Plasminogen: an enigmatic zymogen.

Plasminogen is an abundant plasma protein that exists in various zymogenic forms. Plasmin, the proteolytically active form of plasminogen, is known for its essential role in fibrinolysis. To date, therapeutic targeting of the fibrinolytic system has been for 2 purposes: to promote plasmin generation for thromboembolic conditions or to stop plasmin to reduce bleeding. However, plasmin and plasminogen serve other important functions, some of which are unrelated to fibrin removal. Indeed, for >40 years, the antifibrinolytic agent tranexamic acid has been administered for its serendipitously discovered skin-whitening properties. Plasmin also plays an important role in the removal of misfolded/aggregated proteins and can trigger other enzymatic cascades, including complement. In addition, plasminogen, via binding to one of its dozen cell surface receptors, can modulate cell behavior and further influence immune and inflammatory processes. Plasminogen administration itself has been reported to improve thrombolysis and to accelerate wound repair. Although many of these more recent findings have been derived from in vitro or animal studies, the use of antifibrinolytic agents to reduce bleeding in humans has revealed additional clinically relevant consequences, particularly in relation to reducing infection risk that is independent of its hemostatic effects. The finding that many viruses harness the host plasminogen to aid infectivity has suggested that antifibrinolytic agents may have antiviral benefits. Here, we review the broadening role of the plasminogen-activating system in physiology and pathophysiology and how manipulation of this system may be harnessed for benefits unrelated to its conventional application in thrombosis and hemostasis.

Tranexamic acid for haemostasis and beyond: does dose matter?

Tranexamic acid (TXA) is a widely used antifibrinolytic agent that has been used since the 1960's to reduce blood loss in various conditions. TXA is a lysine analogue that competes for the lysine binding sites in plasminogen and tissue-type plasminogen activator impairing its interaction with the exposed lysine residues on the fibrin surface. The presence of TXA therefore, impairs the plasminogen and tPA engagement and subsequent plasmin generation on the fibrin surface, protecting fibrin clot from proteolytic degradation. However, critical lysine binding sites for plasmin(ogen) also exist on other proteins and on various cell-surface receptors allowing plasmin to exert potent effects on other targets that are unrelated to classical fibrinolysis, notably in relation to immunity and inflammation. Indeed, TXA was reported to significantly reduce post-surgical infection rates in patients after cardiac surgery unrelated to its haemostatic effects. This has provided an impetus to consider TXA in other indications beyond inhibition of fibrinolysis. While there is extensive literature on the optimal dosage of TXA to reduce bleeding rates and transfusion needs, it remains to be determined if these dosages also apply to blocking the non-canonical effects of plasmin.

Tranexamic acid alters the immunophenotype of phagocytes after lower limb surgery.

BACKGROUND: Tranexamic acid (TXA) is an antifibrinolytic agent frequently used in elective surgery to reduce blood loss. We recently found it also acts as a potent immune-modulator in patients undergoing cardiac surgery. METHODS: Patients undergoing lower limb surgery were enrolled into the "Tranexamic Acid in Lower Limb Arthroplasty" (TALLAS) pilot study. The cellular immune response was characterised longitudinally pre- and post-operatively using full blood examination (FBE) and comprehensive immune cell phenotyping by flowcytometry. Red blood cells and platelets were determined in the FBE and levels of T cell cytokines and the plasmin-antiplasmin complex determined using ELISA. RESULTS: TXA administration increased the proportion of circulating CD141+ conventional dendritic cells (cDC) on post-operative day (POD) 3. It also reduced the expression of CD83 and TNFR2 on classical monocytes and levels of circulating IL-10 at the end of surgery (EOS) time point, whilst increasing the expression of CCR4 on natural killer (NK) cells at EOS, and reducing TNFR2 on POD-3 on NK cells. Red blood cells and platelets were decreased to a lower extent at POD-1 in the TXA group, representing reduced blood loss. CONCLUSION: In this investigation we have extended our examination on the immunomodulatory effects of TXA in surgery by also characterising the end of surgery time point and including B cells and neutrophils in our immune analysis, elucidating new immunophenotypic changes in phagocytes as well as NK cells. This study enhances our understanding of TXA-mediated effects on the haemostatic and immune response in surgery, validating changes in important functional immune cell subsets in orthopaedic patients.

Cerebral Amyloid Angiopathy and the Fibrinolytic System: Is Plasmin a Therapeutic Target?

Cerebral amyloid angiopathy is a devastating cause of intracerebral hemorrhage for which there is no specific secondary stroke prevention treatment. Here we review the current literature regarding cerebral amyloid angiopathy pathophysiology and treatment, as well as what is known of the fibrinolytic pathway and its interaction with amyloid. We postulate that tranexamic acid is a potential secondary stroke prevention treatment agent in sporadic cerebral amyloid angiopathy, although further research is required.

Fibrinolysis: A Primordial System Linked to the Immune Response.

The fibrinolytic system provides an essential means to remove fibrin deposits and blood clots. The actual protease responsible for this is plasmin, formed from its precursor, plasminogen. Fibrin is heralded as it most renowned substrate but for many years plasmin has been known to cleave many other substrates, and to also activate other proteolytic systems. Recent clinical studies have shown that the promotion of plasmin can lead to an immunosuppressed phenotype, in part via its ability to modulate cytokine expression. Almost all immune cells harbor at least one of a dozen plasminogen receptors that allows plasmin formation on the cell surface that in turn modulates immune cell behavior. Similarly, a multitude of pathogens can also express their own plasminogen activators, or contain surface proteins that provide binding sites host plasminogen. Plasmin formed under these circumstances also empowers these pathogens to modulate host immune defense mechanisms. Phylogenetic studies have revealed that the plasminogen activating system predates the appearance of fibrin, indicating that plasmin did not evolve as a fibrinolytic protease but perhaps has its roots as an immune modifying protease. While its fibrin removing capacity became apparent in lower vertebrates these primitive under-appreciated immune modifying functions still remain and are now becoming more recognised.

Fibrinolysis and the Immune Response in Trauma.

It has long been known that the fibrinolytic system becomes activated following trauma. At first glance, this is not at all surprising and would appear to be in response to coagulation and the apparent need to remove blood clots and restore blood flow. However, in a bleeding patient, the opposite is what is actually needed. Therefore, one may ask why the fibrinolytic system gets activated in the first place or is there another purpose? Or is it that the waxing and waning of hemostasis in such severely injured patients creates a "moving target" such that the fibrinolytic system itself is constantly responding to changing circumstances? Depending on the injury modalities and the time point post injury, the fibrinolytic system could be either turned on or off. Various theories now abound that offer new insights into the turmoil and paradoxes associated with the fibrinolytic system in this unique setting and the use of antifibrinolytic agents. While this presents one conundrum, there is also another dimension to add to this discussion that has nothing to do with hemostasis per se but rather with the modulation of other critical processes that are also essential for optimal recovery following severe injury. Indeed, overwhelming data are now supporting an important role of the fibrinolytic system in the removal of necrotic tissue (mortolysis) and as a modulator of the innate immune response. Therefore, what is really going on when the fibrinolytic system decides to go into overdrive and generate plasmin, albeit even briefly after a traumatic event? Moreover, what other consequence may occur when antifibrinolytic agents are administered? This review will address this developing story and will outline a hypothesis that places the fibrinolytic system as a gateway to a myriad of processes that are not only linked to fibrin removal but are also broader players in the modulation of innate immunity.

Haemostasis and innate immunity - a complementary relationship: A review of the intricate relationship between coagulation and complement pathways.

Coagulation and innate immunity are linked evolutionary processes that orchestrate the host defence against invading pathogens and injury. The complement system is integral to innate immunity and shares numerous interactions with components of the haemostatic pathway, helping to maintain physiological equilibrium. The term 'immunothrombosis' was introduced in 2013 to embrace this process, and has become an area of much recent interest. What is less apparent in the literature however is an appreciation of the clinical manifestations of the coagulation-complement interaction and the consequences of dysregulation of either system, as seen in many inflammatory and thrombotic disease states, such as sepsis, trauma, atherosclerosis, antiphospholipid syndrome (APS), paroxysmal nocturnal haemoglobinuria (PNH) and some thrombotic microangiopathies to name a few. The growing appreciation of this immunothrombotic phenomenon will foster the drive for novel therapies in these disease states, including anticoagulants as immunomodulators and targeted molecular therapies.

Plasma from patients with vaccine-induced immune thrombotic thrombocytopenia displays increased fibrinolytic potential and enhances tissue-type plasminogen activator but not urokinase-mediated plasminogen activation.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus vector-based COVID-19 vaccines. VITT is associated with markedly raised levels of D-dimer; yet, how VITT modulates the fibrinolytic system is unknown. OBJECTIVES: We aimed to compare changes in fibrinolytic activity in plasma from patients with VITT, patients diagnosed with venous thromboembolism (VTE) after vaccination but without VITT (VTE-no VITT), and healthy vaccinated controls. METHODS: Plasma levels of plasmin-antiplasmin (PAP) complexes, plasminogen, and alpha-2-antiplasmin (α2AP) from 10 patients with VITT, 10 patients with VTE-no VITT, and 14 healthy vaccinated controls were evaluated by enzyme-linked immunosorbent assay and/or Western blotting. Fibrinolytic capacity was evaluated by quantitating PAP levels at baseline and after ex vivo plasma stimulation with 50-nM tissue-type plasminogen activator (tPA) or urokinase for 5 minutes. RESULTS: Baseline PAP complex levels in control and VTE-no VITT individuals were similar but were ∼7-fold higher in plasma from patients with VITT (P < .0001). VITT samples also revealed consumption of α2AP and fibrinogenolysis consistent with a hyperfibrinolytic state. Of interest, VITT plasma produced significantly higher PAP levels after ex vivo treatment with tPA, but not urokinase, compared to the other groups, indicative of increased fibrinolytic potential. This was not due to D-dimer as addition of D-dimer to VTE-no VITT plasma failed to potentiate tPA-induced PAP levels. CONCLUSION: A marked hyperfibrinolytic state occurs in patients with VITT, evidenced by marked elevations in PAP, α2AP consumption, and fibrinogenolysis. An unidentified plasma cofactor that selectively potentiates tPA-mediated plasminogen activation also appears to exist in the plasma of patients with VITT.

Tranexamic acid in a mouse model of cerebral amyloid angiopathy: setting the stage for a novel stroke treatment approach.

Background: Symptomatic intracerebral hemorrhage (sICH) commonly occurs in patients with cerebral amyloid angiopathy (CAA). Amyloid also initiates plasminogen activation and might promote sICH. Objectives: As amyloid-driven plasmin formation can be blocked by tranexamic acid (TXA), we aimed to evaluate the biodistribution and long-term consequences of TXA on brain amyloid-beta (Aß) levels, inflammation, and neurologic function in APP/PS1 mice. Methods: APP/PS1 mice overexpressing the mutant human amyloid precursor protein and wild-type littermates were randomized to TXA (20 mg/mL) or placebo in the drinking water for 6 months. TXA in plasma and various organs was determined by liquid chromatography-mass spectrometry. Plasmin activity assays were performed to evaluate changes in fibrinolytic activity. Neurologic function was evaluated by Y-maze and parallel rod floor testing. Proximity ligation-based immunoassays were used to quantitate changes of 92 biomarkers of inflammation. Brain Aß levels were assessed by immunohistochemistry. Results: Long-term oral TXA administration inhibited fibrinolysis. TXA accumulated in the kidney (19.4 ± 11.2 µg/g) with 2- to 5-fold lower levels seen in the lung, spleen, and liver. TXA levels were lowest in the brain (0.28 ± 0.01 µg/g). Over 6 months, TXA had no discernible effect on motor coordination, novelty preference, or brain Aß levels. TXA reduced plasma levels of epithelial cell adhesion molecule and increased CCL20. Conclusion: Long-term TXA treatment does not alter brain Aß levels or impact neurologic behavior in mice predisposed to amyloid deposition and had minor effects on the levels of inflammatory mediators. This finding supports the safety of TXA and lays the foundation for TXA as a novel treatment to reduce sICH in patients with CAA.

The Fibrinolytic System: Mysteries and Opportunities.

The deposition and removal of fibrin has been the primary role of coagulation and fibrinolysis, respectively. There is also little doubt that these 2 enzyme cascades influence each other given they share the same serine protease family ancestry and changes to 1 arm of the hemostatic pathway would influence the other. The fibrinolytic system in particular has also been known for its capacity to clear various non-fibrin proteins and to activate other enzyme systems, including complement and the contact pathway. Furthermore, it can also convert a number of growth factors into their mature, active forms. More recent findings have extended the reach of this system even further. Here we will review some of these developments and also provide an account of the influence of individual players of the fibrinolytic (plasminogen activating) pathway in relation to physiological and pathophysiological events, including aging and metabolism.

Tranexamic acid rapidly inhibits fibrinolysis, yet transiently enhances plasmin generation in vivo.

Tranexamic acid (TXA) is a lysine analogue that inhibits plasmin generation and has been used for decades as an antifibrinolytic agent to reduce bleeding. Recent reports have indicated that TXA can paradoxically promote plasmin generation. Blood was obtained from 41 cardiac surgical patients randomly assigned to TXA or placebo before start of surgery (preOP), at the end of surgery (EOS), then again on postoperative day 1 (POD-1) as well as POD-3. Plasma levels of tissue-type plasminogen activator (t-PA), urokinase (u-PA), the plasmin-antiplasmin (PAP) complex, as well as t-PA and u-PA-induced clot lysis assays were then determined. Clot lysis and PAP complex levels were also assessed in healthy volunteers before and at various time points after taking 1 g TXA orally. Surgery induced an increase in circulating t-PA, yet not u-PA at EOS. t-PA levels were unaffected by TXA; however, u-PA levels were significantly reduced in patients on POD-3. t-PA and u-PA-induced clot lysis were both inhibited in plasma from TXA-treated patients. In contrast, PAP complex formation, representing plasmin generation, was unexpectedly enhanced in the plasma of patients administered TXA at the EOS time point. In healthy volunteers, oral TXA effectively blocked fibrinolysis within 30 min and blockade was sustained for 8 h. However, TXA also increased PAP levels in volunteers 4 h after administration. Our findings demonstrate that TXA can actually augment PAP complex formation, consistent with an increase in plasmin generation in vivo despite the fact that it blocks fibrinolysis within 30 min. This may have unanticipated consequences in vivo.

Fibrinolysis and COVID-19: A plasmin paradox.

The COVID-19 pandemic has provided many challenges in the field of thrombosis and hemostasis. Among these is a novel form of coagulopathy that includes exceptionally high levels of D-dimer. D-dimer is a marker of poor prognosis, but does this also imply a causal relationship? These spectacularly raised D-dimer levels may actually signify the failing attempt of the fibrinolytic system to remove fibrin and necrotic tissue from the lung parenchyma, being consumed or overwhelmed in the process. Indeed, recent studies suggest that increasing fibrinolytic activity might offer hope for patients with critical disease and severe respiratory failure. However, the fibrinolytic system can also be harnessed by coronavirus to promote infectivity and where antifibrinolytic measures would also seem appropriate. Hence, there is a clinical paradox where plasmin formation can be either deleterious or beneficial in COVID-19, but not at the same time. Hence, it all comes down to timing.

Tissue-Type Plasminogen Activator and Tenecteplase-Mediated Increase in Blood Brain Barrier Permeability Involves Cell Intrinsic Complement.

Background: Tissue-type plasminogen activator (t-PA) has been the mainstay of therapeutic thrombolysis for patients with acute ischaemic stroke (AIS). However, t-PA can cause devastating intracerebral hemorrhage. t-PA can also influence the CNS in part by modulation of BBB permeability. Complement activation also occurs after AIS and has also been reported to increase BBB permeability. The complement components, C3 and C5, can also be activated by t-PA via plasmin formation and cell intrinsic complement may be involved in this process. Tenecteplase (TNK-tPA) is a t-PA variant with a longer plasma half-life, yet the ability of TNK-tPA to modulate the BBB and complement is less clear. Aim: To evaluate the effect of C5 and C5a-receptor 1 (C5aR1) inhibitors on t-PA- and TNK-tPA-mediated opening of the BBB. Methods: We used an in vitro model of the BBB where human brain endothelial cells and human astrocytes were co-cultured on the opposite sides of a porous membrane assembled in transwell inserts. The luminal (endothelial) compartment was stimulated with t-PA or TNK-tPA together with plasminogen, in the presence of PMX205 (a non-competitive C5aR1 antagonist), Avacopan (a competitive C5aR1 antagonist) or Eculizumab (a humanized monoclonal inhibitor of human C5). BBB permeability was assessed 5 and 24 h later. Immunofluorescence was also used to detect changes in C5 and C5aR1 expression in endothelial cells and astrocytes. Results: PMX205, but not Avacopan or Eculizumab, blocked t-PA-mediated increase in BBB permeability at both the 5 and 24 h time points. PMX205 also blocked TNK-tPA-mediated increase in BBB permeability. Immunofluorescence analysis revealed intracellular staining of C5 in both cell types. C5aR1 expression was also detected on the cell surfaces and also located intracellularly in both cell types. Conclusion: t-PA and TNK-tPA-mediated increase in BBB permeability involves C5aR1 receptor activation from cell-derived C5a. Selective inhibitors of C5aR1 may have therapeutic potential in AIS.

Plasmin Generation Potential and Recanalization in Acute Ischaemic Stroke; an Observational Cohort Study of Stroke Biobank Samples.

Rationale: More than half of patients who receive thrombolysis for acute ischaemic stroke fail to recanalize. Elucidating biological factors which predict recanalization could identify therapeutic targets for increasing thrombolysis success. Hypothesis: We hypothesize that individual patient plasmin potential, as measured by in vitro response to recombinant tissue-type plasminogen activator (rt-PA), is a biomarker of rt-PA response, and that patients with greater plasmin response are more likely to recanalize early. Methods: This study will use historical samples from the Barcelona Stroke Thrombolysis Biobank, comprised of 350 pre-thrombolysis plasma samples from ischaemic stroke patients who received serial transcranial-Doppler (TCD) measurements before and after thrombolysis. The plasmin potential of each patient will be measured using the level of plasmin-antiplasmin complex (PAP) generated after in-vitro addition of rt-PA. Levels of antiplasmin, plasminogen, t-PA activity, and PAI-1 activity will also be determined. Association between plasmin potential variables and time to recanalization [assessed on serial TCD using the thrombolysis in brain ischemia (TIBI) score] will be assessed using Cox proportional hazards models, adjusted for potential confounders. Outcomes: The primary outcome will be time to recanalization detected by TCD (defined as TIBI ≥4). Secondary outcomes will be recanalization within 6-h and recanalization and/or haemorrhagic transformation at 24-h. This analysis will utilize an expanded cohort including ~120 patients from the Targeting Optimal Thrombolysis Outcomes (TOTO) study. Discussion: If association between proteolytic response to rt-PA and recanalization is confirmed, future clinical treatment may customize thrombolytic therapy to maximize outcomes and minimize adverse effects for individual patients.

t-PA Suppresses the Immune Response and Aggravates Neurological Deficit in a Murine Model of Ischemic Stroke.

Introduction: Acute ischemic stroke (AIS) is a potent trigger of immunosuppression, resulting in increased infection risk. While thrombolytic therapy with tissue-type plasminogen activator (t-PA) is still the only pharmacological treatment for AIS, plasmin, the effector protease, has been reported to suppress dendritic cells (DCs), known for their potent antigen-presenting capacity. Accordingly, in the major group of thrombolyzed AIS patients who fail to reanalyze (>60%), t-PA might trigger unintended and potentially harmful immunosuppressive consequences instead of beneficial reperfusion. To test this hypothesis, we performed an exploratory study to investigate the immunomodulatory properties of t-PA treatment in a mouse model of ischemic stroke. Methods: C57Bl/6J wild-type mice and plasminogen-deficient (plg-/-) mice were subjected to middle cerebral artery occlusion (MCAo) for 60 min followed by mouse t-PA treatment (0.9 mg/kg) at reperfusion. Behavioral testing was performed 23 h after occlusion, pursued by determination of blood counts and plasma cytokines at 24 h. Spleens and cervical lymph nodes (cLN) were also harvested and characterized by flow cytometry. Results: MCAo resulted in profound attenuation of immune activation, as anticipated. t-PA treatment not only worsened neurological deficit, but further reduced lymphocyte and monocyte counts in blood, enhanced plasma levels of both IL-10 and TNFα and decreased various conventional DC subsets in the spleen and cLN, consistent with enhanced immunosuppression and systemic inflammation after stroke. Many of these effects were abolished in plg-/- mice, suggesting plasmin as a key mediator of t-PA-induced immunosuppression. Conclusion: t-PA, via plasmin generation, may weaken the immune response post-stroke, potentially enhancing infection risk and impairing neurological recovery. Due to the large number of comparisons performed in this study, additional pre-clinical work is required to confirm these significant possibilities. Future studies will also need to ascertain the functional implications of t-PA-mediated immunosuppression for thrombolyzed AIS patients, particularly for those with failed recanalization.