The clinical significance of TAT, PIC, TM, and t-PAIC in vascular events of BCR/ABL-negative myeloproliferative neoplasms.

Predicting the likelihood vascular events in patients with BCR/ABL1-negative myeloproliferative neoplasms (MPN) is essential for the treatment of the disease. However, effective assessment methods are lacking. Thrombin-antithrombin complex (TAT), plasmin-α2- plasmininhibitor complex (PIC), thrombomodulin (TM), and tissue plasminogen activator-inhibitor complex (t-PAIC) are the new direct indicators for coagulation and fibrinolysis. The aim of this study was to investigate the changes of these four new indicators in thrombotic and hemorrhagic events in BCR/ABL1-negative MPN. The study cohort of 74 patients with BCR/ABL negative myeloproliferative disorders included essential thrombocythemia, polycythemia vera, and primary myelofibrosis (PMF). A panel of 4 biomarkers, including TAT, PIC, TM, and t-PAIC were determined using Sysmex HISCL5000 automated analyzers, whereas fibrin/fibrinogen degradation products (FDP), D-dimer and Antithrombin III (ATIII) were analyzed using Sysmex CS5100 coagulation analyzer. A total of 24 (32.4%) patients experienced thrombotic events and hemorrhagic events occurred in 8 patients (10.8%). Compared to patients without hemorrhagic-thrombotic events, patients with thrombotic events had higher fibrinogen (FIB) level, FDP level and lower ATIII activity, while patients with hemorrhagic events had lower white blood cell count and hemoglobin level, higher FDP level (P < 0.05). Patients with a JAK2V617F mutation were more likely to experience thrombotic events (P < 0.05). In addtion, patients with thrombotic events had higher TAT, PIC, TM, and t-PAIC levels than patients without hemorrhagic-thrombotic events (P < 0.05), whereas patients with hemorrhagic events had a lower median value in TAT and TM (no statistical difference, P > 0.05). Patients with higher TAT, TM and t-PAIC were more likely to experience thrombotic events (P < 0.05), and only TAT was positively correlated with thrombotic events (Spearman r =0.287, P = 0.019). TAT, PIC, TM, and t-PAIC combined with ATIII and FDP have a certain value for predicting thrombosis in patients with BCR/ABL1-negative MPN. These 6 parameters are worth further exploration as predictive factors and prognostic markers for early thrombotic events.

Development of a chemiluminescence assay for tissue plasminogen activator inhibitor complex and its applicability to gastric cancer.

BACKGROUND: Venous thromboembolism (VTE), is a noteworthy complication in individuals with gastric cancer, but the current diagnosis and treatment methods lack accuracy. In this study, we developed a t-PAIC chemiluminescence kit and employed chemiluminescence to detect the tissue plasminogen activator inhibitor complex (t-PAIC), thrombin-antithrombin III complex (TAT), plasmin-α2-plasmin inhibitor complex (PIC) and thrombomodulin (TM), combined with D-dimer and fibrin degradation products (FDP), to investigate their diagnostic potential for venous thrombosis in gastric cancer patients. The study assessed variations in six indicators among gastric cancer patients at different stages. RESULTS: The t-PAIC reagent showed LOD is 1.2 ng/mL and a linear factor R greater than 0.99. The reagents demonstrated accurate results, with all accuracy deviations being within 5%. The intra-batch and inter-batch CVs for the t-PAIC reagent were both within 8%. The correlation coefficient R between this method and Sysmex was 0.979. Gastric cancer patients exhibited elevated levels of TAT, PIC, TM, D-D, FDP compared to the healthy population, while no significant difference was observed in t-PAIC. In the staging of gastric cancer, patients in III-IV stages exhibit higher levels of the six markers compared to those in I-II stages. The ROC curve indicates an enhancement in sensitivity and specificity of the combined diagnosis of four or six indicators. CONCLUSION: Our chemiluminescence assay performs comparably to Sysmex's method and at a reduced cost. The use of multiple markers, including t-PAIC, TM, TAT, PIC, D-D, and FDP, is superior to the use of single markers for diagnosing VTE in patients with malignant tumors. Gastric cancer patients should be screened for the six markers to facilitate proactive prophylaxis, determine the most appropriate treatment timing, ameliorate their prognosis, decrease the occurrence of venous thrombosis and mortality, and extend their survival.

α2-Antiplasmin is associated with macrophage activation and fibrin deposition in a macrophage activation syndrome mouse model.

Macrophage activation syndrome (MAS) is a life-threatening condition, characterized by cytopenia, multi-organ dysfunction, and coagulopathy associated with excessive activation of macrophages. In this study, we investigated the roles of alpha2-antiplasmin (α2AP) in the progression of MAS using fulminant MAS mouse model induced by toll-like receptor-9 agonist (CpG) and D-(+)-galactosamine hydrochloride (DG). α2AP deficiency attenuated macrophage accumulation, liver injury, and fibrin deposition in the MAS model mice. Interferon-γ (IFN-γ) is associated with macrophage activation, including migration, and plays a pivotal role in MAS progression. α2AP enhanced the IFN-γ-induced migration, and tissue factor production. Additionally, we showed that fibrin-induced macrophage activation and tumor necrosis factor-α production. Moreover, the blockade of α2AP by neutralizing antibodies attenuated macrophage accumulation, liver injury, and fibrin deposition in the MAS model mice. These data suggest that α2AP may regulate IFN-γ-induced responses and be associated with macrophage activation and fibrin deposition in the MAS progression.

Characterization and Usefulness of Clot-Fibrinolysis Waveform Analysis in Critical Care Patients with Enhanced or Suppressed Fibrinolysis.

INTRODUCTION: Recently, clot-fibrinolysis waveform analysis (CFWA), which is a coagulation and fibrinolysis global assay based on assessing the activated partial thromboplastin time with tissue-type plasminogen activator, was developed. This study aimed to investigate the characteristics of CFWA using plasma samples from patients in the critical care unit. MATERIALS AND METHODS: The fibrinolysis times using CFWA were measured in 298 plasma samples. These samples were divided into three groups based on the reference interval (RI) of fibrinolysis time using CFWA: shortened group, less than RI; within group, within RI; prolonged group, more than RI. The coagulation and fibrinolysis markers, including D-dimer, plasmin-α2 plasmin inhibitor complex (PIC), fibrin monomer complex (FMC), plasmin-α2 plasmin inhibitor (α2-PI), plasminogen (Plg), and fibrinogen (Fbg) were analyzed and compared among the three groups. RESULTS: The FMC level decreased in the order of shortened, within, and prolonged groups, and the decrease was statistically significant among all three group pairs. The opposite tendency was observed for Fbg and fibrinolysis-related markers of α2-PI and Plg, and significant differences were recognized in all pair comparisons except for between within and prolonged groups in Plg. The mean values of the fibrinolysis markers D-dimer and PIC in all three groups were higher than the cut-off values, and the PIC value differed significantly between the within and prolonged groups. CONCLUSION: The fibrinolysis reaction was detected in all three groups, but the status differed. CFWA has the potential to reflect the fibrinolysis status in one global assay.

Increased Levels of Inflammatory and Endothelial Biomarkers in Blood of Long COVID Patients Point to Thrombotic Endothelialitis.

The prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation. Here we demonstrate significantly increased concentrations of von Willebrand factor (VWF), platelet factor 4 (PF4), serum amyloid A (SAA), α-2 antiplasmin (α-2AP), endothelial-leukocyte adhesion molecule 1 (E-selectin), and platelet endothelial cell adhesion molecule (PECAM-1) in the soluble part of the blood. It was noteworthy that the mean level of α-2 antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls. This is alarming if we take into consideration that a significant amount of the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules). We conclude that presence of microclotting, together with relatively high levels of six biomarkers known to be key drivers of endothelial and clotting pathology, points to thrombotic endothelialitis as a key pathological process in Long COVID.

Confusion in the monitoring of coagulation function in pregnant and neonate patients with severe disease: A case reports and brief literature review.

RATIONALE: Different populations have their own unique physiological and pathological characteristics. However, in specialized maternal and child hospitals, there is currently a lack of standardized methods for assessing coagulation dysfunction, both domestically and internationally. PATIENT CONCERNS: A 19-day-old neonate was transferred to neonatal intensive care unit with cyanosis, nasal bleeding for 6 hours, and a consciousness disorder for 5 hours. A 33-year-old woman presented with hydramnios and a 39 + 3week intrauterine pregnancy. All indicators before delivery were normal, but postpartum hemorrhage occurred after delivery. DIAGNOSES: We retrospectively analyzed 1 neonate with pulmonary hemorrhage accompanied by thrombocytopenia and 1 pregnant patient with amniotic fluid embolism. INTERVENTIONS: The new coagulation indicators, such as thrombin-antithrombin complex, plasmin-alpha 2 antiplasmin complex, thrombomodulin, and tissue plasminogen activator-plasminogen activator inhibitor-1 complex, have been indicated to be valuable. In neonates, it is necessary to continuously monitor special items combined with specific therapeutic agents, such as tranexamic acid. In cases where postpartum hemorrhage occurs with low fibrinogen levels, it is essential to effectively identify patients with severe amniotic fluid embolism from a high incidence of specimen clotting. OUTCOMES: The neonate's oxygen saturation stabilized, and after 5 days of treatment with low molecular weight heparin, thrombin-antithrombin complex and plasmin-alpha 2 antiplasmin complex returned to normal levels. The pregnant began to remove the remaining thrombus, the patient's condition recovered, and she had a good prognosis. LESSONS: For pregnant and neonatal critical illnesses, it is necessary to develop personalized coagulation monitoring programs that provide realistic and reasonable treatment recommendations. Such programs should consider the unique physiological and pathological characteristics of different populations to ensure effective management of critically ill patients.

Impaired fibrinolysis and increased clot strength are potential risk factors for thrombosis in lymphoma.

Thrombosis and bleeding are significant contributors to morbidity and mortality in patients with hematological cancer, and the impact of altered fibrinolysis on bleeding and thrombosis risk is poorly understood. In this prospective cohort study, we investigated the dynamics of fibrinolysis in patients with hematological cancer. Fibrinolysis was investigated before treatment and 3 months after treatment initiation. A dynamic clot formation and lysis assay was performed beyond the measurement of plasminogen activator inhibitor 1, tissue- and urokinase-type plasminogen activators (tPA and uPA), plasmin-antiplasmin complexes (PAP), α-2-antiplasmin activity, and plasminogen activity. Clot initiation, clot propagation, and clot strength were assessed using rotational thromboelastometry. A total of 79 patients were enrolled. Patients with lymphoma displayed impaired fibrinolysis with prolonged 50% clot lysis time compared with healthy controls (P = .048). They also displayed decreased clot strength at follow-up compared with at diagnosis (P = .001). A patient with amyloid light-chain amyloidosis having overt bleeding at diagnosis displayed hyperfibrinolysis, indicated by a reduced 50% clot lysis time, α-2-antiplasmin activity, and plasminogen activity, and elevated tPA and uPA. A patient with acute promyelocytic leukemia also displayed marked hyperfibrinolysis with very high PAP, indicating extreme plasmin generation, and clot formation was not measurable, probably because of the extremely fast fibrinolysis. Fibrinolysis returned to normal after treatment in both patients. In conclusion, patients with lymphoma showed signs of impaired fibrinolysis and increased clot strength, whereas hyperfibrinolysis was seen in patients with acute promyelocytic leukemia and light-chain amyloidosis. Thus, investigating fibrinolysis in patients with hematological cancer could have diagnostic value.

PLCL/PCL Dressings with Platelet Lysate and Growth Factors Embedded in Fibrin for Chronic Wound Regeneration.

Introduction: The formation of diabetic ulcers (DU) is a common complication for diabetic patients resulting in serious chronic wounds. There is therefore, an urgent need for complex treatment of this problem. This study examines a bioactive wound dressing of a biodegradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) covered by a thin fibrin layer for sustained delivery of bioactive molecules. Methods: Electrospun PLCL/PCL nanofibers were coated with fibrin-based coating prepared by a controlled technique and enriched with human platelet lysate (hPL), fibroblast growth factor 2 (FGF), and vascular endothelial growth factor (VEGF). The coating was characterized by scanning electron microscopy and fluorescent microscopy. Protein content and its release rate and the effect on human saphenous vein endothelial cells (HSVEC) were evaluated. Results: The highest protein amount is achieved by the coating of PLCL/PCL with a fibrin mesh containing 20% v/v hPL (NF20). The fibrin coating serves as an excellent scaffold to accumulate bioactive molecules from hPL such as PDGF-BB, fibronectin (Fn), and α-2 antiplasmin. The NF20 coating shows both fast and a sustained release of the attached bioactive molecules (Fn, VEGF, FGF). The dressing significantly increases the viability of human saphenous vein endothelial cells (HSVECs) cultivated on a collagen-based wound model. The exogenous addition of FGF and VEGF during the coating procedure further increases the HSVECs viability. In addition, the presence of α-2 antiplasmin significantly stabilizes the fibrin mesh and prevents its cleavage by plasmin. Discussion: The NF20 coating supplemented with FGF and VEGF provides a promising wound dressing for the complex treatment of DU. The incorporation of various bioactive molecules from hPL and growth factors has great potential to support the healing processes by providing appropriate stimuli in the chronic wound.

Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system.

BACKGROUND: Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation. METHODS: Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician. RESULTS: Our long COVID cohort's symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots. CONCLUSION: Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses.

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell-bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

The Role of Fibrinolytic System in Health and Disease.

The fibrinolytic system is composed of the protease plasmin, its precursor plasminogen and their respective activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), counteracted by their inhibitors, plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), protein C inhibitor (PCI), thrombin activable fibrinolysis inhibitor (TAFI), protease nexin 1 (PN-1) and neuroserpin. The action of plasmin is counteracted by α2-antiplasmin, α2-macroglobulin, TAFI, and other serine protease inhibitors (antithrombin and α2-antitrypsin) and PN-1 (protease nexin 1). These components are essential regulators of many physiologic processes. They are also involved in the pathogenesis of many disorders. Recent advancements in our understanding of these processes enable the opportunity of drug development in treating many of these disorders.

Associations between hemostatic markers and mortality in COVID-19 - Compounding effects of D-dimer, antithrombin and PAP complex.

In this single-center cohort study, we applied a panel of laboratory markers to characterize hemostatic function in 217 consecutive patients that underwent testing for COVID-19 as they were admitted to Linköping University Hospital between April and June 2020. In the 96 patients that tested positive for SARS-CoV-2 (COVID-19+), the cumulative incidences of death and venous thromboembolism were 24.0% and 19.8% as compared to 12.4% (p = 0.031) and 11.6% (p = 0.13) in the 121 patients that tested negative (COVID-19-). In COVID-19+ patients, we found pronounced increases in plasma levels of von Willebrand factor (vWF) and fibrinogen. Excess mortality was observed in COVID-19+ patients with the following aberrations in hemostatic markers: high D-dimer, low antithrombin or low plasmin-antiplasmin complex (PAP) formation, with Odds Ratios (OR) for death of 4.7 (95% confidence interval (CI95) 1.7-12.9; p = 0.003) for D-dimer >0.5 mg/L, 5.9 (CI95 1.8-19.7; p = 0.004) for antithrombin (AT) ˂0.85 kIU/l and 4.9 (CI95 1.3-18.3; p = 0.019) for PAP < 1000 µg/L. Compounding increases in mortality was observed in COVID-19+ patients with combined defects in markers of fibrinolysis and coagulation, with ORs for death of 15.7 (CI95 4.3-57; p < 0.001) for patients with PAP <1000 µg/L and D-dimer >0.5 mg/L and 15.5 (CI95 2.8-87, p = 0.002) for patients with PAP <1000 µg/L and AT ˂0.85 kIU/L. We observed an elevated fraction of incompletely degraded D-dimer fragments in COVID-19+ patients with low PAP, indicating impaired fibrinolytic breakdown of cross-linked fibrin.

Assessment of fibrinolytic markers in patients with deep vein thrombosis.

The association of the fibrinolytic markers with deep vein thrombosis (DVT) is still a matter of debate. The present study aimed to investigate the association between fibrinolytic markers and DVT. This observational study recruited 52 adult cases with lower limb DVT and 52 healthy adult volunteers as controls. The quantitative determination of plasminogen activator inhibitor-1 (PAI-1), plasminogen, thrombin activable fibrinolysis inhibitor (TAFI), tissue plasminogen activator (tPA) and α2-antiplasmin (α2-AP) was performed by ELISA. TAFI, plasminogen and t-PA were significantly higher in cases than controls and PAI-1 was significantly lower in cases than controls. In addition, TAFI, plasminogen and t-PA levels were significantly increased in unprovoked and idiopathic DVT cases than controls. The present study suggests that the fibrinolytic markers tested in patients with a remote history of DVT are different than in individuals with no history of DVT and, with further study, may prove useful as screening assays for DVT risk.

Therapeutic Strategies for Disseminated Intravascular Coagulation Associated with Aortic Aneurysm.

Aortic aneurysms are sometimes associated with enhanced-fibrinolytic-type disseminated intravascular coagulation (DIC). In enhanced-fibrinolytic-type DIC, both coagulation and fibrinolysis are markedly activated. Typical cases show decreased platelet counts and fibrinogen levels, increased concentrations of fibrin/fibrinogen degradation products (FDP) and D-dimer, and increased FDP/D-dimer ratios. Thrombin-antithrombin complex or prothrombin fragment 1 + 2, as markers of coagulation activation, and plasmin-α2 plasmin inhibitor complex, a marker of fibrinolytic activation, are all markedly increased. Prolongation of prothrombin time (PT) is not so obvious, and the activated partial thromboplastin time (APTT) is rather shortened in some cases. As a result, DIC can be neither diagnosed nor excluded based on PT and APTT alone. Many of the factors involved in coagulation and fibrinolysis activation are serine proteases. Treatment of enhanced-fibrinolytic-type DIC requires consideration of how to control the function of these serine proteases. The cornerstone of DIC treatment is treatment of the underlying pathology. However, in some cases surgery is either not possible or exacerbates the DIC associated with aortic aneurysm. In such cases, pharmacotherapy becomes even more important. Unfractionated heparin, other heparins, synthetic protease inhibitors, recombinant thrombomodulin, and direct oral anticoagulants (DOACs) are agents that inhibit serine proteases, and all are effective against DIC. Inhibition of activated coagulation factors by anticoagulants is key to the treatment of DIC. Among them, DOACs can be taken orally and is useful for outpatient treatment. Combination therapy of heparin and nafamostat allows fine-adjustment of anticoagulant and antifibrinolytic effects. While warfarin is an anticoagulant, this agent is ineffective in the treatment of DIC because it inhibits the production of coagulation factors as substrates without inhibiting activated coagulation factors. In addition, monotherapy using tranexamic acid in cases of enhanced-fibrinolytic-type DIC may induce fatal thrombosis. If tranexamic acid is needed for DIC, combination with anticoagulant therapy is of critical importance.

Distinctive Biomarker Features in the Endotheliopathy of COVID-19 and Septic Syndromes.

BACKGROUND: Endotheliopathy is a key element in COVID-19 pathophysiology, contributing to both morbidity and mortality. Biomarkers distinguishing different COVID-19 phenotypes from sepsis syndrome remain poorly understood. OBJECTIVE: To characterize circulating biomarkers of endothelial damage in different COVID-19 clinical disease stages compared with sepsis syndrome and normal volunteers. METHODS: Patients with COVID-19 pneumonia (n = 49) were classified into moderate, severe, or critical (life-threatening) disease. Plasma samples were collected within 48 to 72 h of hospitalization to analyze endothelial activation markers, including soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), von Willebrand Factor (VWF), A disintegrin-like and metalloprotease with thrombospondin type 1 motif no. 13 (ADAMTS-13) activity, thrombomodulin (TM), and soluble TNF receptor I (sTNFRI); heparan sulfate (HS) for endothelial glycocalyx degradation; C5b9 deposits on endothelial cells in culture and soluble C5b9 for complement activation; circulating dsDNA for neutrophil extracellular traps (NETs) presence, and α2-antiplasmin and PAI-1 as parameters of fibrinolysis. We compared the level of each biomarker in all three COVID-19 groups and healthy donors as controls (n = 45). Results in critically ill COVID-19 patients were compared with other intensive care unit (ICU) patients with septic shock (SS, n = 14), sepsis (S, n = 7), and noninfectious systemic inflammatory response syndrome (NI-SIRS, n = 7). RESULTS: All analyzed biomarkers were increased in COVID-19 patients versus controls (P < 0.001), except for ADAMTS-13 activity that was normal in both groups. The increased expression of sVCAM-1, VWF, sTNFRI, and HS was related to COVID-19 disease severity (P < 0.05). Several differences in these parameters were found between ICU groups: SS patients showed significantly higher levels of VWF, TM, sTNFRI, and NETS compared with critical COVID-19 patients and ADAMTS-13 activity was significantly lover in SS, S, and NI-SIRS versus critical COVID-19 (P < 0.001). Furthermore, α2-antiplasmin activity was higher in critical COVID-19 versus NI-SIRS (P < 0.01) and SS (P < 0.001), whereas PAI-1 levels were significantly lower in COVID-19 patients compared with NI-SIRS, S, and SS patients (P < 0.01). CONCLUSIONS: COVID-19 patients present with increased circulating endothelial stress products, complement activation, and fibrinolytic dysregulation, associated with disease severity. COVID-19 endotheliopathy differs from SS, in which endothelial damage is also a critical feature of pathobiology. These biomarkers could help to stratify the severity of COVID-19 disease and may also provide information to guide specific therapeutic strategies to mitigate endotheliopathy progression.

α2-antiplasmin positively regulates endothelial-to-mesenchymal transition and fibrosis progression in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN), is microvascular complication of diabetes causes to kidney dysfunction and renal fibrosis. It is known that hyperglycemia and advanced glycation end products (AGEs) produced by hyperglycemic condition induce myofibroblast differentiation and endothelial-to-mesenchymal transition (EndoMT), and exacerbate fibrosis in DN. Recently, we demonstrated that α2-antiplasmin (α2AP) is associated with inflammatory response and fibrosis progression. METHODS: We investigated the role of α2AP on fibrosis progression in DN using a streptozotocin-induced DN mouse model. RESULTS: α2AP deficiency attenuated EndoMT and fibrosis progression in DN model mice. We also showed that the high glucose condition/AGEs induced α2AP production in fibroblasts (FBs), and the reduction of receptor for AGEs (RAGE) by siRNA attenuated the AGEs-induced α2AP production in FBs. Furthermore, the bloackade of α2AP by the neutralizing antibody attenuated the high glucose condition-induced pro-fibrotic changes in FBs. On the other hand, the hyperglycemic condition/AGEs induced EndoMT in vascular endothelial cells (ECs), the FBs/ECs co-culture promoted the high glucose condition-induced EndoMT compared to ECs mono-culture. Furthermore, α2AP promoted the AGEs-induced EndoMT, and the blockade of α2AP attenuated the FBs/ECs co-culture-promoted EndoMT under the high glucose condition. CONCLUSIONS: The high glucose conditions induced α2AP production, and α2AP is associated with EndoMT and fibrosis progression in DN. These findings provide a basis for clinical strategies to improve DN.

Alpha2-antiplasmin deficiency affects depression and anxiety-like behavior and apoptosis induced by stress in mice.

OBJECTIVES: Depression is a psychiatric disorder that affects about 10% of the world's population and is accompanied by anxiety. Depression and anxiety are often caused by various stresses. However, the etiology of depression and anxiety remains unknown. It has been reported that alpha2-antiplasmin (α2AP) not only inhibits plasmin but also has various functions such as cytokine production and cell growth. This study aimed to determine the roles of α2AP on the stress-induced depression and anxiety. METHODS: We investigated the mild repeated restraint stress-induced depressive and anxiety-like behavior in the α2AP+/+ and α2AP-/- mice using the social interaction test (SIT), sucrose preference test (SPT), and elevated plus maze (EPM). RESULTS: The stresses such as the mild repeated restraint stress suppressed α2AP expression in the hippocampus of mice, and the treatment of fluoxetine (selective serotonin reuptake inhibitor [SSRI]) recovered the stress-caused α2AP suppression. We also showed that α2AP deficiency promoted the mild restraint stress-stimulated depression-like behavior such as social withdrawal and apathy and apoptosis in mice. In contrast, α2AP deficiency attenuated the mild restraint stress induced the anxiety-like behavior in mice. CONCLUSIONS: α2AP affects the pathogenesis of depression and anxiety induced by stress.

Fibrinogen and Antifibrinolytic Proteins: Interactions and Future Therapeutics.

Thrombus formation remains a major cause of morbidity and mortality worldwide. Current antiplatelet and anticoagulant therapies have been effective at reducing vascular events, but at the expense of increased bleeding risk. Targeting proteins that interact with fibrinogen and which are involved in hypofibrinolysis represents a more specific approach for the development of effective and safe therapeutic agents. The antifibrinolytic proteins alpha-2 antiplasmin (α2AP), thrombin activatable fibrinolysis inhibitor (TAFI), complement C3 and plasminogen activator inhibitor-2 (PAI-2), can be incorporated into the fibrin clot by FXIIIa and affect fibrinolysis by different mechanisms. Therefore, these antifibrinolytic proteins are attractive targets for the development of novel therapeutics, both for the modulation of thrombosis risk, but also for potentially improving clot instability in bleeding disorders. This review summarises the main properties of fibrinogen-bound antifibrinolytic proteins, their effect on clot lysis and association with thrombotic or bleeding conditions. The role of these proteins in therapeutic strategies targeting the fibrinolytic system for thrombotic diseases or bleeding disorders is also discussed.

Pre-Operative Plasma Extracellular Vesicle Proteins are Associated with a High Risk of Long Term Secondary Major Cardiovascular Events in Patients Undergoing Carotid Endarterectomy.

OBJECTIVE: Patients undergoing carotid endarterectomy (CEA) maintain a substantial residual risk of major cardiovascular events (MACE). Improved risk stratification is warranted to select high risk patients qualifying for secondary add on therapy. Plasma extracellular vesicles (EVs) are involved in atherothrombotic processes and their content has been related to the presence and recurrence of cardiovascular events. The association between pre-operative levels of five cardiovascular disease related proteins in plasma EVs and the post-operative risk of MACE was assessed. METHODS: In 864 patients undergoing CEA from 2002 to 2016 included in the Athero-Express biobank, three plasma EV subfractions (low density lipoprotein [LDL], high density lipoprotein [HDL], and tiny extracellular vesicles [TEX]) were isolated from pre-operative blood samples. Using an electrochemiluminescence immunoassay, five proteins were quantified in each EV subfraction: cystatin C, serpin C1, serpin G1, serpin F2, and CD14. The association between EV protein levels and the three year post-operative risk of MACE (any stroke, myocardial infarction, or cardiovascular death) was evaluated using multivariable Cox proportional hazard regression analyses. RESULTS: During a median follow up of three years (interquartile range 2.2 - 3.0), 137 (16%) patients developed MACE. In the HDL-EV subfraction, increased levels of CD14, cystatin C, serpin F2, and serpin C1 were associated with an increased risk of MACE (adjusted hazard ratios per one standard deviation increase of 1.30, 95% confidence interval [CI] 1.15-1.48; 1.22, 95% CI 1.06-1.42; 1.36, 95% CI 1.16-1.61; and 1.29, 95% CI 1.10-1.51; respectively), independently of cardiovascular risk factors. No significant associations were found for serpin G1. CD14 improved the predictive value of the clinical model encompassing cardiovascular risk factors (net re-classification index = 0.16, 95% CI 0.08-0.21). CONCLUSION: EV derived pre-operative plasma levels of cystatin C, serpin C1, CD14, and serpin F2 were independently associated with an increased long term risk of MACE after CEA and are thus markers for residual cardiovascular risk. EV derived CD14 levels could improve the identification of high risk patients who may benefit from secondary preventive add on therapy in order to reduce future risk of MACE.