Molecular Biomarkers for In Vitro Thrombogenicity Assessment of Medical Device Materials.

To develop standardized in vitro thrombogenicity test methods for evaluating medical device materials, three platelet activation biomarkers, beta-thromboglobulin (ß-TG), platelet factor 4 (PF4), soluble p-selectin (CD62P), and a plasma coagulation marker, thrombin-antithrombin complex (TAT), were investigated. Whole blood, drawn from six healthy human volunteers into Anticoagulant Citrate Dextrose Solution A was recalcified and heparinized over a concentration range of 0.5-1.5 U/mL. The blood was incubated with test materials with different thrombogenic potentials for 60 min at 37°C, using a 6 cm2/mL material surface area to blood volume ratio. After incubation, the blood platelet count was measured before centrifuging the blood to prepare platelet-poor plasma (PPP) and platelet-free plasma (PFP) for enzyme-linked immunosorbent assay analysis of the biomarkers. The results show that all four markers effectively differentiated the materials with different thrombogenic potentials at heparin concentrations from 1.0 to 1.5 U/mL. When a donor-specific heparin concentration (determined by activated clotting time) was used, the markers were able to differentiate materials consistently for blood from all the donors. Additionally, using PFP instead of PPP further improved the test method's ability to differentiate the thrombogenic materials from the negative control for ß-TG and TAT. Moreover, the platelet activation markers were able to detect reversible platelet activation induced by adenosine diphosphate (ADP). In summary, all three platelet activation markers (ß-TG, PF4, and CD62P) can distinguish thrombogenic potentials of different materials and detect ADP-induced reversible platelet activation. Test consistency and sensitivity can be enhanced by using a donor-specific heparin concentration and PFP. The same test conditions are applicable to the measurement of coagulation marker TAT.

Predictive value of thrombin-antithrombin III complex and tissue plasminogen activator-inhibitor complex biomarkers in assessing the severity of early-stage acute pancreatitis.

BACKGROUND AND AIM: The development of acute pancreatitis (AP) is strongly linked to blood clotting and fibrinolysis issues. Modern clinical practices now utilize advanced blood markers like thrombin-antithrombin III complex (TAT), plasmin-α2-plasmin inhibitor complex, thrombomodulin (TM), and tissue plasminogen activator-inhibitor complex (t-PAIC) to assess thrombosis risk. Our study used a highly sensitive chemiluminescence technique to measure these markers in AP patients, aiming to determine their early predictive value for AP severity. METHODS: There were 173 patients with AP, all of whom developed symptoms within 72 h; 102 individuals had onset symptoms within 48 h. The biomarkers were measured upon admission before determining the severity of AP. RESULTS: The levels of TAT, plasmin-α2-plasmin inhibitor complex, TM, and t-PAIC were significantly higher in the severe acute pancreatitis (SAP) group compared with the mild acute pancreatitis and moderate severe acute pancreatitis groups. For the patients within 72 h of onset, TAT, TM, and t-PAIC predicted the occurrence of SAP. For the patients within 48 h of onset, TAT and t-PAIC predicted the occurrence of SAP. The area under the curve (AUC) of prediction models is similar to Bedside Index for Severity in Acute Pancreatitis (BISAP) but significantly higher than C-reactive protein (P < 0.05). Notably, t-PAIC had a larger AUC than TAT, BISAP, and C-reactive protein. CONCLUSION: In the initial 48 h, plasma TAT and t-PAIC levels may predict the development of SAP. Within 72 h, plasma levels of TAT, TM, and t-PAIC may predict the development of SAP, and the TAT + TM + t-PAIC prediction model achieved a maximum AUC of 0.915, comparable to BISAP.

Investigating protease-mediated peptides of inflammation and tissue remodeling as biomarkers associated with flares in psoriatic arthritis.

BACKGROUND: Psoriatic arthritis (PsA) is an inflammatory arthritis associated with psoriasis. PsA disease involves flares, which are associated with increased joint inflammation and tissue remodeling. There is a need for identifying biomarkers related to PsA disease activity and flares to improve the management of PsA patients and decrease flares. The tissue turnover imbalance that occurs during the inflammatory and fibro-proliferative processes during flares leads to an increased degradation and/or reorganization of the extracellular matrix (ECM), where increased proteolysis plays a key role. Hence, protease-mediated fragments of inflammatory and tissue-remodeling components could be used as markers reflecting flares in PsA patients. METHODS: A broad panel of protease-mediated biomarkers reflecting inflammation and tissue remodeling was measured in serum and synovial fluid (SF) obtained from PsA patients experiencing flares (acutely swollen joint[s], PsA-flare). In serum, biomarker levels assessed in PsA-flare patients were compared to controls and in early-diagnosed PsA patients not experiencing flares (referred to as PsA without flare). Furthermore, the biomarker levels assessed in SF from PsA-flare patients were compared to the levels in SF of osteoarthritis (OA) patients. RESULTS: In serum, levels of the PRO-C3 and C3M, reflecting formation and degradation of the interstitial matrix, were found significantly elevated in PsA-flare compared to controls and PsA without flare. The remodeling marker of the basement membrane, PRO-C4, was significantly elevated in PsA-flare compared to PsA without flare. The inflammation and immune cell activity related markers, CRPM, VICM, and CPa9-HNE were significantly elevated in PsA-flare patients compared to controls and PsA without flare. In addition, VICM (AUC = 0.71), CPa9-HNE (AUC = 0.89), CRPM (AUC = 0.76), and PRO-C3 (AUC = 0.86) showed good discriminatory performance for separating PsA-flare from PsA without flare. In SF, the macrophage activity marker, VICM, was significantly elevated whereas the type II collagen formation marker, PRO-C2, was significantly reduced in the PsA-flare compared to OA. The combination of five serum markers reflecting type III and IV collagen degradation (C3M and C4M, respectively), type III and VI collagen formation (PRO-C3 and PRO-C6, respectively), and neutrophil activity (CPa9-HNE) showed an excellent discriminatory performance (AUC = 0.98) for separating PsA-flare from PsA without flares. CONCLUSIONS: The serum biomarker panel of C3M, C4M, PRO-C3, PRO-C6, and CPa9-HNE reflecting synovitis, enthesitis, and neutrophil activity may serve as novel tool for quantitatively monitoring flares in PsA patients.

Coagulation activity and thrombotic risk following high-volume endurance exercise in recreationally active cyclists.

Despite the prognostic effect of physical activity, acute bouts of high-volume endurance exercise can induce cardiac stress and postexercise hypercoagulation associated with increased thrombotic risk. The aim of this study was to explore the effect of high-volume endurance exercise on coagulation and thrombotic activity in recreational cyclists. Thirty-four recreational cyclists completed 4.8 ± 0.3 h of cycling at 45 ± 5% of maximal power output on a bicycle ergometer. Intravenous blood samples were collected preexercise, immediately postexercise, 24 and 48 h postexercise, and analyzed for brain natriuretic peptide (BNP), cardiac troponin (cTn), C-reactive protein (CRP), D-dimer, thrombin-antithrombin (TAT) complex, tissue factor (TF), tissue factor pathway inhibitor (TFPI), and TF-to-TFPI ratio (TF:TFPI). An increase in cTn was observed postexercise (P < 0.001). CRP concentrations were increased at 24 and 48 h postexercise compared with preexercise concentrations (P ≤ 0.001). TF was elevated at 24 h postexercise (P < 0.031) and TFPI was higher immediately postexercise (P < 0.044) compared with all other time points. TF:TFPI was increased at 24 and 48 h postexercise compared with preexercise (P < 0.025). TAT complex was reduced at 48 h postexercise compared with preexercise (P = 0.015), D-dimer was higher immediately postexercise compared with all other time points (P ≤ 0.013). No significant differences were observed in BNP (P > 0.05). High-volume endurance cycling induced markers of cardiac stress among recreational cyclists. However, plasma coagulation and fibrinolytic activity suggest no increase in thrombotic risk after high-volume endurance exercise.NEW & NOTEWORTHY In this study, a high-volume endurance exercise protocol induced markers of cardiac stress and altered plasma coagulation and fibrinolytic activity for up to 48 h in recreationally active cyclists. However, analysis of coagulation biomarkers indicates no increase in thrombotic risk when appropriate hydration and rest protocols are implemented.

Acute Microbial Protease Supplementation Increases Net Postprandial Plasma Amino Acid Concentrations After Pea Protein Ingestion in Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Digestibility is a primary factor in determining the quality of dietary protein. Microbial protease supplementation may be a strategy for improving protein digestion and subsequent postprandial plasma amino acid availability. OBJECTIVES: To assess the effect of co-ingesting a microbial protease mixture with pea protein on postprandial plasma amino acid concentrations. DESIGN: A mixture of 3 microbial protease preparations (P3) was tested for proteolytic efficacy in an in vitro static simulation of gastrointestinal digestion. Subsequently, in a randomized, double-blind, placebo-controlled crossover trial, 24 healthy adults (27 ± 4 y; 12 females, 12 males) ingested 25 g pea protein isolate (20 g protein, 2.2 g fat) with either P3 or maltodextrin placebo (PLA). Blood samples were collected at baseline and throughout a 0‒5 h postprandial period and both the early (0-2 h) iAUC and total (0-5 h) iAUC were examined. RESULTS: Plasma glucose concentrations decreased in both conditions (P < 0.001), with higher concentrations after P3 ingestion compared with PLA (P < 0.001). Plasma insulin concentrations increased for both conditions (P < 0.001) with no difference between conditions (P = 0.331). Plasma total amino acid (TAA) concentrations increased over time (P < 0.001) with higher concentrations observed for P3 compared with PLA (P = 0.010) during the 0‒5 h period. There was a trend for elevated essential amino acid (EAA) concentrations for P3 compared with PLA (P = 0.099) during the 0‒5 h postprandial period but not for leucine (P = 0.282) or branched-chain amino acids (BCAA, P = 0.410). The early net exposure (0‒2 h iAUC) to amino acids (leucine, BCAA, EAA, and TAA) was higher for P3 compared with PLA (all, P < 0.05). CONCLUSIONS: Microbial protease co-ingestion increases plasma TAA concentrations (0-5 h) and leucine, BCAA, EAA, and TAA availability in the early postprandial period (0‒2 h) compared with ingesting pea protein with placebo in healthy adults.

The roles of proteases in prostate cancer.

Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.

Persistent hypercoagulability in dogs envenomated by the European adder (Vipera berus berus).

BACKGROUND: Envenomation by the European adder, Vipera berus berus (Vbb), is a medical emergency. The overall in vivo haemostatic effects of pro- and anticoagulant components in Vbb venom, and the downstream effects of cellular injury and systemic inflammation, are unclear. OBJECTIVES: To longitudinally describe the global coagulation status of dogs after Vbb envenomation and compare to healthy controls. A secondary aim was to investigate differences between dogs treated with and without antivenom. METHODS: Citrated plasma was collected at presentation, 12 hours (h), 24 h, 36 h and 15 days after bite from 28 dogs envenomated by Vbb, and from 28 healthy controls at a single timepoint. Thrombin generation (initiated with and without exogenous phospholipids and tissue factor), thrombin-antithrombin (TAT)-complexes and the procoagulant activity of phosphatidylserine (PS)-expressing extracellular vesicles (EVs), expressed as PS-equivalents, were measured. RESULTS: At presentation the envenomated dogs were hypercoagulable compared to controls, measured as increased thrombin generation, TAT-complexes and PS-equivalents. The hypercoagulability decreased gradually but compared to controls thrombin generation and PS-equivalents were still increased at day 15. The discrepancy in peak thrombin between envenomated dogs and controls was greater when the measurement was phospholipid-dependent, indicating that PS-positive EVs contribute to hypercoagulability. Lag time was shorter in non-antivenom treated dogs, compared to antivenom treated dogs <24 h after envenomation. CONCLUSIONS: Hypercoagulability was measured in dogs up to 15 days after Vbb envenomation. Dogs treated with antivenom may be less hypercoagulable than their non-antivenom treated counterparts. Thrombin generation is a promising diagnostic and monitoring tool for Vbb envenomation.

Activated Factor XI is Increased in Plasma in Response to Surgical Trauma but not to Recombinant Activated FVII-Induced Thrombin Formation.

AIM: Feedback activation of factor XI (FXI) by thrombin is believed to play a critical role in the amplification phase of thrombin generation and to contribute to thrombosis development and hemostasis. However, the activation of FXI by thrombin has been shown in vitro to require a cofactor. In this study, the role of thrombin in activated FXI (FXIa) formation in vivo is investigated. METHODS: The study population comprised probands in whom coagulation activation was triggered by low-dose (15 µg/kg) recombinant activated factor VII (rFVIIa, n=89), of whom 34 with (VTE+) and 45 without a history of venous thromboembolism (VTE-), and patients undergoing major orthopedic surgeries (n=45). FXIa was quantified via an enzyme capture assay using a monoclonal FXI-specific antibody. Thrombin formation was monitored using an oligonucleotide-based enzyme capture assay and the thrombin activation markers prothrombin fragment 1＋2 (F1＋2) and thrombin-antithrombin complex (TAT). RESULTS: In the rFVIIa cohort, FXIa and thrombin remained below their lower limit of quantification of 3.48 and 1.06 pmol/L, respectively. By contrast, during the surgeries, median FXIa levels increased from 3.69 pmol/L pre-operatively to 9.41 pmol/L mid-operatively (P=4·10-4) and remained significantly elevated 24 h thereafter, with 9.38 pmol/L (P=0.001). Peak levels of F1+2 were comparable in the VTE+, VTE-, and surgery cohort (235, 268, and 253 pmol/L), whereas peak TAT levels were higher in the surgery cohort (53.1, 33.9, and 147.6 pmol/L). CONCLUSIONS: Under in vivo conditions, the activation of FXI requires specific local features that are present at the wounded site including potential cofactors of thrombin.

Neutrophil-Derived Proteases Contribute to the Pathogenesis of Early Diabetic Retinopathy.

Purpose: Previous studies indicate that leukocytes, notably neutrophils, play a causal role in the capillary degeneration observed in diabetic retinopathy (DR), however, the mechanism by which they cause such degeneration is unknown. Neutrophil elastase (NE) is a protease released by neutrophils which participates in a variety of inflammatory diseases. In the present work, we investigated the potential involvement of NE in the development of early DR. Methods: Experimental diabetes was induced in NE-deficient mice (Elane-/-), in mice treated daily with the NE inhibitor, sivelestat, and in mice overexpressing human alpha-1 antitrypsin (hAAT+). Mice were assessed for diabetes-induced retinal superoxide generation, inflammation, leukostasis, and capillary degeneration. Results: In mice diabetic for 2 months, deletion of NE or selective inhibition of NE inhibited diabetes-induced retinal superoxide levels and inflammation, and inhibited leukocyte-mediated cytotoxicity of retinal endothelial cells. In mice diabetic for 8 months, genetic deletion of NE significantly inhibited diabetes-induced retinal capillary degeneration. Conclusions: These results suggest that a protease released from neutrophils contributes to the development of DR, and that blocking NE activity could be a novel therapy to inhibit DR.

Neutrophil extracellular traps in patients with liver cirrhosis and hepatocellular carcinoma.

Neutrophil extracellular traps (NETs) are web-like structures consisting of DNA, histones and granule proteins, released from neutrophils in thrombus formation, inflammation, and cancer. We asked if plasma levels of the NET markers myeloperoxidase (MPO)-DNA and citrullinated histone H3 (H3Cit)-DNA, are elevated in liver cirrhosis and hepatocellular carcinoma (HCC) and if the levels correlate with clinical parameters. MPO-DNA, H3Cit-DNA, and thrombin-antithrombin (TAT) complex, as a marker of coagulation activity, were measured using ELISA in plasma from 82 patients with HCC, 95 patients with cirrhosis and 50 healthy controls. Correlations were made to clinical parameters and laboratory data and patients were followed for a median of 22.5 months regarding thrombosis development. H3Cit-DNA was significantly (p < 0.01) elevated in plasma from cirrhosis (66.4 ng/mL) and HCC (63.8 ng/mL) patients compared to healthy controls (31.8 ng/mL). TAT levels showed similar pattern (3.1, 3.7, and 0.0 µg/mL respectively, p < 0.01). MPO-DNA was significantly (p < 0.01) elevated in cirrhosis patients (0.53 O.D.) as compared to controls (0.33 O.D.). Levels of MPO-DNA and H3Cit-DNA correlated positively with Child-Pugh and MELD score. TAT was increased in all Child-Pugh and MELD groups. In multivariable logistic regression, Child B and C liver cirrhosis were independent predictors of elevated H3Cit-DNA in plasma. Levels of MPO-DNA and H3Cit-DNA were similar in patients with or without history of thrombosis, or thrombus formation during follow-up. In conclusion, plasma markers of NET formation are elevated in liver cirrhosis and correlate to the degree of liver dysfunction in patients with liver cirrhosis and/or HCC. The presence of HCC did not further increase the plasma levels of NET markers as compared to patients with cirrhosis only.

COVID-19 infection in severe Alpha 1-antitrypsin deficiency: Looking for a rationale.

The clinical manifestations of COVID-19 are heterogeneous: 46.4% of patients admitted into hospital reported to have at least one comorbidity. Comorbidities such as COPD, diabetes, hypertension and malignancy predispose patients with Covid-19 to adverse clinical outcomes. Alpha 1-antitrypsin deficiency (AATD) is a genetic disorder caused by pathological mutation(s) in the SERPINA1 gene resulting in an imbalance in proteinase activity which may lead to premature emphysema and COPD. Our aim was to investigate whether people with severe AAT deficiency (AATD) have an increased risk of (severe) COVID-19 infection. We collected data on COVID-19 symptoms, laboratory-confirmed infection, hospitalization and treatment by means of a telephone survey, directly administered to Italian severe AATD subjects in May 2020. We then compared our findings with data collected by the Istituto Superiore di Sanità on the total population in Italy during the same period. We found an higher frequency of SARS-CoV-2 infection in our cohort (3.8%) compared to national data regarding infection, thus giving severe AATD a relative risk of 8. 8 (95%CI 5.1-20,0; p<0.0001) for symptomatic SARS-CoV-2 infection. Moreover, the relative risk (RR) was higher in AATD patients with pre-existing lung diseases (RR 13.9; 95%CI 8.0-33.6; p<0.001), but with a similar death rate (1 in 8, 12.5%) compared to the general population (13.9%; RR 0.9). These preliminary findings highlight the importance of close surveillance in the spread of COVID-19 in patients with severe AATD and underlines the need for further studies into the role of the antiprotease shield in preventing SARS-Cov-2 infection.

Impact of Transcatheter Aortic Valve Implantation on Thrombin Generation and Platelet Function.

BACKGROUND: Transcatheter aortic valve implantation (TAVI) is an evolving treatment of severe aortic valve stenosis. However, thromboembolic events such as stroke are common, predominantly early after TAVI. Optimal periprocedural antithrombotic regime is unknown. Especially, as antithrombotic medication enhances bleeding risk, thrombin generation and platelet function are crucial in the pathogenesis of ischemic events. However, the impact of the TAVI procedure on thrombin formation and platelet reactivity is not known by now. METHODS: We evaluated thrombin levels using thrombin-antithrombin (TAT) complexes and prothrombin fragments (PTFs) using enzyme-linked immunosorbent assay. Furthermore, platelet reactivity was measured via light transmission aggregometry before and 2 hours after TAVI in 198 patients. RESULTS: TAT complexes and PTF F1 + 2 substantially increased during TAVI. Postprocedurally, TAT complexes and PTF were significantly higher after TAVI compared with percutaneous coronary intervention due to acute myocardial infarction, while preprocedural TAT complexes and PTF F1 + 2 did not differ. In contrast, platelet reactivity was not altered early after TAVI. Only adenosine diphosphate-induced aggregation was reduced, reflecting preprocedural loading with clopidogrel. CONCLUSION: In this pilot study, we were able to demonstrate that thrombin generation is significantly increased early after TAVI, while platelet function is not affected. Increased thrombin concentrations may contribute to the high risk of postprocedural thromboembolic events. This leads to the hypothesis that extended peri-interventional anticoagulation early after TAVI may be an approach to reduce thromboembolic events.

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.

Blood Coagulation Changes With or Without Direct Oral Anticoagulant Therapy Following Transcatheter Aortic Valve Implantation.

Thromboembolic events remain clinically unresolved after transcatheter aortic valve implantation (TAVI). The use of direct oral anticoagulant (DOAC) to reduce thrombosis associated with TAVI remains controversial. This study aimed at investigating the periprocedural change in blood coagulation and thrombolysis parameters in 199 patients undergoing transfemoral TAVI. Prothrombin activation fragment 1 + 2 (F1 + 2), thrombin-antithrombin complex (TAT), soluble fibrin monomer complex (SFMC), and fibrin/fibrinogen degradation product (FDP) levels were measured before and 1 hour after TAVI and 1, 2, and 7 days postoperatively. Of the 199 patients, 49 were treated with DOAC (apixaban in 32, edoxaban in 10, and rivaroxaban in 7). The F1 + 2 and TAT levels immediately increased 1 hour after TAVI and then gradually decreased in both groups. The SFMC level also significantly increased with a peak on day 1. The FDP level gradually increased, peaking on day 2. The values of F1 + 2, TAT, SFMC, and FDP in patients who used DOAC were significantly lower than those who did not use DOAC at 1 hour after TAVI in F1 + 2 (600 [452 to 765] vs 1055 [812 to 1340] pmol/L; p < 0.001), TAT (21.4 [16.2 to 37.0] vs 38.7 [26.4 to 58.7] µg/mL; p < 0.001) and on day 1 in SFMC (18.2 [9.4 to 57.9] vs 113.4 [70.9 to 157.3] µg/mL; p < 0.001) and day 2 in FDP (6.0 [4.7 to 10.0] vs 12.6 [8.2 to 17.4] µg/mL; p < 0.001). Ischemic stroke within 30 days after TAVI occurred in 3 patients (1.5%), who were not treated with DOAC. Coagulation cascade activation was observed after TAVI. DOAC could reduce transient hypercoagulation following TAVI.

Diagnostic and Prognostic Value of TAT, PIC, TM, and t-PAIC in Malignant Tumor Patients With Venous Thrombosis.

BACKGROUND: Venous thromboembolism (VTE) is an important complication in patients with malignant tumors. Its exact diagnosis and treatment are still lacking. We used a high-sensitive chemiluminescence method to detect thrombin-antithrombin III complex (TAT), plasmin-α2-plasmininhibitor complex (PIC), thrombomodulin (TM), and tissue plasminogen activator-inhibitor complex(t-PAIC) in combination with D-dimer and fibrin degradation product (FDP) to analyze their diagnostic and prognostic value in patients with malignant tumors. METHODS: In total, 870 patients with confirmed malignant tumors were included, 82 of whom had diagnosed VTE; 200 healthy individuals were classified as the control group. The TAT, PIC, TM, and t-PAIC were detected using Sysmex HISCL5000 automated analyzers, whereas FDP and D-dimer were detected using Sysmex CS5100 coagulation analyzer. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic efficiency. Survival probabilities were determined using Kaplan-Meier analysis, and multivariate analyses were performed using a Cox regression model. RESULTS: Compared with healthy controls, patients with malignant tumors showed significantly elevated TAT, PIC, TM, t-PAIC, D-dimer, and FDP. Similarly, compared with patients in the non-thrombosis group, those in the thrombosis group showed significantly elevated levels of the above mentioned markers. Logistic regression analysis showed that TAT, PIC, TM, t-PAIC, D-Dimer, and FDP were all associated with VTE. ROC analysis showed that "TAT+PIC+TM+t-PAIC+D-dimer+FDP"showed the highest sensitivity and specificity. Patients with elevated TAT, PIC, TM, and t-PAIC had a significantly shorter survival. Multivariate Cox survival analysis showed that TM and t-PAIC were significantly associated with poor prognosis. In addition, the incidence of VTE was significantly lower in patients with malignant tumors who were treated with low-molecular-weight heparin (LMWH), and their survival period was significantly longer than that of patients with malignant tumors who were not treated with LMWH. CONCLUSION: TAT, PIC, TM, and t-PAIC combined with D-dimer and FDP were better than the application of a single marker in the diagnosis of VTE in patients with malignant tumors. TAT and PIC can be used as sensitive markers in the diagnosis of VTE but not as prognostic markers. TM and t-PAIC might be independent prognostic indicators in patients with malignant tumors, regardless of the state of thrombus.

Viridicatol Isolated from Deep-Sea Penicillium Griseofulvum Alleviates Anaphylaxis and Repairs the Intestinal Barrier in Mice by Suppressing Mast Cell Activation.

Viridicatol is a quinoline alkaloid isolated from the deep-sea-derived fungus Penicillium griseofulvum. The structure of viridicatol was unambiguously established by X-ray diffraction analysis. In this study, a mouse model of ovalbumin-induced food allergy and the rat basophil leukemia (RBL)-2H3 cell model were established to explore the anti-allergic properties of viridicatol. On the basis of the mouse model, we found viridicatol to alleviate the allergy symptoms; decrease the levels of specific immunoglobulin E, mast cell protease-1, histamine, and tumor necrosis factor-α; and promote the production of interleukin-10 in the serum. The treatment of viridicatol also downregulated the population of B cells and mast cells (MCs), as well as upregulated the population of regulatory T cells in the spleen. Moreover, viridicatol alleviated intestinal villi injury and inhibited the degranulation of intestinal MCs to promote intestinal barrier repair in mice. Furthermore, the accumulation of Ca2+ in RBL-2H3 cells was significantly suppressed by viridicatol, which could block the activation of MCs. Taken together, these data indicated that deep-sea viridicatol may represent a novel therapeutic for allergic diseases.

The Risk Factors of VTE and Survival Prognosis of Patients With Malignant Cancer: Implication for Nursing and Treatment.

Venous thromboembolism (VTE) is very common in patients with malignant cancer. We aimed to conduct a retrospective analysis on the risk factors of VTE and its survival prognosis of patients with malignant cancer, to provide evidence into the management of VTE. Patients with malignant cancer treated in our hospital were selected. The characteristic of patients and related lab detection results including activated partial thromboplastin time (APTT), plasma prothrombin time (PT) and thrombin coagulation time (TT), fibrinogen (FIB), thrombin AT-Ⅲ complex (TAT) and D-dimer (D-D) were collected and analyzed. And logistic regression analyses were performed to identify the potential risk factors. And ROC curves were established to evaluate their predictive ability of VTE for patients with malignant cancers. A total of 286 patients were included, of which 63 patients had VTE, the incidence of VTE in patients with malignant cancers was 22.03%. There were significant differences on the D-D, TAT level between VTE and no VTE patients (all P < 0.05). The survival condition of VTE patients was significantly worse than that of no VTE patients(P = 0.017). D-D (RR7.895, 3.228∼19.286) and TAT (6.122, 2.244∼16.695) were risk factors of VTE for patients with cancers (all P < 0.05). The area under the curve (AUC) of D-D, TAT and combined use was 0.764, 0.698, 0.794 respectively, and the cutoff value for D-D, TAT was 1.835mg/L and 4.58µg/L respectively. For cancer patients with D-D >1.835 mg/L and TAT >4.58 µg/L, early interventions are needed for the prophylaxis of VTE.

Designed Parasite-Selective Rhomboid Inhibitors Block Invasion and Clear Blood-Stage Malaria.

Rhomboid intramembrane proteases regulate pathophysiological processes, but their targeting in a disease context has never been achieved. We decoded the atypical substrate specificity of malaria rhomboid PfROM4, but found, unexpectedly, that it results from "steric exclusion": PfROM4 and canonical rhomboid proteases cannot cleave each other's substrates due to reciprocal juxtamembrane steric clashes. Instead, we engineered an optimal sequence that enhanced proteolysis >10-fold, and solved high-resolution structures to discover that boronates enhance inhibition >100-fold. A peptide boronate modeled on our "super-substrate" carrying one "steric-excluding" residue inhibited PfROM4 but not human rhomboid proteolysis. We further screened a library to discover an orthogonal alpha-ketoamide that potently inhibited PfROM4 but not human rhomboid proteolysis. Despite the membrane-immersed target and rapid invasion, ultrastructural analysis revealed that single-dosing blood-stage malaria cultures blocked host-cell invasion and cleared parasitemia. These observations establish a strategy for designing parasite-selective rhomboid inhibitors and expose a druggable dependence on rhomboid proteolysis in non-motile parasites.

High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma.

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.

Short-Term Oral Anticoagulation Versus Antiplatelet Therapy Following Transcatheter Left Atrial Appendage Closure.

BACKGROUND: The impact of antithrombotic therapy on coagulation system activation after left atrial appendage closure (LAAC) remains unknown. This study sought to compare changes in coagulation markers associated with short-term oral anticoagulation (OAC) versus antiplatelet therapy (APT) following LAAC. METHODS: Prospective study including 78 atrial fibrillation patients undergoing LAAC with the Watchman device. F1+2 (prothrombin fragment 1+2) and TAT (thrombin-antithrombin III) were assessed immediately before the procedure, and at 7, 30, and 180 days after LAAC. RESULTS: Forty-eight patients were discharged on APT (dual: 31, single: 17) and 30 on OAC (direct anticoagulants: 26, vitamin K antagonists: 4), with no differences in baseline-procedural characteristics between groups except for higher spontaneous echocardiography contrast in the OAC group. OAC significantly reduced coagulation activation within 7 days post-LAAC compared with APT (23% [95% CI, 5%-41%] versus 82% [95% CI, 54%-111%] increase for F1+2, P=0.007; 52% [95% CI, 15%-89%] versus 183% [95% CI, 118%-248%] increase for TAT, P=0.048), with all patients in both groups progressively returning to baseline values at 30 and 180 days. Spontaneous echocardiography contrast pre-LAAC was associated with an enhanced activation of the coagulation system post-LAAC (144 [48-192] versus 52 [24-111] nmol/L, P=0.062 for F1+2; 299 [254-390] versus 78 [19-240] ng/mL, P=0.002 for TAT). Device-related thrombosis occurred in 5 patients (6.4%), and all of them were receiving APT at the time of transesophageal echocardiography (10.2% versus 0% if OAC at the time of transesophageal echocardiography, P=0.151). Patients with device thrombosis exhibited a greater coagulation activation 7 days post-LAAC (P=0.038 and P=0.108 for F1+2 and TAT, respectively). CONCLUSIONS: OAC (versus APT) was associated with a significant attenuation of coagulation system activation post-LAAC. Spontaneous echocardiography contrast pre-LAAC associated with enhanced coagulation activation post-LAAC, which in turn increased the risk of device thrombosis. These results highlight the urgent need for randomized trials comparing OAC versus APT post-LAAC.