.

Process of Fibrin Clot Formation and Fibrinolysis

The injury of vessel endothelia initiates extrinsic pathway by releasing tissue factor into the circulation. Meanwhile, thrombin is generated through a sequence of enzymatic cascade interactions. Finally, accompanied by thrombin and factor XIII, fibrinogen polymerizes to form a fibrin network. The fibrin network characteristics is determined by the distinctions in the fibrinogen molecules, which influences fibrin polymerization rate and its susceptibility to be dissolved. Fibrinogen molecules contain six paired polypeptide chains (AαBβγ)₂, E domain, D domain, coiled-coil, and αC regions.¹⁸ The central E region, the site where thrombin cleaves, includes the amino acid termini of polypeptide chains.

The D domain is formed by the carboxyl termini of the Bβ and γ chains and attaches to the E region by 2 α-helical coiled-coil domains. αC-regions are composed of the carboxyl termini of the Aα chains. In the presence of thrombin, fibrin fiber is formed by fibrinogen polymerization through the lateral and vertical aggregation, accompanied by the release of fibrinopeptide A (FpA) and fibrinopeptide B (FpB).¹⁹ This leads to the formation of fibrin monomers and initiation of polymerization. Then, fibrin fiber further crosslinks γ chains or α chains to from neighboring fibrin molecules through factor XIII crosslinking catalyzed by thrombin, which results in increased stability of the fibrin network.^20,21

Fibrinolysis is an opposite process to fibrin polymerization, which degrades fibrin. In the initial stage of fibrinolysis, PLG is converted to plasmin by both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Fibrin acts as major substrate in fibrinolysis, being both a binding surface for PLG as well as t-PA, and also a substance boosting plasmin generation. The affinity between t-PA and PLG is low without fibrin, but increases notably in fibrin existence.²² Studies further found that the major fibrin-binding sites lie in the finger domain and Kringle 2 of t-PA, and one or more of the five Kringle domains lie in PLG.²² Those binding sites contain lysine-binding sites, thus can be blocked by lysine analogs, such as ɛ-ACA and thrombin-activatable fibrinolysis inhibitor (TAFI).²³ Conversely, components of the fibrinolytic inhibition system, such as PLG activator inhibitor (PAI) and inhibitors of plasmin, namely α₂-antiplasmin (α₂-AP), inhibit the fibrinolysis process.²⁴ PAI has two subtypes, PAI-1 inhibits fibrinolysis by suppressing both t-PA and u-PA, PAI-2 exhibits equivalent inhibitory efficiency on both two-chain t-PA and two-chain u-PA, but not single-chain t-PA and pro-urokinase (Fig. 1).²⁵

FIG. 1.

The formation of fibrin network and fibrinolysis. The extrinsic coagulation pathway is triggered by the release of TF, when endothelial vessels are damaged. Then, TF binds to FVII and converts FVII into FVIIa to form the extrinsic tenase complex (TF-FVIIa), which activates FX into FXa. In the presence of Ca²⁺, FXa further combines with the activated factor Va to from the prothrombinase complex (FXa-FVa), resulting in the conversion of prothrombin to thrombin. Eventually, fibrin network is formed through a sequence of enzymatic reactions. On the other hand, plasminogen is converted to plasmin mediated by t-PA and u-PA on the surface of fibrin to degrade fibrin during fibrinolysis, and the inhibitor of fibrinolysis, including PAI-1 (inhibit t-PA and u-PA) and α2-AP. AP, antiplasmin; Ca²⁺, calcium ion; FVII, factor VII; PAI-1, plasminogen activator inhibitor; TF, tissue factor; t-PA, tissue-type plasminogen activator; u-PA, urokinase-type plasminogen activator. Color images are available online.

The Role of Fibrinolysis in Osteogenesis

Immediately after fracture, blood coagulation cascade is activated, developing a hematoma that resulted from fibrin polymerization. The physiological processes of bone healing include three consecutive phages: reactive phase, reparative phase, and remodeling phase.²⁶ During reactive phase, the formation of hematoma is recognized as a crucial part for early bone healing, as it constitutes the early healing microenvironment at the bone injury site.²⁷ Hematoma (fibrin clot) then results from the coagulation cascade to seal blood vessels thus preventing further blood loss, which also stimulates the local inflammatory response and affects the recruitment of mesenchymal progenitor cells in the early bone regeneration process.⁹

Importantly, the intrinsic conformation of fibrin network at fracture sites is crucial for the subsequent fibrinolysis, thus bone healing. There are two different phenotypes of fibrin network defined by researchers, one is described as loose and porous fibrin structure, which is made of thicker fibers, the other is dense and impermeable fibrin structure formed by thinner fibers.^10,28 Studies have shown that, compared with thicker fibers, thinner fibers reduce the generation of plasmin mediated by t-PA, thereby hampering the whole fibrinolytic process.²⁹ The degradation of fibrin clots is a complicated process, where not all fibrin fibers are cleaved by plasmin. There is a sequence of pathways where individual fibers are cleared, including transverse cleavage, bucking, fiber bunding, and fiber collapse.³⁰ Thicker fiber undergoes an alteration of shape before it disappears, such as an increase in diameter and bending, whereas thinner fibers stay in the same shape.³¹ On the whole, thicker fibers have a slower dissolution rate compared with thinner fiber, although there seems to exist more t-PA-binding sites on thicker fibers.

At the network level, the fibrinolysis process was proceeded by transverse cutting (layer by layer) rather than by progressive cleavage uniformly around the fiber, and the lysis front (a dynamic line observed when fibrins are being degraded) of tissue PLG distribution is broader as a result of binding more plasmin in loose fibrin network structure and moves quickly compared with dense fibrin network structure, leading to loose fibrin network susceptible to fibrinolysis.^30,31

Consequently, the porous fibrin structure is “optimal” as it is susceptible to fibrinolysis by plasmin, hence sustaining the constant supplying of growth factors from surrounding tissues and supporting cellular infiltration, whereas dense fibrin structure is “suboptimal” since it is relatively resistant to fibrinolysis thus hindering tissue regeneration.^31–33 This may be one of the explanations that small bone defect (diameter of 1 mm) is capable of self-healing without any intervention, but large bone defect (diameter of 5 mm) is unable to completely self-heal in the native state (Figs. 2 and 3).^34,35

FIG. 2.

Different phenotypes of fibrin network. Clots composed of thinner fibrin fibers result in hematoma with a dense fibrin network, which has poor permeability and resistant to fibrinolysis, thus delaying bone healing (A); clots consisting of thicker fibrin fibers leads to a loose structure, which has higher permeability and are susceptible to fibrinolysis, causing normal bone healing (B). Color images are available online.

FIG. 3.

The dissolution of fibrin clot is layer by layer but not progressive cutting. When fibrinolysis starts, lysis front forms and move laterally. Fibrin clots consist of thick fibers, which present loose conformation that have a broader (bind more plasmin) and quickly moving binding front, resulting in fibrin clots susceptible to fibrinolysis, thus releasing more growth factors to facilitate bone healing. On the contrary, clots composed of thin fibers, which presents dense structure have a narrow (bind less plasmin) and slowly moving lysis front, resulting in slowly dissolved clots thus releasing less growth factors and damaging normal bone healing. Color images are available online.

Besides, the sustained fibrin can damage the normal bone healing process by inhibiting normal cell migration, and a failure of efficient fibrinolysis was found to hamper normal bone regeneration.^6,36 Moreover, normal bone healing occurred after the removal of fibrin in the fracture site, which suggests that the timely fibrinolysis is vital for bone healing.⁷ Under the circumstance of PLG deficiency, fibrin remains in the cartilage callus, and no vascularity exists in cartilage callus, thus primary bone healing is ruined; whereas the removal of fibrin clot in the PLG-deficient bone defect area allows for vascular invasion and the completion of primary bone regeneration (Fig. 4).⁷ This indicates that delayed fibrinolysis is harmful for normal bone healing.

FIG. 4.

The sustained fibrins delay bone healing. In the event of plasminogen deficiency (fibrin persists in the fracture site) in mice, fibrin remains in the cartilage callus, leading to blood vessels failing to invade into the cartilage callus thus impairing bone healing (A). Conversely, the knockout of fibrin results in blood vessels' reinvasion into the cartilage callus, thus facilitating bone healing (B). This indicates that the persistence of fibrins in fracture site is harmful for normal bone healing. Color images are available online.

Wong et al. found that the use of low-magnitude high-frequency vibration (LMHFV) to accelerate fibrinolysis can further enhance the vascular formation, thus promoting fracture healing in animal metaphyseal fracture model.³⁷ Moreover, the application of highly effective fibrinolytic inhibitors, such as aprotinin and ɛ-ACA, resulted in excessively concentrated fibrin, thus severely injuring the regeneration of skin, liver, and bone.⁸ Therefore, fibrinolysis is vital for osteogenesis, and factors facilitating fibrinolysis are considered to enhance bone regeneration.

Fibrinolysis System Components Modulate Osteogenesis

PLG and plasmin

PLG is converted to the active serine protease plasmin by PAs at the early stage of fibrinolysis, which participates in the removal of fibrin. The deficiency of PLG led to decreased bone mineral density and osteoprotegerin expression in osteoblasts, as well as an increased osteoclastogenesis in vitro.¹⁵ In a mouse femur defect model, the deficiency of Plg gene declined cartilage and bone formation, damaged bone formation, reduced osteoblast cell numbers, and the transforming growth factor-β (TGF-β1) levels on day 4, by suppressing the recruitment of macrophages (a cell plays critical roles in repair of many tissues, such as bone and lung) in the fracture site.¹⁶ Intriguingly, the local treatment of vascular endothelial growth factor (VEGF) at the bone defect significantly reversed delayed bone regeneration in Plg-deficiency mice, which further suggested that PLG affects angiogenesis by decreasing the production of VEGF.¹⁶

PLG also enhances mesenchymal stem cell (MSC) survival and persistence, which induces paracrine effects, boosts postischemic neovascularization and tissue repair by activating cysteine-rich protein 61.³⁸ Plasmin regulated bone remodeling by cleaving both free and hydroxyapatite-bound osteocalcin (through binding one of the three plasmin-susceptible sites (lys19-arg20, arg43-arg44, arg44-phe45) within the osteocalcin molecule), which is secreted by osteoblast and negatively affects mineralization by directly mediating bone resorption.³⁹ Those findings indicate that PLG not only affects bone metabolism, but also regulates the proliferation and differentiation of osteogenesis-associated cells.

Tissue-type plasminogen activator

The two PAs, t-PA and u-PA, exert their function during fibrinolysis by converting the inactive proenzyme PLG to plasmin.⁴⁰ Normal osteoblasts and osteoclasts are capable of producing both t-PA and u-PA, which play important roles in bone regeneration.⁴¹ Both t-PA and u-PA are involved in the dissolving of noncollagen proteins from the organic matrix of bone, probably leading to the release of TGF-β1, insulin-like growth factors, and other growth factors from bone matrix.^42,43 Furthermore, interleukin (IL)-1α revealed to facilitate the releasing of t-PA and as u-PA from osteoclasts, thus stimulating bone matrix turnover.⁴⁴

In a femoral bone defect model, the area of bone defect in t-PA-deficiency mice was three-fold larger compared with the controls (t-PA^+/+ mice), and t-PA was found to promote osteoblast proliferation through annexin 2 and extracellular signal-regulated kinase1/2 (ERK1/2), which also facilitated angiogenesis by stimulating the production of VEGF and hypoxia-inducible factor-1α (HIF-1α) at the fracture site.⁴⁵

In addition, t-PA mobilizes CD11b⁺ cells from the bone marrow and upregulates the expression of neoangiogenesis-associated genes, VEGF-A, thereby locally promoting angiogenesis during tissue regeneration.⁴⁶ In a study, myeloid-derived t-PA was shown to enhance macrophage migration through focal adhesion kinase, ras-related C3 botulinum toxin substrate 1 (Rac-1), and nuclear factor kappa-B (NF-κB) signaling pathway.⁴⁷ Moreover, t-PA is conducive to the recruitment of macrophage and (macrophage inflammatory protein-1α) at the bone fracture site, thus resulting in an enhanced bone repair.⁴⁸ In a study, the topical thrombolysis with recombinant tissue plasminogen activator (rt-PA) was beneficial in preventing epidural fibrosis and arachnoiditis after spine surgery in a rat model.⁴⁹

Urokinase-type plasminogen activator

The deficiency of u-PA in mice exhibited decreased accumulation of macrophage, hence severely impaired tissue regeneration.^50,51 It has been reported that u-PA boosted angiogenesis and remodeling of the fracture cartilage at the fracture site in vivo.⁵² Researchers found that u-PA exerted a protective role in arthritis models with “wound healing-like” processes following local injury, probably through u-PA/plasmin-mediated fibrinolysis.⁵³ Delayed early bone healing accompanied with declined macrophage accumulation and phagocytic activity were detected in an u-PA gene-deficient mice model, while the deficiency of t-PA gene injured the late-stage bone regeneration.^45,54

Urokinase-type plasminogen activator receptor (u-PAR) are expressed by osteoblasts and osteoclasts, and its association with β1-integrin, thus promoting the migration of human mesenchymal stem, which occurs in MSC that originated from bone marrow or adipose tissue.⁵⁵ Moreover, u-PAR has been revealed to promote osteoclast differentiation through increasing macrophage colony-stimulating factor secretion from osteoblasts, as well as initiating the phosphatidylinositol 3-kinase/Akt/NF-κB (PI3K/Akt/NF-κB) pathway.⁵⁶ In a uPAR-deficiency mice model, osteoblast was found to have increased proliferative capacity, alkaline phosphatase (ALP) activity, bone matrix protein expression level, and a promoted mineralization, indicating that u-PAR may inhibit osteoblast functions.⁵⁷ Furthermore, u-PAR has also been suggested to mediate osteogenic differentiation of MSCs through the complement C5a receptor.⁵⁸

Angiogenesis is vital for bone tissue engineering as the regeneration of bone requires vascularity to provide essential nutrition and eliminate waste products, along with establishing biocommunications.⁵⁹ The migration of endothelial cells is regarded as an essential step in angiogenesis process, which demands the proteolysis of the underlying basement membrane matrix.^60,61 It is, therefore, of considerable interest for cells to produce proteolytic enzymes allowing cleavage of matrix proteins for favoring cell migration. Substantial evidence has shown that t-PA, u-PA, and matrix metalloproteinases (MMPs) are involved in the degradation of matrix proteins.^62,63 The t-PA modulates fibrin homeostasis while the u-PA primarily affects cell migration and tissue remodeling.⁶⁴ Moreover, u-PA can modulate the construction of self-assembled network, which contributes to the process of prevascularization.⁶²

MMPs are known to perform specific functions during bone repair, and MMPs at low or high levels can result in abnormal bone remodeling.⁶⁵ The deficiency of u-PA was shown to additionally exacerbate the influence of MMP9 deficiency on bone regeneration, and compensatory upregulation of u-PA bioactivity has been observed in MMP9-deficient mice, which indicates that u-PA regulates bone regeneration by controlling MMP9 production.⁶⁶ Intriguingly, the absence of both u-PAR and MMP9 did not delay the wound repairing process, suggesting that u-PA and u-PAR function independently during wound healing in the case of MMP9 deficiency (Fig. 5).⁶⁶

FIG. 5.

The regulatory role of fibrinolysis system in osteogenesis. Plasminogen can be converted into plasmin, which binds with osteocalcin to decompose osteocalcin into two parts to inhibit bone resorption. Moreover, plasminogen contributes to MSC differentiation through Cyr61 and promotes the release of VEGF, thus enhancing the formation of blood vessels at the fracture site. On the other hand, t-PA boosts angiogenesis by VEGF and HIF-1α, facilitates osteoblast through ERK1/2 and annexin 2 signaling pathway, and participates in the migration of macrophage through FAK, Rac-1, and NF-κB signaling pathway. u-PA exerts its role predominantly through its receptor u-PAR, which influences angiogenesis and macrophage migration as well as the formation of osteoclast through M-CSF and PI3K/Akt/NF-κB signaling pathways. Cyr61, cysteine-rich protein 61; ERK1/2, extracellular signal-regulated kinase1/2; FAK, focal adhesion kinase; HIF-1α, hypoxia-inducible factor-1α; NF-κB, nuclear factor kappa-B; MSC, mesenchymal stem cell; M-CSF, macrophage colony-stimulating factor; u-PAR, urokinase-type plasminogen activator receptor; VEGF, vascular endothelial growth factor. Color images are available online.

The Crosstalk Between Fibrinolytic Inhibitory System and Osteogenesis

The fibrinolytic inhibitory system consists of PAI (for example, PAI-1) and inhibitor of plasmin (such as α₂-AP), which is essential for bone repair. PAI-1, an inhibitor for t-PA and u-PA, exerts an array of biological functions, which is involved in the regulation of extracellular matrix degradation, cell migration, apoptosis, and cell proliferation through fibrinolysis-dependent or independent pathways.⁶⁷ PAI-1 has been shown to be essential for bone metabolism. It induces bone loss and delays bone healing as a result of hampering osteoblast differentiation in a streptozotocin (STZ)-induced diabetes model in mice.⁶⁸ In another research, PAI-1 deficiency not only defended STZ-induced bone loss, but also reduced the fluctuation of Runx2, osterix, and ALP in tibia as well as serum osteocalcin levels suppressed by the diabetic state in female mice.⁶⁹

Besides, the addition of small-molecule PAI-1 inhibitor (iPAI-1) into an estrogen deficiency-induced osteoporosis model significantly increased the trabecular bone volume compared with the control group (without adding iPAI-1).⁷⁰ Moreover, the absence of PAI-1 caused a larger fracture callus at 7 and 14 days through boosting extracellular matrix remodeling in a fractured femur model of mice.⁷¹ One of the possible mechanisms was that PAI-1 can cause bone loss through decreasing accumulation and phagocytosis of macrophages at the fracture site during early bone healing.⁷²

In addition, PAI-1 affects the osteogenesis-associated cells (such as MSCs, osteoblasts, osteoclasts, etc.) directly or indirectly. In a diabetic mouse femoral bone defect model, the deficiency of PAI-1 induced a significantly increasing number of osteoblasts and facilitated bone formation.⁷³ Besides, the impaired osteoblast differentiation caused by the upregulation of PAI-1 is involved in the pathogenesis of congenital kyphoscoliosis.⁷⁴ Chondrogenic differentiation of naive induced pluripotent stem cells (iPSCs) was suppressed by adding a PAI-1 inhibitor in the process of MSC induction.⁷⁵ PAI-1 exerts vital role in the differentiation of MSCs into osteoblasts, as the deficiency of PAI-1 can significantly blunt the expression of osteogenic genes in MSCs, and the same results can be obtained by a reduction in endogenous PAI-1 levels by siRNA in vivo.⁷⁶ Furthermore, the mutation of activin type I receptor/ALK2 in fibrodysplasia ossificans progressiva patient-derived iPSCs induced the expression of PAI-1, resulting in the phosphorylation of Smad1 and Smad5, and thus activating bone morphogenetic proteins (BMP) signaling without ligand stimulation.^75,77

α₂-AP, another physiological blocker of fibrinolysis system, was shown to contribute to wound healing. The α₂-AP-deficiency mice displayed an accelerated wound repairing compared with the wild-type mice.⁷⁸ Intriguingly, significantly upregulated VEGF mRNA level was observed in wound lesions compared with the wild-type mice.⁷⁸ Those findings suggest that α₂-AP promotes the expression of VEGF, thus resulting in an accelerated wound closure. In addition, α₂-AP stimulates the generation of type I collagen (which is crucial for bone mineralization by not only providing structural sites for osteocalcin, but also combining with noncollagenous proteins, such as osteocalcin to form a network scaffold) and is involved in the synthesis of TGF-β1 (which facilitates bone regeneration through coupling osteogenesis with angiogenesis).^79,80 Furthermore, α₂-AP negatively affects osteoblast differentiation by suppressing the Wnt/β-catenin pathway.⁸¹

Fibrin(ogen) regulates migration and adhesion of leukocyte

Blood clot acts as a provisional matrix for leukocyte adhesion and migration during bone healing process.⁸² Leukocyte migration is a process where leukocytes are recruited from the circulation to the injured tissue, which is an integral part of the inflammatory response.⁸³ Countless evidence revealed that fibrin(ogen) exerts essential roles in the migration and adhesion of leukocytes.^84,85 There are two pathways involved in leukocyte migration induced by fibrin(ogen). One pathway is that fibrinogen bridges leukocyte to the endothelium through binding to leukocyte integrin receptor Mac-1 and endothelial cell receptor intercellular adhesion molecule, and the site of Mac-1was related to the fibrin gamma chain residues 390–396.^85–87 It has been shown that the existence of fibrinogen increases two- to five-fold of the adhesion of leukocyte to endothelium.⁸⁶

The other pathway is through the very-low-density lipoprotein receptor (VLDLR), a binding site that exists in vascular endothelium.^86,88,89 However, the existing phenomenon is that the specific inhibitors of fibrin–VLDLR interaction could not completely obstruct leukocyte transmigration induced by fibrin, indicating that other pathways participate in this process, whereas the effects of them are much lower.⁹⁰

Fibrin(ogen) regulates the releasing of cytokines and chemokines

Fibrinogen and fibrin are recognized as proinflammatory factors. A significant body of basic science literature found that fibrinogen and fibrin contribute to inflammation not only by indirectly inducing leukocyte migration, but also by directly modulating the expression of cytokines on monocytes and chemokines on leukocytes.^91–93 For example, fibrin was demonstrated to promote the expression of IL-1β by human peripheral blood mononuclear cells mediated partly by the integrin receptor CD11b/CD18.⁹⁴ The coculture of fibrin(ogen) with human synovial fibroblasts led to the upregulation of IL-1 (IL-8).⁹⁵

Moreover, culturing macrophages on fibrin gels resulted in an increasing secretion of IL-10, and exposure of macrophages to soluble fibrinogen led to the upregulation of tumor necrosis factor alpha (TNF-α).⁹⁶ Fibrin(ogen) can directly stimulate the release of IL-6 and monocyte chemoattractant protein-1 (MCP-1) in vivo.⁹³ Fibrin prevents the activation of macrophage inflammation stimulated by fibrinogen, lipopolysaccharide, and interferon-γ.⁹⁶ Research recommended that Mac-1 on the monocytes may act as a fibrin(ogen) receptor in the release of cytokines chemokines.⁹⁷

Another study has shown that fibrin(ogen) stimulates macrophage chemokine secretion through toll-like receptor 4,⁹⁸ but the conclusion is controversial.⁹⁹ Importantly, in the case of fibrin(ogen) knockout, macrophage adhesion capacity was suppressed and the production of IL-6 was reduced.⁹³ On the other hand, fibrin(ogen) can upregulate the secretion of metalloproteinase-9, which promotes monocyte coagulation through an autocrine mechanism.¹⁰⁰ Fibrin(ogen) may act as a crucial switch in regulating macrophage polarization, which has the potential to supply valuable immunomodulatory strategy for tissue regeneration.

Other Fibrinolysis Regulators Promote Osteogenesis

There are many factors that contribute to the architecture and properties of fibrin network. Shiu et al. used carboxyl and alkyl functional groups to modulate the surface chemistry of biomaterial, thereby altering the properties of blood clot formed on biomaterial surface and making the clot susceptible to fibrinolysis thus enhancing bone healing in vitro.¹³ Evidence showed that the implantation of β-tricalcium phosphate (bone substitute biomaterial) into rat mandibular bone defect model resulted in dense fibrin network formed around the implanted substitute, leading to delayed bone healing; on the other hand, the use of s-nitrosoglutathione (GSNO) to modify fibrin structure contributed to bone regeneration in vivo.⁹ There are other factors that regulate fibrin structure, such as ionic strengths, thrombin concentration, and dextran.^101,102 The increasing of plasma ionic strengths leads to a shift from thicker to thinner fiber, especially at an ionic strength of 0.35, and hematoma cannot be degraded for hours in vitro.¹⁰³ Thrombin concentration has been shown to influence fibrin thickness and density.

Low thrombin concentrations (<1 nM) lead to fibrin meshwork consisting of thicker fibrin fibers and can be easily degraded, whereas high thrombin concentrations result in fibrin meshwork composed of thinner fibrin fibers resistant to fibrinolysis.¹⁰⁴ Blood clots are more turbid and consists of thicker fibers in the presence of dextran, thus facilitating its fibrinolysis.¹⁰³ Additionally, calcium ion (Ca²⁺) boosts fibrin monomer polymerization, thus increasing fiber thickness.¹⁰⁵ The fibrinogen molecule contains 3 high-affinity Ca²⁺-binding sites and 10 low-affinity Ca²⁺-binding sites.^106,107 Evidence suggested that the binding of Ca²⁺ to the high-affinity calcium-binding site of γ-chain was a guarantee of pocket “a” working normally during fibrin polymerization, whereas the mutations of high-affinity calcium-binding site severely damaged the function of pocket “a” thus causing fibrinogen failing to polymerize to form fibrin fiber.^108,109 Whereas chlorine antagonizes lateral aggregation, leading to form thinner fiber; zinc (Zn²⁺) increases fiber diameter and alters overall clot structure.^110,111

Fibrinolysis-related physical factors

The degradation of fibrin clots involves in the clearance of individual fibrin fiber. There are several pathways by which fibrin clots are dissolved, such as transverse cleavage of fibers, fiber bundling, buckling, and collapsing, and those pathways are regulated by the concentration of plasmin.^30,112 Transverse cleavage happens when plasmin cuts fiber at a single site. Buckling is a process where fibers are bent, ascribable to tension change in the fibrin network, as a result of which surrounding fibers are broken. The bunding process is where two or more uncutting fibers move together to form fresh and thicker fiber. Fiber collapsing is characterized by the disappearance of uncutting fibers (folded to the edge) on account of elastic recoil force caused by transverse cleavage of surrounding fibers.

The polymerization of fibrin is under tension, leading to tension distributed on fibrin network in the native state. Studies have shown that if there was no tension on the fibrin network, only ∼60% of the fibrin can be degraded eventually. However, the existing tension (in natural state) results in ∼95% clearance of fibrin finally.³⁰ Under normal circumstance, the tension of the fiber exceeds its equilibrium length by ∼23% during polymerization.³⁰ Those findings indicate that fibrin tension probably acts as one of the targets to modulate fibrinolysis (Fig. 6).

FIG. 6.

Tension has a profound influence on the dissolution of fibrin network. In the case of pre-strain = 0% (no tension) distributed on the fibrin network, the uncutting fiber would not alter its shape when transverse cleavage occurred in the surrounding fibers; but when pre-strain = 100% (a state of tension), fibrin recoil force as a result of the cutting of surrounding fibers leads to uncutting fibers folded to the edge to form a new and thick fiber. Color images are available online.

After being implanted into fracture, biomaterials come into contact with blood to form clots on the surface of the implanted materials. Therefore, the properties of the material surface play crucial roles in the formation of blood clots. The hydrophobic surface of polystyrene material was observed to reduce the volume of blood clots, and the inner conformation of the formed clots are looser compared with clots formed on the hydrophilic polystyrene surfaces, and fibrin clots formed on the hydrophobic surface of polystyrene material are easier to dissolve compared with those developed on the hydrophilic polystyrene surfaces.¹¹³

Fibrinolysis-related chemical factors

The regulation of fibrin clot characteristics by modulating fibrinogen molecule facilitates the controllable fibrinolysis, and the approaches, including oxidation, glycation, acetylation, and carbamylation.

Oxidation

The oxidation of specific methionine residues on the α, β, and γ chains of fibrinogen molecule by hypochlorous acid (probably hinder lateral aggregation of protofibrils in fibrin gels) leads to the formation of denser fibrin network of thinner fibers, inducing delayed fibrinolysis.¹¹⁴ Similar results have been found in other studies, which further elucidated that the hypochlorite oxidizes the D region rather than E region of fibrinogen thus affecting the protofibril aggregation leading to denser fibrin network.^115,116 However, there are contrary findings that hypochlorous acid was observed to reduce fibrin clot formation and attenuate clot retraction and fibrinolysis under normal circumstances.¹¹⁷

Moreover, the fibrin susceptibility to plasmin degradation was reduced after oxidation by concentrations of peroxynitrite higher than 100 μmol/L, whereas a low extent of nitration and oxidation showed no effect on these properties.¹¹⁸ Except for the above oxidation caused by reactive oxygen species, the application of ozone, or illumination to modulate fibrinogen achieved consistent results, resulting in a decreased stiffness, lower permeability, and higher density of fibrin clots.^{115,119–121} Overall, oxidation contributes to denser fibrin meshwork with thinner fibers, which are impermeable, leading to a fibrin network resistant to be dissolved by plasmin, as compared with nonoxidized fibrins.

Acetylation

The acetylation of fibrinogen by applying aspirin showed a significantly decreased rate of polymerization as well as maximum turbidity compared with nonacetylated fibrinogen.^122–124 The diameter of individual fibrin fiber modulated by acetylation was increased, and the formed fibrin network shower higher permeability and lower density, as well as increased susceptibility to fibrinolysis.^122–125 Studies further found that mounts of lysine residues existing in the fibrinogen peptides were involved in the crosslinking of fibrinogen, and those lysine residues could be easily acetylated during fibrin polymerization, which is probably the mechanism by which aspirin enhances fibrin fibrinolysis.¹²⁶

Homocysteinylation

The homocysteinylation of fibrinogen affects fibrin clot structure. Animal study showed that rabbits treated with chronic injections of homocysteine or dietary manipulation (to make hyperhomocysteinemia) were observed to develop fibrin clots that consisted of thinner, more tightly packed fibers compared with the control group.¹²⁷ The adding of homocysteine in the reduced form (0.01–1 mM) into whole human plasma delayed the fibrinolysis process, and the adding of homocysteine thiolactone presented similar results.¹²⁸ In general, the homocysteinylation of fibrinogen not only reduces polymerization rate, but also leads to denser fibrin clots composed of thinner fibers.^127,129,130

Glycation

Studies have shown that glucose molecules are able to form strong H-bonds with fibrinogen, and further affect the fibrin structure and morphology.¹³¹ The glycation of fibrinogen leads to altered fibrin clot structure with smaller pore size and delayed degradation rate during fibrinolysis, and deglycosylation of fibrinogen promotes fibrin clot permeability and degradation rates.^132,133 In addition, nonenzymatic glycosylation of fibrinogen impairs the clot susceptibility to fibrinolysis by plasmin.¹³⁴ The accumulated evidence suggests that the glycosylated fibrinogen results in delayed fibrinolysis process.^131–136

Carbamylation

Additionally, although it has been studied only marginally, the carbamylation of fibrinogen was found to alter fibrin polymerization kinetics and impair crosslinking, forming fibrin network with thinner fibers, higher density, and delayed fibrinolysis compared with the control group.¹³⁷

Fibrinolysis-related drugs

Tetranectin is a protein consisting of four identical, noncovalently bound and 181-amino-acid polypeptide chains, which participates in fibrinolysis and proteolysis by binding to PLG and fibrin.¹³⁸ Although its specific mechanism still remains unclear, it is considered to play important roles in mineralization during osteogenesis in vivo and in vitro.¹³⁹ In addition, pentamidine inhibits fibrinolysis by interacting with plasmin in a dose-dependent manner.¹⁴⁰

Platelet regulates fibrinolysis

Platelets play a central role in hemostasis by affecting fibrin polymerization to safeguard vascular integrity as well as accelerating wound healing. Platelets are previously known as an antifibrinolytic regulator by inducing clot retraction of the formed fresh fibrin meshwork and unleashing high concentrations of PAI-1 from their α-granules.^141,142 Clot retraction is a process where clot volume is compressed and excess fluid is extruded mediated by platelets, and platelets tighten the fibrin meshwork surrounding the injured area through expressing integrin αIIbβ3 by α-granules on platelets.¹⁴³ The activated platelets initiate conformational change of αIIbβ3, leading to the activation of αIIbβ3-mediated outside-in signaling.¹⁴⁴ The retraction force that draws the edges of the wound together is generated from the fibrin–αIIbβ3–myosin complex, which is considered to act as an axis to support clot retraction.¹⁴⁵

Retracted clots are more resistant to fibrinolytic degradation compared with nonretracted clots, due to condensing of crosslinked α₂-AP and decreased binding of t-PA to fibrin.^146,147 Furthermore, the fibrinolysis of clot is divided into internal fibrinolysis and external fibrinolysis, and both are affected by clot contraction induced by platelets, as clot contraction initiated by platelets results in approximately two-fold enhancement of internal fibrinolysis rate, but approximately four-fold slower external fibrinolysis rate compared with uncontracted clots.¹⁴⁸

There are reports indicating that platelets exert diverse roles in regulating fibrinolysis. Under physiological flow rates, PLG associates with phosphatidylserine-exposing platelets to initiate fibrinolysis.¹⁴⁹ Moreover, platelets contribute to fibrinolysis mediated by single-chain urokinase-type plasminogen activator (scu-PA).¹⁵⁰ Activated platelet surfaces are anchored by plasma TAFI, to attenuate PLG accumulation and fibrinolysis, resulting in thrombogenicity under flow.¹⁵¹ Platelets contain three types of secretory granules, namely, the α-granules, dense granules, and lysosomes. Those secretory granules secrete amounts of proteins to affect fibrinolysis, such as PLG, PAI-1, α₂-AP, and polyphosphate.¹⁵²

The Important Role of Early Inflammation in Bone Regeneration

Bone has a natural ability to repair spontaneously, which is closely associated with a sequential process consisting of inflammatory, reparative, and remodeling phases similar to other tissues.^153,154 Following a fracture, blood flows out of the injured vessel to form hematoma, and inflammatory cells are recruited to evoke an acute inflammation, which are known to play crucial roles in bone healing cascade. Inflammation process is thought to activate the coagulation cascade.¹⁵⁵ Under conditions of compromised coagulation, the deposition of fibrin and microvascular failure can enhance the inflammatory response.^156,157 Inflammation is considered to affect both bone resorption and formation processes through the recruitment of inflammatory cells (neutrophils, macrophages, lymphocytes) and the release of various cytokines and growth factors.¹⁵⁸

Early inflammation is regarded to trigger regeneration cascade through initiating angiogenesis, recruiting and boosting differentiation of MSCs, and promoting extracellular matrix synthesis.^159–161 A certain level of inflammation is necessary for normal bone repairing as the application of anti-inflammatory substances, such as cyclooxygenase-2 (a strong inducer of inflammation released after fracture) inhibitors, are able to significantly damage normal bone healing.¹⁶²

During the processes of the polymerization and degradation of fibrin(ogen), various peptides are released, including FpA, FpB, D-dimers, fragment-X, fragment-Y, fragment-D, and fragment-E, as well as minor fragments such as the peptide Bβ15–42.¹⁶³ Those peptides are recognized to influence inflammation by affecting cell response and cytokine release during early bone healing.^91,164–166 Elucidating the function of fibrinogen in inflammation is warranted as fibrin degradation product are derived from fibrinogen and possess similar biological functions with fibrinogen.

Fibrinolysis Products Regulate Early Inflammation

Fragment X

Fibrinogen is a 340-kDa plasma protein that consists of three pairs of polypeptide chains (α, β, and γ), to present an extended globular structure.¹⁶⁷ Fragment X originates from fibrinogen molecule by separating the COOH-terminal section from the Aα chain, which is an early fibrinogen degradation product mediated by plasmin. The clots formed from fragment X mediated by thrombin are more fragile and present a loose fibrin conformation, as compared with fibrin network that originated from complete fibrinogen molecules as a result of decreased branching of fibers and fewer interconnections.¹⁶⁸ Those clots, which consist of fragment X, are more susceptible to fibrinolysis.¹⁶⁹ Furthermore, in the company of t-PA, the combination of fragment X with fibrin meshwork results in clots susceptible to fibrinolysis.¹⁶⁹

There are two mechanisms involved in the phenomenon where the adding of fragment X promotes fibrinolysis, one is the presence of fragment X into clots raising maximum turbidity, leading to the formation of thicker fibers that are easily degraded, and the other is the existence of fragment X rendering clots more susceptible to plasmin.^169,170 Those data indicate that fragment X may act as a regulator to promote lysis, thereby promising to enhance bone regeneration.

Fragment D and D-dimer

Fragment D and fragment E are soluble fibrin degradation products produced from the plasmin-mediated degradation of crosslinked fibrin. Fragment D holds the independently folded C-terminal domains of the Bβ- and γ-chains. Purified fragment D has the ability to boost human hemopoietic cell proliferation, which also enhances the effect of IL-3 on primitive hematopoietic progenitors in vitro, and those effects are the same as fibrinogen.^171,172 D-dimer, a covalently bound dimer generated by fragment D, is considered as the marker of fibrinolysis and many fibrinolysis-related diseases. D-dimer is capable of inducing the monocyte/macrophage lineage to secrete essential components, such as IL-6, u-PA, and PAI-2, to affect blood coagulation, fibrinolysis, and inflammation.¹⁷ Fragment D and D-dimer are capable of promoting the secretion of IL-1α, whereas only D-dimer can stimulate the release of IL-1β.¹⁷

Fragment E

Fragment E is the central part of fibrinogen molecule that consist of the N-termini of the three polypeptide chains. Fragment E can enhance the secretion of IL-1α, which stimulates IL-6 release in rat peritoneal macrophages.^17,173 Fragment E potentiates TGF-β-induced fibroblast development thus facilitating wound recovery through forming stable complexes with αVβ3 integrin.¹⁷⁴

Bβ15–42

Bβ15–42 is a small fragment of the N-terminal β chain produced from fibrin cleavage (by plasmin). Bβ15–42 was shown to regulate platelet and endothelial cell migration, fibroblast proliferation, and new blood vessel formation through binding to heparin.²⁰ It can also boost capillary tube formation and angiogenesis by interacting with vascular endothelial (VE)-cadherin.²⁰ Furthermore, Bβ15–42 responds to endothelial cell receptor VE-cadherin and leukocyte integrin CD11c thus promoting leukocyte transmigration.^175–178 Studies have further demonstrated that Bβ15–42-regulated leukocyte transmigration is associated with the activation of the VLDLR-dependent pathway and the Scr kinase Fyn.^179,180

FpB

FpB is a peptide of 14 amino acids released from N-terminal end of B-beta chains of fibrinogen molecules by thrombin in the polymerization of hematoma fibrin network.^181,182 FpB has been shown to perform important functions in many diseases, for example, it can attract macrophages to the intima, which accelerates the early atherosclerotic lesion formation.¹⁸³ FPB has also been recognized as a maker of many other diseases, such as myocardial infarction, chronic liver, etc. However, the chemotaxis effect of FPB was rarely noticed until researches found FPB could act as chemokines for inflammation cells.^164,184 It has been reported that human fibrinopeptide B (hFpB) contributes to the migration of neutrophils and fibroblasts at an optimum concentration of 10^–8 M, and this chemotactic activity can be blocked by applying antiserum to hFpB.¹⁸²

Researchers further found that the chemotaxis of hFpB for neutrophils was similar to that of anaphylatoxin from the fifth component of human complement (C5a), leukotriene B4.¹⁸² The injection of purified FpA and FpB in rat air pouches showed that the number of leukocytes and monocyte chemoattractant protein-1 (MCP-1) levels were increased.^155,185 Researches further found that it was FpB rather than FpA attracting inflammatory cells.¹⁸³

Diseases with retarded fibrinolysis affect bone healing

Delayed fracture healing is usually found in patients with chronic diseases such as diabetes mellitus (DM) and obesity. Moreover, patients with those complications show poor capacity to remove fibrin or low fibrinolytic activity.^186–188 For example, patients who suffer from DM develop fibrin clots that is composed of thicker fibers and denser fibrin network with fewer branch points, resulting in slower fibrinolysis process and significantly delayed wound healing.¹⁸⁹ Additionally, people with obesity showed slower fibrin lysis rate and damaged capacity to recover from long bone defect compared with the control group.^190,191 Furthermore, in a model of obese mice, bone healing was notably decreased compared with the control group, and a reduction of growth factors, such as fibroblast growth factor, TGF-β1, and TNF-α, were observed.¹⁹² One explanation for this may be that the delayed fibrinolysis led to the slow release of growth factors from the dense fibrin clots, thus injuring bone healing.

Additionally, people who are burdened with chronic kidney disease (CKD) have less clot permeability and prolong the fibrin lysis time.^193,194 In addition, in calvaria and femoral defect model of animal, mice with CKD were observed to show an adverse impact on bone regeneration,¹⁹⁵ and patients who were affected by chronic obstructive pulmonary disease (COPD) presented decreased fibrin clot permeability and prolonged fibrinolysis time compared with healthy people.¹⁹⁶ Those patients with COPD showed an impaired bone metabolism and were easily afflicted with fragility fracture as well as had a delayed recovery rate from fracture, such as neck fracture.^197,198 Other diseases, such as inflammatory bowel disease,¹⁹⁹ sickle cell disease,²⁰⁰ and chronic cardiovascular disease,²⁰¹ noticeably alter patient's clot structure and damage the ability to clear fibrin. As far as those diseases, they are believed to have an unsatisfactory recovery ability from fracture as a consequence of the impeded fibrinolysis, although few studies have directly elucidated the influence of those diseases on bone healing.

Conclusion and Future Perspective

Fibrinolysis is a pivotal step for osteogenesis, and is thought to be a target to promote bone healing. The application of various drugs (such as GSNO), ion strength (such as Ca²⁺), and enzyme concentration (such as thrombin) to alter conformation of clots to facilitate fibrinolysis have shown positive effects on bone healing. Furthermore, modifications indicated by the material interfaces (such as change surface wettability) to generate fibrin clots that are easy to dissolve results in enhancement of osteogenesis.¹¹³

Many chronic diseases, such as obesity and DM, which exhibit retarded fibrinolysis and damaged bone healing ability, assign meaning with regard to fibrinolysis, act as a target to regulate osteogenesis. More attention should be paid to the above chronic diseases, with a purpose to further elucidate the underlying mechanism that causes the alterations in fibrinolysis and to intervene the fibrinolysis-related and inefficient bone healing. We propose that future investigations for the research of fibrinolysis/osteogenesis-based strategies should be considered, but not be confined only to aspects that facilitate fibrinolysis, thus promoting bone repairing, such as: (i) new and efficient methods or techniques to alter the inner structure of fibrin clots thus enhancing fibrin degradation in the fracture sites; (ii) the further effects that products of fibrinolysis exert on early inflammation and bone regeneration. It is expected that the understanding of the mechanism that fibrinolysis promote osteogenesis is promising to develop new strategies (similar to LMHFV or other drugs) to alter fibrinolysis process, thus enhancing bone healing.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 86060620, 31960209, and 31760266), Guizhou Science and Technology Fund Project (Grant No. [2020]1Y093), the “Thousand” Level Project of Training High-Level Innovative Talents in Guizhou Province (Grant No. fzc20200612), the 2018 Zunyi “15851 Talent Elite Project” (Grant No. National Natural Science Foundation of China 31760266), and the Zunyi Science and Technology Fund Project (Grant No. Zun Shi Ke He HZ Zi 2021-40). The Doctoral Science Research Startup Funding of Zunyi Medical University (Grant Nos. F-934 and 2017-01).

References

, Zhou

, Lin

, Shi

, and Lin

Nanomaterials for craniofacial and dental tissue engineering. J Dent Res, 96, 725, 2017.

, Liu

, Tian

, et al. Bioswitchable delivery of microRNA by framework nucleic acids: application to bone regeneration. Small, 17, e2104359, 2021.

Liao

, Tian

, Shi

, et al. The fabrication of biomimetic biphasic CAN-PAC hydrogel with a seamless interfacial layer applied in osteochondral defect repair. Bone Res, 5, 17018, 2017.

Sivakumar

P.M.

, Yetisgin

A.A.

, Sahin

S.B.

, Demir

, and Cetinel

Bone tissue engineering: anionic polysaccharides as promising scaffolds. Carbohydr Polym, 283, 119142, 2022.

Yang

, Zhou

, Wei

, and Xiao

Blood clot formed on rough titanium surface induces early cell recruitment. Clin Oral Implants Res, 27, 1031, 2016.

Yuasa

, Mignemi

N.A.

, Nyman

S., et al. Fibrinolysis is essential for fracture repair and prevention of heterotopic ossification. J Clin Invest, 125, 3117, 2015.

O'Keefe

R.J.

Fibrinolysis as a target to enhance fracture healing. N Engl J Med, 373, 1776, 2015.

Krishnan

L.K.

, Vijayan Lal

, Uma Shankar

P.R.

, and Mohanty

Fibrinolysis inhibitors adversely affect remodeling of tissues sealed with fibrin glue. Biomaterials, 24, 321, 2003.

Wang

, Luo

, Yang

, et al. Alteration of clot architecture using bone substitute biomaterials (beta-tricalcium phosphate) significantly delays the early bone healing process. J Mater Chem B, 6, 8204, 2018.

10.

Wang

, Zhang

, Ji

, and Ao

Categorising bone defect hematomas: enhance early bone healing. Med Hypotheses, 113, 77, 2018.

11.

Shiu

H.T.

, Goss

, Lutton

, Crawford

, and Xiao

Formation of blood clot on biomaterial implants influences bone healing. Tissue Eng Part B Rev, 20, 697, 2014.

12.

Wang

, Friis

T.E.

, Masci

P.P.

, Crawford

R.W.

, Liao

, and Xiao

Alteration of blood clot structures by interleukin-1 beta in association with bone defects healing. Sci Rep, 6, 35645, 2016.

13.

Shiu

H.T.

, Goss

, Lutton

, Crawford

, and Xiao

Controlling whole blood activation and resultant clot properties by carboxyl and alkyl functional groups on material surfaces: a possible therapeutic approach for enhancing bone healing. J Mater Chem B, 2, 3009, 2014.

14.

Yang

, and Xiao

Biomaterials regulating bone hematoma for osteogenesis. Adv Healthc Mater e2000726, 2020.

15.

Kanno

, Ishisaki

, Kawashita

, et al. Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function. J Biol Chem, 286, 8952, 2011.

16.

Kawao

, Tamura

, Okumoto

, et al. Plasminogen plays a crucial role in bone repair. J Bone Miner Res, 28, 1561, 2013.

17.

Hamaguchi

, Morishita

, Takahashi

, Ogura

, Takamatsu

, and Saito

FDP D-dimer induces the secretion of interleukin-1, urokinase-type plasminogen activator, and plasminogen activator inhibitor-2 in a human promonocytic leukemia cell line. Blood, 77, 94, 1991.

18.

Mosesson

M.W.

Fibrinogen and fibrin polymerization: appraisal of the binding events that accompany fibrin generation and fibrin clot assembly. Blood Coagul Fibrinolysis, 8, 257, 1997.

19.

Hsieh

Thrombin interaction with fibrin polymerization sites. Thromb Res, 86, 301, 1997.

20.

Mosesson

M.W.

Fibrinogen and fibrin structure and functions. J Thromb Haemost, 3, 1894, 2005.

21.

Duval

, Allan

, Connell

D., Ridger, V.C., Philippou, H., and Ariëns, R.A. Roles of fibrin α- and γ-chain specific cross-linking by FXIIIa in fibrin structure and function. Thromb Haemost, 111, 842, 2014.

22.

Longstaff

, and Kolev

Basic mechanisms and regulation of fibrinolysis. J Thromb Haemost, 13 Suppl 1, S98, 2015.

23.

de Vries

, Veerman

, Nesheim

M.E.

, and Pannekoek

Kinetic characterization of tissue-type plasminogen activator (t-PA) and t-PA deletion mutants. Thromb Haemost, 65, 280, 1991.

24.

Cesarman-Maus

, and Hajjar

K.A.

Molecular mechanisms of fibrinolysis. Br J Haematol, 129, 307, 2005.

25.

Thorsen

, Philips

, Selmer

, Lecander

, and Astedt

Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2. Eur J Biochem, 175, 33, 1988.

26.

Cottrell

J.A.

, Turner

J.C.

, Arinzeh

T.L.

, and O'Connor

J.P.

The biology of bone and ligament healing. Foot Ankle Clin, 21, 739, 2016.

27.

Shiu

H.T.

, Leung

P.C.

, and Ko

C.H.

The roles of cellular and molecular components of a hematoma at early stage of bone healing. J Tissue Eng Regen Med, 12, e1911, 2018.

28.

de Vries

J.J.

, Snoek

C.J.M.

, Rijken

D.C.

, and de Maat

M.P.M.

Effects of post-translational modifications of fibrinogen on clot formation, clot structure, and fibrinolysis: a systematic review. Arterioscler Thromb Vasc Biol, 40, 554, 2020.

29.

Elgue

, Sanchez

, Fatah

, Olsson

, and Blombäck

The effect of plasma antithrombin concentration on thrombin generation and fibrin gel structure. Thromb Res, 75, 203, 1994.

30.

Cone

S.J.

, Fuquay

A.T.

, Litofsky

J.M.

, Dement

T.C.

, Carolan

C.A.

, and Hudson

N.E.

Inherent fibrin fiber tension propels mechanisms of network clearance during fibrinolysis. Acta Biomater, 107, 164, 2020.

31.

Collet

J.P.

, Park

, Lesty

, et al. Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy. Arterioscler Thromb Vasc Biol, 20, 1354, 2000.

32.

Peters

, Schell

, Bail

J., et al. Standard bone healing stages occur during delayed bone healing, albeit with a different temporal onset and spatial distribution of callus tissues. Histol Histopathol, 25, 1149, 2010.

33.

Collen

, Koolwijk

, Kroon

, and van Hinsbergh

V.W.

Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells. Angiogenesis, 2, 153, 1998.

34.

Cameron

, Milner

, Lee

, Cheng

, Fang

, and Jasiuk

Employing the biology of successful fracture repair to heal critical size bone defects. Cur Top Microbiol Immunol, 367, 113, 2013.

35.

Mehta

, Schell

, Schwarz

, et al. A 5-mm femoral defect in female but not in male rats leads to a reproducible atrophic non-union. Arch Orthop Trauma Surg, 131, 121, 2011.

36.

Schlag

, Redl

, Turnher

, and Dinges

Importance of fibrin in wound repair. Rev Laryngol Otol Rhinol (Bord), 108, 5, 1987.

37.

Wong

R.M.Y.

, Choy

V.M.H.

, Li

, et al. Fibrinolysis as a target to enhance osteoporotic fracture healing by vibration therapy in a metaphyseal fracture model. Bone Joint Res, 10, 41, 2021.

38.

Duan

, He

, Lin

, et al. Plasminogen regulates mesenchymal stem cell-mediated tissue repair after ischemia through Cyr61 activation. JCI Insight, 5, e131376, 2020.

39.

Novak

J.F.

, Hayes

J.D.

, and Nishimoto

S.K.

Plasmin-mediated proteolysis of osteocalcin. J Bone Miner Res, 12, 1035, 1997.

40.

Castellino

F.J.

, and Ploplis

V.A.

Structure and function of the plasminogen/plasmin system. Thromb Haemost, 93, 647, 2005.

41.

de Bart

A.C.

, Quax

P.H.

, Löwik

C.W.

, and Verheijen

J.H.

Regulation of plasminogen activation, matrix metalloproteinases and urokinase-type plasminogen activator-mediated extracellular matrix degradation in human osteosarcoma cell line MG63 by interleukin-1 alpha. J Bone Miner Res, 10, 1374, 1995.

42.

Cheng

S.L.

, Shen

, and Peck

W.A.

Regulation of plasminogen activator and plasminogen activator inhibitor production by growth factors and cytokines in rat calvarial cells. Calcif Tissue Int, 49, 321, 1991.

43.

Lijnen

H.R.

Plasmin and matrix metalloproteinases in vascular remodeling. Thromb Haemost, 86, 324, 2001.

44.

Fujisaki

, Tanabe

, Suzuki

, et al. The effect of IL-1alpha on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in osteoblastic ROS 17/2.8 cells. Life Sci, 78, 1975, 2006.

45.

Kawao

, Tamura

, Okumoto

, et al. Tissue-type plasminogen activator deficiency delays bone repair: roles of osteoblastic proliferation and vascular endothelial growth factor. Am J Physiol Endocrinol Metab, 307, E278, 2014.

46.

Ohki

, Ohki

, Ishihara

, et al. Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration. Blood, 115, 4302, 2010.

47.

Lin

, Jin

, Mars

W.M.

, Reeves

W.B.

, and Hu

Myeloid-derived tissue-type plasminogen activator promotes macrophage motility through FAK, Rac1, and NF-κB pathways. Am J Pathol, 184, 2757, 2014.

48.

Mantuano

, Azmoon

, Brifault

, et al. Tissue-type plasminogen activator regulates macrophage activation and innate immunity. Blood, 130, 1364, 2017.

49.

Kemaloglu

, Ozkan

, Yilmaz

, et al. Prevention of spinal epidural fibrosis by recombinant tissue plasminogen activator in rats. Spinal Cord, 41, 427, 2003.

50.

Bryer

S.C.

, Fantuzzi

, Van Rooijen

, and Koh

T.J.

Urokinase-type plasminogen activator plays essential roles in macrophage chemotaxis and skeletal muscle regeneration. J Immunol, 180, 1179, 2008.

51.

Pajarinen

, Lin

, Gibon

, et al. Mesenchymal stem cell-macrophage crosstalk and bone healing. Biomaterials, 196, 80, 2019.

52.

Popa

N.L.

, Wergedal

J.E.

, Lau

K.H.

, Mohan

, and Rundle

C.H.

Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling. J Bone Miner Metab, 32, 124, 2014.

53.

De Nardo

C.M.

, Lenzo

J.C.

, Pobjoy

, Hamilton

J.A.

, and Cook

A.D.

Urokinase-type plasminogen activator and arthritis progression: contrasting roles in systemic and monoarticular arthritis models. Arthritis Res Ther, 12, R199, 2010.

54.

Kawao

, Tamura

, Horiuchi

, et al. The tissue fibrinolytic system contributes to the induction of macrophage function and CCL3 during bone repair in mice. PLoS One, 10, e0123982, 2015.

55.

Chabot

, Dromard

, Rico

, et al. Urokinase-type plasminogen activator receptor interaction with β1 integrin is required for platelet-derived growth factor-AB-induced human mesenchymal stem/stromal cell migration. Stem Cell Res Ther, 6, 188, 2015.

56.

Kalbasi Anaraki

, Patecki

, Tkachuk

, Kiyan

, Haller

, and Dumler

Urokinase receptor mediates osteoclastogenesis via M-CSF release from osteoblasts and the c-Fms/PI3K/Akt/NF-κB pathway in osteoclasts. J Bone Miner Res, 30, 379, 2015.

57.

Furlan

, Galbiati

, Jorgensen

N.R.

, et al. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function. J Bone Miner Res, 22, 1387, 2007.

58.

Kalbasi Anaraki

, Patecki

, Larmann

, et al. Urokinase receptor mediates osteogenic differentiation of mesenchymal stem cells and vascular calcification via the complement C5a receptor. Stem Cells Dev, 23, 352, 2014.

59.

Diomede

, Marconi

G.D.

, Fonticoli

, et al. Functional relationship between osteogenesis and angiogenesis in tissue regeneration. Int J Mol Sci, 21, 3242, 2020.

60.

Dalal

P.J.

, Muller

W.A.

, and Sullivan

D.P.

Endothelial cell calcium signaling during barrier function and inflammation. Am J Pathol, 190, 535, 2020.

61.

Lamalice

, Le Boeuf

, and Huot

Endothelial cell migration during angiogenesis. Circ Res, 100, 782, 2007.

62.

, Daculsi

, Bareille

, Bourget

, and Amedee

uPA and MMP-2 were involved in self-assembled network formation in a two dimensional co-culture model of bone marrow stromal cells and endothelial cells. J Cell Biochem, 114, 650, 2013.

63.

Hallström

, Haupt

, Kraiczy

, et al. Complement regulator-acquiring surface protein 1 of Borrelia burgdorferi binds to human bone morphogenic protein 2, several extracellular matrix proteins, and plasminogen. J Infect Dis, 202, 490, 2010.

64.

Garcia-Touchard

, Henry

T.D.

, Sangiorgi

, et al. Extracellular proteases in atherosclerosis and restenosis. Arterioscler Thromb Vasc Biol, 25, 1119, 2005.

65.

Paiva

K.B.S.

, and Granjeiro

J.M.

Matrix metalloproteinases in bone resorption, remodeling, and repair. Prog Mol Biol Transl Sci, 148, 203, 2017.

66.

Lund

I.K.

, Nielsen

B.S.

, Almholt

, et al. Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling. Dev Biol, 358, 56, 2011.

67.

Declerck

P.J.

, and Gils

Three decades of research on plasminogen activator inhibitor-1: a multifaceted serpin. Semin Thromb Hemost, 39, 356, 2013.

68.

Mao

, Kawao

, Tamura

, et al. Plasminogen activator inhibitor-1 is involved in impaired bone repair associated with diabetes in female mice. PLoS One, 9, e92686, 2014.

69.

Tamura

, Kawao

, Okada

, et al. Plasminogen activator inhibitor-1 is involved in streptozotocin-induced bone loss in female mice. Diabetes, 62, 3170, 2013.

70.

Jin

, Aobulikasimu

, Piao

, et al. A small-molecule PAI-1 inhibitor prevents bone loss by stimulating bone formation in a murine estrogen deficiency-induced osteoporosis model. FEBS Open Bio, 8, 523, 2018.

71.

Rundle

C.H.

, Wang

, Wergedal

J.E.

, Mohan

, and Lau

K.H.

Fracture healing in mice deficient in plasminogen activator inhibitor-1. Calcif Tissue Int, 83, 276, 2008.

72.

Shimoide

, Kawao

, Tamura

, et al. Role of macrophages and plasminogen activator inhibitor-1 in delayed bone repair in diabetic female mice. Endocrinology, 159, 1875, 2018.

73.

Daci

, Verstuyf

, Moermans

, Bouillon

, and Carmeliet

Mice lacking the plasminogen activator inhibitor 1 are protected from trabecular bone loss induced by estrogen deficiency. J Bone Miner Res, 15, 1510, 2000.

74.

Ishiwata

, Iizuka

, Sonoda

, et al. Upregulated miR-224-5p suppresses osteoblast differentiation by increasing the expression of Pai-1 in the lumbar spine of a rat model of congenital kyphoscoliosis. Mol Cell Biochem, 475, 53, 2020.

75.

Matsumoto

, Ikeya

, Hino

, et al. New protocol to optimize iPS cells for genome analysis of fibrodysplasia ossificans progressiva. Stem Cells, 33, 1730, 2015.

76.

Takafuji

, Tatsumi

, Ishida

, et al. Plasminogen activator inhibitor-1 deficiency suppresses osteoblastic differentiation of mesenchymal stem cells in mice. J Cell Physiol, 234, 9687, 2019.

77.

Fukuda

, Kohda

, Kanomata

, et al. Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva. J Biol Chem, 284, 7149, 2009.

78.

Kanno

, Hirade

, Ishisaki

, et al. Lack of alpha2-antiplasmin improves cutaneous wound healing via over-released vascular endothelial growth factor-induced angiogenesis in wound lesions. J Thromb Haemost, 4, 1602, 2006.

79.

Kanno

, Kuroki

, Okada

, et al. Alpha2-antiplasmin is involved in the production of transforming growth factor beta1 and fibrosis. J Thromb Haemost, 5, 2266, 2007.

80.

Tang

, Hu

, Xu

, et al. Megakaryocytes promote bone formation through coupling osteogenesis with angiogenesis by secreting TGF-β1. Theranostics, 10, 2229, 2020.

81.

Kanno

, Ishisaki

, Kuretake

, Maruyama

, Matsuda

, and Matsuo

α2-antiplasmin modulates bone formation by negatively regulating osteoblast differentiation and function. Int J Mol Med, 40, 854, 2017.

82.

Clark

R.A.

Fibrin and wound healing. Ann N Y Acad Sci, 936, 355, 2001.

83.

Ghasemzadeh

, and Hosseini

Intravascular leukocyte migration through platelet thrombi: directing leukocytes to sites of vascular injury. Thromb Haemost, 113, 1224, 2015.

84.

Languino

L.R.

, Duperray

, Joganic

K.J.

, Fornaro

, Thornton

G.B.

, and Altieri

D.C.

Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition. Proc Natl Acad Sci U S A, 92, 1505, 1995.

85.

Altieri

D.C.

Regulation of leukocyte-endothelium interaction by fibrinogen. Thromb Haemost, 82, 781, 1999.

86.

Languino

L.R.

, Plescia

, Duperray

, et al. Fibrinogen mediates leukocyte adhesion to vascular endothelium through an ICAM-1-dependent pathway. Cell, 73, 1423, 1993.

87.

Flick

M.J.

, Du

, and Degen

J.L.

Fibrin(ogen)-alpha M beta 2 interactions regulate leukocyte function and innate immunity in vivo. Exp Biol Med (Maywood), 229, 1105, 2004.

88.

Yakovlev

, Mikhailenko

, Cao

, Zhang

, Strickland

D.K.

, and Medved

Identification of VLDLR as a novel endothelial cell receptor for fibrin that modulates fibrin-dependent transendothelial migration of leukocytes. Blood, 119, 637, 2012.

89.

Yakovlev

, and Medved

Interaction of fibrin with the very low-density lipoprotein (VLDL) receptor: further characterization and localization of the VLDL receptor-binding site in fibrin βN-domains. Biochemistry, 56, 2518, 2017.

90.

Yakovlev

, and Medved

Effect of fibrinogen, fibrin, and fibrin degradation products on transendothelial migration of leukocytes. Thromb Res, 162, 93, 2018.

91.

Hurley

J.V.

Substances promoting leukocyte emigration. Ann N Y Acad Sci, 116, 918, 1964.

92.

Barnhart

M.I.

, Riddle

J.M.

, Bluhm

G.B.

, and Quintana

Fibrin promotion and lysis in arthritic joints. Ann Rheum Dis, 26, 206, 1967.

93.

Szaba

F.M.

, and Smiley

S.T.

Roles for thrombin and fibrin(ogen) in cytokine/chemokine production and macrophage adhesion in vivo. Blood, 99, 1053, 2002.

94.

Perez

R.L.

, and Roman

Fibrin enhances the expression of IL-1 beta by human peripheral blood mononuclear cells. Implications in pulmonary inflammation. J Immunol, 154, 1879, 1995.

95.

Liu

, and Piela-Smith

T.H.

Fibrin(ogen)-induced expression of ICAM-1 and chemokines in human synovial fibroblasts. J Immunol, 165, 5255, 2000.

96.

Hsieh

J.Y.

, Smith

T.D.

, Meli

V.S.

, Tran

T.N.

, Botvinick

E.L.

, and Liu

W.F.

Differential regulation of macrophage inflammatory activation by fibrin and fibrinogen. Acta Biomater, 47, 14, 2017.

97.

Trezzini

, Schüepp

, Maly

F.E.

, and Jungi

T.W.

Evidence that exposure to fibrinogen or to antibodies directed against Mac-1 (CD11b/CD18; CR3) modulates human monocyte effector functions. Br J Haematol, 77, 16, 1991.

98.

Smiley

S.T.

, King

J.A.

, and Hancock

W.W.

Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4. J Immunol, 167, 2887, 2001.

99.

Adams

R.A.

, Bauer

, Flick

M.J.

, et al. The fibrin-derived gamma 377–395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease. J Exp Med, 204, 571, 2007.

100.

Kaneider

N.C.

, Mosheimer

, Günther

, Feistritzer

, and Wiedermann

C.J.

Enhancement of fibrinogen-triggered pro-coagulant activation of monocytes in vitro by matrix metalloproteinase-9. Thromb J, 8, 2, 2010.

101.

Wang

, Friis

, Glatt

, Crawford

, and Xiao

Structural properties of fracture haematoma: current status and future clinical implications. J Tissue Eng Regen Med, 11, 2864, 2017.

102.

Lugovskoĭ

E.V.

, Gritsenko

P.G.

, Skurskiĭ

S.I.

, and Komissarenko

S.V.

[The role of calcium ions in fibrin polymerization]. Ukr Biokhim Zh (1999), 74, 5, 2002.

103.

Carr

M.E.

Jr. , and Alving

B.M.

Effect of fibrin structure on plasmin-mediated dissolution of plasma clots. Blood Coagul Fibrinolysis, 6, 567, 1995.

104.

Wolberg

A.S.

Thrombin generation and fibrin clot structure. Blood Rev, 21, 131, 2007.

105.

Kanaide

, Uranishi

, and Nakamura

Effects of divalent cations on the conversion of fibrinogen to fibrin and fibrin polymerization. Am J Hematol, 13, 229, 1982.

106.

Nieuwenhuizen

, van Ruijven-Vermeer

I.A.

, Nooijen

W.J.

, Vermond

, Haverkate

, and Hermans

Recalculation of calcium-binding properties of human and rat fibrin(ogen) and their degradation products. Thromb Res, 22, 653, 1981.

107.

Everse

S.J.

, Spraggon

, Veerapandian

, Riley

, and Doolittle

R.F.

Crystal structure of fragment double-D from human fibrin with two different bound ligands. Biochemistry, 37, 8637, 1998.

108.

Remijn

J.A.

, Lounes

K.C.

, Hogan

K.A.

, et al. Mutations on fibrinogen (gamma 316–322) are associated with reduction in platelet adhesion under flow conditions. Ann N Y Acad Sci, 936, 444, 2001.

109.

Ikeda

, Kobayashi

, Arai

, et al. Recombinant γT305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole ‘A’ and calcium binding sites. Thromb Res, 134, 518, 2014.

110.

Di Stasio

, Nagaswami

, Weisel

J.W.

, and Di Cera

Cl- regulates the structure of the fibrin clot. Biophys J, 75, 1973, 1998.

111.

Henderson

S.J.

, Xia

, Wu

, et al. Zinc promotes clot stability by accelerating clot formation and modifying fibrin structure. Thromb Haemost, 115, 533, 2016.

112.

Collet

J.P.

, Lesty

, Montalescot

, and Weisel

J.W.

Dynamic changes of fibrin architecture during fibrin formation and intrinsic fibrinolysis of fibrin-rich clots. J Biol Chem, 278, 21331, 2003.

113.

Ruhoff

A.M.

, Hong

J.K.

, Gao

, et al. Biomaterial wettability affects fibrin clot structure and fibrinolysis. Adv Healthc Mater, 10, e2100988, 2021.

114.

Weigandt

K.M.

, White

, Chung

, et al. Fibrin clot structure and mechanics associated with specific oxidation of methionine residues in fibrinogen. Biophys J, 103, 2399, 2012.

115.

Yurina

, Vasilyeva

, Indeykina

, et al. Ozone-induced damage of fibrinogen molecules: identification of oxidation sites by high-resolution mass spectrometry. Free Radic Res, 53, 430, 2019.

116.

Yurina

L.V.

, Vasilyeva

A.D.

, Bugrova

A.E.

, et al. Hypochlorite-induced oxidative modification of fibrinogen. Dokl Biochem Biophys, 484, 37, 2019.

117.

Misztal

, Golaszewska

, Tomasiak-Lozowska

M.M.

, et al. The myeloperoxidase product, hypochlorous acid, reduces thrombus formation under flow and attenuates clot retraction and fibrinolysis in human blood. Free Radic Biol Med, 141, 426, 2019.

118.

Nowak

, Zbikowska

M., Ponczek, M., Kolodziejczyk, J., and Wachowicz, B. Different vulnerability of fibrinogen subunits to oxidative/nitrative modifications induced by peroxynitrite: functional consequences. Thromb Res, 121, 163, 2007.

119.

Bychkova

A.V.

, Vasilyeva

A.D.

, Bugrova

A.E.

, et al. Oxidation-induced modification of the fibrinogen polypeptide chains. Dokl Biochem Biophys, 474, 173, 2017.

120.

Rosenfeld

M.A.

, Shchegolikhin

A.N.

, Bychkova

A.V.

, et al. Ozone-induced oxidative modification of fibrinogen molecules. Biochemistry (Mosc), 78, 1171, 2013.

121.

Piryazev

A.P.

, Aseichev

A.V.

, and Azizova

O.A.

Effect of oxidation-modified fibrinogen on the formation and lysis of fibrin clot in the plasma. Bull Exp Biol Med, 148, 881, 2009.

122.

Bjornsson

T.D.

, Schneider

D.E.

, and Berger

Jr . Aspirin acetylates fibrinogen and enhances fibrinolysis. Fibrinolytic effect is independent of changes in plasminogen activator levels. J Pharmacol Exp Ther, 250, 154, 1989.

123.

Antovic

, Perneby

, Ekman

G.J.

, et al. Marked increase of fibrin gel permeability with very low dose ASA treatment. Thromb Res, 116, 509, 2005.

124.

, Bark

, Wang

, Svensson

, and Blombäck

Effects of acetylsalicylic acid on increase of fibrin network porosity and the consequent upregulation of fibrinolysis. J Cardiovasc Pharmacol, 53, 24, 2009.

125.

Undas

, and Zabczyk

Antithrombotic medications and their impact on fibrin clot structure and function. J Physiol Pharmacol, 69, 2018.

126.

Svensson

, Bergman

A.C.

, Adamson

, Blombäck

, Wallén

, and Jörneskog

Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: a mass spectrometric and isotope labeling study. Biochem Biophys Res Commun, 421, 335, 2012.

127.

Sauls

D.L.

, Lockhart

, Warren

M.E.

, Lenkowski

, Wilhelm

S.E.

, and Hoffman

Modification of fibrinogen by homocysteine thiolactone increases resistance to fibrinolysis: a potential mechanism of the thrombotic tendency in hyperhomocysteinemia. Biochemistry, 45, 2480, 2006.

128.

Malinowska

, Nowak

, and Olas

Comparison of the effect of homocysteine in the reduced form, its thiolactone and protein homocysteinylation on hemostatic properties of plasma. Thromb Res, 127, 214, 2011.

129.

Lauricella

, Quintana

, Castañon

, Sassetti

, and Kordich

Influence of homocysteine on fibrin network lysis. Blood Coagul Fibrinolysis, 17, 181, 2006.

130.

Sauls

D.L.

, Warren

, and Hoffman

Homocysteinylated fibrinogen forms disulfide-linked complexes with albumin. Thromb Res, 127, 576, 2011.

131.

Hood

J.E.

, Yesudasan

, and Averett

R.D.

Glucose concentration affects fibrin clot structure and morphology as evidenced by fluorescence imaging and molecular simulations. Clin Appl Thromb Hemost, 24, 104s, 2018.

132.

Pieters

, Covic

, van der Westhuizen

F.H.

, et al. Glycaemic control improves fibrin network characteristics in type 2 diabetes: a purified fibrinogen model. Thromb Haemost, 99, 691, 2008.

133.

Kanska

, Omar

M.S.

, Budzynska

, et al. Antileukemic activity of glycated fibrinogen-methotrexate conjugates. Anticancer Res, 25, 2229, 2005.

134.

Brownlee

, Vlassara

, and Cerami

Nonenzymatic glycosylation reduces the susceptibility of fibrin to degradation by plasmin. Diabetes, 32, 680, 1983.

135.

Norton

D.G.

, Fan

N.K.

, Goudie

M.J.

, Handa

, Platt

M.O.

, and Averett

R.D.

Computational imaging analysis of glycated fibrin gels reveals aggregated and anisotropic structures. J Biomed Mater Res Part A, 105, 2191, 2017.

136.

Mirmiranpour

, Bathaie

S.Z.

, Khaghani

, Nakhjavani

, and Kebriaeezadeh

Investigation of the mechanism(s) involved in decreasing increased fibrinogen activity in hyperglycemic conditions using L-lysine supplementation. Thromb Res, 130, e13, 2012.

137.

Binder

, Bergum

, Jaisson

, et al. Impact of fibrinogen carbamylation on fibrin clot formation and stability. Thromb Haemost, 117, 899, 2017.

138.

Kluft

, Jie

A.F.

, Los

, de Wit

, and Havekes

Functional analogy between lipoprotein(a) and plasminogen in the binding to the kringle 4 binding protein, tetranectin. Biochem Biophys Res Commun, 161, 427, 1989.

139.

Wewer

U.M.

, Ibaraki

, Schjørring

, Durkin

M.E.

, Young

M.F.

, and Albrechtsen

A potential role for tetranectin in mineralization during osteogenesis. J Cell Biol, 127, 1767, 1994.

140.

Al-Horani

R.A.

, Clemons

, and Mottamal

The in vitro effects of pentamidine isethionate on coagulation and fibrinolysis. Molecules, 24, 2146, 2019.

141.

Samson

A.L.

, Alwis

, Maclean

J.A.A.

, et al. Endogenous fibrinolysis facilitates clot retraction in vivo. Blood, 130, 2453, 2017.

142.

Mosnier

L.O.

, Buijtenhuijs

, Marx

P.F.

, Meijers

J.C.

, and Bouma

B.N.

Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood, 101, 4844, 2003.

143.

Huang

, Li

, Shi

, et al. Platelet integrin αIIbβ3: signal transduction, regulation, and its therapeutic targeting. J Hematol Oncol, 12, 26, 2019.

144.

Tucker

K.L.

, Sage

, and Gibbins

J.M.

Clot retraction. Methods Mol Biol, 788, 101, 2012.

145.

Kasahara

, Kaneda

, Miki

, et al. Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts. Blood, 122, 3340, 2013.

146.

Rijken

D.C.

, and Uitte de Willige

Inhibition of fibrinolysis by coagulation factor XIII. Biomed Res Int, 2017, 1209676, 2017.

147.

Kohro

, Yamakage

, Sato

I., and Namiki, A. Intermittent pneumatic foot compression can activate blood fibrinolysis without changes in blood coagulability and platelet activation. Acta Anaesthesiol Scand, 49, 660, 2005.

148.

Tutwiler

, Peshkova

, Le Minh

, et al. Blood clot contraction differentially modulates internal and external fibrinolysis. J Thromb Haemost, 17, 361, 2019.

149.

Whyte

C.S.

, Swieringa

, Mastenbroek

T.G.

, et al. Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow. Blood, 125, 2568, 2015.

150.

Baeten

K.M.

, Richard

M.C.

, Kanse

S.M.

, Mutch

N.J.

, Degen

J.L.

, and Booth

N.A.

Activation of single-chain urokinase-type plasminogen activator by platelet-associated plasminogen: a mechanism for stimulation of fibrinolysis by platelets. J Thromb Haemost, 8, 1313, 2010.

151.

Suzuki

, Sano

, Mochizuki

, Honkura

, and Urano

Activated platelet-based inhibition of fibrinolysis via thrombin-activatable fibrinolysis inhibitor activation system. Blood Adv, 4, 5501, 2020.

152.

Whyte

C.S.

, Mitchell

J.L.

, and Mutch

N.J.

Platelet-mediated modulation of fibrinolysis. Semin Thromb Hemost, 43, 115, 2017.

153.

Mantovani

, Biswas

S.K.

, Galdiero

M.R.

, Sica

, and Locati

Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol, 229, 176, 2013.

154.

Zhang

, Tian

, and Lin

Functionalizing framework nucleic acid-based nanostructures for biomedical application. Adv Mater e2107820, 2021.

155.

Jennewein

, Tran

, Paulus

, Ellinghaus

, Eble

J.A.

, and Zacharowski

Novel aspects of fibrin(ogen) fragments during inflammation. Mol Med, 17, 568, 2011.

156.

Schouten

, Wiersinga

W.J.

, Levi

, and van der Poll

Inflammation, endothelium, and coagulation in sepsis. J Leukoc Biol, 83, 536, 2008.

157.

Levi

, van der Poll

, and Büller

H.R.

Bidirectional relation between inflammation and coagulation. Circulation, 109, 2698, 2004.

158.

Gruber

Osteoimmunology: inflammatory osteolysis and regeneration of the alveolar bone. J Clin Periodontol, 46 Suppl 21, 52, 2019.

159.

Gerstenfeld

L.C.

, Cullinane

D.M.

, Barnes

G.L.

, Graves

D.T.

, and Einhorn

T.A.

Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem, 88, 873, 2003.

160.

Kon

, Cho

T.J.

, Aizawa

, et al. Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res, 16, 1004, 2001.

161.

Claes

, Recknagel

, and Ignatius

Fracture healing under healthy and inflammatory conditions. Nat Rev Rheumatol, 8, 133, 2012.

162.

Cottrell

, and O'Connor

J.P.

Effect of non-steroidal anti-inflammatory drugs on bone healing. Pharmaceuticals (Basel), 3, 1668, 2010.

163.

Yatsenko

T.A.

, Rybachuk

V.M.

, Yusova

O.I.

, Kharchenko

S.M.

, and Grinenko

T.V.

Effect of fibrin degradation products on fibrinolytic process. Ukr Biochem J, 88, 16, 2016.

164.

Kay

A.B.

, Pepper

D.S.

, and McKenzie

The identification of fibrinopeptide B as a chemotactic agent derived from human fibrinogen. Br J Haematol, 27, 669, 1974.

165.

Colvin

R.B.

, Johnson

R.A.

, Mihm

M.C.

Jr ., and Dvorak

H.F.

Role of the clotting system in cell-mediated hypersensitivity. I. Fibrin deposition in delayed skin reactions in man. J Exp Med, 138, 686, 1973.

166.

Ono

, and Takayanagi

Osteoimmunology in bone fracture healing. Curr Osteoporos Rep, 15, 367, 2017.

167.

Weisel

J.W.

Fibrinogen and fibrin. Adv Protein Chem, 70, 247, 2005.

168.

Weisel

J.W.

, and Papsun

D.M.

Involvement of the COOH-terminal portion of the alpha-chain of fibrin in the branching of fibers to form a clot. Thrombosis Res, 47, 155, 1987.

169.

Schaefer

A.V.

, Leslie

B.A.

, Rischke

J.A.

, Stafford

A.R.

, Fredenburgh

J.C.

, and Weitz

J.I.

Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin. Biochemistry, 45, 4257, 2006.

170.

Gorkun

O.V.

, Veklich

Y.I.

, Medved

L.V.

, Henschen

A.H.

, and Weisel

J.W.

Role of the alpha C domains of fibrin in clot formation. Biochemistry, 33, 6986, 1994.

171.

Zhou

Y.Q.

, Levesque

J.P.

, Hatzfeld

, et al. Fibrinogen potentiates the effect of interleukin-3 on early human hematopoietic progenitors. Blood, 82, 800, 1993.

172.

Hatzfeld

J.A.

, Hatzfeld

, and Maigne

Fibrinogen and its fragment D stimulate proliferation of human hemopoietic cells in vitro. Proc Natl Acad Sci U S A, 79, 6280, 1982.

173.

Lee

M.E.

, Rhee

K.J.

, and Nham

S.U.

Fragment E derived from both fibrin and fibrinogen stimulates interleukin-6 production in rat peritoneal macrophages. Mol Cells, 9, 7, 1999.

174.

Fuchs

, Calitz

, Pavlović

, et al. Fibrin fragment E potentiates TGF-β-induced myofibroblast activation and recruitment. Cell Signal, 72, 109661, 2020.

175.

Bach

T.L.

, Barsigian

, Yaen

C.H.

, and Martinez

Endothelial cell VE-cadherin functions as a receptor for the beta15–42 sequence of fibrin. J Biol Chem, 273, 30719, 1998.

176.

Petzelbauer

, Zacharowski

P.A.

, Miyazaki

, et al. The fibrin-derived peptide Bbeta15–42 protects the myocardium against ischemia-reperfusion injury. Nat Med, 11, 298, 2005.

177.

Zacharowski

, Zacharowski

, Reingruber

, and Petzelbauer

Fibrin(ogen) and its fragments in the pathophysiology and treatment of myocardial infarction. J Mol Med (Berl), 84, 469, 2006.

178.

Martinez

, Ferber

, Bach

T.L.

, and Yaen

C.H.

Interaction of fibrin with VE-cadherin. Ann N Y Acad Sci, 936, 386, 2001.

179.

Gröger

, Pasteiner

, Ignatyev

, et al. Peptide Bbeta(15–42) preserves endothelial barrier function in shock. PLoS One, 4, e5391, 2009.

180.

Ludwig

, and Rossaint

Fibrin degradation product β15-42-new insights in an old pathway. Thromb Haemost, 119, 1719, 2019.

181.

Okumura

, Haneishi

, and Terasawa

Citrullinated fibrinogen shows defects in FPA and FPB release and fibrin polymerization catalyzed by thrombin. Clin Chim Acta, 401, 119, 2009.

182.

Senior

R.M.

, Skogen

W.F.

, Griffin

G.L.

, and Wilner

G.D.

Effects of fibrinogen derivatives upon the inflammatory response. Studies with human fibrinopeptide B. J Clin Invest, 77, 1014, 1986.

183.

Singh

T.M.

, Kadowaki

M.H.

, Glagov

, and Zarins

C.K.

Role of fibrinopeptide B in early atherosclerotic lesion formation. Am J Surg, 160, 156, 1990.

184.

Richardson

D.L.

, Pepper

D.S.

, and Kay

A.B.

Chemotaxis for human monocytes by fibrinogen-derived peptides. Br J Haematol, 32, 507, 1976.

185.

Persson

, Russell

, Mörgelin

, and Herwald

The conversion of fibrinogen to fibrin at the surface of curliated Escherichia coli bacteria leads to the generation of proinflammatory fibrinopeptides. J Biol Chem, 278, 31884, 2003.

186.

Meade

T.W.

, Chakrabarti

, Haines

A.P.

, North

W.R.

, and Stirling

Characteristics affecting fibrinolytic activity and plasma fibrinogen concentrations. Br Med J, 1, 153, 1979.

187.

Stępień

, Miszalski-Jamka

, Kapusta

, Tylko

, and Pasowicz

Beneficial effect of cigarette smoking cessation on fibrin clot properties. J Thromb Thromb, 32, 177, 2011.

188.

Pandolfi

, Cetrullo

, Polishuck

, et al. Plasminogen activator inhibitor type 1 is increased in the arterial wall of type II diabetic subjects. Arterioscler Thromb Vasc Biol, 21, 1378, 2001.

189.

de Vries

, Hoppenbrouwers

, Martinez-Torres

, et al. Effects of diabetes mellitus on fibrin clot structure and mechanics in a model of acute neutrophil extracellular traps (NETs) formation. Int J Mol Sci, 21, 7107, 2020.

190.

Brzezińska-Kolarz

, Kolarz

, Wałach

, and Undas

Weight reduction is associated with increased plasma fibrin clot lysis. Clin Appl Thromb Hemost, 20, 832, 2014.

191.

Thorud

J.C.

, Mortensen

, Thorud

J.L.

, Shibuya

, Maldonado

Y.M.

, and Jupiter

D.C.

Effect of obesity on bone healing after foot and ankle long bone fractures. J Foot Ankle Sur, 56, 258, 2017.

192.

Gao

, Lv

T.R.

, Zhou

J.C.

, and Qin

X.D.

Effects of obesity on the healing of bone fracture in mice. J Orthop Surg Res, 13, 145, 2018.

193.

Sjøland

J.A.

, Sidelmann

J.J.

, Brabrand

, et al. Fibrin clot structure in patients with end-stage renal disease. Thromb Haemost, 98, 339, 2007.

194.

Undas

, Nycz

, Pastuszczak

, Stompor

, and Zmudka

The effect of chronic kidney disease on fibrin clot properties in patients with acute coronary syndrome. Blood Coagul Fibrinolysis, 21, 522, 2010.

195.

Liu

, Kang

, Seriwatanachai

, et al. Chronic kidney disease impairs bone defect healing in rats. Sci Rep, 6, 23041, 2016.

196.

Undas

, Kaczmarek

, Sladek

, et al. Fibrin clot properties are altered in patients with chronic obstructive pulmonary disease. Beneficial effects of simvastatin treatment. Thrombosis and haemostasis, 102, 1176, 2009.

197.

Lai

K.H.

, Chiang

C.Y.

, Yang

R.S.

, Yang

K.C.

, and Wu

C.C.

Conservative treatment of recurrent symptoms of an incomplete, atypical femoral fracture associated with glucocorticoid, bisphosphonate, and denosumab therapy in a patient with chronic obstructive pulmonary disease. Acta Clin Belg, 74, 370, 2019.

198.

Eisler

, Cornwall

, Strauss

, Koval

, Siu

, and Gilbert

Outcomes of elderly patients with nondisplaced femoral neck fractures. Clin Orthop Relat Res, 399, 52, 2002.

199.

Owczarek

, Cibor

, Sałapa

, Głowacki

M.K.

, Mach

, and Undas

Reduced plasma fibrin clot permeability and susceptibility to lysis in patients with inflammatory bowel disease: a novel prothrombotic mechanism. Inflamm Bowel Dis, 19, 2616, 2013.

200.

Faes

, Ilich

, Sotiaux

, et al. Red blood cells modulate structure and dynamics of venous clot formation in sickle cell disease. Blood, 133, 2529, 2019.

201.

Kietsiriroje

, Ariëns

A. S., and Ajjan, R. A. Fibrinolysis in acute and chronic cardiovascular disease. Semin Thromb Hemost, 47, 490, 2021.