A self-elastic chitosan sponge integrating active and passive hemostatic mechanisms for effectively managing uncontrolled coagulopathic hemorrhage.

Developing a self-elastic sponge integrating active and passive hemostatic mechanisms for the effective management of uncontrolled coagulopathic hemorrhage remains a challenge. We here developed a chitosan-based sponge by integrating freeze-drying, chemical decoration of alkyl chains and phosphate groups, and physical loading of thrombin. The sponge exhibited high mechanical strength, self-elasticity, and rapid shape recovery. The sponge facilitated blood cell adhesion, aggregation, and activation through hydrophobic and electrostatic interactions, as well as accelerated blood clotting. The sponge exhibited higher efficacy than commercial gauze and gelatin sponge in managing uncontrolled hemorrhage from heparinized rat tail amputation, liver superficial injury, and liver perforating wound models. In addition, the sponge exhibited favorable biodegradability and biocompatibility. These findings revealed that the developed sponge holds great potential as a novel hemostat for effectively managing uncontrolled coagulopathic hemorrhage from superficial and perforating wounds.

Radial artery pseudoaneurysm a rare complication after coronary angiography: A case report and systematic review of the reported cases.

This is a case of 83 years old male who had radial artery pseudoaneurysm after cardiac catheterization. The diagnosis was through Doppler ultrasound and the patient was treated with thrombin injection and reported good outcomes. The literature also included 41 cases of pseudoaneurysm after catheterization. The mean age of patients was 68.5 years with a male prevalence of 49%. Onset of pseudoaneurysm ranged from 0 days (directly after the catheterization) to 150 days with a median of 5 days. The treatment of patients was mainly surgical (19 cases) followed by compression (either manual or TR band) (12 cases), thrombin injection (four cases), compression then surgery (three cases), compression then thrombin injection (one case), percutaneous endovascular repair using a covered stent (one case) and not reported in one case. All cases recovered well.

Clinical value of TAT, PIC and t-PAIC as predictive markers for severe sepsis in pediatric patients.

Objective: Sepsis in pediatric patients can progress to severe sepsis, and identifying biomarkers of this progression may permit timely intervention to prevent it. This study aimed to investigate the ability of thrombin-antithrombin complex (TAT), α2-plasmininhibitor-plasmin complex (PIC) and tissue-type plasminogen activator-inhibitor complex (t-PAIC) to predict severe sepsis in pediatrics early. Methods: 148 eligible pediatric sepsis patients were enrolled in this study, and were then divided into those who progressed to severe sepsis (n = 50) or not (n = 98). Serum levels of TAT, PIC, and t-PAIC were analysed, and simplified pediatric critical illness score (PCIS) and DIC score were calculated on the day of pediatric sepsis diagnosis. Results: Compared with sepsis patients, severe sepsis patients had higher levels of TAT, PIC and t-PAIC. Correlation analysis revealed that TAT, PIC and t-PAIC were significantly correlated with simplified PCIS and DIC score. ROC curve analysis suggested that TAT, PIC and t-PAIC could serve as biomarkers for predicting severe sepsis with the AUC up to 0.862, 0.759 and 0.851, respectively. Stratified analysis demonstrated that the patients with increased levels of TAT, PIC and t-PAIC had worse illness severity and clinical outcome. Univariate logistic regression analysis revealed that TAT, PIC and t-PAIC were all risk factors for severe sepsis, yet only TAT and t-PAIC were independent risk factors in multivariate model. Conclusions: TAT, PIC and t-PAIC could serve as biomarkers for predicting severe sepsis, and correlated with illness severity in pediatrics, what's more, serum levels of TAT and t-PAIC may be independent risk factors for pediatric severe sepsis.

Evaluating the Efficacy and Safety of Hemofence (Thorombin Cross-linked Sodium Hyaluronate Gel Matrix) in Hemostasis for Intractable Exudative Bleeding in Spinal Surgery: A Multicenter, Randomized, Phase III Clinical Trial.

Objective: To demonstrate the non-inferiority of the novel hemostatic agent, Hemofence® (BMI Korea Co. Ltd., Jeju Korea, thrombin cross-linked sodium hyaluronate gel matrix) compared to the established agent, Floseal Hemostatic Matrix (Baxter, thrombin-gelatin matrix) in achieving hemostasis for spinal surgeries, with secondary objectives to assess additional efficacy and safety. Methods: This clinical trial was a multicenter, randomized, subject-blinded, active-controlled, parallel-group, phase 3 study. Investigational drugs were administered to the first and second bleeding sites of each participant (or only to the first site if a second site was absent), evaluating hemostasis success rate within 10 minutes and the time to achieve hemostasis. Subsequent visits were conducted for safety assessments. For non-inferiority test, a 97.5% one-sided confidence interval was used; the test group was deemed non-inferior if the lower limit exceeded -10%. Results: This trial showed a 97.10% success rate in the test group and 96.05% in the control group for primary efficacy. The 95% confidence interval (-4.90%, 7.44%) confirmed the test drug's non-inferiority. Time to hemostasis showed no significant difference between groups. All adverse events, adverse drug reactions, and serious adverse events were statistically similar between groups (p=1.0000, p=0.2427, and p=0.9663, respectively). Conclusion: A novel hemostatic agent, Hemofence®, demonstrated an efficacy and safety profile comparable to that of Floseal.

Designing Rigid DNA Origami Templates for Molecular Visualization Using Cryo-EM.

DNA origami, a method for constructing nanostructures from DNA, offers potential for diverse scientific and technological applications due to its ability to integrate various molecular functionalities in a programmable manner. In this study, we examined the impact of internal crossover distribution and the compositional uniformity of staple strands on the structure of multilayer DNA origami using cryogenic electron microscopy (cryo-EM) single-particle analysis. A refined DNA object was utilized as an alignment framework in a host-guest model, where we successfully resolved an 8 kDa thrombin binding aptamer (TBA) linked to the host object. Our results broaden the spectrum of DNA in structural applications.

Antithrombin supplementation attenuates heparin resistance in plasma spiked with Gla-domainless factor Xa S195A in vitro.

BACKGROUND: Andexanet alfa is a Gla-domainless mutant (S195A) factor Xa (GDXa) approved for acute reversal of oral factor Xa inhibitors. Cardiac surgery patients exposed to andexanet before cardiopulmonary bypass often exhibit severe heparin resistance. There is a paucity of data on the effectiveness and optimal dosage of antithrombin use in this setting. The objective of this study was to evaluate the in vitro effect of increased heparin with antithrombin levels on attenuating heparin resistance induced by GDXa. METHODS: Heparinised normal pooled plasma and cardiopulmonary bypass plasma were spiked with GDXa 4 µM. Tissue factor-activated thrombin generation was used to assess heparin reversal effects of GDXa and restoration of anticoagulation with additional heparin with and without antithrombin. Serum thrombin-antithrombin complex, antithrombin activity, and tissue factor pathway inhibitor were also measured in tissue factor-activated, recalcified cardiopulmonary bypass plasma spiked with GDXa. RESULTS: In normal pooled plasma, GDXa-induced heparin reversal was mitigated by maintaining a high heparin concentration (12 U ml-1) and supplementing antithrombin (1.5-4.5 µM) based on peak and velocity of thrombin generation. Heparin reversal by GDXa was also demonstrated in cardiopulmonary bypass plasma, but supplementing both heparin (8 U ml-1) and antithrombin (3 µM) attenuated GDXa-induced changes in peak and velocity of thrombin generation by 72.5% and 72.2%, respectively. High heparin and antithrombin levels attenuated thrombin-antithrombin complex formation in tissue factor-activated, GDXa-spiked cardiopulmonary bypass plasma by 85.7%, but tissue factor pathway inhibitor remained depleted compared with control cardiopulmonary bypass plasma. CONCLUSIONS: Simultaneous supplementation of heparin and antithrombin mitigate GDXa-induced heparin resistance by compensating for the loss of tissue factor pathway inhibitor.

Performance of direct oral anticoagulant (DOAC) testing by hemostasis laboratories: The Australasian/Asia-Pacific experience.

INTRODUCTION: Direct oral anticoagulants (DOACs) reflect anticoagulation agents given to treat or prevent thrombosis, having largely replaced vitamin K antagonists (VKAs) such as warfarin. DOACs are given in fixed daily doses and generally do not need monitoring. However, there may be a variety of reasons that justify measurement of plasma DOAC levels in individual patients. METHODS: We report updated findings for DOAC testing in our geographic region, using recent data from the RCPAQAP, an international external quality assessment (EQA) program, currently with some 40-60 participants in each of the different DOAC (rivaroxaban, apixaban, dabigatran) modules, to assess laboratory performance in this area. Data has been assessed for the past 5 years (2019-2023 inclusive), with 20 samples each per DOAC. RESULTS: Data shows a limited repertoire of assays in use, and mostly consistency in reported numerical values when assessing proficiency samples. Available assays mostly comprised reagents from four manufacturing suppliers. There was good consistency across what participants identified as 'DOAC detected', but some variability when participants attempted to grade DOAC levels as low vs moderate vs high. Inter-laboratory/method coefficient of variation (CVs) were generally <15% for each DOAC, when present at >100 ng/mL. CONCLUSION: We hope our findings, reflecting on mostly consistent reporting of DOAC levels and interpretation provides reassurance for clinicians requesting these measurements, and helps support their implementation in regions where there is a paucity of test availability.

An electrochemical biosensor for the amplification of thrombin activity by perylene-mediated photoinitiated polymerization.

BACKGROUND: Thrombin, a coagulation system protease, is a key enzyme involved in the coagulation cascade and has been developed as a marker for coagulation disorders. However, the methods developed in recent years have the disadvantages of complex operation, long reaction time, low specificity and sensitivity. Meanwhile, thrombin is at a lower level in the pre-disease period. Therefore, to accurately diagnose the disease, it is necessary to develop a fast, simple, highly sensitive and specific method using signal amplification technology. RESULTS: We designed an electrochemical biosensor based on photocatalytic atom transfer radical polymerization (photo-ATRP) signal amplification for the detection of thrombin. Sulfhydryl substrate peptides (without carboxyl groups) are self-assembled to the gold electrode surface via Au-S bond and serve as thrombin recognition probes. The substrate peptide is cleaved in the presence of thrombin to generate -COOH, which can form a carboxylate-Zr(IV)-carboxylate complex via Zr(IV) and initiator (α-bromophenylacetic acid, BPAA). Subsequently, an electrochemical biosensor was prepared by introducing polymer chains with electrochemical signaling molecules (ferrocene, Fc) onto the electrode surface by photocatalytic (perylene, Py) mediated ATRP using ferrocenylmethyl methacrylate (FMMA) as a monomer. The concentration of thrombin was evaluated by the voltammetric signal generated by square wave voltammetry (SWV), and the result showed that the biosensor was linear between 1.0 ng/mL âˆ¼ 10 fg/mL, with a lower detection limit of 4.0 fg/mL (∼0.1 fM). Moreover, it was shown to be highly selective for thrombin activity in complex serum samples and for thrombin inhibition screening. SIGNIFICANCE: The biosensor is an environmentally friendly and economically efficient strategy while maintaining the advantages of high sensitivity, anti-interference, good stability and simplicity of operation, which has great potential for application in the analysis of complex samples.

The thrombin receptor PAR4 supports visceral adipose tissue inflammation.

Thrombin inhibition suppresses adiposity, WAT inflammation and metabolic dysfunction in mice. Protease-activated receptor (PAR)1 does not account for thrombin-driven obesity, so we explored the culprit role of PAR4 in this context. Male WT and PAR-4-/- mice received a high fat diet (HFD) for 8 weeks, WT controls received standard chow. Body fat was quantified by NMR. Epididymal WAT was assessed by histology, immunohistochemistry, qPCR and lipase activity assay. 3T3-L1 preadipocytes were differentiated ± thrombin, acutely stimulated ± PAR4 activating peptide (AP) and assessed by immunoblot, qPCR and U937 monocyte adhesion. Epicardial adipose tissue (EAT) from obese and lean patients was assessed by immunoblot. PAR4 was upregulated in mouse WAT under HFD. PAR4-/- mice developed less visceral adiposity and glucose intolerance under HFD, featuring smaller adipocytes, fewer macrophages and lower expression of adipogenic (leptin, PPARγ) and pro-inflammatory genes (CCL2, IL-1ß) in WAT. HFD-modified activity and expression of lipases or perilipin were unaffected by PAR4 deletion. 3T3-L1 adipocytes differentiated with thrombin retained Ki67 expression, further upregulated IL-1ß and CCL2 and were more adhesive for monocytes. In mature adipocytes, PAR4-AP increased phosphorylated ERK1/2 and AKT, upregulated Ki67, CCl2, IL-ß and hyaluronan synthase 1 but not TNF-α mRNA, and augmented hyaluronidase-sensitive monocyte adhesion. Obese human EAT expressed more PAR4, CD68 and CD54 than lean EAT. PAR4 upregulated in obesity supports adipocyte hypertrophy, WAT expansion and thrombo-inflammation. The emerging PAR4 antagonists provide a therapeutic perspective in this context beyond their canonical antiplatelet action.

Electrochemical Monitoring in Anticoagulation Therapy.

The process of blood coagulation, wherein circulating blood transforms into a clot in response to an internal or external injury, is a critical physiological mechanism. Monitoring this coagulation process is vital to ensure that blood clotting neither occurs too rapidly nor too slowly. Anticoagulants, a category of medications designed to prevent and treat blood clots, require meticulous monitoring to optimise dosage, enhance clinical outcomes, and minimise adverse effects. This review article delves into the various stages of blood coagulation, explores commonly used anticoagulants and their targets within the coagulation enzyme system, and emphasises the electrochemical methods employed in anticoagulant testing. Electrochemical sensors for anticoagulant monitoring are categorised into two types. The first type focuses on assays measuring thrombin activity via electrochemical techniques. The second type involves modified electrode surfaces that either directly measure the redox behaviours of anticoagulants or monitor the responses of standard redox probes in the presence of these drugs. This review comprehensively lists different electrode compositions and their detection and quantification limits. Additionally, it discusses the potential of employing a universal calibration plot to replace individual drug-specific calibrations. The presented insights are anticipated to significantly contribute to the sensor community's efforts in this field.

Characterization of Biomarkers of Thrombo-Inflammation in Patients with First-Diagnosed Atrial Fibrillation.

Patients with first-diagnosed atrial fibrillation (FDAF) exhibit major adverse cardiovascular events (MACEs) during follow-up. Preclinical models have demonstrated that thrombo-inflammation mediates adverse cardiac remodeling and atherothrombotic events. We have hypothesized that thrombin activity (FIIa) links coagulation with inflammation and cardiac fibrosis/dysfunction. Surrogate markers of the thrombo-inflammatory response in plasma have not been characterized in FDAF. In this prospective longitudinal study, patients presenting with FDAF (n = 80), and 20 matched controls, were included. FIIa generation and activity in plasma were increased in the patients with early AF compared to the patients with chronic cardiovascular disease without AF (controls; p < 0.0001). This increase was accompanied by elevated biomarkers (ELISA) of platelet and endothelial activation in plasma. Pro-inflammatory peripheral immune cells (TNF-α+ or IL-6+) that expressed FIIa-activated protease-activated receptor 1 (PAR1) (flow cytometry) circulated more frequently in patients with FDAF compared to the controls (p < 0.0001). FIIa activity correlated with cardiac fibrosis (collagen turnover) and cardiac dysfunction (NT-pro ANP/NT-pro BNP) surrogate markers. FIIa activity in plasma was higher in patients with FDAF who experienced MACE. Signaling via FIIa might be a presumed link between the coagulation system (tissue factor-FXa/FIIa-PAR1 axis), inflammation, and pro-fibrotic pathways (thrombo-inflammation) in FDAF.

Ubiquitin-driven G Protein-Coupled Receptor Inflammatory Signaling at the Endosome.

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-ß-arrestin signaling. Besides phosphorylation, GPCRs are also post-translationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses. In this mini-review, we discuss emerging discoveries that provide critical insight into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

Exploring the effects of Factor Xa inhibitors on thrombin generation in people with haemophilia.

Optimal management of cardiovascular disease (CVD) in people with haemophilia (PWH) is a growing issue, given the continuing improvement in life expectancy among PWH. The evolving treatment paradigms targeting higher trough levels and the advent of non-factor replacement therapies (NFRT) means much of the 'protection' PWH were thought to have against CVD may be lost. There is a paucity of evidence regarding the safety of using anticoagulants in PWH. We designed a study assessing the thrombin generation (TG) of PWH of different severities and treatments, compared to non-haemophilia patients receiving a Factor Xa (FXa) inhibitor (apixaban or rivaroxaban), healthy controls, and assessing TG parameters of adding FXa inhibitor to the plasma of PWH receiving emicizumab prophylaxis. In total, 40 patients were included. TG was initiated with 5pM tissue factor (TF) using the calibrated automated thrombinoscope. Compared to those with mild haemophilia, patients receiving a FXa inhibitor had higher endogenous thrombin potential (ETP) (1278.42 vs 1831.36) and velocity index (40.71 vs 112.56), but both had a similar peak height (154.0 vs 262.63) and time to peak (both 5.83). People with severe haemophilia receiving emicizumab had significantly improved TG parameters compared to those not receiving emicizumab - ETP 1678.11 vs 809.96 and peak height 233.8 vs 92.05; however, when FXa inhibitor was added their TG parameters deteriorated to the severe haemophilia range (ETP 1179.60 and peak height 103.05). TG may provide additional useful information regarding the use of anticoagulants in PWH.

The therapeutic effect of proteinase-activated receptor-1 antagonist on colitis-associated carcinogenesis.

BACKGROUND & AIMS: Inflammatory bowel disease is associated with carcinogenesis, which limits the prognosis of the patients. The local expression of proteinases and proteinase-activated receptor 1 (PAR1) increases in the inflammatory bowel disease. The present study investigated the therapeutic effects of PAR1 antagonism on colitis-associated carcinogenesis. METHODS: A colitis-associated carcinogenesis model was prepared in mice by treatment with azoxymethane (AOM) and dextran sulfate sodium (DSS). PAR1 antagonist, E5555, was administered in long- and short-term protocol, starting on the day of AOM injection and 1 week after completing AOM/DSS treatment, respectively. The fecal samples were collected for metagenome analysis of gut microbiota. The intestinal myofibroblast of the Crohn's disease patients were used to elucidate underlying cellular mechanisms. Caco-2 cells were used to investigate a possible source of PAR1 agonist proteinases. RESULTS: AOM/DSS model showed weight loss, diarrhea, tumor development, inflammation, fibrosis, and increased production of inflammatory cytokines. The ß-diversity, but not α-diversity, of microbiota significantly differed between AOM/DSS and control mice. E5555 alleviated these pathological changes and altered the microbiota ß-diversity in AOM/DSS mice. The thrombin expression was upregulated in tumor and non-tumor areas, while PAR1 mRNA expression was higher in tumor areas compared to non-tumor areas. E5555 inhibited thrombin-triggered elevation of cytosolic Ca2+ concentration and ERK1/2 phosphorylation, as well as IL6-induced STAT3 phosphorylation in intestinal myofibroblasts. Caco-2 cell-conditioned media contained immunoreactive thrombin, which cleaved the recombinant protein containing the extracellular domain of PAR1 at the thrombin cleavage site. CONCLUSIONS: PAR1 antagonism is proposed to be a novel therapeutic strategy for treatment of inflammatory bowel disease and it associated carcinogenesis.

Two cases of Factor XI deficiency: Use of Thrombin Generation Assays (TGA) to detect a non-bleeding phenotype.

Factor XI deficiency is a rare disorder of hemostasis. Previously also known as "hemophilia C", this defect has been regarded as a risk factor for bleeding. However, it has been known for long that bleeding tendency and severity of bleeding are not related to the residual factor XI activity in symptomatic patients. Moreover, a large proportion of patients with even severe factor XI deficiency are clinically unremarkable and do not show any signs of abnormal bleeding. Here, we present two cases of factor XI deficiency with a non-bleeding phenotype. Adequate diagnostic work-up and evaluation of the bleeding risk are reported and discussed with focus on thrombin generation assays (TGA) for the prediction of bleeding in affected patients. This is of high relevance in affected patients, particularly in the context of surgery.

Andexanet Alfa Neutralizes the Anticoagulant Effects of Unfractionated Heparin of Bovine, Ovine and Porcine Origin Almost as Protamine Sulfate.

INTRODUCTION: Andexanet alfa (AA) - zhzo, recombinant coagulation factor Xa, is an approved antidote for oral Xa inhibitors (apixaban and rivaroxaban). Unfractionated heparin (UFH) is commonly used for therapeutic, interventional, and surgical indications. Protamine sulfate (PrSO4) is frequently used to neutralize UFH. This study aimed to investigate the comparative neutralization profiles of AA and PrSO4 for heparins of bovine, ovine, and porcine origin. MATERIALS AND METHODS: The neutralization effect of PrSO4 at 25 µg/ml and AA at 100 µg/ml was studied on an approximate surgical/interventional concentration of heparin by supplementing whole blood with each of the heparins at 25 µg/ml. For the clotting profile (activated partial thromboplastin time: aPTT), amidolytic (anti-Xa and anti-IIa), and thrombin generation assay each of the heparin were supplemented from -10-0.62 µg/ml. RESULTS: In the whole blood ACT studies, all three heparins produced strong anti-coagulant effects (400-450 seconds) compared to saline (130-150 seconds). Both AA and PrSO4 almost fully neutralized the anti-coagulant effects of heparins (140-160 seconds). Both antidotes completely reversed the anticoagulant effects of all three heparins in the aPTT and thrombin generation assay. However, PrSO4 was more effective in neutralizing the anti-Xa, and anti-IIa effects than AA, which only partially neutralized these effects. CONCLUSION: Andexanet alfa at 100 µg/ml effectively neutralizes the therapeutic and surgical/interventional concentrations of heparins in in-vitro settings. While differences in the anti-Xa, and anti-IIa effects between heparins were noted, anti-coagulant effect of these agents in the aPTT assay were comparable. A similar neutralization profile was observed in the ACT and thrombin generation assays by both agents.

Protein disulfide isomerase uses thrombin-antithrombin complex as a template to bind its target protein and alter the blood coagulation rates.

During inflammation and situations of cellular stress Protein Disulfide Isomerase (PDI) is released in the blood plasma from the platelet and endothelial cells to influence thrombosis. The addition of exogenous PDI makes the environment pro-thrombotic by inducing disulfide bond formation in specific plasma protein targets like vitronectin, factor V, and factor XI. However, the mechanistic details of PDI interaction with its target remain largely unknown. A decrease in the coagulation time was detected in activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) in addition to the purified recombinant PDI (175nM). The coagulation time can be controlled using an activator (quercetin penta sulfate, QPS) or an inhibitor (Quercetin 3-rutinoside, Q3R) of PDI activity. Likewise, the PDI variants that increase the PDI activity (H399R) decrease, and the variant with low activity (C53A) increases the blood coagulation time. An SDS-PAGE and western blot analysis showed that the PDI does not form a stable complex with either thrombin or Antithrombin (ATIII) but it uses the ATIII-thrombin complex as a template to bind and maintain its activity. A complete inhibition of thrombin activity on the formation of ATIII-thrombin-PDI complex, and the complex-bound PDI-catalyzed disulfide bond formation of the target proteins may control the pro- and anti-thrombotic role of PDI.

Challenges to Laboratory Monitoring of Direct Oral Anticoagulants.

Direct oral anticoagulants (DOACs) exert anticoagulation effect by directly inhibiting Factor Xa (rivaroxaban, apixaban, and edoxaban) or thrombin (dabigatran). Though DOACs are characterized by fixed-dose prescribing and generally do not require routine laboratory drug-level monitoring (DLM), circumstances may arise where the DLM may aid in clinical decision-making, including DOAC dose adjustment, anticoagulant class change, or decisions to withhold or administer reversal agents. We review the current literature that describes high-risk patient groups in which DLM may be beneficial for improved patient anticoagulation management and stewardship. The review also summarizes the limitations of conventional coagulation testing and discuss the emerging utility of quantitative methods for routine and rapid emergent evaluation of DOAC drug levels-in particular, the Anti-Xa activity to detect Factor Xa Inhibitors (rivaroxaban, apixaban, and edoxaban). Both technical and regulatory barriers to widespread DLM implementation are limiting factors to further clinical research that must be overcome, in order to propose universal DOAC DLM strategies and provide clinical-laboratory correlation to formally classify high-risk patient groups.

Relationship between plasma tissue Factor Pathway Inhibitor (TFPI) levels, thrombin generation and clinical risk of bleeding in patients with severe haemophilia A or B.

INTRODUCTION: Bleeding severity in severe haemophilic patients, with low thrombin generation (TG) capacity, can vary widely between patients, possibly reflecting differences in tissue factor pathway inhibitor (TFPI) level. AIM: To compare free TFPI (fTFPI) levels in patients with severe haemophilia A (sHA) and severe haemophilia B (sHB) and to investigate in these patients as a whole the relationships between bleeding and TG potential, between TG potential and fTFPI level and between fTFPI level and bleeding tendency. METHODS: Data on bleeding episodes retrospectively recorded during follow-up visits over 5-10 years were collected and used to calculate the annualised joint bleeding rate (AJBR). fTFPI levels and basal TG parameters were determined in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) using calibrated automated tomography (CAT). RESULTS: Mean fTFPI levels did not differ significantly between sHA (n = 34) and sHB (n = 19) patients. Mean values of endogenous thrombin potential (ETP) and thrombin peak (peak) in PPP and PRP were two-fold higher when fTFPI levels < 9.4 versus > 14.3 ng/mL. In patients treated on demand, ETP and peak in PRP were doubled when AJBR was ≤ 4.9 $ \le 4.9$ , AJBR being halved in patients with a low fTFPI level (9.4 ng/mL). In patients on factor prophylaxis, no association was found between TG parameters and either fTFPI level or AJBR. CONCLUSION: In patients treated on demand, bleeding tendency was influenced by fTFPI levels, which in turn affected basal TG potential. In patients on prophylaxis, bleeding tendency is probably determined primarily by the intensity of this treatment.

Multicenter evaluation of the haemostatic activity of emicizumab in patients with severe haemophilia A.

BACKGROUND: Emicizumab has been approved for the prophylaxis of patients with hemophilia A with or without inhibitors. However, spontaneous and trauma-induced breakthrough bleeds have been reported in patients on emicizumab prophylaxis and no laboratory assay is validated to evaluate the hemostatic activity of emicizumab . OBJECTIVES: The thrombin generation assay (TGA) could be a surrogate marker of the hemostatic efficacy of emicizumab. The correlation between TGA and the methods used to measure emicizumab blood concentration was evaluated in this study. METHODS: TGA was modified by the use of a trigger reagent combining a very low concentration of tissue factor (TF) and activated factor XI (FXIa). Emicizumab quantification was performed by three methods, the modified one-step factor VIII (FVIII) assay, and two methods based on liquid chromatography and mass spectrometry (LC-MS). RESULTS: Using TF/FXIa-triggered TGA and platelet-poor plasma, a relationship was observed between the area under the thrombin generation curve (ETP) and the clinical response of patients to emicizumab. The ultrastructure of fibrin clots was consistent with ETP results and showed that emicizumab had a hemostatic activity equivalent to 20-30 IU/dL of factor VIII. Finally, pharmacokinetic/pharmacodynamic analyses showed no correlation between ETP and LC-MS nor with modified one-stage FVIII assay, but a statistically significant correlation between the LC-MS methods and the time to peak results of TGA. CONCLUSION: Using a modified TGA, this study showed that patients who experienced breakthrough bleeds while on emicizumab had a lower thrombin generating capacity compared to others with good clinical response to emicizumab.