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Background: The ISTH-SSC guidelines for lupus anticoagulant (LA) testing recommend using in-house determined cut-off values, pooled normal plasma (PNP) for ratio normalization, and a ratio for the mixing test interpretation. They strongly support the mixing step role in the diagnostic process., Objectives: To investigate and compare the LA testing results and interpretations obtained following the ISTH-SSC guidelines or the available alternatives., Patients/methods: Blood samples for LA testing from 462 consecutive patients were evaluated for screening, mixing and confirmatory tests. The analysis focused on the interpretation differences between using (1) the in-house cut-off values versus the manufacturer's cut-off values, (2) a normalized ratio calculated using PNP at each run versus the mean of the reference interval, (3) a normalized ratio versus the index of circulating anticoagulant to interpret the mixing step, and (4) a two-step versus three-step procedure., Results: LA testing outcomes were comparable when using the in-house and manufacturer's cut-off values. More positive dilute Russell's viper venom (DRVV) time results were obtained with the normalized ratio based on PNP than with the mean of the reference interval. Overall, the mixing test results obtained with the normalized ratio and the index of circulating anticoagulant showed a good agreement. Among the 97 DRVV Screen test-positive samples, 33 and 89 were classified as LA-positive with the 3-step and the 2-step procedure, respectively., Conclusions: The cut-off value used and the way to normalize ratios had a limited impact. Conversely, it is important to understand the mixing test characteristics to maximize its diagnostic potential., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Continuous intravenous unfractionated heparin (UFH) is a mainstay of therapeutic anticoagulation in the acute setting. The two most common laboratory tests for monitoring UFH are the activated partial thromboplastin time (aPTT) and antifactor Xa (anti-Xa) heparin assay. We reviewed the available evidence to evaluate if the choice of monitoring test for UFH therapy is associated with a difference in the clinical outcomes of bleeding, thrombosis, or mortality., Materials and Methods: MEDLINE, Cochrane database, and conference abstracts from the Society of Critical Care Medicine, the American Society of Hematology, and the American College of Clinical Pharmacy were searched for all studies comparing aPTT and anti-Xa monitoring for therapeutic UFH that evaluated outcomes for bleeding, thrombotic events, or mortality. Risk of bias was assessed with the Cochrane Risk of Bias Tool and Newcastle Ottawa Scale. Pooled relative risk ratios were calculated using an inverse variance-weighted random-effects model., Results: Ten studies (n = 6677) were included for analysis. The use of anti-Xa compared to aPTT was not associated with an increased risk of bleeding (RR 1.03; 95% CI 0.8-1.22 I 2  = 4%) or an increased risk of thrombotic events (RR 0.99; 95% CI 0.76-1.30, I 2  = 3%). There was no difference in mortality within individual studies but the data were not suitable for pooled analysis., Conclusions: Pooled data comparing aPTT vs. anti-Xa for monitoring therapeutic UFH did not suggest differences in the outcomes of bleeding or thrombosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: Bleeding after major hepatobiliary pancreatic (HBP) surgery may be serious. Although postoperative abnormality of the hemostatic system are important elements that affect bleeding, routine activated partial thromboplastin time (APTT) assessment is considered inadequate to predict massive bleeding (MB). Recently, APTT-clot waveform analysis (CWA) was reported to be useful for detecting coagulation disorders., Methods: APTT-CWA was performed using the ACL-TOP analyzer in 188 patients who underwent four major HBP surgeries (distal pancreatectomy, hepatectomy, subtotal stomach-preserving pancreatoduodenectomy (SSPPD), and SSPPD with combined resection and reconstruction of the portal vein) to analyze its usefulness in predicting the risk of bleeding., Results: Seventy (37.2%) patients developed MB and the incidence of MB was highest among patients who underwent hepatectomy. There were no significant differences in routine APTT, the first derivative peak (DP) time and 1/2 fibrin formation peak time between patients with MB and those without MB, throughout the postoperative course. On the other hand, the first and second DP heights were significantly lower in patients with MB than in those without MB and lowest in patients who underwent hepatectomy., Conclusion: APTT-CWA was able to detect the detailed changes in the hemostatic system after major HBP surgery. The patterns of APTT-CWA after major HBP surgery differed among various surgical procedures according to invasiveness. The lower first and the second DP height, which were frequently observed in hepatectomy patients, may be useful for predicting the risk of MB., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas. To do so, a side-by-side evaluation of the extrinsic and intrinsic pathway of coagulation was performed by means of PT, APTT, and CAT measurements on (diluted) pooled plasmas from goats, pigs, rabbits, rats, mice, and humans. Plasma anti-FXa activity was assessed by determining the rate of recombinant hFXa inhibition through chromogenic activity analyses and immunoblotting. In general, rabbit, rat, and mouse plasmas exhibited robust clotting upon stimulation of both the extrinsic and intrinsic pathway, produced more thrombin during CAT upon plasma dilution, and displayed relatively high hFXa inhibitory activities. By comparison, goat, porcine, and human plasma displayed a similar profile in PT and APTT assays, produced less thrombin during CAT upon plasma dilution, and displayed comparable hFXa inhibitory activities. In conclusion, the observed differences in clotting parameters and anti-hFXa activity point to a higher anticoagulant threshold in plasma from rabbits, rats, and particularly in mice relative to human, goat, and porcine plasma. Finally, rat plasma was found to be more relevant to the preclinical assessment of human FX(a) in comparison to murine plasma., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Several routine coagulation tests have been developed to give insight in the coagulation pathway. The laboratory diagnostical process consists of 3 phases, the pre-analytical, analytical and post-analytical phase; however, the pre-analytical phase is most sensitive to errors. The amount of time blood is stored, can affect the measurements of these coagulation tests and result in an incorrect conclusion. Therefore, we performed experiments to determine the maximal storage time, centrifuged blood samples can be reliably measured., Methods: Citrated whole blood from hospital patients, who were tested for routine coagulation, was collected in 2.7 mL citrate tubes. These whole blood samples were centrifuged and the plasma was stored on top of the cell pellet at room temperature. After 2 h, 4 h, 6 h, 12 h and 24 h, the prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen concentration, antithrombin activity, D-dimer concentration and thrombin time were measured using Sysmex CS2100 coagulation analysers., Results and Conclusion: Analytical evaluation of routine coagulation tests resulted in various significant differences and large variations between the various time intervals. Our results indicated that the PT and INR can be measured up till 24 h of storage. Centrifuged blood for measuring the fibrinogen concentration, antithrombin activity, D-dimer concentration and thrombin time can be stored up to 4 h, while 2 h of storage might already be too long for obtaining reliable aPTT measurements., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Rare coagulation disorders (RCDs) are a group of diseases due to coagulation factors deficiency leading to life-long bleeding diathesis. The diagnosis of RCDs is challenging due to the limited knowledge of these disorders and the large heterogeneity of their bleeding patterns. The clinical symptoms of RCDs are extremely diverse in terms of bleeding type, site, severity, age at onset, and duration. The strength of the association between clotting factor activity level in plasma and clinical symptoms is also variable within each RCD. The clinical evaluation of RCDs starts with a detailed collection of clinical history and has been facilitated by bleeding assessment tools, however their effectiveness in diagnosing RCDs requires further investigation. The following laboratory diagnosis of RCDs involves coagulation screening tests, including activated partial thromboplastin time, prothrombin time, and thrombin time. After ruling out the presence of an inhibitor by mixing studies, in case of abnormal results, the specific deficiency is identified by performing one-stage clotting assays using the specific factor-depleted plasmas as substrate. In fibrinogen and FXIII deficiencies coagulation screening tests are not informative, therefore additional tests are needed. Global assays have been developed and are thought to aid in patient management, however, they are not well standardized yet. In addition to outlining the principles of clinical and laboratory diagnosis, this review explores molecular basis of RCDs and laboratory techniques for genetic analysis, and discusses the importance and effectiveness of quality control programs to ensure standardized laboratory results., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Direct oral anticoagulants targeting factor Xa (DXaIs) are administered as prophylaxis for various venothrombotic diseases without routine monitoring required. However, assessment of their anticoagulant effects is necessary to prevent severe events, including major bleeding and/or refractory thrombosis., Objectives: We examined the correlation of ratio of inhibited thrombin generation (RITG), determined using a novel assay based on dilute prothrombin time (dPT), with coagulant markers and laboratory test results to show drug effects. In addition, RITG usefulness as a confirmation test for DXaI therapy was investigated., Methods: Citrated plasma samples were obtained from patients treated with rivaroxaban (n = 882), apixaban (n = 1214), or edoxaban (n = 820) at 4 different institutions in Japan. Laboratory tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimer, and plasma concentrations of DXaIs, were conducted, with drug concentrations divided into peak and trough groups, within and after 5 h of administration., Results: In each DXaI group, RITG was positively correlated with PT, APTT, and drug concentration, and negatively with D-dimer. RITG fluctuation during the peak and trough periods reflected the anticoagulant activity characteristic of each DXaI, which was different from blood concentration fluctuations. RITG showed a significant decrease in cases with thrombosis, while that was increased in those with hemorrhage., Conclusion: We developed RITG, a novel measurement method based on dPT. RITG represents residual coagulation ability in plasma samples, and is useful for assessment of bleeding and thrombotic tendencies in DXaI patients. RITG can be utilized to confirm the effectiveness of oral anticoagulation therapy with DXaI agents., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Malayan pit vipers (Calloselasma rhodostoma) are major health hazard in Southern Thailand causing systemic bleeding by defibrination and thrombocytopenia. Twenty minute whole blood clotting test (20WBCT) is a useful and informative bedside test recommended by WHO for diagnosis of significant coagulopathy following snakebite envenoming since it to some extent predicts the need for anti-venom therapy. Prothrombin time (PT), international normalized ratio (INR) and activated partial thromboplastin time (APTT) are standard clotting assays that are widely used for diagnosis of coagulopathy. The purpose of this study is to compare 20WBCT with the standard clotting assays (PT, INR, APTT, fibrinogen) in management of Malayan pit viper bitten-patients. We studied 296 victims prospectively during a 3-year period. In total, 115 (38.9%) cases showed a positive 20WBCT (un-clot) suggesting systemic envenomation. Thrombocytopenia (platelet count less than 100,000/μL), prolonged PT, prolonged APTT, INR ≥ 1.2, and fibrinogen level <100 mg/dl were found in 12.2%, 47.6%, 18.9%, 34.8% and 40.9%, respectively. Significant predictors for thrombocytopenia by multivariate analysis included systemic bleeding (p < 0.001), higher INR (p < 0.001), 20WBCT showing unclotted blood (p = 0.049), and longer bite-to-hospital time (p = 0.031). When a fibrinogen level below 100 mg/dl was determined as hypofibrinogenemic condition, INR had the highest area under ROC curve followed by 20WBCT and APTT, respectively, to identify such as hypofibrinogenemic condition. The diagnostic accuracy of 20WBCT and INR at or more than 1.155 were better than APTT at any cut-off points. INR >1.155 had a sensitivity of 78.5% and specificity of 90.3% while 20WBCT had sensitivity of 81.0% and specificity of 90.3%. CONCLUSIONS: Both 20WBCT and INR are useful to assess coagulation abnormality in Malayan pit viper-bitten patients., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In Asia, acute kidney injury (AKI) induced by wasp stings is common; however, the pathophysiological mechanisms involved remain unclear. To evaluate the mechanisms associated with AKI induced by wasp stings, we conducted a retrospective cohort study that assessed blood and urinary samples from 112 patients with hospital admissions resulting from wasp stings. These samples were divided into those with AKI and without AKI as described in the Kidney Disease Improving Global Outcomes (KDIGO) database. Of the patients, 48.2% presented with an elevated number of leukocytes (median 19.9 vs. 15.8 × 10 9 /L), serum creatinine (median 122.0 vs. 66.0 μmol/L), alanine aminotransferase (ALT) (median 176 vs. 32 U/L), aspartate aminotransferase (AST) (median 402 vs. 37 U/L), lactate dehydrogenase (LDH) (median 3076.0 vs. 300.0 U/L), creatine kinase (CK) (median 9990.0 vs. 261.0 U/L), creatine kinase myocardial band (CK-mb) (median 200.0 vs. 29.5 U/L), activated partial thromboplastin time (APTT) (median 70.0 vs. 42.5s), prothrombin time (PT) (median 15.0 vs. 12.5s), myoglobin (median 2200.0 vs. 78.0 ng/mL), proteinuria (51.9% vs. 17.2% ≥ 1+), and urinary monocyte chemotactic protein-1 (MCP-1) (median 432.0 vs. 177.0 pg/mL), and subsequently developed AKI. As determined by multivariate logistic regression analysis, elevated leukocytes (>10 × 10 9 /L) [OR 1.12 (95% CI 1.02-1.23)], high myoglobin (>1200 ng/mL) [OR 18.51 (95% CI 1.51-132.27)], and high urinary MCP-1 (>200 pg/mL) [OR 5.42 (95% CI 1.27-30.39)] on admission were independent risk factors for AKI. At admission, baseline values for ALT, aspartate aminotransferase (AST), LDH, CK-mb, APTT, PT, and proteinuria were higher for those who later died as well as for those who developed end-stage renal disease (ESRD). No patients without AKI died or developed ESRD. The present study explored the pathophysiology of AKI induced by wasp stings based on the findings of risk factors as well as factors related to outcomes. An understanding of AKI induced by wasp stings allows better treatment options and clinical management for wasp stings patients., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Lupus anticoagulant (LA)-detection is performed by testing plasma with activated partial thromboplastin time (APTT)-derived and dilute-Russell-viper-venom (dRVV)-derived tests. Results are interpreted by comparison to cut-off values determined by testing plasma from healthy-subjects. Several issues are concerned with the determination of LA cut-offs. Among them, the identification/rejection of outliers (i.e., aberrant values) prior statistical analysis is one of the most important and poorly defined., Objectives: The primary aim of this collaborative study was to evaluate whether outliers' identification/removal by using three algorithms prior cut-off calculation would have any effect on the between-laboratory variability of cut-offs. As a secondary aim, we evaluated whether and to what extent outliers' removal would affect LA-detection rate., Methods: Data sets stemming from 120 plasmas from healthy-donors, collected and tested at each of 11 laboratories were evaluated by three algorithms for outliers' identification. Furthermore, a well characterized set of plasmas certified as being positive at increasing LA-potency, were concomitantly tested and their results used to assess any effect of outlier detection/rejection on LA-detection rate., Results: Relatively few outliers were identified and their elimination (regardless of algorithms) showed no appreciable effects on the inter-laboratory variability of cut-offs nor on the LA-detection rate, indicating that outliers are not the main cause of the inter-laboratory variability of cut-offs for LA-detection., Conclusions: These results strengthen the recommendation that cut-offs should be determined locally after outlier removal (to avoid inclusion of gross, obvious outliers) and that they cannot be interchangeably used in other laboratories even when using the same platform., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest relative to this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Current guidelines recommend performing laboratory tests aimed at monitoring unfractionated heparin (UFH) treatments within a delay not exceeding 1 to 2 h(s) after sampling when blood is collected into citrated tubes. As such a short delay could be an issue, we evaluated the potential impact of longer delays. For that purpose, two citrated tubes were obtained from patients on UFH: one was centrifuged and tested for anti-Xa activity and aPTT within 1 h after collection (T1 h) and one was stored for 4 h at room temperature (T4 h) before being processed. A total of 123 paired tubes were investigated. Anti-Xa activity was significantly lower at T4 h than at T1 h, with a mean bias, calculated according to Bland-Altman, of 0.05 IU/mL. Considering 0.30 to 0.70 IU/mL as the therapeutic range, there were 12 cases of discrepant test results (9.8%). Most of them being around the lower limit of the therapeutic range had no impact on patients' management. APTT was significantly shortened (p < 0.0001) at T4 h vs. T1 h, with a mean bias of -7.9 s. Considering anti-Xa correlated aPTT therapeutic range, 29 cases of discrepant test results (23.6%) were found, 10% would have induce dosage changes. The concordance between anti-Xa activities measured at T4 h and T1 h was excellent (kappa = 0.813) and good for aPTT (kappa = 0.661). In conclusion, extending the delay between blood collection and measurement of tests prescribed for monitoring UFH therapy up to 4 h was found to lead to a systematic reduction in both anti-Xa activity and aPTT in unspun citrated tubes. As changes at T4 h were limited and had few clinically relevance than the ones observed with aPTT testing, a 4 h-delay was found to be acceptable for anti-Xa activity. The maximum delay for aPTT should remain around 1-2 h as changes were more relevant., (Copyright © 2019. Published by Elsevier Ltd.)

Preterm newborns are considered at risk of acquired coagulopathy and are often prophylactically infused with fresh frozen plasma (FFP) even in the absence of bleeding. To assess the coagulation asset of preterm neonates and the biological plausibility of such infusions, we investigated at birth 87 very low birth weight (≤1500 g) preterm (gestational age <35 weeks) newborns and 64 full-term newborns. Preterm neonates were also investigated at different time-points up to 30 days after birth. Plasma from preterm and full-term neonates were subjected to the measurement of prothrombin and activated partial thromboplastin time (PT, APTT), pro- and anticoagulant factors as well as to thrombin-generation procedures both with and without thrombomodulin. PT and APTT of preterm newborns were longer than those of full-term neonates [PT: 15.9 s (11.7-51.2)-vs-13.8 (11.0-25.4), p < 0.001. APTT: 59.0 (37.8-97.5)-vs- 47.3 (28.1-71.9), p < 0.001] and tended to shortening after 30 days from birth. Thrombin-generation defined as endogenous thrombin potential (ETP) was increased in preterm as compared to full-term neonates at birth [1322 nM·min (474-2384)-vs-1006 (697-1612), p < 0.001] and did not change appreciably over time up to 30 days from birth. In conclusion, plasma from preterm neonates displays a procoagulant imbalance at birth as shown by increasing ETP, despite the prolongation of PT and APTT. The results define preterm newborns as having hyper- rather than hypo-coagulability and argue against the infusion of FFP when given prophylactically and/or based solely on prolongation of PT or APTT., (Copyright © 2019. Published by Elsevier Ltd.)

Objectives: Presence of lupus anticoagulants (LA) in haemophilia and their interference in coagulation assays is well-known. Factor VIII (FVIII) inhibitors are generally time and temperature dependent whereas LAs are immediate acting inhibitors (IAIs). The present study reports the challenges in laboratory detection of both progressive and non-progressive, specific FVIII inhibitors in the presence of LA., Methods: From 2012 through 2015, 4900 HA patients were screened for inhibitors. APTT based inhibitor screening tests and Nijmegen-modified Bethesda assay (NBA) were done in all samples. LA test and FVIII inhibitors by ELISA were done in patients with IAIs., Results: Out of 451 patients positive for inhibitors in the initial screening tests, classical and progressive FVIII inhibitors were observed in 398 patients while 53 had IAIs showing no/partial correction in 1:1 mixtures of NPP and patient plasma. In 27 patients, both FVIII and FIX activity levels were <1%, resulting in difficulty in diagnosis. In 48 HA patients with IAIs, 42 were LA positive. 4 patients were found to have only LA with false positive results in NBA while 38 had a combination of LA and FVIII inhibitors. Six patients were LA negative and had only FVIII IAIs. Five (62.5%) of 8 HA patients initiated on immune tolerance induction (ITI) also were positive for IAIs., Conclusion: The findings emphasizes the presence of specific FVIII inhibitors in congenital HA with absence of time dependent inactivation kinetics in a small proportion of cases. ELISA or chromogenic assays along with LA testing can offer accurate laboratory diagnosis in patients with coexisting LA., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: In patients who are receiving dabigatran, a direct oral anticoagulant, measuring the anticoagulant effect before surgery may be needed in certain circumstances. Although the dilute thrombin time (dTT) can reliably measure dabigatran levels, it is not consistently available. More commonly used coagulation tests, including the activated partial thromboplastin time (aPTT) and thrombin time (TT) might have clinical utility but their accuracy is uncertain., Methods: 103 patients stopped dabigatran 1-4 days before an elective surgery/procedure as part of a standardized dabigatran interruption protocol. With a blood sample taken just before surgery, we assessed the accuracy of five aPTT assays (Actin FS, Stago PTT, C.K. PREST, HemosIL aPTT-SP, SynthASil) and TT to measure the residual anticoagulant effect of dabigatran. We determined the sensitivity, specificity and other accuracy indices of these assays to predict a dabigatran level > 30 ng/mL as determined by a reference standard test, the dTT (Hemoclot)., Results: Of five aPTT reagents, four assays had excellent (100%) and one assay had good (93%) sensitivity to detect a level of dabigatran > 30 ng/mL, but all had insufficient specificity (50-74%). A TT > 90 s had good sensitivity (93%) and excellent specificity (100%)., Conclusion: Five aPTT assays had good sensitivity but poor specificity to detect low levels of dabigatran (≤30 ng/mL) after standardized dabigatran interruption before an elective surgery/procedure, thereby limiting the use of aPTT as an alternative to the dTT in preoperative settings., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bleeding heterogeneity observed in haemophilia A (HA) may attribute to that the available monitoring methods cannot appropriately reflect the coagulation profile. The present study aimed to develop a global approach by changing the clotting initiation way in rotational thromboelastometry (ROTEM) assay. ROTEM was run in Factor VIII (FVIII)-immune-depleted plasma to which different concentrations of recombinant VIII (rFVIII) had been added, and also in 31 patients with HA. The clotting activators were APTT reagent (1.2 × 10 -3 of the dose used in the original APTT method) and recombinant tissue factor (0.02 pmol/L). In FVIII-immune-depleted plasma spiked with rFVIII, maximum velocity of coagulation reliably mirrored the rFVIII levels. This dose-response disappeared after the samples were pre-incubated with an antibody against TFPI, protein S, activated prothrombin complex concentrate or rFVIIa known to favour the extrinsic activation. In the HA patients with FVIII 0-0.21 IU/mL, APTT and ROTEM outcomes varied in significant correlations to FVIII activity; however, this correlation became non-significant when only samples with FVIII 0-0.05 IU/mL were included. Conclusions: The decreased coagulation in HA mostly result from deficiency/absence of FVIII; other pro-/anti-thrombotic proteins are also influential. The multiple effects may cause a mismatch between bleeding phenotype and FVIII concentrations. The ROTEM assay with the clotting activators i.e., tiny doses of APTT reagent and TF are more effective than the original APTT method as regards the assay sensitivity to influence by VIII activity and also to that by other pro-/anti-thrombotic proteins, showing the whole coagulation picture behind the phenotypic heterogeneity in HA., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Micrurus venoms are known to induce mainly neurotoxicity in victims. However, other manifestations, including hemorrhage, edema, myotoxicity, complement activation, and hemostatic activity have been reported. In order to develop a more complete pharmacological profile of these venoms, inflammatory responses and hemostasis were evaluated in C57BL/6 mice treated with a sub-lethal dose of M. t. tener (Mtt) venom (8 μg/mouse), inoculated intraperitoneally. The venom induced moderate bleeding into the abdominal cavity and lungs, as well as infiltration of leukocytes into the liver. After 30 min, the release of pro-inflammatory mediators (TNF-α, IL-6, and NO) were observed, being most evident at 4 h. There was a decrease in hemoglobin and hematocrit levels at 72 h, a prolongation in coagulation times (PT and aPTT), a decrease in the fibrinogen concentration and an increase in fibrinolytic activity. In this animal model, it was proposed that Mtt venom induces inflammation with the release of mediators such as TNF-α, in response to the toxins. These mediators may activate hemostatic mechanisms, producing systemic fibrinolysis and hemorrhage. These findings suggest alternative treatments in Micrurus envenomations in which neurotoxic manifestations do not predominate., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Saliva of hematophagous animals, such as ticks, is an excellent source of anticoagulant proteins and polypeptides. Here we describe the identification and characterization of two thrombin inhibitors named as haemathrin 1 and 2 from the salivary gland of tick Haemaphysalis bispinosa using genomic approach. Haemathrins are cysteine-less peptide anticoagulants, which share about 65-70% identity with madanins, and belong to inhibitor I53 superfamily of inhibitors of the MEROPS database. Haemathrins were overexpressed in E. coli and characterized to understand its mechanism of anticoagulant activity. Recombinant haemathrins (rHaemathrins) delayed the thrombin time, prothrombin time, activated partial thromboplastin time and fibrinogen clotting time. Selectivity screening against serine proteases of coagulation cascade reveals that rHaemathrins 1 and 2 specifically inhibit thrombin with an IC 50 of 46.13±0.04μM and 40.05±0.05μM respectively. Similar to madanin, rHaemathrin 1 and 2 were cleaved by thrombin and consequently lost their inhibitory function over time. Analyses of the cleavage products revealed that the first cleavage, which occurs at the C-terminal end of rHaemathrins, drastically reduced their inhibitory activity. The synthetic peptides corresponding to the cleaved fragments showed significant loss in their ability to prolong plasma clotting times and to inhibit the amidolytic activity of thrombin. Thus haemathrins are the first cleavable thrombin inhibitors characterized from the salivary glands of H. bispinosa., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: C1-inhibitor (C1-INH), a serine protease inhibitor in plasma plays a central role in the cross-talk among the complement, coagulation, fibrinolytic and kallikrein-kinin systems. However, previous reports indicate thrombotic risks in children following supraphysiological dosing with C1-INH., Objective: To investigate the role of supraphysiological C1-INH concentrations in clot development with and without addition of Escherichia coli (E. coli) in fresh human whole blood using thromboelastometry., Materials and Methods: Blood was collected in citrate tubes, and C1-INH (3.0 to 47.6μM) or human serum albumin (HSA) was added as a control. Activated partial thromboplastin time (aPTT) was analysed in the plasma. The analyses non-activated thromboelastometry (NATEM), extrinsic (EXTEM) or intrinsic thromboelastometry (INTEM) were performed using rotational thromboelastometry., Results: C1-INH increased aPTT 1.8-fold (p< 0.05), whereas HSA had no effect. C1-INH increased NATEM clotting time (CT) from 789s to 2025 s (p< 0.05) in a dose-dependent manner. C1-INH reduced the NATEM alpha angle from 47 to 28° (p<0.05) and increased the NATEM clot formation time from 261s to 595s (p< 0.05). E. coli significantly reduced the NATEM CT after 120min of incubation. C1-INH prevented E. coli-induced activation (p< 0.05). C1-INH significantly increased the INTEM CT (p< 0.05), but had no effect on EXTEM CT. C1-INH (47.6μM) significantly reduced fibrinolysis measured as NATEM and EXTEM lysis indices LI60., Conclusions: Supraphysiological C1-INH concentrations have dose-dependent anticoagulant effects in human whole blood in vitro. At very high levels C1-INH also inhibits fibrinolysis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Lupus anticoagulant (LA) is an antibody that interferes with in vitro coagulation reactions. The mixing test is considered useful for LA diagnosis and is also recommended to differentiate between acquired hemophilia A (AHA) and factor deficiency. However, there has been little study to differentiate between LA and AHA. Our aims are to investigate whether we can differentiate LA and AHA by the mixing test and to establish new formulas for the mixing test to differentiate these samples clearly., Materials and Methods: We examined 27 LA-positive, 29 coagulation factor deficient, 24 unfractionated heparin and 48 AHA samples. Index of circulating anticoagulant (ICA) values, calculated from the clotting times without incubation and after 2h incubation, were defined as ICA immediate (ICAi) and ICA delayed (ICAd) respectively. ICAd/ICAi and ICAd-ICAi were also calculated to compare the sensitivity and specificity., Results: ICAd/ICAi and ICAd-ICAi for AHA samples were significantly higher than those of the other sample groups. The sensitivities to AHA in ICAi, ICAd, ICAd/ICAi and ICAd-ICAi were 66.7%, 81.3%, 93.8% and 91.7% respectively, while the specificities for AHA were 45.0%, 66.3%, 85.0% and 98.8% respectively. ICAd/ICAi and ICAd-ICAi showed high sensitivity and specificity., Conclusions: ICAd/ICAi and ICAd-ICAi were useful for LA and AHA diagnosis, because these could differentiate between LA and AHA samples. These new formulas can contribute to the rapid diagnosis and treatment of LA and AHA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A two-fold prolongation of activated partial thromboplastin time (APTT) is established as therapeutic range for therapy with unfractionated heparin, hirudin and argatroban. The international normalized ratio (INR) of 2 to 3 is required to maintain anticoagulation in the therapeutic range of vitamin K antagonists. The therapeutic range of anti-factor Xa activity during therapy with low-molecular weight heparins and danaparoid are less well and of direct oral anticoagulants (DOAC) poorly defined. The relation of aPTT and INR values to thrombotic and bleeding events are well established despite a large variation of values in affected patients. The relation of coagulation values of the other anticoagulants to clinical events is open. The value of determination in cancer patients is higher because of the increased risk for thrombotic and bleeding events of this patient group. Several activities are currently undertaken to certify methods for in vitro diagnostic testing for DAOCs., (© 2016 Elsevier Ltd. All rights reserved.)

Introduction: Recent guidelines for lupus anticoagulant (LA) detection recommend mixing test interpretation with either a mixing test-specific cut-off (MTC) or index of circulating anticoagulant (ICA). Few studies directly compare efficacy of these approaches. We retrospectively applied MTC and ICA assessment to raw data of 350 LA-positive plasmas from non-anticoagulated patients to compare detection rates of inhibition., Materials and Methods: Screen and confirm dRVVT and dilute APTT assays were performed on undiluted plasma and 1:1 mixtures with normal pooled plasma. Samples were considered LA-positive if one or both screening test ratios were elevated and corrected by ≥10% with the confirmatory test. Mixing tests were assessed against locally derived cut-offs for MTC (dRVVT >1.13, dAPTT >1.15) and ICA (dRVVT >11.9%, dAPTT >13.2%)., Results: 105 of 350 (30%) were positive in dRVVT and dAPTT, 109/350 (31.1%) were dRVVT positive only and 136/350 were dAPTT positive only (38.9%), from undiluted plasma results. Of the 214 dRVVT positive plasmas, 53 (24.8%) were negative for inhibition by MTC and 65 (30.4%) negative by ICA. Of the 241 dAPTT positive plasmas, 48 (19.2%) were negative by MTC and 97 (40.2%) negative by ICA., Conclusion: Whilst integrated testing often detects LA without mixing tests they are diagnostically useful in certain circumstances. Thus, it is valuable to maximise mixing test interpretation as the dilution can lead to false-negative results. These data on a large cohort of LA-positive plasmas reveal that, with the reagents and equipment employed, MTC is superior to ICA in detecting the in vitro inhibition of LA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: We encountered two patients with hypodysfibrinogenemia and designated them as Okayama II and Otsu I. Although the affected residue(s) in Okayama II and Otsu I overlapped, functionally determined fibrinogen levels and the ratio of functionally to immunologically determined plasma fibrinogen levels were markedly different., Methods: DNA sequence and functional analyses were performed for purified plasma fibrinogen. A recombinant protein was synthesized in Chinese hamster ovary (CHO) cells to determine the secretion of variant fibrinogens., Results: A heterozygous A>G in FGG, resulting in γ320Asp>Gly for Okayama II, and a heterozygous deletion of AATGAT in FGG, resulting in the deletion of γAsn319 and γAsp320 (γΔN319-ΔD320) for Otsu I, were obtained. SDS-PAGE and Coomassie staining revealed that the variant γ-chain was not clear in Okayama II, but was clearly present in Otsu I. The lag period for the fibrin polymerization of Okayama II was slightly slower than that of the normal control, whereas Otsu I fibrinogen indicated no polymerization within 30 min. Both variant γ-chains were synthesized in CHO cells and assembled into fibrinogen; however, the fibrinogen concentration ratio of the medium/cell lysate of γ320Gly was six-fold lower than that of γΔN319-ΔD320., Conclusions: We concluded that the plasma fibrinogen of Okayama II, constituted by a lower ratio of the variant γ-chain, led to the almost normal functioning of fibrin polymerization. However, the plasma fibrinogen of Otsu I, with a higher ratio of the variant γ-chain, led to marked reductions in fibrin polymerization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Thrombin generation assay (TGA) is useful as a global functional test for assessing bleeding or thrombotic risk and its modification with therapy. We investigated TGA to assess anticoagulation status compared with the international normalized ratio (INR) system in patients with primary thrombophilia receiving and not undergoing thromboprophylaxis., Materials and Methods: We studied 50 patients with at least one thrombotic event and a confirmed diagnosis of inherited thrombophilia. Thrombin generation was measured in platelet-poor plasma by calibrated automated thrombography (CAT)., Results: Patients in optimal anticoagulation (INR: 2.0-3.0) showed an endogenous thrombin potential (ETP) of 14-56% of normal and a peak of 18-55% of normal. A significant inverse relationship between INR and thrombin generation parameters (ETP, peak and velocity index) and a linear correlation for lag time was found in patients treated with vitamin-K antagonists (VKA). Receiver-operating characteristics (ROC) analysis showed that the optimal cutoff for ETP was 1600.2 nM · min (111.6% of normal, with a sensitivity of 96.6% and a specificity of 92.9%) and for the peak was 298.3 nM (112.1% of normal, with a sensitivity of 96.4% and a specificity of 100%). According to this analysis, ETP was able to identify patients with increased thrombotic and hemorrhagic risk, correlating with severe clinical complications., Conclusion: TGA showed excellent sensitivity and specificity for assessing anticoagulation status in patients with primary thrombophilia receiving VKA, with significant advantages with regard to INR. Clinical data strongly support ETP as a valuable indicator of thrombotic or hemorrhagic risk in patients receiving or not receiving thromboprophylaxis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Dabigatran (Dabi) is not routinely monitored. However, in emergency cases quantitative assessment is required and laboratories must provide suitable tests at all hours. Little is known about Dabi effects on thrombin generation., Materials and Methods: Patient samples (n=241) were analyzed for functional Dabi concentrations (Dabi-TT) using a combination of the Hemoclot Thrombin Inhibitors assay (HTI®) and, for samples with low levels, undiluted thrombin time (TT). Results were compared to prothrombin time (PT) and activated partial thromboplastin time (APTT). In 49 samples Dabi effects were further investigated with Calibrated Automated Thrombogram (CAT®) for thrombin generation and with Russell's viper venom time (RVVT), prothrombinase-induced clotting time (PiCT®), chromogenic Anti-IIa® and ecarin clotting assay (ECA®). Fibrinogen and D dimer were assessed to reflect the coagulation status of the patient. A subset of these samples (n=21) were also analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: Dabi-TT correlated with RVVT (R(2)=0.49), PiCT® (R(2)=0.73), ECA® (R(2)=0.89), Anti-IIa® (R(2)=0.90) and LC-MS/MS (R(2)=0.81). APTT correlated curvi-linearly with Dabi-TT (R(2)=0.71), but was normal in many cases (18/70) despite Dabi-TT>40ng/mL. There was no association between Dabi-TT and fibrinogen or D dimer levels. Increasing Dabi concentrations prolonged lag time (R(2)=0.54) and, surprisingly, elevated the ETP and Peak of CAT® (p<0.001)., Conclusions: Thrombin-specific tests measure Dabi accurately, whereas coagulation time based assays depend more on other factors. The enhanced thrombin generation in Dabi-treated patients may predict clinically relevant hypercoagulability and warrants further investigation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Injuries caused by aquatic animals in Brazil in most cases are provoked by marine and freshwater catfish. Pimelodus maculatus is a freshwater catfish very common in Brazilian basins that causes frequent accidents mainly amongst fishermen, and whose venom characteristics and pathological mechanisms of the venom are poorly known. In the present study for the first time, we have characterized the main pathophysiological mechanisms associated with the clinical manifestation (pain, local inflammation and edema) of the envenomations caused by P. maculatus crude venom. It was estimated that the crude venom of one P. maculatus stinger contains approximately 100 μg of protein, likely the quantity involved in the envenomation. P. maculatus crude venom induced marked nociceptive and edematogenic effects and caused vascular permeability alterations at doses from 30 to 100 μg/animal. Additionally, P. maculatus crude venom caused a decrease in the contraction force in in situ frog heart, did not cause hemorrhage or alterations in clotting times (prothrombin time and activated partial thromboplastin time), but induced significant changes in the levels of CK and its isoenzyme CK-MB in mice. In the present work, we present a correlation between the effects obtained experimentally and the main symptoms observed in the human accidents provoked by P. maculatus., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background and Objectives: Apixaban is a direct oral anticoagulant (DOAC) targeting factor Xa and thus quenching thrombin generation and clot formation. However, little information is available on the influence that apixaban may have on the parameters of thrombin generation., Methods: Aliquots of a pooled normal plasma have been added with increased concentrations of purified apixaban and were used to assess the degree of modification brought about by the drug on the basic tests of coagulation prothrombin and activated partial thromboplastin time (PT and APTT) and on thrombin generation parameters., Results: The study shows that while apixaban has little effect on PT or APTT it does affect all the parameters of thrombin generation, including the lag-time (which is increased), the endogenous thrombin potential (ETP) and thrombin-peak (both decreased although to a different extent), and the velocity index (decreased). Interestingly, the above effects were more pronounced when the measurements were recorded in the presence of thrombomodulin, thus making the ratio (with/without thrombomodulin) to decrease consistently as a function of the apixaban concentrations., Conclusions: These findings support the antithrombotic properties of apixaban and can help to understand the mechanism(s) of action of this drug. Thrombin generation could be used as a convenient laboratory tool to assess the anticoagulant activity of other drugs and to make between-DOAC comparison., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Patients with cirrhosis have substantial alterations in their hemostatic system, which are paradoxically associated with the risk of both bleeding and thrombotic complications. However, it still remains difficult to predict those risks, because results from conventional coagulation tests, such as the prothrombin time (PT) and activated partial thromboplastin time (APTT), do not reflect the complex hemostatic changes in these patients. More sophisticated global hemostasis tests, such as thrombin generation assays, are not standardized for routine use yet. Here we examined the spatial clot growth in plasma from patients with cirrhosis using the novel Thrombodynamics assay, which uses a fundamentally new approach to test plasma hemostatic capacity., Materials and Methods: Thrombodynamics assays were performed in plasma from thirty-one patients with cirrhosis and twenty-five healthy controls. Results were compared to results with thrombin generation testing and PT/APTT test results., Results: Rates of clot growth, clot size, and clot density from the Thrombodynamics assay were comparable between patients and controls. Thrombin generation in the presence of thrombomodulin was increased in the patients, despite prolonged PT and APTT test results. There was little correlation between parameters derived from the Thrombodynamics assay and the PT, APTT, or thrombin generation data., Conclusions: The Thrombodynamics assay showed preserved clot formation in plasma from patients with cirrhosis, which is in line with the results of the thrombin generation assay in this study and previously reported by others., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Dilute Russell Viper Venom Time (DRVV-T) might be useful in urgent settings for screening patients on Non-VKA Oral Anticoagulants (NOACs)., Aim: To compare the accuracy of DRVV-T with gold standard assays for the assessment of pharmacodynamics of dabigatran, rivaroxaban and vitamin K antagonist (VKA) in plasma samples from patients., Methods: Sixty rivaroxaban, 48 dabigatran and 50 VKA samples from patients were included. DRVV-T was performed in all groups using STA®-Staclot®DRVV-Screen and -Confirm. For NOACs, PT and aPTT were performed using different reagents while plasma drug concentrations were measured by liquid mass-spectrometry (LC-MS/MS). For VKA, INR was performed using RecombiPlasTin 2G®., Results: For NOACs, correlations between calibrated STA®-Staclot®DRVV-Confirm and LC-MS/MS (rs=0.88 and 0.97 for rivaroxaban and dabigatran, respectively) were higher than the ones obtained with STA®-Staclot®DRVV-Screen (rs=0.87 and 0.91), PT (rs=0.83 to 0.86) or aPTT (rs=0.84 to 0.89). Bland Altman analyses showed that calibrated DRVV-T methods tend to overestimate plasma concentrations of NOACs. ROC curves revealed that cut-off to exclude supra-therapeutic levels at Ctrough (i.e. 200ng/mL) are different for dabigatran and rivaroxaban. Neither STA®-Staclot®DRVV-Screen nor -Confirm correlated sufficiently with the intensity of VKA therapy (rs=0.35 and 0.52)., Conclusions: STA®-Staclot®DRVV-Confirm provides a rapid estimation of the intensity of anticoagulation with rivaroxaban or dabigatran without specific calibrators. At Ctrough, thresholds for rivaroxaban and dabigatran can be used to identify supra-therapeutic plasma level. However, this test cannot differentiate the nature of the NOACs. The development of a point-of-care device optimising this method would be of particular interest in emergency situations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Acquired haemophilia A (AHA) is an autoimmune disease that potentially leads to severe bleeding and has a high rate of mortality. This collaborative study aimed to assess the efficacy of the co-administration of FVIII and low-dose rFVIIa in patients with AHA., Materials and Methods: This study retrospectively compared the combined FVIII/low-dose rFVIIa therapy (initial dose range of 25-55μg/Kg) with the combined FVIII/PCC therapy and low-dose rFVIIa monotherapy. Adverse drug reactions and recurrent bleeding episodes were also monitored. Crude comparisons and the exact conditional logistic regression were performed to compare the outcomes between three treatment groups., Results: First bleeding episodes of 56 consecutive patients from 5 centres were analyzed, and 37 bleeding episodes (66.1%) were determined to be severe. Specifically, the rate of bleeding control was significantly higher with the FVIII/low-dose rFVIIa therapy compared to that of the low-dose rFVIIa alone therapy or the FVIII/PCC therapy (58.3% vs. 41.7% vs. 95.0%, respectively). Analyzing of total 236 bleeding episodes showed a clear positive association between the early initiation of haemostatic treatment and efficacy. No therapy-related adverse events in which thrombosis predominated were reported., Conclusions: The combination of FVIII and low-dose rFVIIa offers an ideal haemostatic cover and may be promoted as a feasible and safe therapy protocol for patients with AHA., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Back Ground: It has recently been reported that factor XIa can activate factor X directly and can bypass factors VIII-IX. We evaluated the consequences for factor analysis with the one-stage APTT., Methods: APTT was performed with the Actin FS reagent with ellagic acid as the standard. Silica, high lipid (PTT-A) or low lipid (PTT-LA) were also tested. Factor depleted and deficient plasma's were obtained from commercial sources., Results: The APTT clotting times in factor XII, XI, High Molecular Weight Kininogen, factor X and factor V deficient plasma's were all significantly longer (>100s) than the clotting times of factor VIII- and IX-depleted or deficient plasma's (<100s). That the shorter times for factor VIII and IX deficient plasmas were due to contact activation was supported by biphasic inhibition of the clotting times with addition of Corn Trypsin Inhibitor and Trasylol. The role of factor XI and the by-passing of factor VIII/IX was shown by the use of quenching antibodies towards factor XI and VIII. Enriching factor VIII or IX depleted plasma with purified factor XI and addition of factor XIa showed a strong dependence on factor XI level. Calibration curves for factor analysis were steeper for factors FXII, HMWK, FX and FV, compared to those of both factors VIII and IX. Curves for VIII/IX were found steeper by the use of APTT-A/silica-based, 50% diluted substrate plasma and low factor XI in the substrate plasma., Conclusions: In factors VIII and IX deficient plasmas, the APTT shows an activity which can be attributed to contact activation of factor X by factor XIa. This direct activity is lower with silica reagent compared to ellagic acid, dilution of plasma and low factor XI in substrate plasma., (Copyright © 2014. Published by Elsevier Ltd.)

The antiphospholipid syndrome is characterized by venous or arterial thrombosis and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies. These antibodies cause activation of endothelial and other cell types leading to the release of microparticles with procoagulant and pro-inflammatory properties. The aims of this study were to characterize the levels of endothelial cell, monocyte or platelet derived, and tissue factor-bearing microparticles in patients with antiphospholipid antibodies, to determine the association of circulating microparticles with anticardiolipin and anti-β2-glycoprotein antibodies, and to define the cellular origin of microparticles that express tissue factor. Microparticle content within citrated blood from 47 patients with antiphospholipid antibodies and 144 healthy controls was analyzed within 2hours of venipuncture. Levels of Annexin-V, CD105 and CD144 (endothelial derived), CD41 (platelet derived) and tissue factor positive microparticles were significantly higher in patients than controls. Though levels of CD14 (monocyte-derived) microparticles in patient plasma were not significantly increased, increased levels of CD14 and tissue factor positive microparticles were observed in patients. Levels of microparticles that stained for CD105 and CD144 showed a positive correlation with IgG (R=0.60, p=0.006) and IgM anti-beta2-glycoprotein I antibodies (R=0.58, p=0.006). The elevation of endothelial and platelet derived microparticles in patients with antiphospholipid antibodies and their correlation with anti-β2-glycoprotein I antibodies suggests a chronic state of vascular cell activation in these individuals and an important role for β2-glycoprotein I in development of the pro-thrombotic state associated with antiphospholipid antibodies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Although dabigatran, an oral direct thrombin inhibitor, does not require routine monitoring, high plasma concentration of dabigatran (PDC) at trough level is shown to be a high risk for bleeding in patients with nonvalvular atrial fibrillation (NVAF). As dabigatran prolongs the activated partial thromboplastin time (APTT), we examined relationships of PDC at trough with APTT and clinical features to identify patients at high risk for major bleeding during dabigatran treatment., Materials and Methods: In the consecutive 48 patients with NVAF taking dabigatran at a daily dose of 220mg (n=32) or 300mg (n=16), we measured PDC using HEMOCLOT Thrombin Inhibitor assay and APTT ratio to control before (trough) and 2hours after taking dabigatran., Results: PDC was positively correlated with APTT ratio (R(2)=0.64, p<0.0001). Using this regression equation and values of median trough PDC 116 (46.7-269) ng/mL observed in patients with major bleeding in the RE-LY trial, we calculated the expected value of APTT ratio corresponding to the 10th percentile of trough PDC (46.7). It was 1.20. There was a significant increase in trough PDC with increasing CHA2DS2-VASc score (p=0.01) and with increasing HAS-BLED score (p=0.01), especially in CHA2DS2-VASc score ≥4 and in HAS-BLED score ≥3, respectively. The highest trough PDC was obtained in patient group with CHA2DS2-VASc score ≥4, HAS-BLED score ≥3, or creatinine clearance ≤80, each combined with trough APTT ratio ≥1.20., Conclusions: This study provides an important clinical implication for identifying patients at high risk for major bleeding during dabigatran treatment in clinical practice., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Edoxaban is an oral, once-daily direct factor Xa (FXa) inhibitor. Although rapidly cleared, strategies to reverse edoxaban-mediated effects on anticoagulation are needed in cases of excessive bleeding or emergency. This study evaluated the effect of two prohemostatic agents, recombinant factor VIIa (rFVIIa) and factor VIII inhibitor bypass activity (FEIBA), on the anticoagulatory effects of supratherapeutic concentrations of edoxaban in human whole blood ex vivo., Materials and Methods: Blood samples were collected from six healthy volunteers. Edoxaban (500 or 1000 ng/mL), alone or followed by rFVIIa (0.8 or 1.8μg/mL) or FEIBA (0.75 or 1.5 U/mL), was added to an aliquot of each sample. Biomarkers, including prothrombin time (PT), activated partial thromboplastin time (aPTT), extrinsic FXa activity (anti-FXa), intrinsic factor X activity, and D-dimer, were assessed at 0.25, 0.5, 1, 2, and 4 hours after adding rFVIIa or FEIBA., Results: Decreases in measures of PT (p<0.0001), aPTT (p<0.0001), and anti-FXa (p<0.0001) were observed when rFVIIa or FEIBA was added to edoxaban-containing blood samples. Intrinsic FX activity was increased up to 20% and 31% of normal in the presence of edoxaban by rFVIIa and FEIBA, respectively. The impact of these agents on the anticoagulant effects of edoxaban were observed within 15 minutes and remained relatively unchanged at each timepoint thereafter., Conclusions: The findings of this ex vivo study suggest that rFVIIa and FEIBA rapidly reversed edoxaban-mediated anticoagulation effects based on PT and aPTT, but had minimal effect based on intrinsic FX activity. No dose response was observed for rFVIIa or FEIBA., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Trauma-induced coagulopathy following severe injury is associated with increased bleeding and mortality. Injury may result in alteration of cellular phenotypes and release of cell-derived microparticles (MP). Circulating MPs are procoagulant and support thrombin generation (TG) and clotting. We evaluated MP and TG phenotypes in severely injured patients at admission, in relation to coagulopathy and bleeding., Methods: As part of the Prospective Observational Multicenter Major Trauma Transfusion (PROMMTT) study, research blood samples were obtained from 180 trauma patients requiring transfusions at 5 participating centers. Twenty five healthy controls and 40 minimally injured patients were analyzed for comparisons. Laboratory criteria for coagulopathy was activated partial thromboplastin time (APTT) ≥ 35 sec. Samples were analyzed by Calibrated Automated Thrombogram to assess TG, and by flow cytometry for MP phenotypes [platelet (PMP), erythrocyte (RMP), leukocyte (LMP), endothelial (EMP), tissue factor (TFMP), and Annexin V positive (AVMP)]., Results: 21.7% of patients were coagulopathic with the median (IQR) APTT of 44 sec (37, 53), and an Injury Severity Score of 26 (17, 35). Compared to controls, patients had elevated EMP, RMP, LMP, and TFMP (all p<0.001), and enhanced TG (p<0.0001). However, coagulopathic PROMMTT patients had significantly lower PMP, TFMP, and TG, higher substantial bleeding, and higher mortality compared to non-coagulopathic patients (all p<0.001)., Conclusions: Cellular activation and enhanced TG are predominant after trauma and independent of injury severity. Coagulopathy was associated with lower thrombin peak and rate compared to non-coagulopathic patients, while lower levels of TF-bearing PMPs were associated with substantial bleeding., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Lupus anticoagulant (LA) is an antibody that interferes with one or more in vitro coagulation reactions, which are dependent on interactions with protein-phospholipid complexes. For LA diagnosis, a mixing test is considered useful for differentiating the inhibitor from a factor deficiency. However, the usefulness and the index of circulating anticoagulant (ICA) in a mixing test with activated partial thromboplastin time (APTT) has not been adequately investigated, and there is scant information regarding the effects of warfarin, heparin, and hemophilia plasma on ICA. We evaluated the usefulness of ICA by investigating the correlation of that index with international normalized ratio (INR), heparin concentration, and factor VIII activity in hemophilia patients., Materials and Methods: We examined samples from 28 patients positive for LA, 23 receiving warfarin, 19 receiving unfractionated heparin, and 29 with hemophilia A, as well as 61 normal samples. APTT-SLA, Actin FSL, APTT-SP, and PTT-LA were used as reagents in this study., Results: The correlation coefficient values between ICA and INR, heparin concentration, and factor VIII activity ranged from 0.031-0.342, 0.764-0.843, and 0.564-0.754, respectively, with the 4 reagents. The ICA values for the LA-positive samples were significantly higher than for the normal, warfarin, heparin, and hemophilia samples with all APTT reagents. Samples with a high heparin concentration above approximately 0.5U/ml showed ICA values greater than 15., Conclusion: ICA was able to distinguish LA-positive samples from the normal, warfarin, and hemophilia samples, but not heparin samples. ICA calculated from APTT clotting time is useful for LA diagnosis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Hypercoagulability due to high coagulation factor levels resulting from host inflammatory response to cancer contributes to an increased risk of venous thromboembolism (VTE) in cancer patients. Central venous catheters (CVCs) further heighten this risk. Activated partial thromboplastin time (aPTT) can be used to broadly screen for elevated levels of relevant coagulation factors. Our objective was to determine if a shortened aPTT ratio (coagulation time of test- to- reference plasma) was a predictor of CVC-associated VTE in cancer patients., Materials and Methods: We performed a retrospective case-control study on cancer patients undergoing tunneled CVC insertion at our center from 1999 to 2006 and identified 40 patients who had CVC-associated VTE. VTE was confirmed with color duplex ultrasonography or computed tomography scan. For each case, we obtained 5 controls that had the same cancer diagnosis and were matched on the following factors: age, chemotherapy, hormone therapy (if applicable), tobacco use, TNM staging and year of diagnosis. All patients had aPTT testing within 30 days prior to surgery. We compared aPTT and aPTT ratio between cases and controls using Wilcoxon two sample test., Results: aPTT ratio was significantly shorter in patients with CVC-related VTE as compared to controls [0.86 (95% confidence interval (CI) 0.78, 0.94) vs. 0.98 (0.94, 1.01), p=0.0003]. Mean aPTT was also significantly shorter. [25.6 seconds (95% CI 23.2, 27.9) vs. 28.1 (26.9, 29.3), p=0.001] aPTT ratios of the controls tended to spread across larger aPTT ratio values whereas those of cases tended to clustered around the mean., Conclusions: Cancer patients undergoing catheter placement who develop CVC-associated VTE have a shorter aPTT and aPTT ratio than those who do not develop VTE. aPTT, a simple and inexpensive test might be useful as a predictor of CVC-associated VTE risk in cancer patients., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This work provides a graphic description of the time course of hemostasis tests results during spontaneous evolution of Echis envenoming and correction of hemostasis disorders with antivenom therapy. The dynamics of fibrinogenemia (g L(-1)), prothrombin time (PT, %), activated partial thromboplastin time (aPTT, patient/normal ratio) and platelet count (Giga L(-1)) were collected from coagulopathic envenomed patients of a 12 years prospective study in Africa. Sixty patients were included. 47 of them (78%) received an antivenom (33 ± 12 ml) and 13 did not. Thirty patients (50%) presented bleeding. Only one patient died. The time for fibrinogen to be more than 1 g L(-1) was 181 ± 116 h (7.5 days) in the spontaneous evolution group versus 40 ± 21 h in the antivenom group (p < 0.0001). The times for reaching a PT above 50% were 140 ± 64 min (5.8 days) versus 25 ± 15 h (p < 0.00001) and for reaching an aPTT less than 1.5 times the normal values, 116 ± 76 h (4.7 days) versus 10 ± 9 h respectively (p < 0.0002). Thrombopenia was not a common feature of Echis envenomation. This study is the first one to provide a chart of the evolution of the hemostatic tests during envenomation caused by Echis bites. The plots enable to estimate that, in Echis envenomation, in the absence of antivenom administration, hemostasis remains severely affected until the 8-10th day of evolution. On the contrary, efficient antivenom against African vipers corrects clotting functions within a few hours., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: Quercetin is widely distributed in plants and has been reported to have effects of anti-inflammation and anti-thrombosis. In this study, we evaluated the protective effect of quercetin on LPS-induced experimental DIC in rabbits, and tried to clarify its mechanism against DIC., Materials and Methods: LPS-induced DIC model in rabbits was established through continuous infusion of 100 ug · kg(-1) · h(-1) LPS for a period of 6h. Six groups were divided: quercetin-treated groups (0.5, 1.0, and 2.0 mg·kg(-1) · h(-1), respectively), LPS-control group, heparin-control group (100 IU · kg(-1) · h(-1)), and saline-control group. APTT, PT, and plasma FIB level were measured, the plasma levels of ALT, BUN, and TNF-α were detected, and the activity of Protein C and ATIII was recorded., Results: A continuous injection of LPS induced a gradual impairment of hemostatic parameters, a rise in plasma level of TNF-α, and damage in renal and hepatic function. The intravenous administration of quercetin significantly attenuated the increase of APTT, PT, ALT, BUN, and TNF-α, and the decrease of plasma FIB level and activity of Protein C and ATIII., Conclusion: Quercetin may have a protective effect against LPS-induced DIC in rabbits through anti-inflammation and anticoagulation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: A sedentary lifestyle is a major risk factor for cardiovascular and thrombotic complications. While habitual endurance activity will reduce the risk of these adverse events, the influence of habitual resistance exercise is less clear. This study examined coagulation and fibrinolytic responses to an acute exhaustive resistance exercise test (AERET) in both resistance-trained (RT, min 2 yr, 5 men and 5 women) and untrained (UT, 5 men and 5 women) subjects., Methods: The AERET consisted of six sets of 10 repetitions of squats at 80% of 1-repetition maximum. Venous blood was collected pre-exercise, immediate post exercise (IP), and +15, +60, and +120 minutes post exercise., Results: Compared to UT, RT exhibited a lower capacity to form a clot as seen by activated partial Thromboplastin time (aPTT) integrated area under the curve over time (iAUC) levels, lower pre-exercise and 120 min post-exercise plasminogen activator inhibitor -1 (PAI-1) activity, and higher tissue plasminogen activator (tPA) activity immediately post-exercise. There were no significant differences between RT and UT for fibrinogen, prothrombin fragment 1+2 (PTF 1+2), and thrombin-antithrombin complexes (TAT)., Conclusion: These results suggest that habitual resistance exercise training may provide an enhanced fibrinolytic state., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Here, we investigated the anticoagulant effects of darexaban in mice and human plasma in vitro, effects of darexaban in thrombosis and bleeding models in mice, and reversal effects of anti-inhibitor coagulant complex (ACC) and recombinant factor VIIa (rFVIIa) on anticoagulant effects of darexaban. In mice, darexaban inhibited FXa activity in plasma with an ED50 value of 24.8 mg/kg. Both darexaban and warfarin prolonged prothrombin time (PT) at 3 mg/kg and 0.3 mg/kg/day, respectively. PT and activated partial thromboplastin time (aPTT) prolonged by darexaban were dose-dependently reversed by intravenously-administered rFVIIa, significantly so at 1 mg/kg. In a pulmonary thromboembolism (PE) mouse model, both darexaban and warfarin dose-dependently reduced the mortality rate, significantly so at 10 mg/kg and 3 mg/kg/day, respectively. In a FeCl3-induced venous thrombosis (VT) mouse model, darexaban (0.3-10 mg/kg) dose-dependently decreased the thrombus protein content, significantly so at doses of 3 mg/kg or higher. In a tail-transection mouse model, darexaban had no significant effect on the amount of blood loss at doses up to 10 mg/kg, while warfarin showed a dose-dependent increase in blood loss, significantly so from 1 mg/kg/day. Darexaban and its metabolite darexaban glucuronide significantly prolonged PT and aPTT in human plasma in vitro, and while rFVIIa concentration-dependently reversed the prolonged PT in this plasma, ACC dose-dependently reversed both PT and aPTT changes prolonged by darexaban. Taken together, these results suggest that darexaban has a potential to be an oral anticoagulant with a better safety profile than warfarin, and that rFVIIa and ACC may be useful as antidotes to darexaban in cases of overdose., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: The experiment was designed to analyze whether and how nitric oxide (NO) microbubbles facilitates deep vein thrombosis (DVT) resolution in a rat model., Methods and Results: DVT was induced by ligation of the inferior vena cava (IVC) and left common iliac vein (LCIV) in a rat model. The rats were sacrificed at day 2 and day 8 after ligation. NO was wrapped in lipidic microbubbles which were injected (1.6ml/kg) via tail vein twice a day. Thrombi isolated from IVC were measured by weight (g) and weight length ratio (g/cm).The histological analysis of LCIV indicated that platelets and inflammatory cells aggregation were reduced. The expression of vascular cell adhesion molecule-1 (VCAM-1) was quantified by immunohistochemical (IHC) staining and western blot. The coagulation functions including prothrombin time (PT), thrombin time (TT),activated partial thromboplastin time (APTT) and fibrinogen (FIB) were tested as well.Vein walls from IVC were processed by real-time polymerase chain reaction (RT-PCR) for several endothelial genes including matrix metalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9), thrombomodulin (TM), and endothelial nitric oxide synthase (eNOS). The thrombus weight and the expression of VCAM-1 significantly decreased after NO microbubbles treatment. The expression of these endothelial genes were significantly up-regulated by NO micribubbles. There was no statistical difference among the groups in terms of PT, APTT, and TT., Conclusion: NO microbubbles significantly facilitate DVT resolution in a rat model. The antithrombotic properties of NO microbubbles may be associated with reduced platelets and inflammatory cells aggregation, enhanced collagen turnover and stimulus to an anticoagulant condition of endothelium., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: The thrombin-activatable fibrinolysis inhibitor (TAFI) is a potent inhibitor of fibrinolysis. However, the time course of TAFI and its activated form (TAFIa) following trauma, in particular in patients suffering trauma-induced coagulopathy, has been poorly examined., Methods: A total of 26 severely injured trauma patients were prospectively enrolled. TAFI and TAFIa levels were measured upon arrival and through hospital days one to 10. Trauma-induced coagulopathy was defined as elevated international normalized ratio (INR), and/or prolonged activated partial thromboplastin time (aPTT) and/or thrombocytopenia within one day of admission., Results: TAFIa and TAFI levels showed the largest decrease on days one and two, respectively, with a progressive increase thereafter. Overall, 11 patients developed coagulopathy. No statistically significant differences were found for TAFI levels between the two groups. For TAFIa, however, coagulopathic patients experienced significantly lower levels on admission and on days six to eight (all p<0.05). Statistically significant correlations were found between TAFIa level on admission and the amount of packed red blood cells (p=0.011; Spearman's correlation coefficient=-0.5) and fresh frozen plasma (p=0.044; Spearman's correlation coefficient=-0.405) transfused within the initial 24hours., Conclusion: Depletion of TAFIa may contribute to the development of trauma-induced coagulopathy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Although no laboratory monitoring is needed for new anticoagulants, the measurement of their activity is required in special clinical situations. Standardised tests have been developed for rivaroxaban and dabigatran which allow the measurement of the patient's response to the drug at Cmax (2 to 3 hours after intake) or at trough (before repeated administration). The results can be expressed in mg per ml of plasma and compared to the expected concentrations. The influence of the new anticoagulants of coagulation assays has been determined. Several clinical cases of major bleeding have been reported and a severe coagulopathy was found in these patients (prolonged PT and aPTT, increased drug concentration in plasma). These observations raise the question regarding the potential benefit of laboratory coagulation monitoring from time to time. Trials are needed to determine the relationship of assay results with bleeding or thrombotic complications. Pros and Cons laboratory measurements are discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: Prothrombin G20210A (PT20210) carriers have increased prothrombin levels and increased risk for venous thrombosis. We hypothesized PT20210 carriers would have decreased PT, aPTT, and dRVVT clotting times., Methods: We reviewed 1186 thrombotic risk panels that included PT, aPTT, dRVVT, and PT20210 genotype with potential confounding variables, excluding samples consistent with anticoagulant therapy or lupus anticoagulant presence. We examined relationships of PT20210 with PT, aPTT, and dRVVT correcting for covariates using multivariate regression. We confirmed associations in 1876 separate panel results and a group of homozygotes for PT20210 and used general linear models to determine if associated tests predict PT20210 status., Results: Neither PT, aPTT, nor dRVVT was shorter in PT20210 carriers. Contrary to our hypothesis, PT20210 was significantly associated with higher dRVVT (p=0.001), but not PT or aPTT. dRVVT differences were significant in a replicate sample p=0.035 and an additional sample of PT20210 homozygotes (p=0.02). Of all variables available, only dRVVT predicted PT20210 carrier status (p=0.0008, AUC=0.64)., Conclusions: We observed an association between longer dRVVT and the prothrombin G20210A mutation in a retrospective observational study. These findings merit further study in large well-characterized clinical cohorts and laboratory research experiments., (Copyright © 2012 Elsevier Ltd. All rights reserved.)