Thrombin-activatable Fibrinolysis Inhibitor Research Articles

SARS-CoV-2 infection results in upregulation of Plasminogen Activator Inhibitor-1 and Neuroserpin in the lungs, and an increase in fibrinolysis inhibitors associated with disease severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in coagulation activation although it is usually not associated with consumption coagulopathy. D-dimers are also commonly elevated despite systemic hypofibrinolysis. To understand these unusual features of coronavirus disease 2019 (COVID-19) coagulopathy, 64 adult patients with SARS-CoV-2 infection (36 moderate and 28 severe) and 16 controls were studied. We evaluated the repertoire of plasma protease inhibitors (Serpins, Kunitz, Kazal, Cystatin-like) targeting the fibrinolytic system: Plasminogen Activator Inhibitor-1 (PAI-1), Tissue Plasminogen Activator/Plasminogen Activator Inhibitor-1 complex (t-PA/PAI-1), α-2-Antiplasmin, Plasmin-α2-Antiplasmin Complex, Thrombin-activatable Fibrinolysis Inhibitor (TAFI)/TAFIa, Protease Nexin-1 (PN-1), and Neuroserpin (the main t-PA inhibitor of the central nervous system). Inhibitors of the common (Antithrombin, Thrombin-antithrombin complex, Protein Z [PZ]/PZ inhibitor, Heparin Cofactor II, and α2-Macroglobulin), Protein C ([PC], Protein C inhibitor, and Protein S), contact (Kallistatin, Protease Nexin-2/Amyloid Beta Precursor Protein, and α-1-Antitrypsin), and complement (C1-Inhibitor) pathways, in addition to Factor XIII, Histidine-rich glycoprotein (HRG) and Vaspin were also investigated by enzyme-linked immunosorbent assay. The association of these markers with disease severity was evaluated by logistic regression. Pulmonary expression of PAI-1 and Neuroserpin in the lungs from eight post-mortem cases was assessed by immunohistochemistry. Results show that six patients (10%) developed thrombotic events, and mortality was 11%. There was no significant reduction in plasma anticoagulants, in keeping with a compensated state. However, an increase in fibrinolysis inhibitors (PAI-1, Neuroserpin, PN-1, PAP, and t-PA/PAI-1) was consistently observed, while HRG was reduced. Furthermore, these markers were associated with moderate and/or severe disease. Notably, immunostains demonstrated overexpression of PAI-1 in epithelial cells, macrophages, and endothelial cells of fatal COVID-19, while Neuroserpin was found in intraalveolar macrophages only. These results imply that the lungs in SARS-CoV-2 infection provide anti-fibrinolytic activity resulting in a shift toward a local and systemic hypofibrinolytic state predisposing to (immuno)thrombosis, often in a background of compensated disseminated intravascular coagulation.

Increased thrombin activatable fibrinolysis inhibitor activity is associated with hypofibrinolysis in dogs with sepsis.

Disorders of coagulation are well-recognized in dogs with sepsis, but data regarding fibrinolysis disorders are limited. We aimed to characterize fibrinolysis in dogs with sepsis compared to healthy controls. We hypothesized that dogs with sepsis would be hypofibrinolytic, and that hypofibrinolysis would be associated with non-survival. This was a prospective observational cohort study. We enrolled 20 client-owned dogs with sepsis admitted to the Cornell University Hospital for Animals and 20 healthy pet dogs. Coagulation and fibrinolytic pathway proteins including antiplasmin activity (AP), antithrombin activity (AT), thrombin activatable fibrinolysis inhibitor activity (TAFI), D-dimer concentration, fibrinogen concentration, and plasminogen activity were measured and compared between groups. Overall coagulation potential, overall fibrinolysis potential, and overall hemostatic potential were calculated from the curve of fibrin clot formation and lysis over time. Compared to healthy controls, dogs with sepsis had lower AT (P = 0.009), higher AP (P = 0.002), higher TAFI (P = 0.0385), and higher concentrations of fibrinogen (P < 0.0001) and D-dimer (P = 0.0001). Dogs with sepsis also had greater overall coagulation potential (P = 0.003), overall hemostatic potential (P = 0.0015), and lower overall fibrinolysis potential (P = 0.0004). The extent of fibrinolysis was significantly negatively correlated with TAFI. No significant differences were observed between survivors and non-survivors. Dogs with sepsis were hypercoagulable and hypofibrinolytic compared to healthy dogs, suggesting potential utility of thromboprophylaxis in this patient population. The association between high TAFI and low overall fibrinolysis potential might provide a potential mechanism for this hypofibrinolysis.

Investigation of cofactor activities of endothelial microparticle-thrombomodulin with liposomal surrogate

Thrombomodulin (TM) is a type I transmembrane glycoprotein mainly expressed on the endothelial cells, where it binds thrombin to form the thrombin-TM complex that can activate protein C and thrombin-activable fibrinolysis inhibitor (TAFI) and induce anticoagulant and anti-fibrinolytic reactions, respectively. Cell activation and injury often sheds microparticles that contain membrane TM, which circulate in biofluids like blood. However, the biological function of circulating microparticle-TM is still unknown even though it has been recognized as a biomarker of endothelial cell injury and damage. In comparison with cell membrane, different phospholipids are exposed on the microparticle surface due to cell membrane ‘‘flip-flop’’ upon cell activation and injury. Liposomes can be used as a microparticle mimetics. In this report, we prepared TM-containing liposomes with different phospholipids as surrogates of endothelial microparticle-TM and investigated their cofactor activities. We found that liposomal TM with phosphatidylethanolamine (PtEtn) showed increased protein C activation but decreased TAFI activation in comparison to liposomal TM with phosphatidylcholine (PtCho). In addition, we investigated whether protein C and TAFI compete for the thrombin/TM complex on the liposomes. We found that protein C and TAFI did not compete for the thrombin/TM complex on the liposomes with PtCho alone and with low concentration (5%) of PtEtn and phosphatidylserine (PtSer), but competed each other on the liposomes with higher concentration (10%) of PtEtn and PtSer. These results indicate that membrane lipids affect protein C and TAFI activation and microparticle-TM may have different cofactor activities in comparison to cell membrane TM.

Fibrinolytic Status and Risk of Death After Acute Pulmonary Embolism.

Acute pulmonary embolism (PE) is a heterogeneous disease process with variable presentation and outcomes. The endogenous fibrinolytic system is a complex framework of regulatory pathways that maintains homeostasis by dissolving overabundant thrombi. We sought to investigate phenotypic profiles of the endogenous fibrinolytic system among patients presenting with acute PE and their impact on mortality. We enrolled all consecutive patients with acute PE in our institutional Pulmonary Embolism Response Team registry. We collected blood samples at the time of PE diagnosis and analyzed concentrations of plasminogen activator inhibitor 1 (PAI-1), thrombin-activatable fibrinolysis inhibitor (TAFI), and alpha-2-antiplasmin (A2A). We assessed the association of concentration of fibrinolytic inhibitors and 1-year all-cause mortality and various echocardiographic markers of right ventricular (RV) dysfunction. There is significant variability of PAI-1, A2A, and TAFI concentrations across the spectrum of PE risk profiles with high PAI-1, low TAFI, and low A2A (herein referred to as a high-risk biomarker profile) correlating with worse PE severity. High-risk biomarker profile correlated with high-risk echocardiographic features of RV dysfunction, including increased RV/left ventricular (LV) ratio, low tricuspid annular plane systolic excursion, and low right ventricular outflow tract velocity time integral. Higher-risk biomarker profile was able to discriminate and independently identify patients at high risk of all-cause mortality (Group 2 HR 6 95% CI 1.3-27.8, Group 3 HR 12, 95% CI 1.7-86). Further studies are needed to assess the exact pathophysiological link between fibrinolytic status and poor outcome after acute PE and to ascertain the impact of anti-inhibitors of the fibrinolytic system on response to therapy and outcomes after acute PE.

Glycemic Control and Plasma Levels of Pro-Inflammatory and Pro-Thrombotic Biomarkers in Diabetic Patients Presenting with Acute Pulmonary Embolism.

Type I and type II diabetes are closely associated with a pro-inflammatory state and to a pro-thrombotic state. The role of glycemic control in pulmonary embolism (PE) is poorly understood and requires additional investigation. The aim of this study is to investigate the relationship between glycemic control and thrombo-inflammatory biomarkers in a PE patient cohort compared to normal samples. Demographic and clinical information for 86 diabetic patients and 106 non-diabetic patients presenting with acute PE was collected via retrospective chart review. Plasma levels of pro-inflammatory (C-reactive protein [CRP], tumor necrosis factor-alpha [TNF-α], interleukin-6 [IL-6]) and pro-thrombotic (d-dimer, plasminogen activator inhibitor-1 [PAI-1], tissue plasminogen activator [tPA], thrombin activatable fibrinolysis inhibitor [TAFI], von-Willebrand factor [vWF], endogenous glycosaminoglycans [GAGs]) biomarkers were drawn within 24 hours of diagnosis of acute PE. Data was also obtained for a population of healthy adult controls. All the pro-inflammatory and pro-thrombotic biomarkers were elevated in diabetic PE patients in comparison to healthy controls. None of the biomarkers were elevated in diabetic PE patients when compared to non-diabetic PE patients. There was no difference in the levels of the pro-inflammatory biomarkers according to glycemic control. The plasma level of TAFI was elevated in diabetic patients with poor glycemic control. Diabetic patients were more likely to have a more severe PE. These studies demonstrate that thrombo-inflammatory biomarkers are elevated in diabetic PE patients with associated comorbidities in comparison to normal individuals. However, there is no difference between the PE cohort alone in comparison to PE with diabetes. The role of TAFI within the continuum of diabetic vascular disease warrants additional investigation.

The Relevance of Thrombo-Inflammatory Biomarkers and Their Relationship with Circulating Glycosaminoglycans in End-Stage Renal Disease Patients

En-stage renal disease (ESRD) is a growing public health problem. The atherosclerotic cardiovascular complications are the leading causes of mortality and morbidity in the ESRD. In this study, we sought to quantify the levels of thrombo-inflammatory biomarkers in an ESRD patients in comparison to healthy controls to determine their relevance in thrombo-inflammation and adverse outcomes. The levels of D-Dimer, C-reactive protein (CRP), plasminogen activator inhibitor 1 (PAI-1) antigen, functional PAI-1, thrombin activatable fibrinolysis inhibitor, tissue plasminogen activator, von Willebrand factor, and anti-PF4 IgG and microparticle (MP) activity were quantified by using commercially available ELISA immunoassays for each of the ESRD (n = 73) and control plasma samples (n = 10). The levels of endogenous glycosaminoglycans (GAGs) were quantified by utilizing a Heparin Red Probe (Redprobes UG, Germany). The collected data were analyzed to demonstrate the relationship between various parameters. All the tested biomarkers were increased in ESRD patients in comparison to healthy controls (p < 0.05). These biomarkers have shown significant correlations within each other except for anti-PF4 Ig G and MPs. The CRP levels were significantly higher in patients who had coronary artery disease (CAD) (p < 0.05), but there was no significant difference in other biomarkers according to the cardiovascular outcomes. In the multivariate analysis, the CRP (odds ratio: 1.19; 95% confidence interval: 1.01-1.41; p: 0.03) value was an independent predictor of CAD. In this study, we demonstrated increased levels of 10 different biomarkers in ESRD patients. The CRP levels can be a good predictor of CAD in ESRD patients.

Structure/characteristics of thrombin activatable fibrinolysis inhibitor (TAFI) and its assay method

Structure/characteristics of thrombin activatable fibrinolysis inhibitor (TAFI) and its assay method

The influence of lower-leg injury and knee arthroscopy on natural anticoagulants and fibrinolysis

BackgroundPatients with lower-leg injuries and those undergoing knee arthroscopy are at increased risk of developing venous thromboembolism. The mechanism is unknown, including the influence of lower-leg injury and knee arthroscopy on natural anticoagulant factors and fibrinolysis. ObjectivesTo study the effect of lower-leg injury and knee arthroscopy on plasma levels of anticoagulant and fibrinolytic factors. MethodsWe applied the following 2 designs to investigate this effect: a cross-sectional study for lower-leg trauma and a before-and-after study for knee arthroscopy. Plasma samples of POT-CAST– and POT-KAST–randomized clinical trial participants (collected shortly after lower-leg trauma or before or after arthroscopy) were analyzed for clot lysis time and levels of antithrombin, tissue factor pathway inhibitor, protein C, free protein S, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, antiplasmin, thrombin activatable fibrinolysis inhibitor, plasmin-antiplasmin, and D-dimer. For the effect of lower-leg injury, samples of 289 patients were compared with preoperative samples of 293 arthroscopy patients, acting as controls using linear regression and adjusting for age, sex, body mass index, comorbidities, and diurnal variation. For the effect of knee arthroscopy, mean changes were calculated for 277 patients using linear mixed models adjusted for diurnal variation. Parameters other than CLT and D-dimer were measured in smaller subsets. ResultsIn lower-leg injury patients, most parameters were stable, whereas D-dimer increased. After arthroscopy, most parameters decreased (especially clot lysis time, D-dimer, plasminogen, and anticoagulant factors), whereas tissue plasminogen activator and thrombin activatable fibrinolysis inhibitor slightly increased. ConclusionIn contrast to lower-leg injury, knee arthroscopy was associated with decreased natural anticoagulant factor levels. Neither lower-leg injury nor knee arthroscopy affected in vivo fibrinolysis.

Sex-dependent balance between thrombin and plasmin generation in the presence of thrombomodulin.

Assessing simultaneous generation of thrombin (TG) and plasmin (PG) is an approach to evaluate the balance between coagulation and fibrinolysis with sensitivity to predict endogenous thrombin and plasmin generation. The addition of thrombomodulin (TM), provides the essential component for thrombin activation of protein C and thrombin-activatable fibrinolysis inhibitor. However, the influence of sex on the balance between TG and PG with and without TM addition has not been investigated to date. To investigate the possible sex-based differences in TG and PG in the presence and absence of TM. Simultaneous TG and PG were measured in plasma samples obtained from 17 males and 17 females upon tissue factor and tissue plasminogen activator addition. Thrombin- and plasmin-specific fluorogenic substrates Z-Gly-Gly-Arg-AMC and Boc-Glu-Lys-Lys-AMC were used in the study. Thrombin and plasmin peak height (TPH and PPH) and production rate (TPR and PPR) values were determined. To evaluate the balance between TG and PG, the ratios between TPH and PPH (TPH/PPH) and TPR and PPR (TPR/PPR) were calculated. TPH between males and females demonstrated significant difference regardless of TM addition. TPR demonstrated differences between males and females only upon TM addition, while PG parameters was not dependent on the sex of the donor. TM significantly lowered TPH/PPH in males, and enhanced TPR/PPR in females. Thus, TPH/PPH and TPR/PPR significantly differed between men and women. Our results indicate that TM may act differently in males and females by shifting the underlying TG/PG balance to fibrinolysis in males and to coagulation in females.

Fibrinolysis in Cardiac Arrest Patients Treated with Hypothermia.

Hypothermia affects coagulation, but the effect of hypothermia on fibrinolysis is not clarified. Imbalance in the fibrinolytic system may lead to increased risk of bleeding or thrombosis. Our aim was to investigate if resuscitated cardiac arrest patients treated with hypothermia had an unbalanced fibrinolysis. A prospective cohort study, including 82 patients were treated with hypothermia at 33°C ± 1°C after out-of-hospital cardiac arrest. Blood samples were collected at 24 hours (hypothermia) and at 72 hours (normothermia). Samples were analyzed for fibrin D-dimer, tissue plasminogen activator (tPA), plasminogen, plasminogen activator Inhibitor-1 (PAI-1), thrombin-activatable fibrinolysis inhibitor (TAFI), and an in-house dynamic fibrin clot formation and lysis assay.Compared with normothermia, hypothermia significantly increased plasminogen activity (mean difference = 10.4%, 95% confidence interval [CI] 7.9-12.9), p < 0.001), PAI-1 levels (mean difference = 275 ng/mL, 95% CI 203-348, p < 0.001), and tPA levels (mean difference = 1.0 ng/mL, 95% CI 0.2-1.7, p = 0.01). No differences between hypothermia and normothermia were found in TAFI activity (p = 0.59) or in the fibrin D-dimer levels (p = 0.08). The fibrin clot lysis curves showed three different patterns: normal-, flat-, or resistant clot lysis curve. At hypothermia 45 (55%) patients had a resistant clot lysis curve and 33 (44%) patients had a resistant clot lysis curve at normothermia (p = 0.047). Comatose, resuscitated, cardiac arrest patients treated with hypothermia express an inhibited fibrinolysis even after rewarming. This could potentially increase the thromboembolic risk. ClinicalTrials.gov ID: NCT02258360.

Markers of Hereditary Thrombophilia with Unclear Significance.

Thrombophilia leads to an increased risk of venous thromboembolism. Widely accepted risk factors for thrombophilia comprise deficiencies of protein C, protein S, and antithrombin, as well as the factor V "Leiden" mutation, the prothrombin G20210A mutation, dysfibrinogenemia, and, albeit less conclusive, increased levels of factor VIII. Besides these established markers of thrombophilia, risk factors of unclear significance have been described in the literature. These inherited risk factors include deficiencies or loss-of-activity of the activity of ADAMTS13, heparin cofactor II, plasminogen, tissue factor pathway inhibitor (TFPI), thrombomodulin, protein Z (PZ), as well as PZ-dependent protease inhibitor. On the other hand, thrombophilia has been linked to the gain-of-activity, or elevated levels, of α2-antiplasmin, angiotensin-converting enzyme, coagulation factors IX (FIX) and XI (FXI), fibrinogen, homocysteine, lipoprotein(a), plasminogen activator inhibitor-1 (PAI-1), and thrombin-activatable fibrinolysis inhibitor (TAFI). With respect to the molecular interactions that may influence the thrombotic risk, more complex mechanisms have been described for endothelial protein C receptor (EPCR) and factor XIII (FXIII) Val34Leu. With focus on the risk for venous thrombosis, the present review aims to give an overview on the current knowledge on the significance of the aforementioned markers for thrombophilia screening. According to the current knowledge, there appears to be weak evidence for a potential impact of EPCR, FIX, FXI, FXIII Val34Leu, fibrinogen, homocysteine, PAI-1, PZ, TAFI, and TFPI on the thrombotic risk.

Differential Fibrinolysis Profile in COVID-19 Patients Dependent on Immunosuppressive Treatment Regimen: A Nested Case-Control Study

Introduction: Severe coronavirus disease 2019 (COVID-19) is associated with thrombotic complications, despite prophylactic and therapeutic anticoagulation. The disease course of critically ill COVID-19 patients has been characterized by severe hypercoagulability and hypofibrinolysis leading to an increased risk of venous thromboembolism. In COVID-19 (low molecular weight) heparins do not provide sufficient protection against venous thromboembolism. Since the start of the COVID-19 pandemic, several immune modulatory interventions have been introduced to improve outcome, including dexamethasone and tocilizumab. Whether dexamethasone and tocilizumab would alter the course of hypercoagulability by modifying inflammation, is unknown. Tocilizumab, that reduces IL-6 binding to its cellular receptors, has been shown to reduce the plasma plasminogen activator inhibitor-1 (PAI-1). On the other hand, dexamethasone can induce PAI-1 release. Data on the net impact of dexamethasone and tocilizumab on fibrinolysis in critically ill COVID-19 patients is lacking. Therefore, we aimed to characterize the fibrinolytic profile of severe COVID-19 patients on different treatment regimens and to unravel underlying mechanisms of the hypofibrinolytic state of these patients. Methods: A nested case-control study within the Maastricht Intensive Care COVID (MaastrICCht) cohort on mechanically ventilated COVID-19 patients was conducted. Patients receiving tocilizumab and dexamethasone (toci group) were age- and sex-matched with patients receiving only dexamethasone (dexa group) and patients receiving neither tocilizumab nor dexamethasone (control group). Whole blood was collected from these patients at three different time points after intubation (week 1, 2, and 3). The dexa and toci group received dexamethasone treatment before intubation and the toci group received one bolus of tocilizumab before or on the day of intubation. All blood samples were collected thereafter. Whole blood tissue plasminogen activator rotational thromboelastometry (tPA-ROTEM) was performed and subsequently inflammatory markers (c-reactive protein (CRP), ferritin, IL-6, fibrinogen), fibrinolysis parameters (tPA, PAI-1, thrombin activatable fibrinolysis inhibitor (TAFI), plasmin-alpha-2-antiplasmin complex (PAP)) and coagulation biomarkers (D-dimer, prothrombin fragment 1.2 (F1.2)) were measured in plasma samples. All markers were analyzed with linear mixed-effects regression models to estimate the differences between treatment groups over time. Results: Matching showed similar groups (Table 1.) for the baseline characteristics. At week 1 the dexagroup's IL-6 level was significantly lower, whereas the toci group's IL-6 level was significantly higher compared to the control group (difference[95%CI]: -198.37 [-350.86- -45.88], p=0.017 and 269.59 [113.91-425.27], p=0.002, respectively). CRP levels were significantly lower at week 1 in both dexa and toci groups compared to the control group (median [IQR] 60.50 [30.75 - 131.25] and 18.50 [6.00 - 60.00] vs. 296.00 [223.75 - 347.00]) suggesting effective immune suppression. Divergent fibrinolysis was seen at week 1 by tPA-ROTEM showing lower lysis time (LT) in the toci group compared to the control group (difference[95%CI]: -1,759.21 [-2752.05 - -766.34; p=0.002), whereas the dexa group did not differ. Interestingly, LT decreased over time in the control group, while an increase was seen in the toci group (Figure 1.). PAI-1 levels were lower in week 1 in the toci group compared to the control group (difference [95%CI] -7.32 [-14.95 - 0.30], p=0.071) and the tocigroup showed an overall increase in PAI-1 levels, while the control and dexa group showed a decrease over time. High TAFI and tPA levels were observed in all groups at week 1 and over time regardless of the treatment regimens. Coagulation biomarkers D-dimer and prothrombin fragment 1.2 were elevated but did not differ or change over time between groups. Conclusions: The differences and changes over time revealed by tPA-ROTEM and the fibrinolysis markers between the three different treatment regimens show the direct interaction between inflammation and fibrinolysis in these patients. The lower LT and PAI-1 levels upon tocilizumab treatment could be indicative for better fibrinolytic activity in these patients. This study was supported by the Dutch Covid and Thrombosis Coalition (DCTC). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Amplified Thrombo-Inflammatory Biomarkers in Pulmonary Embolism with Augmented Responses in Von-Willebrand Factor, Microparticles and Interleukin-6 in Patients with Cancer

Introduction: The multifactorial and complex pathophysiology of pulmonary embolism (PE) involves dysregulation of hemostatic system including fibrinolytic imbalance, endothelial compromise, and generation of thrombotic mediators, along with cellular defects and hemodynamic derangement. These pathophysiologic processes results in the generation of thrombo-inflammatory biomarkers reflecting the endogenous hemostatic imbalance. Cancer is prevalent in PE patients and is considered as an added risk factor for the adverse outcomes. Thrombo-inflammatory biomarker profiling provide means to risk stratify PE patients. This study aimed to profile several of these biomarkers in normal individuals and PE patients and further demonstrate their additional amplification in patients with cancer as a co-morbidity. Materials & Method: Four hundred male and female patients of 18 years or older age, were included through enrolment in conjunction with an IRB approved project by the pulmonary embolism response team (PERT) registry. Diagnosis of PE was confirmed by computed tomography (CT), angiography or ventilation perfusion imaging. Blood samples were drawn with in 24-72 hours of confirmed diagnosis of acute PE. Normal human plasma (NHP) samples were obtained from 50 healthy individuals including 25 male and 25 female and used for referencing purposes. All of the plasma samples were analyzed for D-Dimer, plasminogen activator inhibitor-1 (PAI-1) antigen, tissue-type plasminogen activator (tPA), activated thrombin-activatable fibrinolysis inhibitor (TAFI-a), von Willebrand factor (vWF), c-Reactive Protein (CRP) and interleukin-6 (IL-6) were measured by commercially available ELISA methods. Microparticles were measured by using a chromogenic functional method. Individual biomarkers were compared to normal plasma and further stratified on cancer status and PE severity. Results are presented as mean±SD and fold increase. For the degree of association, GraphPad Prism Software was used. Results: PE patients exhibited varying levels of upregulation of all of the individual biomarkers compared to normal's, D-Dimer (37.05-fold), PAI-1 antigen (3.95-fold), tPA (4.69-fold), TAFI (1.21-fold), vWF (1.89-fold), CRP (35.34-fold), IL-6 (21.62-fold), and microparticles (3.04-fold) increase in PE patients with reference to NHP. When the PE group stratified into cancer and non-cancer patients, most of the biomarkers except D-Dimer and microparticles showed varying degree of modest elevation in the cancer group (figure 1). vWF, IL6 and microparticles levels were found to be further amplified in PE group with cancer. Of these three biomarkers, only vWF remained amplified when stratified by PE severity in patients with cancer, however this change was only observed in the low-grade PE patients (table 1). The IL-6 levels showed increased trend in the low-grade and sub-massive patient group, however because of the wide scatter, it was not significant. Conclusion: These studies demonstrate that thrombo-inflammatory biomarkers are upregulated in PE patients. Furthermore, PE patients with cancer exhibit amplified responses for vWF, IL-6 and microparticles levels suggesting their role in mediating the pathogenesis involving endothelial, inflammatory and thrombogenic mechanisms. Among the biomarkers studied, vWF is central to thrombo-inflammatory process and is distinct in PE cohort with cancer, despite taking into account the lower PE severity. These studies suggest that selective biomarker profiling of thrombo-inflammatory biomarkers may be valuable in the risk stratification and therapeutic management of PE patients at large. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hemorrhagic shock and tissue injury provoke distinct components of trauma-induced coagulopathy in a swine model.

Tissue injury (TI) and hemorrhagic shock (HS) are the major contributors to trauma-induced coagulopathy (TIC). However, the individual contributions of these insults are difficult to discern clinically because they typically coexist. TI has been reported to release procoagulants, while HS has been associated with bleeding. We developed a large animal model to isolate TI and HS and characterize their individual mechanistic pathways. We hypothesized that while TI and HS are both drivers of TIC, they provoke different pathways; specifically, TI reduces time to clotting, whereas, HS decreases clot strength stimulates hyperfibrinolysis. After induction of general anesthesia, 50kg male, Yorkshire swine underwent isolated TI (bilateral muscle cutdown of quadriceps, bilateral femur fractures) or isolated HS (controlled bleeding to a base excess target of -5mmol/l) and observed for 240min. Thrombelastography (TEG), calcium levels, thrombin activatable fibrinolysis inhibitor (TAFI), protein C, plasminogen activator inhibitor 1 (PAI-1), and plasminogen activator inhibitor 1/tissue-type plasminogen activator complex (PAI-1-tPA) were analyzed at pre-selected timepoints. Linear mixed models for repeated measures were used to compare results throughout the model. TI resulted in elevated histone release which peaked at 120min (p = 0.02), and this was associated with reduced time to clot formation (R time) by 240min (p = 0.006). HS decreased clot strength at time 30min (p = 0.003), with a significant decline in calcium (p = 0.001). At study completion, HS animals had elevated PAI-1 (p = 0.01) and PAI-1-tPA (p = 0.04), showing a trend toward hyperfibrinolysis, while TI animals had suppressed fibrinolysis. Protein C, TAFI and skeletal myosin were not different among the groups. Isolated injury in animal models can help elucidate the mechanistic pathways leading to TIC. Our results suggest that isolated TI leads to early histone release and a hypercoagulable state, with suppressed fibrinolysis. In contrast, HS promotes poor clot strength and hyperfibrinolysis resulting in hypocoagulability.

Changes in fibrinolytic activity and coagulation factors after epicardial left atrial appendage closure in patients with atrial fibrillation.

The left atrial appendage (LAA) is known to be the primary source of thrombus formation in atrial fibrillation (AF). We investigate whether epicardial LAA occlusion (LAAO) from the cardiovascular system has an effect on coagulation and prothrombotic status in AF. Twenty-two patients with nonvalvular AF, who were not currently receiving oral anticoagulation (OAC) therapy, participated in a single-center prospective study. We measured fibrinogen and plasminogen levels along with plasma fibrin clot permeability, clot lysis time (CLT) and endogenous thrombin potential (ETP) before the LAAO procedure, at discharge and 1 month afterward. One month after the LAAO procedure, plasma fibrin clot permeability improved by 39.3% as measured by clots prepared from peripheral blood (P=0.019) and also after adjustment for fibrinogen (P=0.027). Higher plasma fibrin clot permeability was associated with improved clot susceptibility to lysis (r=-0.67, P=0.013). CLT was reduced by 10.3% (P=0.0020), plasminogen activator inhibitor-1 antigen levels were reduced by 52% (P=0.023) and plasminogen activity was increased by 8.9% (P=0.0077). A trend toward decreased thrombin generation, reflected by a decreased ETP and peak thrombin generated was also observed 1 month after LAAO procedure (P=0.072 and P=0.087, respectively). No differences were observed in tissue-type plasminogen activator and thrombin-activatable fibrinolysis inhibitor plasma levels (both P>0.05). Obtained results seem to confirm that LAA plays a key role in thrombogenesis. Elimination of LAA from the circulatory system may improve fibrin clot permeability and susceptibility to fibrinolysis in peripheral blood.

Visualization of Domain- and Concentration-Dependent Impact of Thrombomodulin on Differential Regulation of Coagulation and Fibrinolysis.

Thrombomodulin (TM) functions as a dual modulator-anticoagulant and antifibrinolytic potential-by the thrombin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). Activated TAFI cleaves the C-terminal lysine of partially degraded fibrin and inhibits both plasminogen binding and its activation on the fibrin surface. We have reported previously that activated platelets initiate fibrin network formation and trigger fibrinolysis after the accumulation of tissue-type plasminogen activator and plasminogen. To analyze the effects of domain-deletion variants of TM on coagulation and fibrinolysis at different concentrations. Domain-deletion variants of TM, such as D123 (all extracellular regions), E3456 (minimum domains for thrombin-dependent activation of protein C and TAFI), and E456 (minimum domains for that of protein C but not TAFI), were used at 0.25 to 125 nM for turbidimetric assay to determine the clotting time and clot lysis time and to visualize fibrin network formation and lysis in platelet-containing plasma. A low concentration of either D123 or E3456, but not of E456, prolonged clot lysis time, and delayed the accumulation of fluorescence-labeled plasminogen at the activated platelets/dense fibrin area due to effective TAFI activation. Conversely, only the highest concentrations of all three TM variants delayed the clotting time, though fibrin network formation in the vicinity of activated platelets was almost intact. TAFI activation might be affected by attenuation in thrombin activity after the clot formation phase. These findings suggest that the spatiotemporal balance between the anticoagulant and antifibrinolytic potential of TM is controlled in domain- and concentration-dependent manners.

Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation.

The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a representative example, we performed a preparative purification of this enzyme. The purified protein is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput functional and structural studies.

Serum thrombin-activatable fibrinolysis inhibitor levels and its relation with pathogenesis and bleeding and prognosis in patients with Crimean Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is a viral hemorrhagic fever, which is common in Turkey and globally. The pathogenesis of coagulation disorders, which is seen in viral hemorrhagic fevers remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) has a key role in this process In this study, we aimed to evaluate whether TAFI levels contributed to bleeding and whether it is related to prognosis in CCHF patients.Eighty-four patients older than 15 years of age, who were admitted to our hospital who had positive immunoglobulin M (enzyme-linked immunosorbent assay [ELISA]) and/or polymerase chain reaction test results for CCHF between 2009 and 2010, were included in the study. The control group included 30 healthy adults. The plasma TAFI levels were compared between patients and controls, and also between patients with bleeding and no bleeding, and between patients with mild-moderate and severe disease. The mean TAFI levels were lower in patients (mean: 87.82 ng/ml, median: 61.69 ng/ml (interquartile range [IQR] 30.49-537.95) than controls (mean: 313.5 ng/ml with a median: 338.5 ng/ml (IQR 182-418). However, median TAFI levels were significantly higher in patients with bleeding compared to those without bleeding (78.99 and 50.28 ng/ml, respectively; p = 0.032). Median IQR TAFI levels were similar between patients with mild-moderate and severe disease (64.72 (41.37-113.85), and, 58.66 (42.44-118.93) ng/ml, respectively; p = 0.09) and survivors and nonsurvivors (86.14 ± 77.98 and 103.48 ± 69.92, respectively; p = 0.3). Although TAFI levels were lower in the patients with CCHF compared to healthy controls, it does not seem to be a major player in the prognosis.

The suboptimal fibrinolytic response in COVID‐19 is dictated by high PAI‐1

BackgroundSevere COVID‐19 disease is associated with thrombotic complications and extensive fibrin deposition. This study investigates whether the hemostatic complications in COVID‐19 disease arise due to dysregulation of the fibrinolytic system. MethodsThis prospective study analyzed fibrinolytic profiles of 113 patients hospitalized with COVID‐19 disease with 24 patients with non‐COVID‐19 respiratory infection and healthy controls. Antigens were quantified by Ella system or ELISA, clot lysis by turbidimetric assay, and plasminogen activator inhibitor‐1 (PAI‐1)/plasmin activity using chromogenic substrates. Clot structure was visualized by confocal microscopy. ResultsPAI‐1 and its cofactor, vitronectin, are significantly elevated in patients with COVID‐19 disease compared with those with non‐COVID‐19 respiratory infection and healthy control groups. Thrombin activatable fibrinolysis inhibitor and tissue plasminogen activator were elevated in patients with COVID‐19 disease relative to healthy controls. PAI‐1 and tissue plasminogen activator (tPA) were associated with more severe COVID‐19 disease severity. Clots formed from COVID‐19 plasma demonstrate an altered fibrin network, with attenuated fiber length and increased branching. Functional studies reveal that plasmin generation and clot lysis were markedly attenuated in COVID‐19 disease, while PAI‐1 activity was elevated. Clot lysis time significantly correlated with PAI‐1 levels. Stratification of COVID‐19 samples according to PAI‐1 levels reveals significantly faster lysis when using the PAI‐1 resistant (tPA) variant, tenecteplase, over alteplase lysis. ConclusionThis study shows that the suboptimal fibrinolytic response in COVID‐19 disease is directly attributable to elevated levels of PAI‐1, which attenuate plasmin generation. These data highlight the important prognostic potential of PAI‐1 and the possibility of using pre‐existing drugs, such as tenecteplase, to treat COVID‐19 disease and potentially other respiratory diseases.

A missense mutation in lectin domain of thrombomodulin causing functional deficiency

Thrombomodulin (TM) functions in coagulation, fibrinolysis and inflammation by its cofactor activity for protein C, thrombin-activatable fibrinolysis inhibitor (TAFI) activation and high mobility group box 1 (HMGB1) degradation induced by thrombin. It has been widely reported that mutations in TM are related to thromboembolic diseases but hardly in lectin domain. Here we report our findings about the functional deficiencies in TM caused by substitution of aspartate with tyrosine at residue 126. Three patients suffering from recurrent thromboembolic diseases were identified with this mutation and their plasma soluble TM levels were decreased. Transfected cells expressing wild-type TM or the variant and corresponding proteins were used to examine TM functions in vitro. The cofactor activity of the mutant for protein C, TAFI activation was reduced to approximately 50% and 60% respectively. Loss in anti-inflammation due to weakened HMGB1 degradation was also observed. And the study with thrombosis models of mice suggested the decreased inhibition of thrombus development of the mutant. Together the results showed deleterious changes on TM function caused by this mutation, which may explain the thrombophilia tendency of the patients. This work provided supportive evidence that mutation in lectin domain of TM might be related to thrombotic diseases and may help us better understand the physiological roles of TM.