Value of circulating neutrophil elastase for detecting recurrence of differentiated thyroid cancer.

Objective: The inflammatory microenvironment has been implicated in differentiated thyroid cancer (DTC). Inflammatory stimuli induce the release of components of neutrophils into extracellular space, leading to formation of neutrophil extracellular trap (NET), which can stimulate growth and progression of cancer. Generation of activated factor XII and thrombin is also involved in cancer progression. This study attempted to determine whether the level of circulating markers of NET, activated factor XII, and endogenous thrombin potential may be useful for detecting the recurrence of DTC. Methods: A total of 122 patients with DTC were recruited during the postoperative follow-up period. Measurement of the levels of circulating markers of NET (neutrophil elastase, histone-DNA complex, cell-free dsDNA), activated factor XII, and endogenous thrombin potential was performed. Results: A significantly elevated level of neutrophil elastase was detected in patients with recurrence (n = 12) compared to those without recurrence (n = 110), while significant elevation of the levels of other markers was not observed. The value for area under the curve (0.717, P = 0.018) of neutrophil elastase for detecting recurrence of DTC was superior to that (0.661, P = 0.051) of serum thyroglobulin. An elevated level of neutrophil elastase was significantly associated with recurrence of DTC independent of serum thyroglobulin. Conclusions: Because an elevated level of neutrophil elastase was detected in patients with recurrence of DTC and showed a significant association with recurrence of DTC, it can be proposed as a novel biomarker for use in detecting recurrence of DTC along with other tests.

Distinct mutational pattern of T-cell large granular lymphocyte leukemia combined with pure red cell aplasia: low mutational burden of STAT3.

T-cell large granular lymphocyte leukemia (T-LGL) is often accompanied by pure red cell aplasia (PRCA). A high depth of next generation sequencing (NGS) was used for detection of the mutational profiles in T-LGL alone (n = 25) and T-LGL combined with PRCA (n = 16). Beside STAT3 mutation (41.5%), the frequently mutated genes included KMT2D (17.1%), TERT (12.2%), SUZ12 (9.8%), BCOR (7.3%), DNMT3A (7.3%), and RUNX1 (7.3%). Mutations of the TERT promoter showed a good response to treatment. 3 of 41 (7.3%) T-LGL patients with diverse gene mutations were revealed as T-LGL combined with myelodysplastic syndrome (MDS) after review of bone marrow slide. T-LGL combined with PRCA showed unique features (low VAF level of STAT3 mutation, low lymphocyte count, old age). Low ANC was detected in a STAT3 mutant with a low level of VAF, suggesting that even the low mutational burden of STAT3 is sufficient for reduction of ANC. In retrospective analysis of 591 patients without T-LGL, one MDS patient with STAT3 mutation was revealed to have subclinical T-LGL. T-LGL combined with PRCA may be classified as unique subtype of T-LGL. High depth NGS can enable sensitive detection of concomitant MDS in T-LGL. Mutation of the TERT promoter may indicate good response to treatment of T-LGL, thus, its addition to an NGS panel may be recommended.

Comparison of the International Normalized Ratio Between a Point-of-Care Test and a Conventional Laboratory Test: the Latter Performs Better in Assessing Warfarin-induced Changes in Coagulation Factors.

Background: Point-of-care testing (POCT) coagulometers are increasingly used for monitoring warfarin therapy. However, in high international normalized ratio (INR) ranges, significant discrepancy in the INR between POCT and conventional laboratory tests occurs. We compared the INR of POCT (CoaguChek XS Plus; Roche Diagnostics, Mannheim, Germany) with that of a conventional laboratory test (ACL TOP 750; Instrumentation Laboratory SpA, Milan, Italy) and explored possible reasons for discrepancy. Methods: Paired POCT and conventional laboratory test INRs were analyzed in 400 samples from 126 patients undergoing warfarin therapy after cardiac surgery. Coagulation factor and thrombin generation tests were compared using the Mann-Whitney U test. Correlations between coagulation factors and INRs were determined using Pearson correlation coefficients. Results: The mean difference in the INR between the tests increased at high INR ranges. Endogenous thrombin potential levels were decreased at INR <2.0 for CoaguChek XS Plus and 2.0< INR <3.0 for ACL TOP 750 compared with those at INR <2.0 for both tests, indicating a better performance of ACL TOP 750 in assessing thrombin changes. The correlation coefficients of coagulation factors were stronger for ACL TOP 750 INR than for CoaguChek XS Plus INR. Vitamin K-dependent coagulation factors were found to contribute to the INR discrepancy. Conclusions: Decreases in vitamin K-dependent coagulation and anticoagulation factors can explain the significant discrepancy between the two tests in high INR ranges. Since conventional laboratory test INR values are more reliable than POCT INR values, a confirmatory conventional laboratory test is required for high INR ranges.

Assessment of Rotational Thromboelastometry and Thrombin Generation Assay to Identify Risk of High Blood Loss and Re-Operation After Cardiac Surgery.

Introduction: We aimed to investigate parameters for prediction of post-operative blood loss and re-operation in patients who underwent cardiopulmonary bypass. Methods: Thrombin generation assay, activated partial thromboplastin time, activated clotting time and rotational thromboelastometry (ROTEM) tests were performed at 4 time points in 65 patients: before skin incision (T1), after heparin injection (T2), after protamine reversal (T3) and before skin closure (T4). Results: Pre-operative endogenous thrombin potential (ETP) and peak thrombin levels were significantly lower in patients with high post-operative blood loss (≥ 800 mL) within 24 h than in those with low blood loss (< 800 mL). Clotting time (CT), maximal clotting firmness, clotting firmness time and alpha angle values of ROTEM measured at T2, T3 or T4 were significant predictors for high post-operative blood loss. An increase in CT-EXTEM over 4 time points was significant in patients who had a re-operation within 48 h compared to their counterparts. Conclusions: This study indicates that pre-operative ETP could predict high post-operative blood loss and that intra-operative ROTEM also helps to stratify risks of high post-operative blood loss and re-operation.

High Circulating Levels of Neutrophil Extracellular Traps Parameters Predicting Poor Outcome in COVID-19.

OBJECTIVE: Exploration of biomarkers to predict the severity of COVID-19 is important to reduce mortality. Upon COVID-19 infection, neutrophil extracellular traps (NET) are formed, which leads to a cytokine storm and host damage. Hence, the extent of NET formation may reflect disease progression and predict mortality in COVID-19. METHODS: We measured 4 NET parameters - cell-free double stranded DNA (cell-free dsDNA), neutrophil elastase, citrullinated histone H3 (Cit-H3), and histone - DNA complex - in 188 COVID-19 patients and 20 healthy controls. Survivors (n=166) were hospitalized with or without oxygen supplementation, while non-survivors (n=22) expired during in-hospital treatment. RESULTS: Cell-free dsDNA was significantly elevated in non-survivors in comparison with survivors and controls. The survival rate of patients with high levels of cell-free dsDNA, neutrophil elastase, and Cit-H3 was significantly lower than that of patients with low levels. These three markers significantly correlated with inflammatory markers (absolute neutrophil count and C-reactive protein). CONCLUSION: Since the increase in NET parameters indicates the unfavourable course of COVID-19 infection, patients predisposed to poor outcome can be rapidly managed through risk stratification by using these NET parameters.

Real-world evidence of lupus anticoagulant testing: simultaneous positivity of diluted Russell's viper venom time and silica clotting time increases thrombotic risk prediction.

Lupus anticoagulant (LA) is composed of heterogeneous autoantibodies, which have a close association with thrombotic events. Due to its heterogeneity, two methods for increasing sensitivity are recommended for LA. An investigation of the thrombotic risk and anticardiolipin (aCL) and anti-ß2-glycoprotein I (aB2GPI) antibody profiles was conducted based on the results of using two parallel methods (dilute Russell viper venom time (dRVVT), silica clotting time (SCT)) in a real world clinical laboratory. Of 5120 patients, 684 patients (13%) were LA positive, and 422 patients (8%) experienced thrombotic events including pregnancy complication. Development of thrombotic events was more likely to occur in patients who were positive for both dRVVT and SCT compared with those who were positive for dRVVT or SCT only. In addition, significantly higher positive rates of aCL and aB2GPI and the persistently positive rate of LA at intervals of 12 weeks or longer were observed in patients who were positive for both dRVVT and SCT compared with those who were positive for dRVVT or SCT only. Considering three laboratory tests (LA, aCL, and aB2GPI), high thrombotic risk was observed for patients with both dRVVT and SCT positive LA results. A report on LA results that divides LA positive into two types (LA-single positive and LA-both positive) may be beneficial to clinicians in detection of high-risk thrombotic patients.

Contact system activation in disseminated intravascular coagulation: activities of prekallikrein and high-molecular-weight kininogen are significant risk factors.

The contact system activation can play a role in microthrombus formation of disseminated intravascular coagulation (DIC). This study investigated whether the activity of prekallikrein and high-molecular-weight kininogen (HMWK) correlated DIC progression. Contact system factors (prekallikrein, HMWK, activated factor XII), coagulation factors (IX, XI, XII) and tissue factor were measured in 140 patients who clinically suspected of having DIC. Prekallikrein and HMWK activity levels showed significant linear relationships with DIC score and antithrombin level, whereas prekallikrein and HMWK antigen levels did not. The activated factor XII, factor XII, factor XI and tissue factor were significant risk factors of overt-DIC. This finding suggests that consumption of prekallikrein and HMWK contributes to microvascular thrombosis in DIC. Measurements of prekallikrein and HMWK activity could be used as potential diagnostic markers for overt-DIC.

Diagnostic and prognostic role of circulating neutrophil extracellular trap markers and prekallikrein in patients with high-grade serous ovarian cancer.

Objective: Tumor-promoting inflammation is among the hallmarks of cancer. Prekallikrein is among the acute-phase reactants in the inflammatory response; moreover, neutrophils release nuclear contents into the extracellular space to create neutrophil extracellular traps (NET). We aimed to investigate the diagnostic and prognostic utilities of circulating plasma NET markers and prekallikrein for high-grade serous ovarian cancer (HGSOC). Methods: Circulating levels of three NET markers (histone-DNA complex, cell-free DNA, and neutrophil elastase) and prekallikrein were measured in 75 patients with HGSOC and 23 healthy controls. We used an area under the receiver operating characteristic curve (AUC) analysis to investigate their diagnostic and prognostic utilities for HGSOC. Results: Compared with healthy controls, patients with HGSOC showed significantly higher levels of the three NET markers and prekallikrein. Patients with advanced-stage HGSOC showed significantly higher levels of the cell-free DNA (87.4 vs. 79.5 ng/ml; P = 0.013), compared with those with early-stage HGSOC. Further, the levels of histone-DNA complex, neutrophil elastase, and prekallikrein did not significantly differ according to the cancer stage. All markers showed significant diagnostic utility. Notably, a logistic regression-based model that comprised all four markers showed the strongest diagnostic power (AUC, 0.966; 95% confidence interval [CI], 0.933-1.000). Specifically, neutrophil elastase was identified as an independent poor prognostic factor for overall survival (adjusted hazard ratio [aHR], 10.17; 95% CI, 1.09-94.97; P = 0.042) and progression-free survival (aHR, 14.47; 95% CI, 1.52-137.35; P = 0.020) in patients with HGSOC. Conclusions: The levels of the three NET markers and prekallikrein might be novel diagnostic and prognostic markers for HGSOC.

Activation of Factor XII and Kallikrein-Kinin System Combined with Neutrophil Extracellular Trap Formation in Diabetic Retinopathy.

BACKGROUND: In diabetic retinopathy (DR), neutrophil extracellular traps (NET) and kallikrein-kinin system are considered as contributing factors. However, the detail activation mechanisms has not been fully understood. Since the NET could provide negative-charged surface for factor XII activation and the activated factor XII (XIIa) can initiate kallikrein-kinin system, this study investigated whether patients with DR show activation of NET, factor XII and kallikrein-kinin system. METHODS: The markers related to NET (DNA-histone complex) and kallikrein-kinin system (high-molecular-weight kininogen, prekallikrein, bradykinin) and factor XIIa were measured in 253 patients with diabetes. To access ex vivo effect of glucose, DNA-histone complex and factor XIIa were measured in whole blood stimulated by glucose. RESULTS: The circulating level of DNA-histone complex and factor XIIa were significantly higher in patients with DR than those without DR. In logistic regression analysis, DNA-histone complex, factor XIIa, and high-molecular-weight kininogen were the risk factors of DR. In recursive partitioning analysis, among patients with diabetes duration less than 10 years, patients with high level of DNA-histone complex (>426 AU) showed high risk of DR. In ex vivo experiment, glucose significantly elevated both DNA-histone complex and factor XIIa. CONCLUSION: Our findings suggest that activation of factor XII and kallikrein-kinin system combined with NET formation actively occur in patients with DR and circulating levels of DNA-histone complex, factor XIIa and HMWK can be potential biomarkers to estimate the risk of DR. Strategies against factor XII activation may be beneficial to inhibit DR.

Thrombotic Risk of Non-Criteria Anti-Phospholipid Antibodies Measured by Line Immunoassay: Superiority of Anti-Phosphatidylserine and Anti-Phosphatidic Acid Antibodies.

BACKGROUND: Increasing interest has focused on the development of new assays more specific for APS and application of multiple combinations of anti-phospholipid antibodies (aPLs). This study explored the thrombotic risk of non-criteria aPLs measured by a new line immunoassay (LIA) and the benefit of additional non-criteria aPLs results to the APS diagnosis. METHODS: LIA were performed to detect 9 aPLs in 180 patients requested for lupus anticoagulant (LA) measurement. Antibodies against anti-cardiolipin (CL), ß2 glycoprotein I (GPI), and ß2GPI domain I were measured by ELISA. RESULTS: The agreement percentages for IgG/IgM anti-CL and anti-ß2GPI between ELISA and LIA (anti-CL 68.2% and 82.6%; anti-ß2GPI 71.7% and 93.2%, respectively). Among 9 aPLs measured by LIA, single presence IgG of anti-phosphatidylserine (odds ratio (OR) 16.477) and anti-phosphatidic acid (OR 9.625) predicted higher thrombotic risk than anti-ß2GPI (OR 5.538). Other aPLs measured by LIA (anti-prothrombin, anti-annexin V, anti-phosphatidylinositol, phosphatidylethanolamine, and anti-phosphatidylglycerol) did not show any significant thrombotic risk. Addition of the 2 non-criteria aPLs (anti-phosphatidylserine and anti-phosphatidic acid) to the established APS criteria increased the diagnostic specificity and accuracy for thrombosis. The positive rates of anti-ß2GPI and anti-phosphatidylserine measured by LIA were quite high in patients with positive anti-ß2GPI domain I. CONCLUSIONS: The anti-phosphatidylserine and anti-phosphatidic acid among non-criteria aPLs have a high likeli-hood as new markers for thrombotic prediction.

Contact System Activation and Neutrophil Extracellular Trap Markers: Risk Factors for Portal Vein Thrombosis in Patients With Hepatocellular Carcinoma.

Although portal vein thrombosis (PVT) commonly occurs in patients with hepatocellular carcinoma (HCC), the hypercoagulability mechanism in patients with HCC is not entirely clear. Recently, tumor-induced formation of neutrophil extracellular traps (NET) has been shown to trigger contact system activation, and contact system activation has been shown to be a new mechanism of thrombosis. Therefore, we investigated whether contact system activation and NET formation occurred in relation to PVT in HCC patients. The circulating levels of NET formation markers (DNA-histone complex, double-stranded DNA, neutrophil elastase) and contact system activation markers (factor XIIa and high-molecular-weight kininogen) were measured in 177 patients who had been diagnosed with HCC and 48 healthy controls. Presence of PVT was confirmed in 77 HCC patients. The levels of NET formation and contact system activation markers were significantly higher in patients than in healthy controls and they increased significantly with the increase in the model for end-stage liver disease (MELD) scores. Of note, these markers were significantly higher in HCC patients with PVT than in those without PVT. These NET formation markers and the contact system activation markers were significant thrombotic risk factors in HCC patients. The well-known liver injury markers (alanine transaminase, prothrombin time) significantly contributed to factor XIIa level. Contact system activation and NET formation are well correlated with liver disease severity and the markers of these can be used as thrombotic risk factors in HCC patients. In addition, therapeutics inhibiting the contact system can be potentially used to manage PVT in HCC patients.

Elevated extracellular trap formation and contact system activation in acute leukemia.

Leukemic cells release their nuclear contents into the extracellular space upon activation. The released nuclear contents, called extracellular traps, can activate the contact system of coagulation. This study accessed the extent of contact system activation, the levels of extracellular traps, and coagulation activation in hematologic malignancies including acute leukemia. In 154 patients with hematologic malignancies (acute leukemia, n = 29; myelodysplastic syndrome, n = 20; myeloproliferative neoplasms, n = 69; plasma cell myeloma, n = 36) and 48 normal controls, the levels of coagulation factors (fibrinogen and factor VII, VIII, IX, and XII), D-dimer, thrombin generation, extracellular trap markers (histone-DNA complex, cell-free dsDNA, leukocyte elastase), and contact system markers (activated factor XII [XIIa], high-molecular-weight kininogen, prekallikrein, bradykinin) were measured. Patients with acute leukemia showed the highest levels of peak thrombin, extracellular trap markers, and factor XIIa. Factor XIIa level was significantly associated with the presence of acute leukemia. The histone-DNA complex and cell-free dsDNA were revealed as significant associated factors with the factor XIIa level. Three markers of extracellular traps and two markers of thrombin generation significantly contributed to the hemostatic abnormalities in hematologic malignancies. Contact system was activated in acute leukemia and its activation was significantly associated with the extent of extracellular trap formation. This finding suggests that extracellular traps might be a major source of contact system activation and therapeutic strategies targeting extracellular trap formation or contact system activation may be beneficial in acute leukemia.

Thrombin Generation Assay Detects Moderate-Intensity Statin-Induced Reduction of Hypercoagulability in Diabetes.

Statins not only have a lipid-lowering effect but also reduce inflammation and have an antithrombotic effect. Since hypercoagulability assessed by thrombin generation assay (TGA) and increased formation of neutrophil extracellular traps (NET) were demonstrated in diabetes, we investigated whether statin therapy in diabetes modifies coagulation status and NET formation. Twenty-five consecutive patients with diabetes were recruited. Global coagulation assays (prothrombin time [PT], activated partial thromboplastin time [aPTT], and TGA) and NET markers (DNA-histone complex, cell-free DNA, and neutrophil elastase) were measured before and after 3-month moderate-intensity statin therapy. In addition, all coagulation factors and 3 anticoagulation factors were measured. Statin therapy significantly reduced endogenous thrombin potential (ETP) value and blood lipids but did not change the PT and aPTT values or NET formation markers. Statin significantly decreased not only coagulation factors (II, V, VIII, IX, and X) but also the anticoagulation factor antithrombin. Statin-induced reduction of factor V and X significantly contributed to the reduction of ETP value. The extent of reduction in coagulation factors correlated with that of anticoagulation factors, but not that of cholesterol. It is possible to use TGA as a global coagulation assay that can detect coagulation status modified by statin therapy. Additional studies are needed to evaluate the clinical implications of statin-induced simultaneous reduction of coagulation and anticoagulation factors.

Increased plasma viscosity in plasma cell dyscrasia and whole blood viscosity in polycythemia vera.

BACKGROUND: Although hyperviscosity syndrome in plasma cell dyscrasia (PCD) and thrombosis in myeloproliferative neoplasm (MPN) are major causes of morbidity and mortality, blood viscosity measurements are often underutilized. OBJECTIVE: This study aimed to characterize whether whole blood viscosity (WBV) or plasma viscosity (PV) could be predictive of hyperviscosity syndrome in PCD and could be elevated in subgroups of MPN. METHODS: A total of 75 patients with hematologic diseases: PCD (n = 26), MPN (n = 25) including polycythemia vera (P. vera) and lymphoma (n = 24) were enrolled along with 104 healthy controls. Both WBV and PV were measured using a capillary tube viscometer. Hyperviscosity syndrome was defined as having 2 or more hyperviscosity symptoms. RESULTS: Patients with PCD showed significantly higher PVs at high and low shear rates when compared to healthy controls, especially in those with hyperviscosity syndrome. The sensitivity and specificity of WBV and PV in detecting hyperviscosity syndrome were 28.6% and 94.1%, and 71.4% and 66.7%, respectively. Patients with P. vera exhibited high WBV and RBC counts compared to healthy controls. CONCLUSION: PV is predictive of hyperviscosity syndrome in PCD and WBV is elevated in patients with P. vera. It suggests that hemorheologic disturbances exist in patients with PCD and MPN and that tests of viscosity may be helpful in detecting hemorheological disturbances.

Benefits of Thromboelastography and Thrombin Generation Assay for Bleeding Prediction in Patients With Thrombocytopenia or Hematologic Malignancies.

BACKGROUND: Thromboelastography (TEG) provides comprehensive information on the whole blood clot formation phases, whereas thrombin generation assay (TGA) reveals the endogenous thrombin levels in plasma. We investigated the potential significance of TEG and TGA parameters for prediction of clinical bleeding in hematologic patients on the basis of the patient's platelet levels. METHODS: TEG and TGA were performed in 126 patients with thrombocytopenia or hematologic malignancies. The bleeding tendencies were stratified on the basis of the World Health Organization bleeding grade. RESULTS: Maximum amplitude (MA) and clot formation in TEG and endogenous thrombin potential (ETP) in TGA showed significant associations with high bleeding grades (P=0.001 and P=0.011, respectively). In patients with platelet counts ≤10×109/L, low MA values were strongly associated with a high bleeding risk. For bleeding prediction, the area under the curve (AUC) of MA (0.857) and ETP (0.809) in patients with severe thrombocytopenia tended to be higher than that of platelets (0.740) in all patients. Patients with platelet counts ≤10×109/L displayed the highest AUC of the combined MA and ETP (0.929). CONCLUSIONS: Both TEG and TGA were considered to be good predictors of clinical bleeding in patients with severe thrombocytopenia. Combination of the ETP and MA values resulted in a more sensitive bleeding risk prediction in those with severe thrombocytopenia.

Contact system activation and high thrombin generation in hyperthyroidism.

BACKGROUND: Hyperthyroidism is associated with increased thrombotic risk. As contact system activation through formation of neutrophil extracellular traps (NET) has emerged as an important trigger of thrombosis, we hypothesized that the contact system is activated along with active NET formation in hyperthyroidism and that their markers correlate with disease severity. SUBJECTS AND METHODS: In 61 patients with hyperthyroidism and 40 normal controls, the levels of coagulation factors (fibrinogen, and factor VII, VIII, IX, XI and XII), D-dimer, thrombin generation assay (TGA) markers, NET formation markers (histone-DNA complex, double-stranded DNA and neutrophil elastase) and contact system markers (activated factor XII (XIIa), high-molecular-weight kininogen (HMWK), prekallikrein and bradykinin) were measured. RESULTS: Patients with hyperthyroidism showed higher levels of fibrinogen (median (interquartile range), 315 (280-344) vs 262 (223-300), P = 0.001), D-dimer (103.8 (64.8-151.5) vs 50.7 (37.4-76.0), P < 0.001), peak thrombin (131.9 (102.2-159.4) vs 31.6 (14.8-83.7), P < 0.001) and endogenous thrombin potential (649 (538-736) vs 367 (197-1147), P = 0.021) in TGA with 1 pM tissue factor, neutrophil elastase (1.10 (0.39-2.18) vs 0.23 (0.20-0.35), P < 0.001), factor XIIa (66.9 (52.8-87.0) vs 73.0 (57.1-86.6), P < 0.001), HMWK (6.11 (4.95-7.98) vs 3.83 (2.60-5.68), P < 0.001), prekallikrein (2.15 (1.00-6.36) vs 1.41 (0.63-2.22), P = 0.026) and bradykinin (152.4 (137.6-180.4) vs 118.3 (97.1-137.9), P < 0.001) than did normal controls. In age- and sex-adjusted logistic regression analysis, fibrinogen, factor VIII, IX and XIIa, D-dimer, peak thrombin, neutrophil elastase, HMWK and bradykinin showed significant odds ratios representing hyperthyroidism's contribution to coagulation and contact system activation. Free T4 was significantly correlated with factors VIII and IX, D-dimer, double-stranded DNA and bradykinin. CONCLUSION: This study demonstrated that contact system activation and abundant NET formation occurred in the high thrombin generation state in hyperthyroidism and were correlated with free T4 level.

Porcine endothelium induces DNA-histone complex formation in human whole blood: a harmful effect of histone on coagulation and endothelial activation.

BACKGROUND: Neutrophils play a role in xenograft rejection. When neutrophils are stimulated, they eject the DNA-histone complex into the extracellular space, called neutrophil extracellular traps (NET). We investigated whether NET formation actively occurs in the xenograft and contributes to coagulation and endothelial activation. METHODS: Human whole blood was incubated with porcine aortic endothelial cells (pEC) from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. In the supernatant plasma from human blood, the level of the DNA-histone complex was measured by ELISA, and thrombin generation was measured using a calibrated automated thrombogram. Histone-induced tissue factor and adhesion molecule expression were measured by flow cytometry. RESULTS: pEC from both wild-type and GTKO pigs significantly induced DNA-histone complex formation in human whole blood. The DNA-histone complex produced shortened the thrombin generation time and clotting time. Histone alone dose-dependently induced tissue factor and adhesion molecule expression in pEC. Aurintricarboxylic acid pretreatment partially inhibited pEC-induced DNA-histone complex formation. CONCLUSIONS: DNA-histone complex actively generated upon xenotransplantation is a novel target to inhibit coagulation and endothelial activation. To prevent tissue factor and adhesion molecule expression, a strategy to block soluble histone may be required in xenotransplantation.

Histones Induce the Procoagulant Phenotype of Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-Regulation.

The high circulating levels of histones found in various thrombotic diseases may compromise the anticoagulant barrier of endothelial cells. We determined how histones affect endothelial procoagulant tissue factor (TF) and anticoagulant thrombomodulin (TM). Surface antigens, soluble forms, and mRNA levels of TF and TM were measured by flow cytometry, ELISA, and real-time RT-PCR, respectively. TF and TM activity were measured using procoagulant activity, thrombin generation, or chromogenic assays. Involvement of the toll-like receptor (TLR) was assessed using the neutralizing antibodies. Histones dose-dependently induced surface antigens, activity and mRNA levels of endothelial TF. Histone-treated endothelial cells significantly shortened the lag time and enhanced the endogenous thrombin potential of normal plasma, which was normalized by a TF neutralizing antibody. Histones induced phosphatidylserine and protein-disulfide isomerase expression in endothelial cells. Histones also reduced the surface antigen, activity, and mRNA levels of endothelial TM. Polysialic acid and heparin reversed the histone-induced TF up-regulation and TM down-regulation. Activated protein C did not affect the TF up-regulation, but interrupted TM down-regulation. TLR2, and TLR4 inhibitors partially blocked the TF up-regulation. Histones induced the endothelial procoagulant phenotype through TF up-regulation and TM down-regulation. The effects of histones were partly mediated by TLR2, TLR4. Strategies to inhibit the harmful effects of histones in endothelial cells may be required in order to prevent a thrombotic environment.