Platelet activation near point-like source of agonist: Experimental insights and computational model.

Disorders of hemostasis resulting in bleeding or thrombosis are leading cause of mortality in the world. Blood platelets are main players in hemostasis, providing the primary response to the vessel wall injury. In this case, they rapidly switch to the activated state in reaction to the exposed chemical substances such as ADP, collagen and thrombin. Molecular mechanisms of platelet activation are known, and detailed computational models are available. However, they are too complicated for large-scale problems (e.g. simulation of the thrombus growth) where less detailed models are required, which still should take into account the variation of agonist concentration and heterogeneity of platelets. In this paper, we present a simple model of the platelet population response to a spatially inhomogeneous stimulus. First, computational nodes modeling platelets are placed randomly in space. Each platelet is assigned the specific threshold for agonist, which determines whether it becomes activated at a given time. The distribution of the threshold value in a population is assumed to be log-normal. The model was validated against experimental data in a specially designed system, where the photorelease of ADP was caused by localized laser stimulus. In this system, a concentration of ADP obeys 2-dimensional Gaussian distribution which broadens due to the diffusion. The response of platelets to the point-like source of ADP is successfully described by the presented model. Our results advance the understanding of platelet function during hemostatic response. The simulation approach can be incorporated into larger computational models of thrombus formation.

In vivo thrombin activity in the diatom Phaeodactylum tricornutum: biotechnological insights.

Diatoms are responsible for 20% of global carbon dioxide fixation and have significant potential in various biotechnological and industrial applications. Recently, the pennate diatom Phaeodactylum tricornutum has emerged as a prominent platform organism for metabolic engineering and synthetic biology. The availability of its genome sequence has facilitated the development of new bioengineering tools. In this study, we used in silico analyses to identify sequences potentially encoding thrombin-like proteins, which are involved in recognizing and cleaving the thrombin sequence LVPRGS in P. tricornutum. Protein structure prediction and docking studies indicated a similar active site and ligand positioning compared to characterized human and bovine thrombin. The evidence and efficiency of the cleavage were determined in vivo using two fusion-protein constructs that included YFP to measure expression, protein accumulation, and cleavage. Western blot analysis revealed 50-100% cleavage between YFP and N-terminal fusion proteins. Our findings suggest the existence of a novel thrombin-like protease in P. tricornutum. This study advances the application of diatoms for the synthesis and production of complex proteins and enhances our understanding of the functional role of these putative thrombin sequences in diatom physiology. KEY POINTS: • Protein structure predictions reveal thrombin-like active sites in P. tricornutum. • Validated cleavage efficiency of thrombin-like protease on fusion proteins in vivo. • Study advances bioengineering tools for diatom-based biotechnological applications.

Impact of Temperature on the Self-Assembly of Fibrinogen in Thrombin-Free Solutions.

Self-assembly of thrombin-free solutions of fibrinogen can be triggered not only by a drop in the ionic strength but also by an appropriate decrease in temperature. Accordingly, an in situ study of self-assembly of fibrinogen in saline buffered solution is carried out by means of time-resolved light scattering providing the molar mass, geometric size, and hydrodynamic radius of the growing intermediates. The resulting data provide access to the morphology of the intermediates and to the mechanism in which these intermediates grow during the early stages of self-assembly. Modeling the results of concentration dependent experiments based on temperature gradients in terms of a chain growth mechanism leads to the corresponding molar standard enthalpy and entropy of aggregation.

Reconfigurable DNA Nanocage for Protein Encapsulation and Regulation.

Creating nanomachines capable of precisely capturing, organizing, and regulating the activity of target biomolecules holds profound significance for advancing nanotechnology and therapeutics. Here, we develop a multistage reconfigurable DNA nanocage that can enclose and modulate proteins through multivalent interactions, activated by specific molecular signals. By strategically designing and manipulating the strut architecture of the DNA nanocages, we can achieve precise control over their reconfiguration among pyramid, square, and linear branch shapes. Additionally, we demonstrated its ability to capture thrombin and effectively inhibit its coagulation activity by incorporating two thrombin-targeting aptamers into the designed arms of the DNA nanocage. The activity of thrombin can be recovered by rearranging the conformation of the DNA nanocage and exposing the protein, thereby activating the coagulation process. This approach enriches the design toolbox for dynamic nanomachines and inspires a new strategy for protein encapsulation and regulation with potential future therapeutic applications.

Ultrasonic Control of Protein Splicing by Split Inteins.

Utilizing ultrasound as an external stimulus to remotely modulate the activity of proteins is an important aspect of sonopharmacology and establishes the basis for the emerging field of sonogenetics. Here, we describe an ultrasound-responsive protein splicing system that enables spatiotemporal control of split-intein-mediated protein ligation. The system utilizes engineered split inteins that are caged and can be activated by thrombin released from a high molar mass DNA-based carrier under focused ultrasound sonication. This approach represents a general method for controlling the functions of proteins of interest by ultrasound, as demonstrated here by the controlled synthesis of the superfolder green fluorescence protein (GFP) and calcitonin. Furthermore, calcitonin receptor-mediated signal transduction in cells was triggered by this system in vitro without harming cell viability. By expanding the sonogenetic toolbox with protein splicing technologies, this study provides a possible pathway to deploy ultrasound for remotely controlling a variety of protein functions in deep tissue in the future.

Modelling the effects of 4-factor prothrombin complex concentrate for the management of factor Xa-associated bleeding.

The management of factor Xa (FXa) inhibitor-associated bleeding remains a clinical challenge. Massive bleeding is often associated with complex coagulopathy and, thus, the sole reversal of FXa inhibitors might not be sufficient to restore hemostasis, requiring instead a multimodal approach. Four-factor prothrombin complex concentrate (4F-PCC) is widely recognized as a viable treatment option for FXa inhibitor-associated bleeding. Here, we applied computational models to explore the effect 4F-PCC has on the coagulation cascade and restoration of thrombin generation in a system that simulates a patient that has received a FXa inhibitor. The coagulation model is largely based on a previously developed model with modifications incorporated from various other published sources. The model was calibrated and validated using data from a phase 3 clinical trial of vitamin K antagonist reversal with 4F-PCC. Using the parameters and initial conditions determined during the calibration and validation process, the prothrombin time (PT) test simulations predicted a PT of 11.4 seconds. The model successfully simulated the effects of rivaroxaban and apixaban on total thrombin concentration and showed that 4F-PCC increased thrombin generation in the presence of rivaroxaban or apixaban.

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.

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.

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.

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.

Activated platelets retain and protect most of their factor XIII-A cargo from proteolytic activation and degradation.

ABSTRACT: Platelet factor XIII-A (FXIII-A) is a major cytoplasmic protein (∼3% of total), representing ∼50% of total circulating FXIII. However, mobilization of FXIII-A during platelet activation is not well defined. To determine mechanisms mediating the retention vs release of platelet FXIII-A, platelets from healthy humans and mice (F13a1-/-, Fga-/-, Plg-/-, Stim1fl/flPf4-Cre, and respective controls) were stimulated with thrombin, convulxin plus thrombin, or calcium ionophore (A23187), in the absence or presence of inhibitors of transglutaminase activity, messenger RNA (mRNA) translation, microtubule rearrangement, calpain, and Rho GTPase. Platelet releasates and pellets were separated by (ultra)centrifugation. FXIII-A was detected by immunoblotting and immunofluorescence microscopy. Even after strong dual agonist (convulxin plus thrombin) stimulation of human platelets, >80% platelet FXIII-A remained associated with the platelet pellet. In contrast, essentially all tissue factor pathway inhibitor, another cytoplasmic protein in platelets, was released to the supernatant. Pellet-associated FXIII-A was not due to de novo synthesis via platelet F13A1 mRNA. The proportion of platelet FXIII-A retained by vs released from activated platelets was partly dependent on STIM1 signaling, microtubule rearrangement, calpain, and RhoA activation but did not depend on the presence of fibrinogen or plasminogen. Immunofluorescence microscopy confirmed the presence of considerable FXIII-A within the activated platelets. Although released FXIII-A was cleaved to FXIII-A∗ and could be degraded by plasmin, platelet-associated FXIII-A remained uncleaved. Retention of substantial platelet-derived FXIII-A by activated platelets and its reduced susceptibility to thrombin- and plasmin-mediated proteolysis suggest platelet FXIII-A is a protected pool with biological role(s) that differs from plasma FXIII.

Filamin A regulates platelet shape change and contractile force generation via phosphorylation of the myosin light chain.

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.

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.

Investigating Protein-Nucleic Acid Binding Interactions with Diethylpyrocarbonate Covalent Labeling-Mass Spectrometry.

Nucleic acids are important biomolecules that facilitate numerous cellular functions and have in recent years become promising candidates for treating disease. Consequently, there is a need for methods to characterize protein interactions with these molecules. Here, we demonstrate that diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry (CL-MS) can provide structural information for protein-nucleic acid binding by characterizing the binding sites of two DNA aptamers specific to thrombin. Reductions in thrombin labeling are observed at the pair's binding interfaces. Furthermore, we find that binding of the aptamers causes changes in labeling at residues in the thrombin active site and known exosites for each aptamer, showcasing the sensitivity of DEPC CL-MS to significant allosteric changes.

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.

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.

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.

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.

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.