Thrombin Inhibitor Research Articles

Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis

Haematophagous leeches express a broad variety of secretory proteins in their salivary glands, among them are hirudins and hirudin-like factors. Here, we describe the identification, molecular and initial functional characterization of Tandem-Hirudin (TH), a novel salivary gland derived factor identified in the Asian medicinal leech, Hirudinaria manillensis. In contrast to the typical structure of hirudins, TH comprises two globular domains arranged in a tandem-like orientation and lacks the elongated C-terminal tail. Similar structures of thrombin inhibitors have so far been identified only in kissing bugs and ticks. Expression of TH was performed in both cell-based and cell-free bacterial systems. A subsequent functional characterization revealed no evidence for a thrombin-inhibitory potency of TH.

Evaluation of a pharmacist-led intervention to improve medication adherence in patients initiating dabigatran treatment: a comparison with standard pharmacy practice in Poland

BackroundDabigatran is a direct thrombin inhibitor used to treat cardiac arrhythmias, and rates of non-adherence to dabigatran in Polish populations are high. The current study examined how a pharmacist-led intervention of counselling with pictogram-enhanced medication instructions, and smartphone medication reminders, can improve adherence to dabigatran.MethodsA 3-month pharmacist-led intervention was conducted in community pharmacies in Poland on 325 men and women filling a dabigatran prescription for the first time. Participating pharmacies were assigned into the Control Group (n = 172 patients) or the Intervention Group (n = 153 patients). The primary outcome of this prospective study was self-reported medication adherence assessed at 3 time points (day 7, day 21, and day 90) after initiation of dabigatran.ResultsPatients in the Intervention Group were significantly more adherent (mean days on Dabigatan/week) than the Control Group at 7 days (6.0 ± 0.9 vs 5.4 ± 1.1, p < 0.0001), 21 days (5.6 ± 1.0 vs 4.9 ± 1.3, p < 0.0001), and 90 days (5.5 ± 1.3 vs 4.4 ± 2.0, p < 0.0001), respectively. The percentage of patients in the Intervention Group who reported taking dabigatran twice/day as prescribed was significantly higher than the Control Group at 7 days (82.7% vs 71.4%, p = 0.0311), at 21 days (84.4% vs 58%, p < 0.0001), and at 90 days (78.4% vs 39.7%, p < 0.0001), respectively. The proportion of patients fully adherent (every day, twice/day) at 90 days was significantly higher in the Intervention Group than in the Control Group (26.1% vs 13.2%, p = 0.0145).ConclusionsOur findings support the role for interventions in community pharmacies in Poland to improve medication adherence, thus providing evidence for the efficacy of a pharmacist-led pictogram and smartphone-based program to support optimal dabigatran treatment.

(1R,3S)-THCCA-Asn: To show the discovery of selective inhibitor of thrombin by successfully combining virtual screening and biological assay

Thrombin is the most potent platelet aggregator. To discover the selective inhibitor of thrombin that is important to curing platelet aggregation-related diseases, docking experiments were performed to dock (1R,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1R,3S)-THCCA], and (1S,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1S,3S)-THCCA], into the p pocket of bovine thrombin. The ideal match supported that (1R,3S)-THCCA could be used as a potential lead compound. In this case 20 natural amino acids were theoretically introduced into the 3-carboxyl of (1R,3S)-THCCA and 20 derivatives, (1R,3S)-THCCA-amino acids, were docked into p pocket of bovine thrombin to perform virtual screening. The screening revealed that comparing to (1R,3S)-THCCA itself the DockScores of 16 derivatives were higher, and (1R,3S)-THCCA-Asn (4j) got the highest DockScore. Thus, 16 derivatives were synthesized for experimental study. The in vitro anti-platelet aggregation assay showed that at 100 μM of concentration the 16 derivatives failed to inhibit the platelet aggregation induced by both adenosine diphosphate and arachidonic acid. On the other hand, however, the IC50 value of the 16 derivatives inhibiting the platelet aggregation induced by platelet activating factor and thrombin ranged from 9.44 μM to 194.64 μM and from 0.07 μM to 9.56 μM, respectively. The in vitro anti-platelet aggregation assay suggested that the 16 derivatives selectively inhibited the platelet aggregation induced by thrombin. In particular, the IC50 of (1R,3S)-THCCA-Asn (4j) had the lowest value. On rat model at 1 nmol/kg of dosage the 16 derivatives effectively prevented thrombus formation. It is worth pointing out that even at 0.01 nmol/kg of dosage, 4j still effectively prevented thrombus formation. 4j hardly has effects on the proliferation of mammalian cells and rat tail bleeding time. In conclusion, the combination of virtual screening and biological assays successfully lead to the discovery of 4j as a promising candidate of selective inhibitor of thrombin.

Pulsed laser irradiation induces the generation of alloy cluster ions for the screening of protease activity

Pulsed laser irradiation can cause the fragmentation of nanoparticles, which generates cluster ions. This allows nanoparticles to be adopted as mass tag/signal amplifiers in laser desorption/ionization mass spectrometry (LDI-MS) bioassays. Herein, we demonstrate the potential of using the signal from alloy cluster ions in bioassays through a fibrin clot model to determine the activity of thrombin. A mixed solution of silver and gold nanoparticles functionalized with fibrinogen (Fg‒Ag NPs/Fg‒Au NPs) treated with thrombin can form clots composed of aggregated fibrin-Au NPs/Ag NPs. These clots analyzed with LDI-MS are noted to form intense Ag–Au alloy cluster ions, especially [Ag2Au]+, which were used to detect thrombin concentration with a dynamic range of 2.5–50 pM in human plasma. This sensing platform was further employed for the screening of direct thrombin inhibitors. This work developed a novel bioassay utilizing metallic gas-phase reactions generated from pulsed laser irradiation of aggregated nanoparticles to monitor enzymatic activity and to screen inhibitors. We believe that LDS-MS can serve as a new platform for gas-phase reaction-based bioassays.

Life without blood: Molecular and functional analysis of hirudins and hirudin‐like factors of the Asian non‐hematophagous leech Whitmania pigra

BackgroundSeveral leech species of the genera Hirudo, Hirudinaria, and Whitmania are widely used in traditional Chinese medicine (TCM) for the oral treatment of disorders associated with blood stasis. Among them, the non‐hematophagous leech Whitmania pigra expresses a variety of components that have the potential to act on the vertebrate blood coagulation system. ObjectiveWhether the thrombin inhibitor hirudin, probably the most prominent leech‐derived anticoagulant, is actually present in Whitmania pigra, is still a matter of debate. To answer that open question was the aim of the study. MethodsWe identified several putative hirudin‐encoding sequences in transcriptome data of Whitmania pigra. Upon gene synthesis and molecular cloning the respective recombinant proteins were expressed in Escherichia coli, purified, processed, and eventually functionally characterized for thrombin‐inhibitory potencies in coagulation assays. ResultsWe were successful in the identification and functional characterization of several putative hirudins in Whitmania pigra. Some, but not all, of these factors are indeed thrombin inhibitors. Whitmania pigra hence expresses both hirudins (factors that inhibit thrombin) and hirudin‐like factors (that do not or only very weakly inhibit thrombin). Furthermore, we revealed the exon/intron structures of the corresponding genes. Coding sequences of some putative hirudins of Whitmania pigra were present also in transcriptome datasets of Hirudo nipponia, a hematophagous leech that is likewise used in TCM. ConclusionsBased on both structural and functional data we provide very strong evidence for the expression of hirudins in Whitmania pigra. This is the first description of hirudins in a non‐hematophagous leech.

Role of protease‐activated receptor‐1 (PAR‐1) in the glomerular filtration barrier integrity

Protease‐activated receptors (PAR) play an important role in the regulation of cellular function by the coagulation system, and they are activated by thrombin. PAR‐1 is expressed in both endothelial cells and podocytes in the kidney. The role of PAR1 in the maintenance of the glomerular filtration barrier is not clear. Anticoagulant‐related nephropathy (ARN) is a kidney disease with glomerular hematuria and red blood cell tubular casts. We validated 5/6 nephrectomy (5/6NE) in rats as a model of ARN and had demonstrated that direct thrombin inhibitor (dabigatran) induces ARN. The aim of this study was to investigate the role of PAR‐1 in the ARN pathogenesis. 5/6NE rats were treated with dabigatran (150 mg/kg/day), PAR‐1 inhibitor SCH79797 (1 and 3 mg/kg/day) and PAR‐1 agonist TFLLR‐NH2 (0.25 and 0.50 µmol/kg/day) for 7 days. Serum creatinine and hematuria were assessed daily. Kidney morphology was evaluated at the end of the study. In 5/6NE rats treated with either dabigatran or combination with a PAR‐1 modulator, there was an elevation in serum creatinine, glomerular hematuria, red blood casts in the tubules, and acute tubular epithelial cell injury. Interestingly, both PAR‐1 modulators in a dose‐depended manner had similar effects on the serum creatinine levels and hematuria as those of dabigatran. Dabigatran‐induced increase in the systolic blood pressure was not affected by PAR‐1 modulators. In conclusion, the normal function of PAR‐1 is crucial to maintain the glomerular filtration barrier integrity. Either activation or blockage of PAR‐1 leads to glomerular hematuria and subsequent acute tubular epithelial cell injury.

Anticoagulant Effects of Dabigatran on Coagulation Laboratory Parameters in Pediatric Patients: Combined Data from Five Pediatric Clinical Trials.

Background Dabigatran etexilate, a direct oral thrombin inhibitor, is approved to treat venous thromboembolism (VTE) in both adults and children. Objectives This population analysis characterized relationships between dabigatran total plasma concentrations and coagulation laboratory parameters (activated partial thromboplastin time [aPTT]; diluted thrombin time [dTT]; ecarin clotting time [ECT]). Methods Data from three phase 2a and one single-arm and one randomized, comparative phase 2b/3 pediatric studies (measurements: aPTT 2,925 [ N = 358]; dTT 2,348 [ N = 324]; ECT 2,929 [ N = 357]) were compared with adult data (5,740 aPTT, 3,472 dTT, 3,817 ECT measurements; N = 1,978). Population models were fitted using nonlinear mixed-effects modeling. Covariates (e.g., sex, age) were assessed on baseline and drug-effect parameters, using a stepwise covariate model-building procedure. Results Overall, relationships between dabigatran, aPTT, dTT, and ECT were similar in children and adults. For children aged <6 months, a higher proportion of baseline samples were outside or close to the upper aPTT and ECT adult ranges. No age-related differences were detected for dTT. With increasing dabigatran concentration, aPTT rose nonlinearly (half the maximum effect at 368 ng/mL dabigatran) while dTT and ECT increased linearly (0.37 and 0.73% change per ng/mL dabigatran, respectively). Mean baseline aPTT (45 vs. 36 seconds) and ECT (40 vs. 36 seconds) were slightly increased for those aged <6 months versus older children. Conclusion The similar relationships of laboratory parameters observed across pediatric age groups suggests that developmental changes in the hemostatic system may have little effect on response to dabigatran.

Evaluation of clinical outcomes in patients treated with heparin or direct thrombin inhibitors during extracorporeal membrane oxygenation: a systematic review and meta-analysis

BackgroundThe number of patients treated with extracorporeal membrane oxygenation (ECMO) devices is increasing. Anticoagulation therapy is crucial to prevent thrombosis during ECMO therapy. Predominantly, heparin has been used as primary anticoagulant but direct thrombin inhibitors (DTI) have been established as alternatives. The aim of this systematic review and meta-analysis was to evaluate clinical outcomes in patients treated with heparin compared to different DTI during ECMO.MethodsA systematic search was conducted. Full scientific articles were sought for inclusion if heparin anticoagulation was compared to DTI (argatroban/bivalirudin) in ECMO patients. Risk of bias was assessed by Newcastle Ottawa scale. Primary endpoint was in-hospital mortality. Bleeding events, thrombotic events, hours of ECMO support, days of hospital stay, percentage of time within therapeutic range and time to therapeutic range were extracted from full texts as secondary endpoints. Results were presented as Forrest-plots. GRADE was used for confidence assessment in outcomes.ResultsSystematic search identified 4.385 records, thereof 18 retrospective studies for a total of 1942 patients, complied with the predefined eligibility criteria:15 studies investigated bivalirudin and 3 studies investigated argatroban versus heparin. Risk of bias was high for most studies. In-hospital mortality, major bleeding events and pump-related thrombosis were less frequent in DTI group as compared to heparin [mortality—OR 0.69, 95% CI 0.54–0.86; major bleeding—OR 0.48, 95% CI 0.29–0.81; pump thrombosis—OR 0.55, 95% CI 0.40–0.76]. Additionally, percentage of time within therapeutic range was higher for DTI [SMD 0.54, 95% CI 0.14–0.94]. GRADE approach revealed a very low level of certainty for each outcome.ConclusionIn this meta-analysis, DTI and especially bivalirudin showed beneficial effects on clinical outcomes in ECMO patients as compared to heparin. However, due to the lack of randomized trials, certainty of evidence is low.Trial RegistrationThis systematic review and meta-analysis was prospectively registered at PROSPERO data base (reference number CRD42021237252).Graphical

A chitosan based multimodal “soft” hydrogel for rapid hemostasis of non-compressible hemorrhages and its mode of action

Uncontrolled hemorrhages during accidents or surgery require non-compressible dressings to halt bleeding and avoid damage to soft tissue and organs. Here, we report the synthesis of a sodium tripolyphosphate crosslinked chitosan-based hemostatic hydrogel dressing incorporating hemostasis agonists like aluminum chloride, an aggregation enhancer and silica nanoparticles, a contact activator, as confirmed by FTIR analyses. Rheology studies displayed G” > G’ values confirming typical soft gel-like behavior that was maintained upto shear rate 10/s. Further, composite had excellent water absorption capacity (737 ± 50%) and showed 6-fold enhanced in vitro hemostasis as compared to bare hydrogel. Platelet aggregation was enhanced with composite as compared to SFLLRN peptide (a thrombin mimic) and adenosine diphosphate (calcium release activator) while it was absent with heparin (thrombin inhibitor). Enhanced activation and release of calcium were observed by composite with a specific fluorescent calcium dye in A549 cell model. The composite was biocompatible and in vivo evaluation of hemostatic efficiency in rat liver injury revealed enhanced blood clotting (16 ± 2.1 s) of hydrogel composite as compared to commercial HemoStat Clear gel (55 ± 5 s). Thus, developed soft hydrogel composite has potential for application as a safe, easy-to-apply, rapid, non-compressible hemostatic agent.

Differential Antivenom and Small-Molecule Inhibition of Novel Coagulotoxic Variations in Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium American Viperid Snake Venoms.

Within Neotropical pit-vipers, the Mexican/Central-American clade consisting of Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium is a wide-ranging, morphologically and ecologically diverse group of snakes. Despite their prevalence, little is known of the functional aspects of their venoms. This study aimed to fill the knowledge gap regarding coagulotoxic effects and to examine the potential of different therapeutic approaches. As a general trait, the venoms were shown to be anticoagulant but were underpinned by diverse biochemical actions. Pseudo-procoagulant activity (i.e., thrombin-like), characterized by the direct cleavage of fibrinogen to form weak fibrin clots, was evident for Atropoides picadoi, Cerrophidion tzotzilorum, Metlapilcoatlus mexicanus, M. nummifer, M. occiduus, M. olmec, and Porthidium porrasi. In contrast, other venoms cleaved fibrinogen in a destructive (non-clotting) manner, with C. godmani and C. wilsoni being the most potent. In addition to actions on fibrinogen, clotting enzymes were also inhibited. FXa was only weakly inhibited by most species, but Cerrophidion godmani and C. wilsoni were extremely strong in their inhibitory action. Other clotting enzymes were more widely inhibited by diverse species spanning the full taxonomical range, but in each case, there were species that had these traits notably amplified relatively to the others. C. godmani and C. wilsoni were the most potent amongst those that inhibited the formation of the prothrombinase complex and were also amongst the most potent inhibitors of Factor XIa. While most species displayed only low levels of thrombin inhibition, Porthidium dunni potently inhibited this clotting factor. The regional polyvalent antivenom produced by Instituto Picado Clodomiro was tested and was shown to be effective against the diverse anticoagulant pathophysiological effects. In contrast to the anticoagulant activities of the other species, Porthidium volcanicum was uniquely procoagulant through the activation of Factor VII and Factor XII. This viperid species is the first snake outside of the Oxyuranus/Pseudonaja elapid snake clade to be shown to activate FVII and the first snake venom of any kind to activate FXII. Interestingly, while small-molecule metalloprotease inhibitors prinomastat and marimastat demonstrated the ability to prevent the procoagulant toxicity of P. volcanicum, neither ICP antivenom nor inhibitor DMPS showed this effect. The extreme variation among the snakes here studied underscores how venom is a dynamic trait and how this can shape clinical outcomes and influence evolving treatment strategies.

Hemoclot Thrombin Inhibitor Assay and Expected Peak-Trough Levels of Dabigatran: A Multicenter Study.

PurposeDabigatran concentrations monitoring are gaining importance of special situations, but limited data are available for the expected peak and trough levels. The hemoclot thrombin inhibitor (HTI) is dabigatran-calibrated quantitative determination of dabigatran concentration. This study aims to validate HTI assay as the quantification choice of dabigatran, and providing the expected peak and trough levels.Materials and MethodsThis is a multi-center methodology validate study, including seven hospitals from Beijing, Shanghai, Henan, Hunan, Chongqing, and Fujian. We retrospectively analyzed plasma samples taken from 118 healthy subjects and 183 patients receiving dabigatran. Dabigatran concentrations were measured with HTI assay and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Linear regression, Spearman correlation and Bland-Altman analysis were used in this study.ResultsThe mean concentration ratio of HPLC-MS/MS and HTI assays was 1.03 and 0.98 at 2 and 12 h, and the acceptance ranges for both the ratio limit as well as the limit of agreement were met, suggesting good agreement between the HTI-derived plasma concentrations and HPLC-MS/MS. The reference detection range of single dose dabigatran 150 mg in healthy subjects was 33–159 ng/ml. About 500 blood samples were taken from 183 patients suggested that the expected peak and trough levels range of dabigatran 110 mg was about 95–196 and 36–92 ng/ml.ConclusionHemoclot thrombin inhibitor assay can be a good quantitative detection method of dabigatran. Expected peak and trough levels provide a basis for the rational use of dabigatran, and provide important Asian population data for the update of the international clinical guidelines for hematological testing.Clinical Trial Registration[https://clinicaltrials.gov], identifier [NCT03161496].

Modified thromboelastometric tests provide improved sensitivity and specificity to direct oral anticoagulants compared to standard thromboelastometric tests in-vitro

BackgroundThe detection of direct oral anticoagulants (DOACs) is still challenging but important in emergency patients. We recently demonstrated that modified thromboelastometry can detect rivaroxaban and dabigatran. Data on the detection rates of modified compared to standard thromboelastometric tests of apixaban and edoxaban, are missing. The aim of this in-vitro dose-effect-study was to add data on these DOACs and to evaluate thromboelastometric tests in-vitro using data of both studies.MethodsThe study was approved by the Ludwig-Maximilians-University ethics committee (No 17-525-2). Written informed consent was obtained from all individuals. Blood samples of healthy volunteers and samples of 10 volunteers for each DOAC were used. Blood samples were spiked with six different concentrations of edoxaban and apixaban (0ng/ml; 31.25ng/ml; 62.5ng/ml; 125ng/ml; 250 ng/ml; 500ng/ml). Modified tests (low-tissue-factor test TFTEM and ecarin-based test ECATEM) as well as standard tests (e.g. FIBTEM) analyzing extrinsic pathway of coagulation were used. Receiver operating characteristics analyzes were performed as well as regression analyzes.ResultsTFTEM CT correlated well with anti-Xa levels of apixaban and edoxaban (apixaban: r2 = 0.8064 p < 0.0001; edoxaban: r2 = 0.8603; p < 0.0001). The detection of direct FXa inhibitors (> 30 ng/mL) was successful with FIBTEM CT with a sensitivity and specificity of 81% and 90%, respectively. As expected, ECATEM CT was not prolonged by direct FXa-inhibitors due to its specificity for direct thrombin inhibitors. Again, TFTEM CT provided the highest sensitivity and specificity for the detection of direct FXa inhibitors with 96% and 95%, respectively. ECATEM test showed 100% sensitivity and 100% specificity for the detection of dabigatran.ConclusionsOur study presents modified thromboelastometric tests with improved detection of even low DOAC concentrations > 30 ng/mL, including apixaban in-vitro. The study thus complements the previously published data on dabigatran and rivaroxaban. Validation studies must confirm the results due to the explanatory design of this study.

Factor Xa inhibitors, Era to replace traditional anticoagulants

The past 15 years have seen increased usage of factor Xa inhibitor anticoagulants such as rivaroxaban, apixaban, and edoxaban, along with dabigatran (competitive direct thrombin inhibitor), which have been used increasingly in various clinical settings, for the prevention and treatment of thrombosis. In many ways Factor Xa inhibitors have replaced the use of warfarin, which requires prudent monitoring . Factor Xa inhibitor short half-lives, compared to warfarin’s, provide some assurance that the drug concentrations will decline rapidly when therapy is discontinued in patients with normal renal function. Good haemostatic efficacy was achieved in 83% patients on apixaban and 80% patients on rivaroxaban . Major bleeding events in nonvalvular atrial fibrillation patients on rivaroxaban were 3.6% per year and on apixaban were 2.13% per year in respective landmark trials conducted. Some drawbacks of Factor Xa inhibitors include uncertainty about dosing in some patient populations (eg, renal dysfunction, marked extremes of body weight), and their higher drug cost.

Pharmacotherapy for pulmonary embolism: new anticoagulants

Background: Pulmonary embolism is associated with reduced survival and considerable economic burden worldwide. In Korea, the incidence of pulmonary embolism has been gradually increasing. Older individuals are at an increased risk for pulmonary embolism and anticoagulation-related bleeding events. Typically, heparin and vitamin K antagonists are employed to treat pulmonary embolism; however, these agents present numerous limitations. Hence, novel anticoagulants with improved safety and efficacy profiles are urgently needed.Current Concepts: Direct oral anticoagulants (DOACs), including direct thrombin (coagulation factor II) inhibitors and selective inhibitors of coagulation factor Xa, have emerged as alternative agents. Phase III, large-scale clinical trials have revealed that DOACs are non-inferior to standard therapy during initial and long-term treatment of pulmonary embolism, considering the safety profile. Evidence-based clinical guidelines recommend that primary care clinicians employ DOACs over warfarin to achieve anticoagulation.Discussion and Conclusion: For over 70 years, the standard therapy for most patients with pulmonary embolism has involved heparin administration, overlapped and followed by a vitamin K antagonist. Recently developed DOACs against coagulation factor Xa or thrombin might overcome limitations of standard therapy, including the need for injection and regular dose adjustment with laboratory monitoring. These limitations hinder the management of patients with pulmonary embolism and negatively impact the patient’s quality of life. Four DOACs, including apixaban, dabigatran, edoxaban, and rivaroxaban, are currently available for treating pulmonary embolism in Korea, which could simplify the therapeutic strategy.

The Effect of Heparin and Its Preparations on Disseminated Intravascular Coagulation Mortality and Hospitalization: A Systematic Review.

Methods The databases of PubMed, Scopus, Embase, and Web of Science were searched systematically up to November 2021. The quality of RCTs was assessed by Cochrane Collaboration's tool and the risk of bias was assessed for cohort studies through NOS score. Results Out of 3288 articles, eight studies were eligible to be included in this study. Our review retrieved six RCTs and two retrospective cohort studies consisting of 950 participants diagnosed by DIC. A significant effect of heparin on DIC mortality was identified in four studies. Furthermore, heparin was used as a control group in three studies. Conclusions We concluded that administration of heparin and its preparations in DIC patients could reduce the mortality rate and duration of hospitalization, especially in the earlier stages of DIC.

The Changing Landscape of Anticoagulation in Pediatric Extracorporeal Membrane Oxygenation: Use of the Direct Thrombin Inhibitors

Bleeding and thrombosis frequently occur in pediatric patients with extracorporeal membrane oxygenation (ECMO) therapy. Until now, most patients are anticoagulated with unfractionated heparin (UFH). However, heparin has many disadvantages, such as binding to other plasma proteins and endothelial cells in addition to antithrombin, causing an unpredictable response, challenging monitoring, development of heparin resistance, and risk of heparin-induced thrombocytopenia (HIT). Direct thrombin inhibitors (DTIs), such as bivalirudin and argatroban, might be a good alternative. This review will discuss the use of both UFH and DTIs in pediatric patients with ECMO therapy.

Performance of a Qualitative Point-of-Care Strip Test to Detect DOAC Exposure at the Emergency Department: A Cohort-Type Cross-Sectional Diagnostic Accuracy Study.

An accurate point-of-care test for detecting effective anticoagulation by direct oral anticoagulants (DOACs) in emergencies is an unmet need. We investigated the accuracy of a urinary qualitative strip test (DOAC Dipstick) to detect relevant DOAC exposure in patients who presented to an emergency department. In this prospective single-center cohort-type cross-sectional study, adults on DOAC treatment were enrolled. We assessed clinical sensitivity and specificity of DOAC Dipstick factor Xa and thrombin inhibitor pads to detect DOAC plasma levels ≥30 ng/mL using urine samples as the testing matrix. Liquid chromatography coupled with tandem-mass spectrometry was used as the reference standard method for plasma and urine measurement of DOAC concentrations. Of 293 patients enrolled, 265 patients were included in the analysis, of whom 92 were treated with rivaroxaban, 65 with apixaban, 77 with edoxaban, and 31 with dabigatran. The clinical sensitivity and specificity of the dipstick on urine samples to detect ≥30 ng/mL dabigatran plasma levels were 100% (95% confidence interval [CI]: 87–100%) and 98% (95% CI: 95–99%), respectively. The sensitivity and specificity of the dipstick to detect ≥30 ng/mL factor Xa inhibitor plasma levels were 97% (95% CI: 94–99%) and 69% (95% CI: 56–79%), respectively. The DOAC Dipstick sensitively identified effective thrombin and factor Xa inhibition in a real-world cohort of patients presenting at an emergency department. Therefore, the dipstick might provide a valuable test to detect relevant DOAC exposure in emergencies, although further studies will be needed to confirm these findings.

Assessing the Role of a Malonamide Linker in the Design of Potent Dual Inhibitors of Factor Xa and Cholinesterases.

The rational discovery of new peptidomimetic inhibitors of the coagulation factor Xa (fXa) could help set more effective therapeutic options (to prevent atrial fibrillation). In this respect, we explored the conformational impact on the enzyme inhibition potency of the malonamide bridge, compared to the glycinamide one, as a linker connecting the P1 benzamidine anchoring moiety to the P4 aryl group of novel selective fXa inhibitors. We carried out structure–activity relationship (SAR) studies aimed at investigating para- or meta-benzamidine as the P1 basic group as well as diversely decorated aryl moieties as P4 fragments. To this end, twenty-three malonamide derivatives were synthesized and tested as inhibitors of fXa and thrombin (thr); the molecular determinants behind potency and selectivity were also studied by employing molecular docking. The malonamide linker, compared to the glycinamide one, does significantly increase anti-fXa potency and selectivity. The meta-benzamidine (P1) derivatives bearing 2′,4′-difluoro-biphenyl as the P4 moiety proved to be highly potent reversible fXa-selective inhibitors, achieving inhibition constants (Ki) in the low nanomolar range. The most active compounds were also tested against cholinesterase (ChE) isoforms (acetyl- or butyrylcholinesterase, AChE, and BChE), and some of them returned single-digit micromolar inhibition potency against AChE and/or BChE, both being drug targets for symptomatic treatment of mild-to-moderate Alzheimer’s disease. Compounds 19h and 22b were selected as selective fXa inhibitors with potential as multimodal neuroprotective agents.

Induction of endothelial barrier dysfunction by serum factors in rats subjected to traumatic brain injury and hemorrhagic shock.

Traumatic brain injury (TBI) has been associated with the development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanisms linking TBI with the development of ARDS, we characterized the effects of serum factors released following TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS. We found that serum samples from animals exposed to both controlled cortical impact (CCI) and HS, but not from sham‐operated rats induced significant barrier dysfunction in human pulmonary artery EC monolayers at 2 days post injury. Thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of the serum‐induced trans‐endothelial resistance (TER) decline caused by CCI‐HS serum, but not in later time points. However, both the early and late phases of CCI‐HS‐induced EC permeability were inhibited by heparin. The barrier disruptive effects of CCI‐HS serum were also prevented by serum preincubation with heparin‐sepharose. Pulmonary EC treated for 3 h with serum from CCI‐HS rats demonstrated a significant decline in expression of EC junctional protein, VE‐Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE‐cadherin antibody. These results suggest that exposure to CCI‐HS causes early and late‐phase barrier disruptive effects in vascular endothelium. While thrombin‐PAR1 signaling has been identified as a mechanism of acute EC permeability increase by CCI‐HS serum, the factor(s) defining long‐term EC barrier disruption in CCI‐HS model remains to be determined.

Direct (New) Oral Anticoagulants (DOACs): Drawbacks, Bleeding and Reversal

Direct (new) Oral Anticoagulants (DOACs) have emerged as a contemporary and promising option in the treatment of thromboses and VTE, while protecting the coagulation cascade against untoward bleeding events. They are used in the management and prophylaxis of Venous Thromboembolism (VTE) and other thrombotic diseases. The most prominent complication of these agents is bleeding. These agents have similar or lower rates of major intracranial hemorrhages, while they had a higher risk of major gastrointestinal bleeding when compared to warfarin. This manuscript is aimed to revise and update the literature findings to outline the side effects of DOACs in various clinical scenarios. A narrative review of currently published studies was performed. Online database searches were performed for clinical trials published before July 2021, on the efficacy and adverse effects attributed to the anticoagulant treatment, especially DOACs. A literature search via electronic databases was carried out, beginning with the usage of the agents in the Western Languages papers. The search terms initially included direct (new) oral anticoagulants, dabigatran, rivaroxaban, apixaban, edoxaban, idarucizumab, andexanet, prothrombin complex concentrates, and fresh frozen plasma. Papers were examined for methodological soundness before being included. Severe bleeding episodes require aggressive interventions for successful management. Therefore, bleeding should be evaluated in special regard to the location and rate of hemorrhage, and total volume of blood loss. Patient's age, weight and organ dysfunctions (e.g., kidney/liver failure or chronic respiratory diseases) directly affect the clinical course of overdose. Management recommendations for hemorrhage associated with DOAC use vary, depending on the class of the culprit agent (direct thrombin inhibitor vs. FXa inhibitor), the clinical status of the patient (mild/ moderate vs. severe/life-threatening), and capabilities of the institution. Specific reversal agents (i.e., idarucizumab and andexanet alfa) can be used if available, while prothrombin complex concentrates, fresh frozen plasma and/ or tranexamic acid can also be employed as nonspecific replacement agents in the management of DOAC-related bleeding diathesis.