N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

The potential impact of Covid-19 on the capacity of routine laboratory tests to detect heparin-induced thrombocytopenia.

In Covid-19, anticoagulation with heparin is often administered to prevent or treat thromboembolic events. Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin treatment, caused by heparin-dependent, platelet activating anti-platelet factor 4 (PF4)/heparin antibodies. Diagnosis of HIT is based on the combination of clinical parameters, allowing to determine the pretest probability, and laboratory testing for anti-PF4/heparin antibodies and confirmatory functional assays, such as the heparin-induced platelet activation (HIPA) test.We report the case of a patient with severe Covid-19 pneumonia requiring ECMO treatment, who developed recurrent clotting of the ECMO filter and a drop in platelet count under heparin treatment. He was therefore suspected to have HIT and the anticoagulation was switched to argatroban. Despite high clinical probability and high titres of anti-PF4/heparin antibodies, the functional HIPA test was negative. Nevertheless, argatroban was continued rather than to reinstate anticoagulation with heparin. Reevaluation 7 days later then demonstrated a strongly positive functional HIPA test and confirmed the diagnosis of HIT. Under anticoagulation with argatroban the patient gradually improved and was finally weaned off the ECMO.In conclusion, this case highlights the critical importance of clinical judgement, exploiting the 4 T score, given that Covid-19 patients may present a different pattern of routine laboratory test results in HIT diagnostics. The possibility of a false negative HIPA test has to be considered, particularly in early phases of presentation. In cases of a discrepancy with high clinical probability of HIT and/or high titre anti-PF4/heparin antibodies despite a negative HIPA test, a reevaluation within 3 to 5 days after the initial test should be considered in order to avoid precipitant reestablishment of unfractionated heparin, with potentially fatal consequences.

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.

Characterisation of Plasma Mitochondrial DNA, MMP-9 and Neutrophil Elastase in Patients Undergoing Coronary Artery Bypass Grafting: Effects of Tranexamic Acid and Postoperative Pneumonia.

BACKGROUND: Postoperative pneumonia is a major cause of morbidity and mortality following cardiac surgery. The inflammatory response to cardiac surgery has been widely studied, but specific mechanisms for postoperative pneumonia have not been determined. Tranexamic acid is renowned for its effect on bleeding but can also modulate inflammatory processes. Cardiac surgery is known to release mitochondrial DAMPs (mtDAMPs) and is linked to postoperative inflammation and atrial fibrillation. We speculated that mtDAMPs might be related to postoperative pneumonia and that this might be modulated by tranexamic acid. METHODS: Forty-one (41) patients from the Aspirin and Tranexamic Acid for Coronary Artery Surgery (ATACAS) trial were studied. Levels of mitochondrial DNA, matrix metallopeptidase 9 (MMP-9) and neutrophil elastase (NE) were determined in plasma preoperatively, at 24 and 72 hours post-surgery and correlated with clinical outcome. RESULTS: mtDNA was significantly elevated postoperatively in the placebo and tranexamic acid (TXA) groups. Neutrophil elastase increased immediately postoperatively and at 24 hours. MMP-9 was elevated in the placebo group early postoperatively and in the TXA group at the immediate postoperative time point and after 24 hours. Six (6) of the 41 (14.6%) patients subsequently developed pneumonia. mtDNA levels were significantly increased at the early postoperative period and the 24-hour time point in patients with pneumonia. CONCLUSIONS: Cardiac surgery releases mtDNA, increases MMP-9 and NE and this was not influenced by TXA. Inflammation postoperatively might be linked to pneumonia since mtDNA was further elevated in these patients. Due to the low number of individuals developing pneumonia, further studies are warranted to clearly identify whether TXA impacts on the inflammatory response in postoperative pneumonia.

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.

Fibrinolysis Shutdown in Trauma: Historical Review and Clinical Implications.

Despite over a half-century of recognizing fibrinolytic abnormalities after trauma, we remain in our infancy in understanding the underlying mechanisms causing these changes, resulting in ineffective treatment strategies. With the increased utilization of viscoelastic hemostatic assays (VHAs) to measure fibrinolysis in trauma, more questions than answers are emerging. Although it seems certain that low fibrinolytic activity measured by VHA is common after injury and associated with increased mortality, we now recognize subphenotypes within this population and that specific cohorts arise depending on the specific time from injury when samples are collected. Future studies should focus on these subtleties and distinctions, as hypofibrinolysis, acute shutdown, and persistent shutdown appear to represent distinct, unique clinical phenotypes, with different pathophysiology, and warranting different treatment strategies.

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.

Plasmin: A Modulator of Immune Function.

Plasmin is the effector protease of the fibrinolytic system, well known for its involvement in fibrin degradation and clot removal. However, plasmin is also recognized as a potent modulator of immunological processes by directly interacting with various cell types including leukocytes (monocytes, macrophages, and dendritic cells) and cells of the vasculature (endothelial cells, smooth muscle cells) as well as soluble factors of the immune system and components of the extracellular matrix. In fact, the removal of misfolded proteins and maintenance of tissue homeostasis seem to be major physiological functions of plasmin. However, a large body of evidence also suggests that excessive plasmin generation frequently contributes to the pathophysiology of acute and chronic inflammatory processes. Hence, one question arising from the broadening effects of plasmin in physiology is whether antifibrinolytic drugs (i.e., tranexamic acid, epsilon aminocaproic acid, or aprotinin) that target plasmin either directly or indirectly and which are commonly used to prevent or treat bleeding might have unintended consequences on the immune response or on other nonfibrinolytic processes in vivo.

Mitochondrial DNA and Toll-Like Receptor-9 Are Associated With Mortality in Critically Ill Patients.

OBJECTIVES: Despite underlying pathologies leading to ICU admittance are heterogeneous, many patients develop a systemic inflammatory response syndrome often in the absence of microbial pathogens. Mitochondrial DNA that shows similarities to bacterial DNA may be released after tissue damage and activates the innate immune system by binding to toll-like receptor-9 on immune cells. The aim of this study was to analyze whether levels of mitochondrial DNA are associated with 30-day survival and whether this predictive value is modified by the expression of its receptor toll-like receptor-9. DESIGN: Single-center, prospective, observational study. SETTING: A tertiary ICU in a university hospital. PATIENTS: Two hundred twenty-eight consecutive patients admitted to a medical ICU between August 2012 and August 2013. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood was taken within 24 hours after ICU admission, and the levels of circulating mitochondrial DNA were quantified by real-time polymerase chain reaction. Toll-like receptor-9 expression in monocytes was measured by flow cytometry. Median acute physiology and chronic health evaluation II score was 20, and 30-day mortality was 25%. Median mitochondrial DNA levels at admission were significantly higher in nonsurvivors when compared with survivors (26.9, interquartile range = 11.2-60.6 ng/mL vs 19.7, interquartile range = 9.5-34.8 ng/mL; p < 0.05). Patients with plasma levels of mitochondrial DNA in the highest quartile (mitochondrial DNA > 38.2 ng/mL) had a 2.6-fold higher risk (p < 0.001) of dying, independently of age, gender, diagnosis, and acute physiology and chronic health evaluation II score. Mitochondrial DNA improved the c-statistic of acute physiology and chronic health evaluation II score (p < 0.05) and showed enhancement in individual risk prediction indicated by a net reclassification improvement of 32.3% (p < 0.05). Stratification of patients according to toll-like receptor-9 expression above/below median demonstrated that only patients with high expression of toll-like receptor-9 showed an increased risk associated with increased mitochondrial DNA levels (odds ratio, 2.7; p < 0.01), whereas circulating mitochondrial DNA was not associated with mortality in patients with low toll-like receptor-9 expression (odds ratio, 1.1; p = 0.98). CONCLUSIONS: Circulating levels of mitochondrial DNA at ICU admission predict mortality in critically ill patients. This association was in particular present in patients with elevated toll-like receptor-9 expression.

Soluble neprilysin and survival in critically ill patients.

BACKGROUND: Critically ill patients admitted to an intensive care unit (ICU) exhibit a high mortality rate irrespective of the initial cause of hospitalization. Neprilysin, a neutral endopeptidase degrading an array of vasoactive peptides became a drug target within the treatment of heart failure with reduced ejection fraction. The aim of this study was to analyse whether circulating levels of neprilysin at ICU admission are associated with 30 day mortality. METHODS AND RESULTS: In this single-centre prospective observational study, 222 consecutive patients admitted to a tertiary ICU at a university hospital were included. Blood was drawn at admission and soluble neprilysin levels were measured using ELISA. In the total cohort, soluble neprilysin levels did not differ according to survival status after 30 days as well as type of admission. However, in patients after surgery or heart valve intervention, 30 day survivors exhibited significantly lower circulating neprilysin levels as compared to those who died within 30 days (660.2, IQR: 156.4-2512.5 pg/mL vs. 6532.6, IQR: 1840.1-10 000.0 pg/mL; P = 0.02). Soluble neprilysin predicted mortality independently from age, gender, and commonly used scores of risk-prediction (EuroSCORE II, STS-score, and SAPS II score). Additionally, soluble neprilysin was markedly elevated in patients with sepsis and septic shock (P < 0.05). CONCLUSION: At the time of ICU admission, circulating levels of neprilysin independently predicted 30 day mortality in patients following cardiac surgery or heart valve intervention, but not in critically ill medical patients. Furthermore, patients suffering from sepsis and septic shock displayed significantly increased circulating neprilysin levels.

Interventional Reperfusion Strategies for Acute Pulmonary Embolism.

Acute pulmonary embolism (APE) is a common, potentially life-threatening cardiovascular emergency, and represents the third leading cause of cardiovascular mortality after myocardial infarction and stroke. Risk stratification is important to guide the management of APE, as an early reperfusion strategy is associated with improved clinical outcomes in specific high-risk conditions. Pulmonary artery reperfusion is commonly achieved by systemic intravenous administration of thrombolytic drugs, but catheter-directed thrombolysis (CDThr) and interventional techniques of catheter-based embolectomy provide novel therapeutic approaches with an improved risk-benefit ratio. Future trials will help to determine when to use these different devices in massive or sub-massive APE, and which patient population is likely to benefit from interventional treatment.

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.

Sex-dependent effects of tranexamic acid on blood-brain barrier permeability and the immune response following traumatic brain injury in mice.

BACKGROUND: Tranexamic acid (TXA) is an anti-fibrinolytic agent used to reduce bleeding in various conditions including traumatic brain injury (TBI). As the fibrinolytic system also influences the central nervous system and the immune response, TXA may also modulate these parameters following TBI. OBJECTIVES: To determine the effect of TXA on blood-brain barrier (BBB) integrity and changes in immune and motor function in male and female mice subjected to TBI. METHODS: Wild-type and plasminogen deficient (plg-/-) mice were subjected to TBI then administered either TXA/vehicle. The degree of BBB breakdown, intracerebral hemorrhage (ICH), motor dysfunction, and changes in inflammatory subsets in blood and brain were determined. RESULTS AND CONCLUSIONS: Tranexamic acid significantly reduced BBB breakdown, and increased blood neutrophils in male mice 3 hours post-TBI. In contrast, TXA treatment of female mice increased BBB permeability and ICH but had no effect on blood neutrophils at the same time-point. TXA improved motor function in male mice but still increased BBB breakdown in female mice 24 hours post-TBI. Brain urokinase-type plasminogen activator (u-PA) antigen and activity levels were significantly higher in injured females compared to males. Because TXA can promote a pro-fibrinolytic effect via u-PA, these sex differences may be related to brain u-PA levels. TXA also increased monocyte subsets and dendritic cells in the injured brain of wild-type male mice 1 week post-TBI. Plg-/- mice of both sexes had reduced BBB damage and were protected from TBI irrespective of treatment indicating that TXA modulation of the BBB is plasmin-dependent. In conclusion, TXA is protective post-TBI but only in male mice.

Toll-like receptor 2 and 9 expression on circulating neutrophils is associated with increased mortality in critically ill patients.

BACKGROUND: Toll-like receptors (TLRs) play an important role in inflammatory processes in critically ill patients by binding to pathogen-associated molecular patterns and danger-associated molecular patterns (DAMPs). Whether neutrophil or monocyte TLR expression patterns are associated with outcome in critical illness is unknown. OBJECTIVES: To answer this question, we conducted a prospective, observational study including 215 consecutive patients admitted to a medical ICU at a tertiary care center. METHODS: Blood was drawn at admission and expression of TLR-2, TLR-4, and TLR-9 on neutrophils and monocytes were analyzed by flow cytometry. RESULTS: Median Acute Physiology and Chronic Health Evaluation II (APACHE II) score was 19, and 30-day mortality was 26%. TLR-2 expression on neutrophils was associated with APACHE II, Simplified Acute Physiology Score II, and Sepsis-related Organ Failure Assessment score. TLR-2 (P < 0.001) and TLR-9 (P < 0.05) expression on neutrophils was significantly higher in nonsurvivors. In contrast, neutrophil TLR-4 expression and monocyte TLR expression were not associated with survival. Neutrophil TLR-2 (odds ratio 3.8; 95% confidence interval 1.4-10.2; P < 0.05) and TLR-9 (odds ratio 4.0; 95% confidence interval 2.0-8.1; P < 0.001) expression in the third tertile predicted mortality independent from APACHE II, serum lactate, serum creatinine, and procalcitonin, respectively. CONCLUSION: We provide evidence for prognostic properties of neutrophil TLR-2 and TLR-9 expression regarding 30-day mortality in unselected critically ill patients, independent from baseline clinical characteristics, and laboratory values. These findings suggest that specific TLR-dependent activation of the innate immune system via neutrophils possibly caused by cell damage and release of otherwise intracellular components may play a significant role in the pathophysiology of critical illness.

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.

Flow Cytometric Evaluation of Traditional and Novel Surface Markers for the Diagnosis of Plasma Cell Dyscrasias.

Increasing interest has been expressed for flow cytometric immunophenotyping for diagnosis and monitoring in plasma cell dyscrasias over the last decades. The aim of this investigation was to compare the expression strength of various cell surface markers used traditionally or currently under investigation on normal and abnormal PC populations. We enrolled 295 consecutive patients undergoing bone marrow aspiration in the workup of monoclonal gammopathies, selecting 54 normal and 241 abnormal PC populations via flow cytometry to characterize the expression of CD45, CD38, CD138, CD19, CD56, CD20, CD27, CD28, CD81, CD117 and CD200 on the cell surface of PCs. We observed significant differences in the expression strength of all assessed markers between normal and abnormal PC populations in all markers except for CD20. While none of them was conclusive on its own, the combination of CD81 positivity and CD117 negativity was present in 98.1% of normal PC populations tested. In contrast, particularly CD117 positivity, but also CD81 negativity was indicative of an abnormal PC phenotype. Our results highlight the descriptive value of CD81 and CD117 for the allocation of bone marrow PCs to a normal or abnormal phenotype.

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.

Activated CD8+ T Cells Cause Long-Term Neurological Impairment after Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) leaves many survivors with long-term disabilities. A prolonged immune response in the brain may cause neurodegeneration, resulting in chronic neurological disturbances. In this study, using a TBI mouse model, we correlate changes in the local immune response with neurodegeneration/neurological dysfunction over an 8-month period. Flow cytometric analysis reveals a protracted increase in effector/memory CD8+ T cells (expressing granzyme B) in the injured brain. This precedes interleukin-17+CD4+ T cell infiltration and is associated with progressive neurological/motor impairment, increased circulating brain-specific autoantibodies, and myelin-related pathology. Genetic deficiency or pharmacological depletion of CD8+ T cells, but not depletion of CD4+ T cells, improves neurological outcomes and produces a neuroprotective Th2/Th17 immunological shift, indicating a persistent detrimental role for cytotoxic T cells post-TBI. B cell deficiency results in severe neurological dysfunction and a heightened immune reaction. Targeting these adaptive immune cells offers a promising approach to improve recovery following TBI.

Tranexamic Acid Influences the Immune Response, but not Bacterial Clearance in a Model of Post-Traumatic Brain Injury Pneumonia.

The antifibrinolytic agent, tranexamic acid (TXA), an inhibitor of plasmin formation, currently is evaluated to reduce bleeding in various conditions, including traumatic brain injury (TBI). Because plasmin is implicated in inflammation and immunity, we investigated the effects of plasmin inhibition on the immune response after TBI in the presence or absence of induced pneumonia. Wild-type mice treated with vehicle or TXA or mice deficient in plasminogen (plg-/-) underwent TBI using the controlled cortical impact model. Mice were then subjected to Staphylococcus aureus induced pneumonia and the degree of immune competence determined. Significant baseline changes in the innate immune cell profile were seen in plg-/- mice with increases in spleen weight and white blood cell counts, and elevation in plasma interleukin-6 levels. The plg-/- mice subjected to TBI displayed no additional changes in these parameters at the 72 h or one week time point post-TBI. The plg-/- mice subjected to TBI did not exhibit any further increase in susceptibility to endogenous infection. Pneumonia was induced by intratracheal instillation of S. aureus. The TBI did not worsen pneumonia symptoms or delay recovery in plg-/- mice. Similarly, in wild type mice, treatment with TXA did not impact on the ability of mice to counteract pneumonia after TBI. Administration of TXA after TBI and subsequent pneumonia, however, altered the number and surface marker expression of several myeloid and lymphoid cell populations, consistent with enhanced immune activation at the 72 h time point. This investigation confirms the immune-modulatory properties of TXA, thereby highlighting its effects unrelated to inhibition of fibrinolysis.

Tranexamic acid modulates the cellular immune profile after traumatic brain injury in mice without hyperfibrinolysis.

BACKGROUND: Traumatic brain injury (TBI) is known to promote immunosuppression, making patients more susceptible to infection, yet potentially exerting protective effects by inhibiting central nervous system (CNS) reactivity. Plasmin, the effector protease of the fibrinolytic system, is now recognized for its involvement in modulating immune function. OBJECTIVE: To evaluate the effects of plasmin and tranexamic acid (TXA) on the immune response in wild-type and plasminogen-deficient (plg-/- ) mice subjected to TBI. METHODS: Leukocyte subsets in lymph nodes and the brain in mice post TBI were evaluated by flow cytometry and in blood with a hemocytometer. Immune responsiveness to CNS antigens was determined by Enzyme-linked Immunosorbent Spot (ELISpot) assay.  Fibrinolysis was determined by thromboelastography and measuring D-dimer and plasmin-antiplasmin complex levels. RESULTS: Plg-/-  mice, but not plg+/+  mice displayed increases in both the number and activation of various antigen-presenting cells and T cells in the cLN 1 week post TBI. Wild-type mice treated with TXA also displayed increased cellularity of the cLN 1 week post TBI together with increases in innate and adaptive immune cells. These changes occurred despite the absence of systemic hyperfibrinolysis or coagulopathy in this model of TBI. Importantly, neither plg deficiency nor TXA treatment enhanced the autoreactivity within the CNS. CONCLUSION: In the absence of systemic hyperfibrinolysis, plasmin deficiency or blockade with TXA increases migration and proliferation of conventional dendritic cells (cDCs) and various antigen-presenting cells and T cells in the draining cervical lymph node (cLN) post TBI. Tranexamic acid might also be clinically beneficial in modulating the inflammatory and immune response after TBI, but without promoting CNS autoreactivity.