Thrombin Activity Research Articles

The Multifaceted Roles of Thrombomodulin: Anti-coagulation, Anti-inflammation, and Anti-tumor Potential.

Thrombomodulin (TM) is a single-chain transmembrane glycoprotein with anticoagulant effects. TM has two forms: membrane type existing on the cell surface and blood type free in plasma and urine. TM functions as an anticoagulant cofactor for thrombin activation of protein C on the surface of vascular endothelial cells. Due to the excellent anti-coagulant effects in modulating the coagulation and fibrinolytic system, the recombinant human soluble TM (rhsTM) has been used for the treatment of disseminated intravascular coagulation (DIC). In addition to anti-coagulation, many studies have shown that TM can also exert anti-inflammatory and anti-tumor effects. TM has a lectin-like domain at its N-terminus that has been shown to exhibit direct anti-inflammatory functions. At the same time, due to its special structure, thrombomodulin plays an important role in vascular-related mechanistic diseases by participating in the regulation of inflammatory pathways, complement, HMGB1, etc. In this article, changes in TM expression in the body after injury, composition of TM structural domains, anticoagulant, anti-inflammatory, and antitumor effects, and related mechanisms of TM were systematically reviewed, to provide a theoretical basis and reference for the potential clinical implications of TM in treating various diseases.

Enhancing Immunomodulation and Osseointegration of Bone Implants via Thrombin-Activated Platelet-Rich Plasma Self-Assembly.

Platelet-rich plasma (PRP) is characterized by elevated concentrations of growth factors that facilitate bone repair. Nonetheless, the effective integration of PRP with bone implants and the sustained release of its active constituents pose significant challenges. In this study, thrombin is grafted onto the surface of polyetheretherketone (PEEK) via an N,N'-Disuccinimidyl Carbonate (DSC) linker and the retained enzymatic activity of thrombin enables the controlled activation of PRP self-assembly, resulting in the formation of a functional bio-gel layer. The optimal thrombin concentration to be 100 U/ mL-1 is determined, at which point both the grafting amount and enzymatic activity of thrombin reaches their peak, with no further increases observed at higher concentrations. PRP solutions with varying platelet enrichment ratios are subsequently activated on the thrombin-grafted PEEK surface, yielding self-assembled bio-gels capable of sustained growth factor release for up to 16 days. The thrombin-activated PRP bio-gel on PEEK surface not only enhances in vitro cell adhesion, proliferation, osteogenic differentiation, vascularization and specific polarization of macrophages, but also effectively facilitates in vivo angiogenesis, immunomodulation and bone formation in a platelet dose-dependent manner. Consequently, the thrombin-activated PRP gel presents a promising strategy for the biological functionalization of PEEK implants in orthopedic applications.

Inflammation and Coagulation in Neurologic and Psychiatric Disorders.

Coagulation factors are intrinsically expressed in various brain cells, including astrocytes and microglia. Their interaction with the inflammatory system is important for the well-being of the brain, but they are also crucial in the development of many diseases in the brain such as stroke and traumatic brain injury. The cellular effects of coagulation are mediated mainly by protease-activated receptors. In this review, we sum up the role of the coagulation cascade in the development of different diseases including psychiatric disorders. In inflammatory diseases such as multiple sclerosis, fibrinogen activates microglia and suppresses the differentiation of oligodendrocytes, leading to axonal damage and suppression of remyelination. In ischemic stroke, thrombin activity is associated with the size of infarction, and the inhibition of either thrombin- or protease-activated receptor 1 promotes neuronal survival and reduces the size of infarction. Patients suffering from Alzheimer's disease express higher levels of thrombin, which in turn damages the endothelium, increases blood-brain barrier permeability, and induces cell apoptosis. In major depressive disorder, a positive correlation is present between prothrombotic states and suicidality. Moreover, both protein S deficiency and antiphospholipid antibodies are associated with schizophrenia and there is an effect of warfarin on psychosis-free intervals. Studying the coagulation in the brain could open a new door in understanding and treating neurological and psychiatric disorders, and extensive research should be conducted in this field.

Exosite crosstalk in thrombin.

Thrombin is the central mediator of hemostasis, where it converts fibrinogen to fibrin, activates upstream factors to promote coagulation, activates factor XIII and thrombin-activatable fibrinolysis inhibitor to stabilize fibrin, mediates anticoagulation, and modulates cellular activity via cell surface receptors. Thus, regulation of thrombin activity is essential to the hemostatic balance. Thrombin is regulated by positively charged surface domains that surround the active site. These exosites bind substrates, inhibitors, cofactors, and receptors that coordinate to direct thrombin to the appropriate location and modulate catalytic activity. Thus, the exosites are essential to the activity and regulation of thrombin. In addition to acting as binding sites, the exosites modulate the active site allosterically. Furthermore, the exosites impact each other, whereby the binding of ligands to one exosite impacts the function of the opposing exosite. Given the integral role that exosites play in the regulation of thrombin, they are attractive targets for the regulation of thrombin; they are attractive targets for the development of new anticoagulants.

Mice with reduced protease-activated receptor 4 reactivity show decreased venous thrombosis and platelet procoagulant activity.

Hypercoagulation and thrombin generation are major risk factors for venous thrombosis. Sustained thrombin signaling through protease-activated receptor (PAR) 4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single nucleotide polymorphism in PAR4 (rs2227376) changes proline to leucine extracellular loop 3, which decreases PAR4 reactivity and is associated with a lower risk for venous thromboembolism (VTE) in a genome wide association studies meta-analysis. The goal of this study was to determine the mechanism for the association of rs2227376 with a reduced risk of VTE using mice with a homologous mutation (PAR4-P322L). Venous thrombosis was examined using our recently generated PAR4-P322L mice in the inferior vena cava stasis and stenosis models. Coagulation and clot stability were measured using rotational thromboelastometry. Thrombin-generating potential was measured in platelet-rich plasma. Phosphatidylserine surface expression and platelet-neutrophil aggregates were analyzed using flow cytometry. Mice heterozygous (PAR4P/L) or homozygous (PAR4L/L) at position 310 had reduced sizes of venous clots at 48 hours. PAR4P/L and PAR4L/L platelets had progressively decreased phosphatidylserine in response to thrombin and convulxin, in addition to decreased thrombin generation and decreased PAR4-mediated platelet-neutrophil aggregation. The leucine allele in extracellular loop 3, PAR4-322L, leads to fewer procoagulant platelets, decreased endogenous thrombin potential, and reduced platelet-neutrophil aggregation. This decreased ability to generate thrombin and bind to neutrophils offers a mechanism for PAR4's role in VTE, highlighting a key role for PAR4 signaling.

Low thrombin inactivation capacity is associated with an increased risk of recurrent ischemic events after ischemic stroke at a young age.

Patients with ischemic stroke at a young age (18-50 years) have an increased long-term risk of recurrent ischemic events. Hypercoagulability may contribute to this high risk. To investigate associations between in and ex vivo hemostatic parameters and recurrent ischemic events after an ischemic stroke or transient ischemic attack at a young age. We included patients with ischemic stroke or TIA between 1980 and 2010 from the prospective FUTURE cohort. Blood samples were collected in 2010 and patients were followed for recurrent ischemic events from 2010 to 2023. Pro- and anticoagulant markers and thrombin generation assay were measured. Thrombin dynamic analysis was used to study underlying pro- and anticoagulant processes. Hazard ratios (HR) per standard deviation increase were assessed with cause-specific hazard models. Of the initial cohort of 581 patients, 332 were eligible. The median time between the index event and 2010 was 7.6 years. During a mean follow-up of 6.5 years, 70 of 332 (21.1%) patients experienced a recurrent ischemic event. Lower antithrombin levels (adjusted HR=0.77; 95% CI 0.60-0.98) and higher fibrinogen levels (HR=1.35; CI 1.04-1.73) were associated with higher risk of recurrent ischemic events. Plasma thrombin generation was not associated with recurrence. However, the thrombin decay constant (HR=0.67; CI 0.51-0.87) was associated with a lower risk of recurrent ischemic events. After an ischemic stroke or TIA at a young age, thrombin decay constant, reflecting reduced protection against thrombin (low antithrombin) and decreased potential to inhibit thrombin (high fibrinogen), is associated with recurrent ischemic events.

Bidirectional Enzyme Inhibition and Activation for In Situ Formation of Injectable Hydrogel Using a Bispecific Aptamer.

In situ injectable hydrogels have been explored for biomedical applications, including regenerative medicine and drug delivery. However, controlling the kinetics of their gelation to facilitate easy injection remains a challenge. The purpose of this study was to demonstrate the potential of using bispecific aptamers and complementary sequences as a bidirectional modulation system for controlling enzyme-mediated hydrogel formation kinetics. The results show that a bispecific thrombin-binding aptamer effectively inhibits thrombin activity and significantly slowed fibrin hydrogel formation. Upon interaction with its complementary sequence, this inhibition could be reversed. As a result, the aptamer-bound thrombin was activated, leading to an acceleration of the fibrin formation kinetics. Thus, bispecific aptamers and complementary sequences can effectively function as dynamic control systems for enzyme-catalyzed in situ injectable hydrogel formation.

Simple and low-cost thrombin activity assay using screen-printed electrodes with portable electrochemiluminescence device

Real-time monitoring of thrombin activity is essential for preventing, diagnosing, and treating coagulation-related diseases. Herein, we present a simple, disposable electrochemiluminescence (ECL) biosensor for thrombin activity assay based on screen-printed electrodes (SPE). The thrombin-specific peptide substrate Mpr (4-Methoxybenzyl group)-GGRK-Fc (ferrocene) was identified from four candidates using molecular docking and specificity constant experiments. Mpr-GGRK-Fc exhibited the highest apparent specificity constant (kcat/KM) of 2.09 × 1012 M−1 s−1, indicating superior affinity and specificity for thrombin. Thrombin catalyzes the cleavage of Mpr-GGRK-Fc, resulting in the detachment of Fc from the electrode surface, which leads to an increase in ECL intensity. Under optimal conditions, the biosensor exhibits a wide linear range from 1.0 × 10−4 U/mL to 1.0 U/mL, with a low detection limit of 4.02 × 10−5 U/mL. The biosensor shows high specificity for thrombin among a range of proteins. Compared to commercial fluorescence kits, our biosensor offers superior sensitivity, selectivity, and stability in detecting thrombin activity in whole blood, making it a promising tool for bedside clinical applications.

Diagnosis of atrial fibrillation as the source of cryptogenic retrieved clots by a novel thrombin secretion assay.

Secondary prevention of acute ischemic stroke depends on identifying the source of cryptogenic clots. We previously reported that secreted thrombin activity from endovascularly retrieved clots is significantly different in atrial fibrillation (AF) vs atherosclerosis (AS), probably due to the invivo biology of the clots. The purpose of this study was to validate and optimize thrombin secretion for clot source diagnosis. Strokes were defined as either AS, AF, or cryptogenic, with the latter group defined as AF or non-AF by long-term clinical follow-up. Artificial venous blood clots were either prewashed or unwashed to model AS and AF sources. Thrombin secretion from the clot was measured fluorometrically by manually transferring the clots between wells or by washing them in an agarose bead column, collecting samples manually, or by using the fraction collector. The secretion pattern was represented as the ratio of maximal initial values to minimal baseline. The new column-based assay was faster and more informative. Significant differences in thrombin secretion ratios were observed between prewashed and unwashed artificial clots, and between AF and AS clots, with a cutoff ratio of 0.52 showing 78.9% specificity and 90.0% sensitivity. This cutoff identified 84% of cryptogenic patients who developed definite AF, and lifetable analysis found significantly more definite AF confirmation by cardiac monitoring in this group than in patients with a probable non-AF ratio (P = .021 by log-rank [Mantel-Cox] test). We developed a rapid and sensitive method for determining cryptogenic clot sources that correlates well with long-term clinical outcomes.

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets.

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

Oxidative stress model of lipopolysaccharide combined with thrombin inducing in broiler chicks.

Lipopolysaccharides (LPS) are commonly used to construct inflammation models. However, poultry have a certain degree of tolerance to LPS due to the lack of thrombin XI and XII in their bodies. Thrombin activation produces clotting factors that can cleave prothrombin to form thrombin. The purpose of this study was to construct a chick oxidative stress model used different concentrations of LPS combined with thrombin in order to screen for the optimal concentration for constructing the oxidative stress model, and to explore the effects of this stimulus on various indicators of chicks. Eighty-one young chicks (4-days-old) were randomly divided into three groups with 27 chicks per group where each group contained 3 replicates with 9 birds each: a control group (physiological saline), a low-dose group (LPS 5 mg/kg thrombin 150 U/kg), and a high-dose group (LPS 10 mg/kg thrombin 300 U/kg). The results indicated that compared with the control group, the low-dose group and the high-dose group significantly increased the content of Malondialdehyde (MDA) in serum and reduced the content of T-AOC, GSH-PX and SOD, respectively. Meanwhile, the levels of NO and inflammatory factors IL-1β, IL-6 and TNF-α TNF-α in the liver were significantly increased in the low-dose and high-dose groups compared with the control group, respectively. Liver and thymus tissue sections from the low- and high-dose groups showed hemorrhage, hemolysis, and a small amount of exudation. In terms of inflammatory effect, the serum MDA content and the levels of NO, IL-1β, IL-6 and TNF-α factors in the liver were significantly increased in the low-dose group compared with the high-dose group. On histopathological observation, tissue damage was more pronounced in the low-dose group than in the high-dose group. In conclusion, LPS combined with thrombin could induce oxidative stress in chicks and the pathological changes of the low-dose effect are more pronounced.

Activation of PAR1 contributes to ferroptosis of Schwann cells and inhibits regeneration of myelin sheath after sciatic nerve crush injury in rats via Hippo-YAP/ACSL4 pathway

ObjectivePeripheral nerve injury (PNI) is characterized by high incidence and sequela rate. Recently, there was increasing evidence that has shown ferroptosis may impede functional recovery. Our objective is to explore the novel mechanism that regulates ferroptosis after PNI. MethodsLC-MS/MS proteomics was used to explore the possible differential signals, while PCR array was performed to investigate the differential factors. Besides, we also tried to activate or inhibit the key factors and then observe the level of ferroptosis. Regeneration of myelin sheath was finally examined in vivo via transmission electron microscopy. ResultsProteomics analysis suggested coagulation signal was activated after sciatic nerve crush injury, in which high expression of F2 (encoding thrombin) and F2r (encoding PAR1) were observed. Both thrombin and PAR1-targeted activator TRAP6 can induce ferroptosis in RSC96 cells, which can be rescued by Vorapaxar (PAR1 targeted inhibitor) in vitro. Further PCR array revealed that activation of PAR1 induced ferroptosis in RSC96 cells by increasing expression of YAP and ACSL4. Immunofluorescence of sciatic nerve confirmed that the expression of YAP and ACSL4 were simultaneously reduced after PAR1 inhibition, which may contribute to myelin regeneration after injury in SD rats. ConclusionInhibition of PAR1 can relieve ferroptosis after sciatic nerve crush injury in SD rats through Hippo-YAP/ACSL4 pathway, thereby regulating myelin regeneration after injury. In summary, PAR1/Hippo-YAP/ACSL4 pathway may be a promising therapeutic target for promoting functional recovery post-sciatic crush injury.

Endothelial NMMHC IIA dissociation from PAR1 activates the CREB3/ARF4 signaling in thrombin-mediated intracerebral hemorrhage

IntroductionThere is an urgent need for cerebroprotective interventions to improve the suboptimal outcomes with intracerebral hemorrhage (ICH). Despite the important role of nonmuscle myosin heavy chain IIA (NMMHC IIA) in the blood–brain barrier (BBB), its function in ICH remains unclear. ObjectivesThe objective of this study is to explore how NMMHC IIA functions in ICH and to evaluate the effectiveness of targeting NMMHC IIA as a treatment for ICH. MethodsWe firstly examined the protein expression of NMMHC IIA in clinical patients and animal models with ICH. The function of NNMMHC IIA was then corroborated by using overexpress or knockdown NMMHC IIA specifically in ECs mice and pBMECs. In addition, we explored protein interacts with NMMHC IIA and signaling pathways after ICH by LC-MS/MS and transcriptomics analysis with an emphasis on the function of PAR1 and the CREB3/ARF4 signaling pathway, and validated them in three kind of animal models. To support the clinical translation of our results, we targeted NMMHC IIA to bicalutamide selected from a library of marketed drugs and examined to validate its ameliorative effect on ICH. ResultsWe observed an upregulation of endothelial NMMHC IIA in the brain following the onset of ICH in both patients and mice, while inhibited NMMHC ⅡA improved ICH induced by thrombin, warfarin or tissue plasminogen activator (tPA) after ischemic stroke. Mechanistically, the head domain of NMMHC IIA interacted with protease-activated receptor 1 (PAR1) at the 380–430 aa region and subsequently dissociated and activated the CREB3/ARF4 signaling pathway. We found that bicalutamide and blebbistatin could bind to NMMHC IIA and effectively protect mice from thrombin-mediated ICH. ConclusionThe findings indicated that NMMHC IIA dissociated from PAR1 and activated CREB3/ARF4 pathway, which aggravated BBB damage induced by thrombin. This suggested that NMMHC IIA was a novel potential therapeutic target for BBB-related diseases.

Thrombin confers chemotherapeutic resistance by promoting transcriptional induction and post-translational stabilization of pro-survival MCL1 in TNBC

The association between idiopathic venous thrombosis and occult cancer is widely recognized. However, the comprehensive understanding of how thrombin, generated during the process of thrombosis, possesses the potential to augment the malignant phenotype is still not well understood. The coagulation protease thrombin mediates its effects by cleaving protease-activated receptor 1 (PAR1), a receptor abundantly expressed on the surface of triple-negative breast cancer (TNBC) cells. While emerging evidence implicates coagulation proteases in facilitating cancer progression, the precise molecular pathways underlying thrombin-mediated induction of chemoresistance remain poorly defined. Here, we demonstrate that thrombin-induced PAR1 activation in TNBC cells promotes the development of a multidrug-resistant phenotype, mechanistically linked to the upregulation of the pro-survival protein MCL1. Genetic ablation of MCL1 sensitizes TNBC cells to cytotoxic drugs despite thrombin exposure, affirming MCL1's functional importance. Chromatin immunoprecipitation analyses reveal thrombin triggers protein kinase A-dependent phosphorylation of serine 133 residue of cAMP-responsive element-binding protein (CREB), enhancing CREB's affinity for the co-activators CBP and p300. Furthermore, thrombin treatment induces the nuclear translocation of CREB-regulated transcription coactivator 2 (CRTC2) in a calcium-dependent manner, which collectively interacts with CREB/CBP-P300. The coordinated action of these transcriptional co-activators facilitates the transcriptional induction of MCL1. We further report that PAR1 activation augments MCL1 binding to the deubiquitinase USP9X, reducing MCL1 turnover. Our pre-clinical breast cancer murine model also shows that genetic deletion of PAR1 sensitizes breast cancer cells to chemotherapeutic drugs in vivo. Collectively, these findings emphasize the thrombin-PAR1 axis as a novel driver of chemoresistance. Utilizing FDA-approved oral anticoagulants for selective blocking of thrombin action may serve as a potential therapeutic adjunct for the treatment of triple-negative breast cancer.

Immunothrombosis in Acute Ischemic Stroke.

Ischemic stroke is one of the leading causes of disability and mortality worldwide. Thrombosis is the main pathological process of stroke and is therefore an important therapeutic target in stroke prevention. In recent years, with the development of endovascular treatment and therefore retrieving the thrombus for further investigation, evidence is accumulating that immune cells are inextricably linked to stroke pathogenesis. Circulating immune cells have been found to induce immunothrombosis, and they actively participate in the formation of the thrombus by promoting platelet recruitment and thrombin activation. Additionally, the formation of thromboinflammation leads to increased instability of atherosclerotic plaques. We review the concepts of stroke immunothrombosis and thromboinflammation and the effect of immune cells on vessel recanalization and patient outcome. In addition, we elaborate on the possible mechanism of immune cells being activated and participating in thrombosis in ischemic stroke.

Coagulopathy and acute pancreatitis: pathophysiology and clinical treatment.

Coagulopathy is a critical pathophysiological mechanism of acute pancreatitis (AP), arising from the complex interplay between innate immune, endothelial cells and platelets. Although initially beneficial for the host, uncontrolled and systemic activation of coagulation cascade in AP can lead to thrombotic and hemorrhagic complications, ranging from subclinical abnormalities in coagulation tests to severe clinical manifestations, such as disseminated intravascular coagulation. Initiation of coagulation activation and consequent thrombin generation is caused by expression of tissue factor on activated monocytes and is ineffectually offset by tissue factor pathway inhibitor. At the same time, endothelial-associated anticoagulant pathways, in particular the protein C system, is impaired by pro-inflammatory cytokines. Also, fibrin removal is severely obstructed by inactivation of the endogenous fibrinolytic system, mainly as a result of upregulation of its principal inhibitor, plasminogen activator inhibitor type 1. Finally, increased fibrin generation and impaired break down lead to deposition of (micro) vascular clots, which may contribute to tissue ischemia and ensuing organ dysfunction. Despite the high burden of coagulopathy that have a negative impact on AP patients' prognosis, there is no effective treatment yet. Although a variety of anticoagulants drugs have been evaluated in clinical trials, their beneficial effects are inconsistent, and they are also characterized by hemorrhagic complications. Future studies are called to unravel the pathophysiologic mechanisms involved in coagulopathy in AP, and to test novel therapeutics block coagulopathy in AP.

Injectable tissue-engineered human cartilage matrix composite fibrin glue for regeneration of articular cartilage defects

Due to the lack of blood vessels and nerves, the ability of cartilage to repair itself is limited, and the injury of articular cartilage urgently needs effective treatment. Currently, the limitation of clinical repair for cartilage defects is that it is difficult to form pure hyaline cartilage repair, and the source of cartilage tissue and cells is limited. To obtain high-purity regenerated hyaline cartilage, we proposed to construct an injectable hydrogel precursor by using human living hyaline cartilage graft (hLhCG) secreted by human chondrocytes as the dispersed phase and fibrinogen solution as the continuous phase, by double injection with thrombin, three-dimensional network hydrogel structure was formed under the action of thrombin to repair joint defects. The component phenotypes of hLhCG and biomechanical properties of composite gel scaffolds were verified. After 12 weeks of injection of the mixed phase at the defect site, the regenerated tissues are similar in composition to adjacent natural tissues and exhibit similar biomechanical properties. The phenotype of regenerated cartilage was verified, confirming the successful regeneration of hyaline cartilage.

Efficacy and Safety of Rivaroxaban, Apixaban, and Edoxaban for Nonvalvular Atrial Fibrillation Based on Blood Coagulation Activity and Drug Plasma Concentration: SETtsu and North Osaka Multicenter Direct Oral AntiCoagulant (SET DOAC) Registry.

Background: The therapeutic effects of oral anticoagulant drugs for nonvalvular atrial fibrillation (NVAF) suggest that the three factor Xa (FXa) inhibitors may have distinct safety profiles, though this is not yet fully conclusive. This study investigated the current dosing of rivaroxaban, apixaban, and edoxaban by monitoring drug plasma concentration (PC) and coagulation activity from the viewpoint of the safety. Methods and results: This multicenter clinical study monitored the drug PC and two coagulation biomarkers (fibrinogen and fibrin monomer complex [FMC]) at peak and trough timing in 268 outpatients taking rivaroxaban (n = 72), apixaban (n = 71), and edoxaban (n = 125) for NVAF. Doses were adjusted based on the dose-adjustment criteria of each drug. Referencing our previous study, peak drug PC remained below the cut-off level for predicting bleeding events except in eight patients (rivaroxaban, n = 3; apixaban, n = 2; edoxaban, n = 3) in whom bleeding events occurred. Among them, two (one each on rivaroxaban and edoxaban) had a peak drug PC below the cut-off level. Drug PCs widely varied from peak to trough, whereas FMC levels, reflecting thrombin activity, remained within the normal range (<6.1 µg/mL) regardless of PC variations. These results indicated that the anticoagulant effects of these drugs persisted throughout the day regardless of the drug PC levels, dosage, and dosing frequency. Regarding the change over time in peak PC, the elevation over time developed more in rivaroxaban (29/57; 50.9%, p < 0.05) than in edoxaban (32/101; 31.7%), and rivaroxaban tended to accumulate more than edoxaban. Conclusions: Although drug PC levels of once-daily FXa inhibitors widely varied from peak to trough, FMC levels were maintained within the normal range without daily variations. Rivaroxaban also tended to accumulate over time. The results indicate the low risk of thrombotic events with once-daily FXa inhibitors and its correspondence to the twice-daily regimen.

Factor XIII Activation Peptide Residues Play Important Roles in Stability, Activation, and Transglutaminase Activity.

A subunit of factor XIII (FXIII-A) contains a unique activation peptide (AP) that protects the catalytic triad and prevents degradation. In plasma, FXIII is activated proteolytically (FXIII-A*) by thrombin and Ca2+ cleaving AP, while in cytoplasm, it is activated nonproteolytically (FXIII-A°) with increased Ca2+ concentrations. This study aimed to elucidate the role of individual parts of the FXIII-A AP in protein stability, thrombin activation, and transglutaminase activity. Recombinant FXIII-A AP variants were expressed, and SDS-PAGE was used to monitor thrombin hydrolysis at the AP cleavage sites R37-G38. Transglutaminase activities were assessed by cross-linking lysine mimics to Fbg αC (233-425, glutamine-substrate) and monitoring reactions by mass spectrometry and in-gel fluorescence assays. FXIII-A AP variants, S19P, E23K, and D24V, degraded during purification, indicating their vital role in FXIII-A2 stability. Mutation of P36 to L36/F36 abolished the proteolytic cleavage of AP and thus prevented activation. FXIII-A N20S and P27L exhibited slower thrombin activation, likely due to the loss of key interdomain H-bonding interactions. Except N20S and P15L/P16L, all activatable FXIII-A* variants (P15L, P16L, S19A, and P27L) showed similar cross-linking activity to WT. By contrast, FXIII-A° P15L, P16L, and P15L/P16L had significantly lower cross-linking activity than FXIII-A° WT, suggesting that loss of these prolines had a greater structural impact. In conclusion, FXIII-A AP residues that play crucial roles in FXIII-A stability, activation, and activity were identified. The interactions between these AP amino acid residues and other domains control the stability and activity of FXIII.

Safety, Tolerability, Pharmacodynamics, and Pharmacokinetics of Recombinant Neorudin, a New Anticoagulant Drug in Patients With Acute Coronary Syndrome.

This study evaluated the safety, tolerability, pharmacodynamics, and pharmacokinetics of recombinant neorudin (EPR-hirudin [EH]) in patients with acute coronary syndrome (ACS), providing a basis for further therapeutic research. This open-label, single-center, nonrandomized, nonblinded, and noncontrolled trial categorized 24 patients with nonprogressive ACS who met the screening criteria into 3 groups. They received an intravenous injection of neorudin (0.4mg/kg), followed by an intravenous drip at doses of 0.15, 0.30, and 0.45mg/kg/h for 3 days in the low-, medium-, and high-dose groups, respectively. The safety, tolerability, pharmacodynamics, and pharmacokinetics of EH were assessed after treatment, indicating that neorudin was safe and well tolerated in nonprogressive ACS. No serious adverse events or clinical composite end points were observed. The activated partial thromboplastin time and thrombin time increased significantly and dose dependently following EH administration across all groups compared to pretreatment values. Conversely, thrombin activity significantly decreased after drug administration but returned to baseline levels shortly after drug withdrawal. Within the administered dose range, neorudin exposure increased with the dose, and its half-life was approximately 2hours. Neorudin was found to be safe and tolerable for treating patients with nonprogressive ACS, demonstrating therapeutic efficacy at doses up to 0.45mg/kg/h over a 3-day period.