Protein-Induced DNA Dumbbell Amplification (PINDA) and its applications to food hazards detection.

Quantification of trace amounts of proteins is technically challenging because proteins cannot be directly amplified like nucleic acids. To improve the analytical sensitivity and to complement conventional protein analysis methods, we developed a highly sensitive and homogeneous detection strategy called Protein-Induced DNA Dumbbell Amplification (PINDA). PINDA combines protein recognition with exponential nucleic acid amplification by using protein binding probes made of DNA strands conjugated to protein affinity ligands. When a pair of probes bind to the same target protein, complementary nucleic acid sequences that are conjugated to each probe are brought into close proximity. The increased local concentration of the probes results in the formation of a stable dumbbell structure of the nucleic acids. The DNA dumbbell is readily amplifiable exponentially using techniques such as loop-mediated isothermal amplification. The PINDA assay eliminates the need for washing or separation steps, and is suitable for on-site applications. Detection of the model protein, thrombin, has a linear range of 10 fM-100 pM and detection limit of 10 fM. The PINDA technique is successfully applied to the analysis of dairy samples for the detection of ß-lactoglobulin, a common food allergen, and Salmonella enteritidis, a foodborne pathogenic bacterium. The PINDA assay can be easily modified to detect other targets by changing the affinity ligands used to bind to the specific targets.

ErpY-like Protein Interaction with Host Thrombin and Fibrinogen Intervenes the Plasma Coagulation through Extrinsic and Intrinsic Pathways.

The survival and proliferation of pathogenic Leptospira within a host are complex phenomena that require careful consideration. The ErpY-like lipoprotein, found on the outer membrane surface of Leptospira, plays a crucial role in enhancing the bacterium's pathogenicity. The rErpY-like protein, in its recombinant form, contributes significantly to spirochete virulence by interacting with various host factors, including host complement regulators. This interaction facilitates the bacterium's evasion of the host complement system, thereby augmenting its overall pathogenicity. The rErpY-like protein exhibits a robust binding affinity to soluble fibrinogen, a vital component of the host coagulation system. In this study, we demonstrate that the rErpY-like protein intervenes in the clotting process of the platelet-poor citrated plasma of bovines and humans in a concentration-dependent manner. It significantly reduces clot density, alters the viscoelastic properties of the clot, and diminishes the average clotting rate in plasma. Furthermore, the ErpY-like protein inhibits thrombin-catalyzed fibrin formation in a dose-dependent manner and exhibits saturable binding to thrombin, suggesting its significant role in leptospiral infection. These findings provide compelling evidence for the anticoagulant effect of the ErpY-like lipoprotein and its significant role in leptospiral infection.

Synergistic Procoagulant Mechanism and Application of Kaolin-Zeolite Composite Hemostat for Effective Hemorrhage Control.

Achieving timely and effective hemorrhage control is imperative for the survival of individuals with severe bleeding. Hemostatic materials, by enhancing the natural cell-based coagulation response, are essential tools in modern and military medical practice for controlling bleeding, especially in emergency and surgical settings. Here, we report a new type of composite hemostatic material with two different aluminosilicate-based components, kaolin and zeolite, which synergistically work together in different stages of the coagulation cascade reactions. Kaolin can effectively activate the clotting factor FXII in the early stage, and zeolite can accumulate and assemble FXa and FVa on its surface and thereafter lead to the formation of highly active thrombin in the later stage. The synergistic action mechanism between kaolin and zeolite significantly boosts the levels of FXIIa and FXa, and it also greatly enhances plateau thrombin activity. For practical application, a kaolin-modified zeolite gauze is fabricated, and it demonstrates excellent hemostatic effectiveness. Compared to the combat gauze currently used in front-line treatment, it reduces blood loss by 75% and shortens hemostasis time by 33% in a rabbit femoral artery injury model. In addition, this kaolin-zeolite gauze has no heat release problem and a nearly zero particle shedding rate, which greatly decreases the safety risk compared to current commercial inorganic-based hemostatic gauzes.

[Study on the Experimental Methodology of Plasma Clot Retraction].

OBJECTIVE: To explore the key factors affecting plasma clot retraction and optimize the experimental method of plasma clot retraction, in order to study the regulation of platelet function and evaluate the modulatory effects of drugs on plasma clot retraction. METHODS: The effects of different concentrations of thrombin, Ca2 + and platelets on plasma clot retraction were studied, and the detection system of plasma clot retraction was optimized. The availability of the detection system was then validated by analyzing the regulatory effects of multiple signaling pathway inhibitors on plasma clot retraction. RESULTS: Through the optimization study of multiple factors, platelet rich plasma (PRP) containing 0.5 mmol/L Ca2 + and 40×109/L platelets was treated with 0.2 U/ml thrombin to perform plasma clot retraction analysis. After treatment with thrombin for 15 min, plasma clot retracted significantly. After treatment with thrombin for 30 min, the percentage of plasma clot retraction was more than 50%. The regulatory effects of multiple signaling pathway inhibitors on plasma clot retraction were studied in this detection system. PKC inhibitor Go 6983 exhibited a significant inhibitory effect on plasma clot retraction, while PI3K inhibitor Ly294002 and p38 MAPK inhibitor SB203580 slightly suppressed plasma clot retraction. CONCLUSION: PRP containing 0.5 mmol/L Ca2 + and 40×109/L platelets can be induced with 0.2 U/ml thrombin to conduct plasma clot retraction analysis, which can be used to study the regulation of platelet function and evaluate the modulatory effects of drugs on plasma clot retraction.

Thrombin Generation Is Associated With Extracellular Vesicle and Leukocyte Lipid Membranes in Atherosclerotic Cardiovascular Disease.

BACKGROUND: Clotting, leading to thrombosis, requires interactions of coagulation factors with the membrane aminophospholipids (aPLs) phosphatidylserine and phosphatidylethanolamine. Atherosclerotic cardiovascular disease (ASCVD) is associated with elevated thrombotic risk, which is not fully preventable using current therapies. Currently, the contribution of aPL to thrombotic risk in ASCVD is not known. Here, the aPL composition of circulating membranes in ASCVD of varying severity will be characterized along with the contribution of external facing aPL to plasma thrombin generation in patient samples. METHODS: Thrombin generation was measured using a purified factor assay on platelet, leukocyte, and extracellular vesicles (EVs) from patients with acute coronary syndrome (n=24), stable coronary artery disease (n=18), and positive risk factor (n=23) and compared with healthy controls (n=24). aPL composition of resting/activated platelet and leukocytes and EV membranes was determined using lipidomics. RESULTS: External facing aPLs were detected on EVs, platelets, and leukocytes, elevating significantly following cell activation. Thrombin generation was higher on the surface of EVs from patients with acute coronary syndrome than healthy controls, along with increased circulating EV counts. Thrombin generation correlated significantly with externalized EV phosphatidylserine, plasma EV counts, and total EV membrane surface area. In contrast, aPL levels and thrombin generation from leukocytes and platelets were not impacted by disease, although circulating leukocyte counts were higher in patients. CONCLUSIONS: The aPL membrane of EV supports an elevated level of thrombin generation in patient plasma in ASCVD. Leukocytes may also play a role although the platelet membrane did not seem to contribute. Targeting EV formation/clearance and developing strategies to prevent the aPL surface of EV interacting with coagulation factors represents a novel antithrombotic target in ASCVD.

Differential effects of physiological agonists on the proteome of platelet-derived extracellular vesicles.

Arterial thrombosis contributes to some of the most frequent causes of mortality globally, such as myocardial infarction and stroke. Platelets are essential mediators of physiological haemostasis and pathological thrombosis. Platelet activation is controlled by a multitude of signalling pathways. Upon activation, platelets shed platelet-derived extracellular vesicles (pEVs). In this Special Issue: Extracellular Vesicles, Moon et al. investigate the impact of various platelet agonists (thrombin, ADP, collagen) on the proteome of pEVs. The study demonstrates that pEVs exhibit an agonist-dependent altered proteome compared to their parent cells, with significant variations in proteins related to coagulation, complement, and platelet activation. The study observes the rapid generation of pEVs following agonist stimulation with specific proteome alterations that underscore an active packaging process. This commentary highlights the implications of their findings and discusses the role of pEV cargo in cardiovascular disease with potential novel therapeutic and diagnostic opportunities.

Studies on in vivo antithrombotic activity of quercetin, a natural flavonoid isolated from a traditional medicinal plant, African eggplant (Solanum indicum).

ETHNOPHARMACOLOGICAL RELEVANCE: Every year, cardiovascular diseases (CVDs) account for about 17.9 million deaths, making them the primary cause of both morbidity and mortality. Conventional drugs, which are often prescribed to treat cardiovascular diseases, are costly and have adverse effects. Consequently, dietary modifications and other medications are needed. Traditional use of Solanum indicum as cardiotonic to treat hypertension and anticoagulant potency has been reported but poorly evaluated scientifically. AIM OF THE STUDY: This study investigated the in vivo anticoagulant activity and mechanism of anticoagulation of quercetin (QC), a bioactive compound isolated from S. indicum (SI) hydroethanolic fruit extract. MATERIALS AND METHODS: Bioassay-guided fractionation (anticoagulant activity) extracted QC from hydroethanolic SI extract. QC was extensively characterized biochemically and pharmacologically. The interaction between QC and thrombin was investigated using spectrofluorometric and isothermal calorimetric methods. Cytotoxicity, antiplatelet, and thrombolytic studies were carried out in vitro. The Swiss albino mice were used to assess the in vivo, anticoagulant, and antithrombotic activities of QC. RESULTS: QC exhibits anticoagulant activity via (i) uncompetitive inhibition of thrombin but not FXa with a Ki value of 33.11 ± 4.2 µM and (ii) a partial inhibition of thrombin-catalyzed platelet aggregation with an IC50 value of 13.2 ± 1.2 µM. The experimental validation of the in silico study's prediction of QC's binding to thrombin was confirmed by spectrofluorometric and isothermal calorimetric analyses. QC was nontoxic to mammalian, non-hemolytic cells and demonstrated thrombolytic activity by activating plasminogen. QC demonstrated in vivo anticoagulant efficacy, preventing k-carrageen-induced thrombus formation in mice's tails. In the acute circulatory stasis paradigm in mice, QC reduces thromboxane B2 (TXB2) and endothelin-1 (ET-1) while increasing nitric oxide synthase (eNOS) and 6-keto prostaglandin F1α (6-keto-PGF1 α). CONCLUSION: Effective in vivo anticoagulant and antithrombotic properties of S. indicum's bioactive component QC point to the plant's potential use as a herbal anticoagulant medication for preventing and treating cardiovascular diseases linked to thrombosis.

Development a novel nano-platform for Thrombolysis acceleration by Thrombin sensitive polymer-peptide hybrid nancapsules.

According to the importance of time in treatment of thrombosis disorders, faster than current treatments are required. For the first time, this research discloses a novel strategy for rapid dissolution of blood clots by encapsulation of a fibrinolytic (Reteplase) into a Thrombin sensitive shell formed by polymerization of acrylamide monomers and bisacryloylated peptide as crosslinker. Degradability of the peptide units in exposure to Thrombin, creates the Thrombin-sensitive Reteplase nanocapsules (TSRNPs) as a triggered release system. Accelerated thrombolysis was achieved by combining three approaches including: deep penetration of TSRNPs into the blood clots, changing the clot dissolution mechanism by altering the distribution pattern of TSRNPs to 3D intra-clot distribution (based on the distributed intra-clot thrombolysis (DIT) model) instead of peripheral and unidirectional distribution of unencapsulated fibrinolytics and, enzyme-stimulated release of the fibrinolytic. Ex-vivo study was carried out by an occluded tube model that mimics in-vivo brain stroke as an emergency situation where faster treatment in short time is a golden key. In in vivo, efficacy of the developed formulation was confirmed by PET scan and laser Doppler flowmetry (LDF). As the most important achievements, 40.0 ± 0.7 (n = 3) % and 37.0 ± 0.4 (n = 3) % reduction in the thrombolysis time (faster reperfusion) were observed by ex-vivo and in-vivo experiments, respectively. Higher blood flow and larger digestion mass of clot at similar times in comparison to non-encapsulated Reteplase were observed that means more effective thrombolysis by the developed strategy.

Polyphenol-stabilized coacervates for enzyme-triggered drug delivery.

Stability issues in membrane-free coacervates have been addressed with coating strategies, but these approaches often compromise the permeability of the coacervate. Here we report a facile approach to maintain both stability and permeability using tannic acid and then demonstrate the value of this approach in enzyme-triggered drug release. First, we develop size-tunable coacervates via self-assembly of heparin glycosaminoglycan with tyrosine and arginine-based peptides. A thrombin-recognition site within the peptide building block results in heparin release upon thrombin proteolysis. Notably, polyphenols are integrated within the nano-coacervates to improve stability in biofluids. Phenolic crosslinking at the liquid-liquid interface enables nano-coacervates to maintain exceptional structural integrity across various environments. We discover a pivotal polyphenol threshold for preserving enzymatic activity alongside enhanced stability. The disassembly rate of the nano-coacervates increases as a function of thrombin activity, thus preventing a coagulation cascade. This polyphenol-based approach not only improves stability but also opens the way for applications in biomedicine, protease sensing, and bio-responsive drug delivery.

Integration of clotting and fibrinolysis: central role of platelets and factor XIIIa.

PURPOSE: The aim of the present study was to establish the role of platelets and activated factor XIIIa (FXIIIa) in the structuring of the fibrin network as well as to clarify the effect of network compaction on clot lysis. METHODS: Turbidimetry was used for the one-stage clotting test where platelet-free plasma (PFP) is regarded as single factor-deficient plasma (platelets as lacking factor) and autologous platelet-rich plasma (PRP) as deficiency corrected plasma. Structural features of the developed and subsequently lysed fibrin network, formed under static and flow conditions, were visualized by confocal microscopy. RESULTS: Thrombin-initiated plasma clotting revealed changes in the shape of the absorption curve, more pronounced in the presence of platelets. These changes correlate with the transformation of the fibrin scaffold during clot maturing. With the combined action of platelets, thrombin and Ca2+, plasma clotting passes through two phases: initial formation of a platelet-fibrin network (first peak in the polymerization curve), and then the compaction of fibrin, driven by FXIIIa (the second peak) which can be further modulate by the contractile action of platelets. These structural changes, mediated by platelets and FXIIIa, have been shown to determine subsequent clot lysis. CONCLUSIONS: Platelet aggregates serve as organizing centers that determine the distribution of fibrin in clot volume. The openwork structure of the platelet-transformed fibrin provides the necessary prerequisites for its timely lysis. The revealed aspects of the interaction of platelets and FXIIIa, which accompanies the maturation of a fibrin clot, may lead to new approaches in the pharmacological correction of disorders associated with both thrombotic episodes and bleeding tendency.

Kinetic analysis of prothrombinase assembly and substrate delivery mechanisms.

Prothrombinase complex, composed of coagulation factors Xa (FXa) and Va (FVa) is a major enzyme of the blood coagulation network that produces thrombin via activation of its inactive precursor prothrombin (FII) on the surface of phospholipid membranes. However, pathways and mechanisms of prothrombinase formation and substrate delivery are still discussed. Here we designed a novel mathematical model that considered different potential pathways of FXa or FII binding (from the membrane or from solution) and analyzed the kinetics of thrombin formation in the presence of a wide range of reactants concentrations. We observed the inhibitory effect of large FVa concentrations and this effect was phospholipid concentration-dependent. We predicted that efficient FII activation occurred via formation of the ternary complex, in which FVa, FXa and FII were in the membrane-bound state. Prothrombin delivery was mostly membrane-dependent, but delivery from solution was predominant under conditions of phospholipid deficiency or FXa/FVa excess. Likewise, FXa delivery from solution was predominant in the case of FVa excess, but high FII did not switch the FXa delivery to the solution-dependent one. Additionally, the FXa delivery pathway did not depend on the phospholipid concentration, being the membrane-dependent one even in case of the phospholipid deficiency. These results suggest a flexible mechanism of prothrombinase functioning which utilizes different complex formation and even inhibitory mechanisms depending on conditions.

Therapeutic efficacy of thrombin-preconditioned mesenchymal stromal cell-derived extracellular vesicles on Escherichia coli-induced acute lung injury in mice.

BACKGROUND: Acute lung injury (ALI) following pneumonia involves uncontrolled inflammation and tissue injury, leading to high mortality. We previously confirmed the significantly increased cargo content and extracellular vesicle (EV) production in thrombin-preconditioned human mesenchymal stromal cells (thMSCs) compared to those in naïve and other preconditioning methods. This study aimed to investigate the therapeutic efficacy of EVs derived from thMSCs in protecting against inflammation and tissue injury in an Escherichia coli (E. coli)-induced ALI mouse model. METHODS: In vitro, RAW 264.7 cells were stimulated with 0.1 µg/mL liposaccharides (LPS) for 1 h, then were treated with either PBS (LPS Ctrl) or 5 × 107 particles of thMSC-EVs (LPS + thMSC-EVs) for 24 h. Cells and media were harvested for flow cytometry and ELISA. In vivo, ICR mice were anesthetized, intubated, administered 2 × 107 CFU/100 µl of E. coli. 50 min after, mice were then either administered 50 µL saline (ECS) or 1 × 109 particles/50 µL of thMSC-EVs (EME). Three days later, the therapeutic efficacy of thMSC-EVs was assessed using extracted lung tissue, bronchoalveolar lavage fluid (BALF), and in vivo computed tomography scans. One-way analysis of variance with post-hoc TUKEY test was used to compare the experimental groups statistically. RESULTS: In vitro, IL-1ß, CCL-2, and MMP-9 levels were significantly lower in the LPS + thMSC-EVs group than in the LPS Ctrl group. The percentages of M1 macrophages in the normal control, LPS Ctrl, and LPS + thMSC-EV groups were 12.5, 98.4, and 65.9%, respectively. In vivo, the EME group exhibited significantly lower histological scores for alveolar congestion, hemorrhage, wall thickening, and leukocyte infiltration than the ECS group. The wet-dry ratio for the lungs was significantly lower in the EME group than in the ECS group. The BALF levels of CCL2, TNF-a, and IL-6 were significantly lower in the EME group than in the ECS group. In vivo CT analysis revealed a significantly lower percentage of damaged lungs in the EME group than in the ECS group. CONCLUSION: Intratracheal thMSC-EVs administration significantly reduced E. coli-induced inflammation and lung tissue damage. Overall, these results suggest therapeutically enhanced thMSC-EVs as a novel promising therapeutic option for ARDS/ALI.

A shape memory cationized chitosan sponge loaded with thrombin for high-effective hemostasis and antibacterial activity.

This study presents a thrombin-loaded cationized chitosan (TCCS) sponge with highly effective hemostatic and antibacterial activity. The TCCS sponge, prepared using a multistep method, features a porous structure, favorable mechanical properties, excellent water absorption ability, and shape recovery triggered by water or blood. The TCCS sponge exhibited strong antibacterial activity against Methicillin-resistant Staphylococcus aureus and Escherichia coli. Additionally, it demonstrated enhanced procoagulant and hemostatic efficacy in rat tail amputation and rat liver perforation wound models compared to commercial hemostats. Furthermore, the sponge exhibited favorable biocompatibility and biosafety. These findings suggest that the TCCS sponge has substantial potential for practical applications in managing severe hemorrhages and bacterial infections.

Neutrophil extracellular traps induce intrahepatic thrombotic tendency and liver damage in cholestatic liver disease.

BACKGROUND: Cholestatic liver diseases induce local and systemic hypercoagulation, with neutrophil extracellular traps (NETs) serving as major drivers. These NETs have been linked to decreased liver function in patients with obstructive jaundice. However, the impact of NETs on liver hypercoagulation in cholestatic liver disease remains unknown. METHODS: We utilized bile duct ligation to create experimental mice and analyzed NETs formation in the liver. Fibrin deposition, tissue factor expression, and inflammation in the liver were visualized through western blot and immunohistochemical techniques. LSECs were incubated with isolated NETs, and we detected endothelial procoagulant activity using coagulation protein production assays and measuring endothelial permeability. In both in vivo and in vitro settings, DNase I was applied to clarify the effect of NETs on intrahepatic hypercoagulability, hepatotoxicity, LSEC, and macrophage activation or injury. RESULTS: Bile duct ligation mice exhibited significantly increased levels of NETs in liver tissue, accompanied by neutrophil infiltration, tissue necrosis, fibrin deposition, and thrombophilia compared to sham mice. Notably, NETs resulted in phosphatidylserine and tissue factor exposure on LSEC, enhancing coagulation Factor Xa and thrombin production. The enhanced procoagulant activity could be reversed by degrading NETs with DNase I. Additionally, NETs-induced permeability changes in LSECs, characterized by increased VE-cadherin expression and F-actin retraction, which could be rescued by DNase I. Meanwhile, NET formation is associated with KC activation and the formation of inflammatory factors. CONCLUSIONS: NETs promote intrahepatic activation of coagulation and inflammation, leading to liver tissue injury. Strategies targeting NET formation may offer a potential therapeutic approach for treating cholestatic liver disease.

Genetic Susceptibility of Thrombin Measurement Levels and the Risk of Colon Adenocarcinoma: A Mendelian Randomization Study.

Aims/Background: This investigation sought to establish a possible correlation between thrombin measurement levels and the risk of developing colon adenocarcinoma (COAD). Methods: Thrombin measurement levels were sourced from a study by Pietzner M (2020, PMID: 33328453) and integrated into the IEU database. Data on COAD were obtained from the FinnGen database (2021, C3_COLON_ADENO). Various analytical methods were used to assess the relationship, including inverse variance weighting (IVW), mendelian randomization-Egger (MR-Egger) regression, as well as weighted median and mode techniques. Sensitivity analyses were performed, including Cochran's Q test, MR-Egger intercept test, mendelian randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO), along with leave-one-out analysis, to ensure the robustness of the results. Results: The IVW analysis indicated a significant inverse association between elevated thrombin levels and the risk of COAD (odds ratio (OR) = 0.76, 95% CI = 0.66-0.88, p = 0.0003). These findings were supported by the weighted median analysis (OR = 0.78, 95% CI = 0.68-0.90, p = 0.0006) and the weighted mode analysis (OR = 0.78, 95% CI = 0.68-0.88, p = 0.0017). Conclusion: This research identified an inverse causal relationship between thrombin measurement levels and the incidence of COAD, suggesting that higher thrombin levels are associated with a reduced risk of developing COAD.

Structural, Biochemical Characterization and Molecular Mechanism of Cerastokunin: A New Kunitz-Type Peptide with Potential Inhibition of Thrombin, Factor Xa and Platelets.

The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Based on its anti-trypsin effect, Cerastokunin, a 7.75 kDa peptide, was purified until homogenity by three steps of chromatography. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with Kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% ß-strands with pI 8.48. Cerastokunin showed a potent anticoagulant effect by inhibiting the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose-response blockade of PARs-dependent pathway platelet once stimulated by thrombin. An increased concentration of Cerastokunin resulted in a larger decrease of tail thrombus in the mice-carrageenan model in an in vivo investigation when compared to the effects of antithrombotic medications. At all Cerastokunin doses up to 6 mg/kg, no in vivo toxicity was seen in challenged mice over the trial's duration.

PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases such as thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 G-protein coupled receptors-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and transient receptor potential canonical 3 (TRPC3) channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells, contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.

[Exploring the mechanism of IgA vasculitis pathogenesis through the interaction of thrombin and inflammatory factors using urinary proteomics]. / è¿ç”¨å°¿è›‹ç™½ç»„å­¦æŠ€æœ¯æŽ¢ç´¢å‡è¡€é ¶ä¸Žç‚Žç—‡å› å­ç›¸äº’ä½œç”¨å‚ä¸ŽIgAè¡€ç®¡ç‚Žå‘ç— çš„æœºåˆ¶.

OBJECTIVES: To explore the evidence, urinary biomarkers, and partial mechanisms of hypercoagulability in the pathogenesis of IgA vasculitis (IgAV). METHODS: Differential expression of proteins in the urine of 10 healthy children and 10 children with IgAV was screened using high-performance liquid chromatography-tandem mass spectrometry, followed by Reactome pathway analysis. Protein-protein interaction (PPI) network analysis was conducted using STRING and Cytoscape software. In the validation cohort, 15 healthy children and 25 children with IgAV were included, and the expression levels of differential urinary proteins were verified using enzyme-linked immunosorbent assay. RESULTS: A total of 772 differential proteins were identified between the IgAV group and the control group, with 768 upregulated and 4 downregulated. Reactome pathway enrichment results showed that neutrophil degranulation, platelet activation, and hemostasis pathways were involved in the pathogenesis of IgAV. Among the differential proteins, macrophage migration inhibitory factor (MIF) played a significant role in neutrophil degranulation and hemostasis, while thrombin was a key protein in platelet activation and hemostasis pathways. PPI analysis indicated that thrombin directly interacted with several proteins involved in inflammatory responses, and these interactions involved MIF. Validation results showed that compared to healthy children, children with IgAV had significantly higher urine thrombin/creatinine and urine MIF/creatinine levels (P<0.05). CONCLUSIONS: Thrombin contributes to the pathogenesis of IgAV through interactions with inflammatory factors. Urinary thrombin and MIF can serve as biomarkers reflecting the hypercoagulable and inflammatory states in children with IgAV.

ITIH4 reversed the effects of thrombin on VSMCs stiffness via JNK and ERK signaling pathway.

Vascular smooth muscle cell (VSMCs) is one of the important cell types in artery. VSMCs stiffening may regulate vascular stiffness and contribute to the development of vulnerable plaques. Thrombin, an enzyme in coagulation system, is involved in pathological processes of atherosclerosis. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) plays an important role in regulating inflammation and may have cardiovascular protective effect. Therefore, the elucidation of the mechanisms underlying ITIH4-mediated VSMCs stiffening helps to provide new ideas and potential targets for the diagnosis and treatment of atherosclerosis. In this study, we used specific ITIH4 expression vector and siRNA methods to transfect VSMCs. Our results found that ITIH4 expression increased VSMCs stiffness, meanwhile, ITIH4 siRNA decreased VSMCs stiffness. ITIH4 increased acetylated α-tubulin and inhibited ERK1/2 and JNK, but not P38 MAPK. ERK inhibitor (PD98059) or JNK inhibitor (SP600125) treatment increased acetylated α-tubulin expression and cell stiffness in VSMCs. ITIH4 was downregulated by thrombin treatment, ITIH4 partly reversed the effect of thrombin on acetylated α-tubulin and VSMCs stiffness. These results indicated that ITIH4 regulated acetylated α-tubulin expression in VSMCs and was against the effects of thrombin on VSMCs stiffness. JNK and ERK signaling pathways were proved to participate in this process.