Maintaining the Balance: The Critical Role of Plasmin Activity in Orthopaedic Surgery Injury Response

Abstract

The musculoskeletal system plays vital roles in the body, facilitating movement, protecting vital structures, and regulating hematopoiesis and mineral metabolism. Injuries to this system are common and can cause chronic pain, loss of range of motion, and disability. The acute phase response (APR) is a complex process necessary for surviving and repairing injured musculoskeletal tissue. To conceptualize the APR, it is useful to divide it into two distinct phases, survival and repair. During the survival-APR, a “damage matrix” primarily composed of fibrin, via thrombin activity, is produced to contain the zone of injury. Once containment is achieved, the APR transitions to the repair phase, where reparative inflammatory cells use plasmin to systematically remove the damage matrix and replace it with new permanent matrices produced by differentiated mesenchymal stem cells. The timing of thrombin and plasmin activation during their respective APR phases is crucial for the appropriate regulation of the damage matrix. This review focuses on evidence indicating that inappropriate exuberant activation of plasmin during the survival-APR can result in an overactive APR, leading to an ‘immunocoagulopathy’ that may cause ‘immunothrombosis’ and death. Conversely, pre-clinical data suggests that too little plasmin activity during the repair-APR may contribute to failed tissue repair, such as a fracture non-union, and chronic inflammatory degenerative diseases like osteoporosis. Future clinical studies are required to affirm these findings. Therefore, the temporal-spatial functions of plasmin in response to musculoskeletal injury and its pharmacological manipulation are intriguing new targets for improving orthopaedic care.

Musculoskeletal System and Orthopaedic Surgery.

The musculoskeletal system is a vital and complex system that performs several crucial functions in the body. It not only facilitates movement and safeguards vital structures, but also regulates hematopoiesis and mineral metabolism. The integration of bones, muscles, tendons, and joints works together to resist gravity and allow for movement. The microenvironment within the bone marrow also governs the production of diverse blood cells, including erythrocytes, lymphocytes, and platelets, rendering the musculoskeletal system an indispensable hematopoietic center. Furthermore, bone contains roughly 99% of calcium and 90% of phosphate in the body(1). For these reasons, a healthy and functioning musculoskeletal system is fundamental to survival.

The musculoskeletal system inevitably undergoes injuries due to its physical demands. Such injuries range from minor injuries from everyday use to acute injuries like muscle tears or bone fractures that can lead to immediate loss of limb function. Likewise, while surgery can be used to improve the function of the musculoskeletal system, it often requires controlled injury to the structures, such as an osteotomy. Injury presents four detriments that all must be resolved before this tissue returns to full function: bleeding, risk of infection, tissue hypoxia, and tissue dysfunction. To overcome these problems, the body has carefully coordinated mechanisms in place to repair tissues, collectively referred to as the acute phase response (APR). If the APR processes fail, chronic complications can arise including chronic pain, loss of range of motion, and disability. In the US, musculoskeletal disease or injury affects one out of every two adults over the age of 18(2), accounting for about two-thirds of the disease conditions listed in the International Classification system of Diseases (ICD-10). For these reasons, the field of orthopaedic surgery is inextricably rooted in the physiology of the APR.

The Acute Phase Response: A Temporal Balancing Act of Thrombin, Fibrin, and Plasmin.

The APR is triggered by injury and typically lasts around 6 weeks, with severity and duration proportional to the magnitude of the injury(3–5). To conceptualize this process, it is useful to divide the APR into two distinct phases: survival and repair. During the survival-APR (Figure 1A), activation of thrombin and subsequent production of a fibrin damage matrix work to contain the zone of injury, providing hemostasis, preventing the spread of microorganisms, and serving as a scaffold for tissue repair(3–5). In addition to these canonical functions, thrombin likewise promotes the production of latent-growth factors, cytokines, and collagenases that support inflammation and tissue repair.

Figure 1: Roles of thrombin and plasmin in the Acute Phase Response (APR).

After an isolated injury, e.g., femur fracture, the APR follows a predictable and quantifiable time-course with minimal risk of adverse outcomes. The biological systems activated during the APR rapidly change and can be generally divided into two biologically distinct phases: ‘survival-APR’ and ‘repair-APR’. Temporally, survival-APR precedes repair-APR, occurring during early convalescence. It involves activation of thrombin, which converts fibrinogen to fibrin, which through cooperation with activated platelets and first responder inflammatory cells, contains the zone of injury. Once containment is achieved, the survival-APR transitions to repair-APR (indicated by circular arrows). In this phase, the serine protease plasmin promotes repair – in its canonical role to remove fibrin (fibrinolysis). Additionally, in its non-canonical role, plasmin it activates reparative inflammatory cells (e.g., M2 macrophages) to degrade and remove damaged matrix and damaged tissue, promotes angiogenesis, and tissue reconstruction (e.g., osteogenesis) by activating growth factors and other proteases (e.g., MMPs) from their precursor forms and egress of mesenchymal stem cells.

Within the zone of injury, the survival-APR is tightly regulated; if damage matrix is excessive or extends beyond the zone of injury, this can lead to thrombosis, but if the damage matrix is insufficient or is removed by inopportune fibrinolysis, this can lead to bleeding. Therefore, mechanisms that regulate coagulation and fibrinolysis are essential during the survival-APR so that the damage matrix is appropriately localized and maintained, proportional to the severity of the injury. Tissue factor (TF) and thrombin activatable fibrinolysis inhibitor (TAFI) are two molecules that play crucial roles in the initiation of coagulation and downregulation of fibrinolysis, respectively,(6) during the survival-APR. Following an injury, TF initiates blood coagulation through the extrinsic pathway, culminating in the activation of prothrombin to thrombin, and the production of the fibrin-laden damage matrix. To help preserve this matrix, thrombin likewise activates latent TAFI(7), which downregulates fibrinolysis by removing the C-terminal lysine residues from fibrin polymers, preventing the binding of plasminogen and subsequent activation(7).

Once hemostasis and containment are achieved, numerous regulatory mechanisms, including antithrombin, protein C, and protein S pathways, work together to regulate coagulation and contain the zone of injury prior to the repair-APR. Antithrombin, a serine protease inhibitor, covalently binds to thrombin and coagulation factors IXa, Xa, Xia, and XIIa to inhibit their enzymatic activity. Meanwhile, activation protein C via the thrombin-thrombomodulin complex works to limit the production of thrombin through inactivation of factors Va and VIIIa. Thrombomodulin contributes by binding thrombin and modifies its substrate to favor activation of protein C. Furthermore, protein S acts as a cofactor for protein C, enhancing the efficiency of activated protein C in cleaving and inactivating factors Va and VIIIa.

As the repair-APR phase begins, many of these same factors contribute to regulating fibrinolysis (Figure 1B). Plasmin activation and transition from survival to reparative inflammation are key to removing the damage matrix and replacing it with new matrices produced by differentiated mesenchymal stem cells(8). During the transition from survival to repair-APR, plasmin capitalizes on thrombin’s prior activity through non-canonical roles within the zone of injury to activate the “reparative permissive niche”. For instance, plasmin’s activity activates growth factors and collagenases, the production of which was previously stimulated by thrombin, leading to tissue revascularization and reconstruction(4). Local activation of plasmin is facilitated by the expression of plasminogen receptors on cell surfaces via free lysine binding sites in the kringle domains of plasminogen(9). Plasminogen binding sites and receptors come in multiple forms, including fibrin within the damage tissues themselves, tailless receptors (no transmembrane domain and/or cytoplasmic tail) or tailed receptors (presence of transmembrane domain and/or cytoplasmic tail)(10). Tailless plasminogen receptors on leukocytes include α-enolase, annexin 2, and amphoterin, while tailed plasminogen receptors include integrin adhesion receptors, such as α_Mβ₂ present on leukocytes which aid in recruiting inflammatory cells to the area.

The regulation of plasmin activity is critical both during the survival and repair-APR. This is primarily achieved by the release of plasminogen activator inhibitor-1 (PAI-1) and fast acting protease inhibitors of plasmin such as alpha-2-antiplasmin (α2AP) and alpha-2- macroglobulin (A2M). Thrombin activity during the survival-APR promotes the release of PAI-1(11, 12), which stops the activation of plasmin via inhibition of two key activators, tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This works to limit excessive fibrinolysis during the survival-APR, which can contribute to bleeding risk. Furthermore, regulation of plasmin activity by PAI-1 has been demonstrated to contribute to regulating inflammation during both the survival and repair-APR(13). Finally, during the repair-APR, PAI-1 also works to stimulate regenerative pathways that support tissue regeneration. Further commentary on the function of PAI-1, especially as it extends to pericellular proteolysis, tissue remodeling and other processes critical for tissue repair, can be found in Sillen & Declerck’s recent review(14).

α2AP and A2M likewise play important roles in regulating plasmin activity and promoting tissue regeneration. α2AP acts as a principal regulator of fibrinolysis by binding to active plasmin, thereby blocking plasmin’s capacity to interact with fibrin. Importantly, α2AP can undergo modifications or be genetically altered in ways that markedly alter the protein function, with detectable clinical consequences. For example, when α2AP is diminished, this can lead to bleeding, while increased levels of α2AP have been associated with increased risk of thrombosis and poor tissue repair(15, 16). A2M is a relatively less specific inhibitor of fibrinolysis that has the capacity to inactivate plasmin, kallikrein, tPA and uPA. Like α2AP, A2M binds to active proteases and signal protease degradation. A2M also has numerous other functions including promoting cellular migration, binding of cytokines and growth factors, and regulating immune cell functions, which together markedly contribute to musculoskeletal tissue repair. A thorough review on this topic can be found in Vandooren and Itoh recent publication(17).

Impact of a dysregulated APR in Orthopaedic Surgery.

The APR is essential for surviving an injury and repairing musculoskeletal tissues, making it a crucial aspect of orthopaedic surgery. Despite being essential for musculoskeletal injury, a maladaptive or failed APR is responsible for most adverse outcomes in orthopaedic surgery. During the survival-APR, orthopaedic patients are susceptible to problems such as bleeding or infection if the first responders and damage matrix provide too diminutive of a response. Alternatively, if the survival-APR is too exuberant this can result in an immunocoagulopathic state which can lead to pathology of immunothrombosis(18) such as deep venous thrombosis (DVT), pulmonary embolus (PE), and multi-organ dysfunction syndrome (MODS)(19, 20). Furthermore, it is now apparent that the inability to transition from the survival to the repair phase of the APR is a leading cause of unfavorable outcomes during late convalescence, such as failed tissue repair and fracture non-unions. Moreover, the presence of persistent damage matrix may trigger inflammatory cells within injured tissues, leading to a chronic pro-inflammatory state and the development of degenerative diseases like osteoporosis and arthritis(21). As such, gaining a deeper comprehension of the factors that contribute to an aberrant APR and persistent damage matrix, as well as finding ways to prevent or mitigate them, is crucial for enhancing orthopaedic care.

The goal of this review article is to provide an overview of the current knowledge on the roles of fibrin and plasmin in response to musculoskeletal injury, including their temporal-spatial functions and pharmacological manipulation currently available. The article specifically discusses evidence indicating that inappropriate early activation of plasmin during the survival-APR following musculoskeletal injury can lead to an overactive APR, while insufficient plasmin activity during the repair-APR can result in failed tissue repair and chronic inflammatory degenerative diseases(4). Thus, these findings emphasize the critical importance of the timing, location, and amount of plasmin activation for surviving orthopaedic injuries and repairing injured tissue.

Inappropriate plasmin activation in survival-APR – Level-1 Trauma.

Following a severe injury or large orthopaedic procedure, systemic activation of plasmin can play a pathologic role in the dysregulation of the survival-APR. Clinically, early activation of plasmin is captured as hyperfibrinolysis, which results in excessive bleeding(22) (Figure 2A). Both level-1 trauma and elective surgery patients can experience significant hyperfibrinolysis, which correlates with marked patient morbidity and mortality (23, 24). While several mechanisms may contribute to hyperfibrinolysis, multiple studies have suggested that severe injuries trigger the release of a bolus tPA from the endothelium(25, 26), overwhelming PAI-1, which results in exuberant and potentially systemic plasmin activation. Clinically, multiple prospective studies have corroborated these mechanisms, showing that hyperfibrinolysis in trauma patients is correlated with increased circulating tPA levels and inversely associated with circulating PAI-1(25, 26).

Figure 2: Dysregulated Acute Phase Response- Level 1 Trauma or High Blood Loss Surgery.

One potential mechanism by which a severe injury, either from a trauma or invasive surgery, provokes a pathologic APR. A) Modeling of these biological responses suggest that plasmin is inappropriately and exuberantly activated early (1–2 hours) during the survival-APR, referred to as hyperfibrinolysis (purple curve), which potentially initiates a complex series of pathophysiology and adverse outcomes including both bleeding and an increasing risk of thrombosis. First, it has been recently discovered that when the initial damage matrix is insufficient, the coagulation and inflammatory systems of the survival-APR are able to upscale their response to produce a more exuberant and fortified ‘damage matrix’. B) This can be achieved through the cooperative interaction of fibrin and DNA NETs produced by first responder cells. It is hypothesized that this fortified damage matrix can better contain the zone of injury. C) when upscaling of the damage matrix occurs systemically, referred to as “immunocoagulopathy”, or if a damage matrix metastasizes from the site of injury, it can lead to the accumulation of damage matrices in other organs. D) Depending on the severity of the immunocoagulopathy, these systemic events may become pathologic, referred to as “immunothrombosis”, which can further propagate inflammation and coagulation responses and drive “secondary organ disease” distant from the site of injury by altering the organ’s vasculature or function. E) Once survival is ensured and the APR transitions to the reparative-APR, if the damage matrix persists within tissues, it has been suggested in preclinical models to be able to drive a chronic inflammatory state, resulting in impaired tissue healing.

In response to hyperfibrinolysis, a fortified damage matrix is produced to better contain the zone of injury if the initial damage matrix is insufficient(27, 28). Recent research has shown that neutrophils, macrophages, and platelets can activate inflammasome signaling pathways(29) to fortify the base fibrin matrix with materials such as DNA and histones (NETs) and polyphosphates(30, 31)(Figure 2B). Changes to the damage matrix are thought to be achieved in a dose-dependent manner to damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) on immune cells and injured tissues(32). In addition to improving containment of the zone of injury, fortification of the damage matrix has been shown ex vivo to also increased hemostatic and anti-septic properties of the damage matrix(31, 33), while also making it more resistant to fibrinolysis. Taken together, it is possible that this fortification of the damage matrix markedly supports survival from a severe injury, though further studies are warranted to fully characterize the molecular pathways driving matrix fortification, the impact of this process during the survival-APR, and clinical translation of these findings.

Historically, the increased activity of leukocytes and production inflammatory cytokines has been referred to as systemic inflammatory response syndrome (SIRS) while the accompanying changes in coagulation were referred to as trauma induced coagulopathy (TIC). The more recent use of the term “immunocoagulopathy” is relevant in modeling these pathologies, especially following severe injury, as it clearly indicates that these molecular responses are interlinked both molecularly and physiologically(34, 35)(Figure 2C).

Following hyperfibrinolysis(36), a period of poor plasmin activity during the survival-APR is many times observed in severely injured patients, which is referred to in literature as “fibrinolytic shutdown”(37, 38). Following the systemic activation of plasmin, patients have been shown to enter a chronic state of hypofibrinolysis where plasmin activity is insufficient(39). This can lead to an increased risk of thrombosis, organ failure, and has been shown to be prognostic clinically for an increased risk of lethal immunothrombotic outcomes during the survival-APR(40, 41). There are several proposed mechanisms for fibrinolytic shutdown, which appear to be an adaptive response to a severe-APR. A detailed discussion on this topic, with a focus on traumatic injury can be found in the recent review by Moore et. al(42). In addition to these proposed mechanisms, it is now better understood that availability of plasminogen in circulation can also be impacted by severe injury, both via consumption during its mass activation to plasmin (i.e., hyperfibrinolysis), depletion of the plasma until hepatic expression of plasminogen can restore blood to physiologic levels(43, 44), and degradation by other proteases such as neutrophil elastase(45). Lastly, as previously mentioned, hyperfibrinolysis can signal the reinforcement of the damage matrix, which has been demonstrated ex vivo to diminish plasmin ability to break down the damage matrix(46).

While this fortification of the damage matrix at the site of injury is theoretically crucial during the survival-APR to combat hyperfibrinolysis, bleeding, and risk of infection, strengthening of the damage matrix could likewise have a multisystem effect. When upscaling of the damage matrix occurs systemically, referred to as “immunocoagulopathy”, or if a damage matrix metastasizes from the site of injury, it can lead to the accumulation of damage matrices in other organs referred to as “immunothrombosis”. Immunothrombosis can further propagate inflammation and coagulation responses(18, 19). Metastasized fortified damage matrix drives “secondary organ disease” distant from the site of injury by altering the organ’s vasculature or function(19, 47, 48) (Figure 2D). As such, immunothrombosis and damage matrix deposition appears to be the main driver of MODS following large injuries such as a level-1 injury, infection (e.g., COVID or sepsis) or big surgeries(47–49).

Targeting Plasmin in Survival-APR.

To combat this series of events leading to immunothrombosis, antifibrinolytic drugs, such as tranexamic acid (TXA) and aminocaproic acid (Amicar), are often used clinically to control excessive plasmin activation, hyperfibrinolysis, and associated bleeding in cases of large surgeries or traumatic injuries(41, 50). TXA, a lysine analogue first developed in Japan as a topical hemostatic agent in 1962, binds to the lysine-binding sites of plasminogen, preventing it from binding to fibrin and becoming activated by tPA(51, 52). Clinical studies have shown that early administration of TXA can reduce bleeding, leading to improved survival and recovery outcomes in patients(53). Specifically, the efficacy of TXA in reducing blood loss and improving outcomes in traumatic injury and noncardiac surgery has been demonstrated in large-scale trials, including the CRASH-2 trial and a multi-center trial of over 9,500 patients(54, 55).

In addition to reducing blood loss, recent studies have also suggested that TXA may have both plasmin-dependent and -independent anti-inflammatory effects that can help mitigate the immunocoagulopathy response in trauma(56, 57). TXA has been shown to inhibit the production of pro-inflammatory cytokines and reduce the activation of immune cells by modulating the activity of key transcription factors involved in the regulation of inflammatory gene expression. By limiting the extent of the inflammatory response and dampening the hyperfibrinolytic response following trauma, TXA may help to improve outcomes in trauma patients. Future studies are necessary to determine the optimal therapeutic timing of TXA administration to support improved outcomes in trauma patients. Furthermore, once plasmin is activated, TXA becomes ineffective at controlling hyperfibrinolysis or subsequent immunocoagulopathy(58). Therefore, alternative therapies that target the regulation of immunocoagulopathy and damage matrix production are needed.

Consequences of insufficient plasmin activity on repair-APR.

As highlighted in the normal repair-APR, plasmin activity is crucial for repairing nearly all musculoskeletal tissues. It is important to note that the molecular requirements of plasmin in tissue repair are based on pre-clinical animal models, and further clinical research is needed to determine whether boosting plasmin activity would benefit patients who are at risk of wound healing issues. However, it is possible that the prolonged fibrinolytic shutdown, fortified damage matrix, or other disease-induced reduction of plasmin activity during the repair-APR could contribute to altered repair outcomes in these patient populations (Figure 2E). Here we will provide examples of the role of plasmin in common musculoskeletal injuries including fractures and muscle repair.

Fracture:

Fractures are a common musculoskeletal injury cared for by orthopaedic surgeons around the world. While most fractures heal without incident, up to 10% of cases fail to heal, leading to significant patient morbidity and increased care costs. During the initial phase of the survival APR, fibrin matrices are deposited at the fracture site to stop bleeding, reduce infection risk by trapping microorganisms, and provide initial biomechanical stability(5). As bone is an extremely vascular organ, achieving hemostasis is crucial for surviving a fracture injury. While fibrin is traditionally thought to be deposited mostly intravascularly, recent research has shown that after vascular injury, the majority of fibrin is deposited in the extravascular space and throughout the damaged tissue (59, 60). This is also true during fracture repair, where fibrin is deposited not only in the disrupted vasculature but throughout the damaged tissue site to contain the zone of injury(61).

Once the zone of injury is successfully contained, the reparative APR phase begins to resolve the strain and hypoxia at the fracture site. As part of the transition from survival to reparative APR, the temporary fibrin matrix is cleared and replaced with more permanent matrices that make up the fracture callus. This transition supports new vascularity followed by new bone formation(5). Previously it was hypothesized that this initial fibrin matrix was critical for fracture healing, however, preclinical studies have now shown that fibrinogen-deficient mice experience no temporal or quantitative differences in fracture healing(61). Alternatively, if fibrin is not removed (i.e., delayed fibrinolysis), this is sufficient to impair fracture healing by blocking angiogenesis, vascular bridging, and subsequent bone union(61)(Figure 3). Confirming the importance of fibrinolysis, when fibrinogen was pharmacologically reduced in this model, fracture healing was restored. Thus, while the fibrin matrix is indispensable for fracture healing, plasmin activity and fibrinolysis are crucial for proper fracture repair.

Figure 3: Fibrinolysis in Fracture Repair.

Fibrin must be removed following fracture injury for revascularization and subsequent ossification to occur. Wild-type mice exhibit robust vascularity at the fracture site 14 days post fracture (Top panel). Colored vessels represent fracture vascularity visualized by microfilm perfusion and overlapped if the radiograph of the associated fracture callus. Warm colors (red and orange) represent larger vessels in diameter whereas cooler colors (green and blue) represent smaller diameter vessels. In plasminogen knockout mice, where fibrinolysis is impaired, angiogenesis is significantly inhibited (Top middle panel). When fibrinogen was depleted in plasminogen knockout mice, angiogenesis is largely restored (Top right panel). Through supplementary histologic analysis, it was observed that fibrin acts as a barrier for bone revascularization, preventing VEGF produced by the hypertrophic chondrocytes from effectively reaching the VEGF receptor on endothelial cells. This ultimately results is bone non-union is plasminogen deficient mice seen in the bottom panel. Yellow arrows indicate site of the fracture, which is fully unionized in wild type animals. Together, these findings demonstrate the importance of completely resolving one phase of repair before a next can begin.

In addition to clearing fibrin, plasmin also supports fracture repair through multiple non-canonical roles. During the survival-APR, plasmin activates pro-inflammatory cytokines and chemokines which supports leukocyte recruitment and activation at the injury site(4). Entrance of phagocytic cells to the zone of injury, specifically macrophages and neutrophils, can aid in removing damaged tissues, making way for angiogenesis and fracture callus formation(62–64). When transitioning to the reparative-APR, plasmin resolves inflammation by converting pro-inflammatory to anti-inflammatory states(65, 66), which is essential for proper fracture healing. Clinically, increased systemic inflammation has been shown to lengthen fracture healing time and increase complication rates(67). Furthermore, by activating various matrix-metalloproteinases, plasmin activity drives the breakdown of extracellular matrix, which likewise supports angiogenesis and fracture callus remodeling(68, 69). However, unlike fibrinolysis, no single metalloproteinase has been shown to be required for fracture repair due to functional redundancy(70, 71). Additionally, recent in vitro studies have suggested that plasmin aids in fracture healing by activating Cyr61 to promote periosteal progenitor proliferation, survival, and migration within the zone of injury(72). Further studies are required to assess the importance of this contribution to fracture healing in vivo. Finally, plasmin likewise supports fracture callus remodeling(73) by supporting osteoclast function. Within a fracture callus, osteoclasts are responsible for bone resorption to remodel excess bone tissue, while osteoblasts continue to deposit new bone tissue. Specifically, plasmin can activate pro-osteoclastogenic factors such as transforming growth factor-beta (TGF-β) and insulin-like growth factor (IGF), which stimulate osteoclast formation and activity(74). This process continues until the bone is restored to its pre-fracture shape and strength.

As highlighted above, a disproportionate APR can lead to complications of survival (hemorrhage, thrombosis, systemic inflammatory response syndrome, infection, death) and/or repair (delayed fracture healing or bone non-union) (Figure 2). Fracture healing can be impaired by various clinical conditions, including diabetes, smoking, severe traumatic injuries, and chronic inflammation-related conditions such as rheumatoid arthritis(4, 5). These conditions have been linked to hypofibrinolysis during the repair-APR and/or the development of a modified damage matrix that can drive a chronic inflammatory state. Given that temporary damage matrix must be cleared to allows for angiogenesis and subsequent ossification, persistent damage matrix may contribute to the elevated incidence of fracture non-union among these populations. For example, in individuals with type 2 diabetes, fibrinogen can become denser and less porous due to glycation(75), while smoking can disrupt angiogenesis and alter fibrin clot formation(76, 77). Likewise, severe injury can result in immunocoagulopathy and the production of robust fibrin-DNA NETs damage matrices, which are difficult to remove, can be pro-inflammatory and have been shown to impede soft tissue repair(78). Further research is needed to determine if immunocoagulopathy and the production of fortified damage matrices can likewise impeded fracture repair.

In summary, this section highlights in detail how plasmin, though both canonical and non-canonical activities can influence fracture repair. However, it is important to note that many of the molecular evidence of plasmin’s contribution to fracture repair are based on pre-clinical animal models. Thus, further clinical research is necessary to determine whether enhancing plasmin activity would benefit patients at risk of fracture repair issues.

Muscle:

When muscle is injured, the survival-APR responds by depositing a fibrin-laden damage matrix to contain the injury, stop bleeding, and prevent infection. Once containment is achieved, plasmin activity is responsible for removing the damage matrix via fibrinolysis, allowing for angiogenesis and muscle regeneration to occur. Specifically, work from the Muñoz-Cánoves lab demonstrated that plasmin activity peaks in the injured muscle within 3–5 days of the injury, where it is responsible for removing fibrin and necrotic tissue via a u-PA dependent mechanism on phagocytic cells(79, 80). In addition to managing damage matrix, plasmin likewise has non-canonical roles in healing muscle where it activates pro-regenerative factors, such as VEGF-A and pro-MMPs, promotes anti-inflammatory signaling, and stimulates satellite stem cells to differentiate into functional myotubules to replace damaged muscle(4).

Together, the canonical and non-canonical activities of plasmin help to establish a “reparative permissive niche” within injured muscle to support timely regeneration. If plasmin activity is insufficient or the deposited damage matrix is resistance to degradation, delayed healing, muscle weakness, and the formation of scar tissue or fibrosis can occur. In severe cases, fibrosis can result in muscle contractures, which can significantly impact patient morbidity and ability to conduct daily activities.

In addition to the above roles in muscle repair, recent preclinical studies have demonstrated that plasmin likewise plays an important role in preventing skeletal muscle calcification through fibrin-independent mechanisms(81, 82) (Figure 4). Specifically, as little as a 50% deficiency in plasminogen and plasmin activity is sufficient to allow for dystrophic calcification to form in skeletal muscle following injury. Complementary studies have demonstrated that dystrophic calcification can be effectively regressed from sites of damage skeletal muscle, allowing for proper regeneration to occur(83). The regression of dystrophic calcification was found to occur through both a macrophage and plasmin activity dependent manner. Importantly, studies likewise demonstrated that if dystrophic calcification was not removed from the site of muscle injury, it was sufficient to drive heterotopic ossification (HO), or the formation of mature bone within soft tissues(83). Thus, plasmin activity plays important roles for both preventing the initial dystrophic calcification, but also regressing dystrophic calcification to prevent HO formation.

Figure 4: Plasmin Prevents Skeletal Muscle Calcification.

A) Following a focal injury to the lower limb, mice that are partially deficient in fibrinolysis (Plg+/−) or fully deficient in fibrinolysis (Plg−/−) develop marked calcification that is detected by microcomputed tomography. B) Similar results were observed when Plg+/− and Plg−/− mice were crossed with a murine model of Duchenne muscular dystrophy (mdx mouse) that lead to chronic muscle injury. In these animals, muscle calcification was observed in a gene dependent manner across the entire body, aligning with the chronic muscle injury phenotype. Importantly, these results were found to occur though fibrin-independent mechanisms, such that Plg+/− crossed with fibrinogen deficient animals still develop marked calcification. Figures represented from the author’s prior work (81).

Formation of HO within skeletal muscle can result in chronic inflammation, pain, and marked tissue dysfunction Contrary to the long-standing notion that HO develops from a “gain of function” mutation of bone-related growth factor signaling pathways (i.e. bone morphogenic protein)(84), current research and genetic studies support that severe injury induced HO occurs in response to altered systemic factors that influence the zone of injury(83, 85). Prior work has demonstrated that a deficiency in plasmin activity is sufficient to allow for dystrophic calcification to form and if not regressed, can lead to HO formation(81). Given that trauma patients can 1) experience impaired healing including HO within injured tissues and 2) have been noted to experience reductions of plasmin activity clinically(44), it is possible that altered plasmin activity in these patients is a driving force of impaired muscle healing and calcification. Future clinical observational studies are warranted to determine if enhancing plasmin activity may improve patient outcomes.

Plasmin Role in Degenerative Disease:

A key principle of fibrin damage matrix production and fortification is its ability to contain an injury and support the survival-APR inflammatory responses through DAMP/PRR pathways and inflammasome signaling(32, 86). While fortification of damage matrices can provide better hemostasis and anti-microbial properties to improve survival, when these fortified damage matrices are deposited throughout the body and persist into the survival-APR, they continue to be capable of propagating inflammatory signaling leading to a vicious cycle of chronic activation of coagulation and inflammation within tissues(19)(Figure 2). When this occurs, it can result in “post-injury syndromes” and organ dysfunction in various organ systems.

In addition to matrices being fortified in response to a severe injury, pre-existing comorbidities such as diabetes, obesity, or aging can lead to a modified damage matrix that have altered capacity to be removed(75, 87). This can occur through inflammasome signaling and post-transcriptional modifications, such as reactive oxidative species (ROS)(88, 89). Regardless of the cause, modification of the damage matrix to make it more resistant to plasmin degradation likely plays a significant role in preventing injuries from being completely resolved by the repair-ARP, leading to fibrosis, chronic inflammation, and organ dysfunction/degeneration. Below are how these diseases may manifest in musculoskeletal degenerative phenotypes of bone and joints.

Osteoporosis:

Osteoporosis is characterized by a reduction in bone density and strength, which increases the risk of fracture healthcare costs, leading to a marked impact on patient mobility and quality of life. Osteoporosis is caused by a combination of factors such as reduced bone formation, increased bone resorption, and chronic inflammation. While current treatment for osteoporosis focuses on reducing inflammation and bone resorption, recent research has suggested that persistent fibrin deposition may also contribute to the development of this condition(90).

Studies in plasminogen-deficient mice have shown that the absence of fibrinolysis leads to the accumulation of fibrin in bones, which activates macrophages and triggers the production of inflammatory cytokines. These cytokines promote the differentiation of bone-resorbing osteoclasts, leading to bone loss and osteoporosis (Figure 5). However, when fibrinogen was genetically removed or the ability of fibrin to signal through the MAC-1 binding site was blocked, bone loss was prevented in plasminogen-deficient animals. Clinical studies have also identified a relationship between elevated circulating fibrinogen and age-related osteoporosis in women(91).

Figure 5: Diminished Fibrinolysis Drive Bone Degeneration.

Plasminogen deficient animals (Plg−/− experience marked osteoporosis, indicated by red arrows, compared to wildtype controls (Plg+/+). Preclinical studies have demonstrated that loss of fibrinolysis leads to persistent fibrin within bone, driving inflammation and subsequent osteoclastogenesis resulting in net bone loss.

It is worth noting that patients with risk factors for osteoporosis, such as obesity, diabetes, smoking, and menopause, are also known to experience hypofibrinolysis and altered damage matrices. Therefore, further research is needed to determine if targeting fibrin deposition in bones may be an effective strategy for preventing or slowing the progression of osteoporosis.

Arthritis:

Previous research has shown that the persistence of damage matrix deposition, particularly fibrinogen, at the articular surface is a major contributing factor to osteoarthritis (OA) damage(92). Although damage matrix deposition is necessary for achieving hemostasis and wound healing, similar to fracture healing, if it is not cleared from the joint space, it can lead to pathologic joint degeneration by promoting inflammatory cell infiltration and increasing local cytokines and chemokines(93). For instance, fibrinogen possesses a binding motif for integrin αMβ2, a leukocyte integrin receptor expressed on the surface of CD11b/CD18 immune cells that recruits inflammatory cells to the area and upregulates TNF-α, IL-1β, and IL-6(64, 94). In a murine model of collagen-induced arthritis (CIA), fibrinogen deficiency reduced the local inflammatory response to the collagen injection and conferred resistance to RA in the paw joints, indicating that fibrin is a pathological driver in this disease(94). In the same model, Fib-γ390–396A mice expressing fibrinogen unable to bind to αMβ2 on macrophages were resistant to CIA. In addition to fibrinogen, recent studies have also shown that production of DNA NETs can directly contribute to cartilage degeneration and arthritis(95–97). These recent studies provide additional evidence for the potential composition of damage matrix and its capacity to impact joint function.

Plasmin likewise plays fibrin-independent roles in joint degeneration, like muscle and bone repair, but whether it is protective, or a driver of joint degeneration varies depending on the joint and time point assessed(4, 98). In rheumatoid arthritis (RA) patients, plasmin activates MMP-9, which significantly contributes to the degradation of joint cartilage. Additionally, RA is marked by persistent acute-phase reactions within the synovial fluid, which cause localized inflammation, fibrin formation, cellular infiltration, and plasmin activation in the synovium. Although acute, local activation of plasmin in damaged tissues promotes the removal of pro-inflammatory fibrin, persistent plasmin activation within joints can paradoxically instigate degeneration of articular cartilage and bone surfaces within joints through aberrant activation of tissue proteases and immune cells. Previous studies have shown that tPA-mediated activation of plasmin resolves fibrin and fibrin-related joint inflammation, while chronic, uPA-mediated activation of plasmin in RA drives progression of joint degeneration(99, 100). Reports vary in the interpretation if plasmin is a cause for degenerative joint disease or activated to help resolve persistent inflammation and damage matrix deposition.

Summary.

In summary, the APR has a pivotal role in addressing orthopaedic injury-related issues through a precisely orchestrated thrombin and plasmin activation that is spatially and temporally balanced. An essential component of the APR is the formation of the damage matrix, which acts as a barrier against microbial invasion and bleeding. However, if the damage matrix remains after surviving the injury, tissue repair and degeneration problems may arise. Insufficient damage matrix production may lead to poor microbial containment and hemostasis, while overactive survival-APR and damage matrix fortification, as seen in immunocoagulopathy, may result in life-threatening immunothrombosis. Moreover, if plasmin activity is inadequate or if the damage matrix is fortified and plasmin-resistant, tissue repair may fail, resulting in chronic inflammation and organ degeneration. Importantly, much of the work on plasmin’s pathological role in the survival-APR is from clinical studies. While several clinical conditions suggest that plasmin’s failure to play canonical or non-canonical roles can significantly affect tissue repair and degeneration, newer findings about plasmin’s role in the repair-APR come from preclinical models and require validation. Thus, it is crucial to maintain a balance in thrombin and plasmin activation and in damage matrix formation and removal to ensure successful tissue repair and prevent adverse outcomes.