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Glioblastoma is an aggressive type of cancer that begins in cells called astrocytes that support nerve cells that can occur in the brain or spinal cord. It can form in the brain or spinal cord. Despite the variety of modern therapies against GBM, it is still a deadly disease. Patients usually have a median survival of approximately 14 to 15 months from the diagnosis. Glioblastoma is also known as glioblastoma multiforme. The pathogenesis contributing to the proliferation and metastasis of cancer involves aberrations of multiple signalling pathways through multiple genetic mutations and altered gene expression. The coagulant factors like thrombin and tissue factor play a noteworthy role in cancer invasion. They are produced in the microenvironment of glioma through activation of protease-activated receptors (PARs) which are activated by coagulation proteases. PARs are members of family G-protein-coupled receptors (GPCRs) that are activated by coagulation proteases. These components play a key role in tumour cell angiogenesis, migration, invasion, and interactions with host vascular cells. Further, the release of neurotransmitters is also found to regulate malignancy in gliomas. Exploration of the interplay between malignant neural circuitry with the normal conditions is also decisive in finding effective therapies for these apparently invasive tumours. The present review discusses the molecular classification of gliomas, activation of PARs by coagulation protease, and its role in metastasis of gliomas. Further, the differential involvement of neurotransmitters in the pathogenesis of gliomas has also been discussed. Targeting these molecules may present a potential therapeutic approach for the treatment of gliomas.

The mechanism underlying thrombus formation in acute coronary syndrome (ACS) involves both platelets and thrombin. While both pathways are targeted in acute care, platelet inhibition has been predominantly administered in the chronic phase, yet thrombin plays a key role in platelet activation and fibrin formation. Among ACS patients, there is also a persistent chronic increase in thrombin generation, which is associated with a higher rate of adverse events. In the setting of post-ACS care with rivaroxaban or vorapaxar, targeting thrombin has been associated with decreased thrombin generation and reduced cardiovascular events, but has been associated with increased bleeding risk. We explored the evidence supporting thrombin generation in the pathophysiology of recurrent events post-ACS and the role of thrombin as a viable therapeutic target. One specific target is factor XI inhibition, which is involved in thrombin generation, but may also allow for the preservation of normal hemostasis., Competing Interests: Declaration of competing interest Dr. Bahit receives modest honorarium from MSD, Pfizer, Bristol-Myers Squibb, CSL Behring, Janssen, Boehringer Ingelheim, Anthos; Dr. Gibson receives research support from Johnson & Johnson and Bristol-Myers Squibb, and consulting support from AstraZeneca, Johnson & Johnson, Janssen & Bayer., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Endothelial nitric oxide synthase (eNOS) is a critical regulatory enzyme that controls vascular tone via the production of nitric oxide. Although thrombin also modulates vascular tone predominantly via the activation of protease-activated receptors (PARs), the time course and mechanisms involved in how thrombin controls eNOS enzymatic activity are unknown. eNOS enzymatic activity is enhanced by the phosphorylation of eNOS-Ser1177 and reduced by the phosphorylation of eNOS-Thr495. In this study, we hypothesized that thrombin regulates vascular tone through the differential phosphorylation of eNOS. Using rat descending aorta, we show that thrombin modulates vascular tone in an eNOS-dependent manner via activated PAR-1. We also show that thrombin causes a temporal biphasic response. Protein kinase C (PKC) is associated with second phase of thrombin-induced response. Western blot analysis demonstrated thrombin phosphorylated eNOS-Ser1177 and eNOS-Thr495 in human umbilical vein endothelial cells. A PKC inhibitor suppressed the thrombin-induced phosphorylation of eNOS-Thr495, but not that of eNOS-Ser1177. Our results suggest that thrombin induces a temporal biphasic vascular response through the differential phosphorylation of eNOS via activated PAR-1. Thrombin causes transient vasorelaxation by the phosphorylation of eNOS-Ser1177, and subsequent attenuation of vasorelaxation by the phosphorylation of eNOS-Thr495 via PKC, leading to the modulation of vascular tone., Competing Interests: Declaration of competing interest The authors indicated no potential conflicts of interest., (Copyright © 2022 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Thrombin is a key regulator of the homeostasis system. Also, it actively participates in progression of various systemic diseases, including atherosclerosis. There is a large amount of experimental and clinical data on the involvement of thrombin in the pathogenesis of ischemic heart disease (IHD). Thus, studying thrombin activity regulation is promising. Also, the question whether it is possible to use biomarkers of thrombin activity as predictors of cardiovascular complications in IHD patients is relevant. The present review focuses on major mechanisms of thrombin functioning, its role in development and progression of atherosclerosis, and available tests for evaluation of thrombin functional activity. Major clinical studies are discussed that evaluated the efficacy of thrombin inhibitors and protease-activated receptor antagonists.

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (< 10 minutes) generates a 5.6-kDa fragment (residues 1-48) with full vasoinhibin activity, that is, it inhibited the proliferation, invasion, and permeability of cultured endothelial cells and promoted the lysis of a fibrin clot in plasma with a similar potency to that of a conventional 14-kDa vasoinhibin (residues 1-123). The R48-G49 cleavage site is highly conserved throughout evolution and precedes the intramolecular disulfide bond (C58-C174), thereby allowing the 5.6-kDa vasoinhibin to be released without a reduction step. Furthermore, the 5.6-kDa vasoinhibin is produced by endogenous thrombin during the clotting process. These findings uncover the smallest vasoinhibin known, add thrombin to the list of PRL-cleaving proteases generating vasoinhibin, and introduce vasoinhibin as a thrombin-activated mechanism for the regulation of hemostasis, vasopermeability, and angiogenesis in response to tissue injury., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Disseminated intravascular coagulation (DIC) is a common and life-threatening complication in sepsis. Sepsis-associated DIC is recognized as the systemic activation in coagulation with suppressed fibrinolysis that leads to organ dysfunction in combination with systemic intravascular inflammation. In this process, thrombin contributes a key role in connecting both coagulation and inflammation. Endothelial injury, a result of sepsis, causes DIC due to the effect of multiple activated factors that include neutrophils, platelets, and damage-associated molecular patterns. Recent advances in the understanding of pathophysiology have made it possible to diagnose sepsis-associated DIC at earlier timing with better accuracy. However, progress in the treatment is still limited, and new therapeutics for sepsis-associated DIC are needed.

Factor XI (FXI) is a serine protease involved in the propagation phase of coagulation and in providing clot stability. Several mutations in the F11 gene lead to FXI deficiency, a rare mild bleeding disorder. Current laboratory methods are unable to assess bleeding risk in FXI-deficient patients, because the degree of bleeding tendency does not correlate with plasma FXI activity as measured by routine coagulometric aPTT-based assays. Bleeding manifestations are highly variable among FXI-deficient patients and FXI replacement therapy can be associated with an increased thrombotic risk. A correct evaluation of the patient hemostatic potential is crucial to prevent under- or overtreatment. In recent years, different research groups have investigated the use of global coagulation assays as alternative for studying the role of FXI in hemostasis and identifying the clinical phenotype of FXI deficiency. This brief review article summarizes the main features of coagulation factor XI and its deficiency and resumes the principle axes of research and methods used to investigate FXI functions., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Neutrophils constitute the major population of infiltrating leukocytes after stroke including intracerebral hemorrhage (ICH), and these cells may exhibit pro-inflammatory and anti-inflammatory phenotypes depending on the external stimuli. Here we constructed an experimental system to evaluate how the properties of neutrophils were influenced by the injured brain tissues. HL60 cells differentiated into neutrophils were added to the culture medium of neonatal rat cortico-striatal slices maintained at liquid-air interface. Thrombin was applied to the cultures to mimic the pathogenic events associated with ICH. HL60 cells responded to thrombin by increasing mRNA expression of pro-inflammatory IL-1β and anti-inflammatory IL-10 with a different time course. Co-presence of cortico-striatal slice cultures significantly enhanced IL-1β mRNA expression, whereas attenuated IL-10 mRNA expression, in HL60 cells. Toll-like receptor 4 (TLR4) agonist lipopolysaccharide synergistically enhanced IL-1β mRNA expression with thrombin, and TLR4 inhibitor TAK-242 abolished thrombin-induced IL-1β mRNA expression in the presence of slice cultures. On the other hand, thrombin-induced cell death in cortico-striatal cultures was attenuated by the presence of HL60 cells. This experimental system may provide a unique platform to elucidate complex cell-to-tissue interactions during ICH pathogenesis., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Intracerebral hemorrhage (ICH) results from blood vessels rupture in the brain, forming a blood clot in the brain parenchyma. Leakage of blood constituents causes detrimental tissue damages, ensuing long-lasting neurological deficits; however, effective therapeutic approaches are not yet developed to date. In this study, leukotriene B 4 (LTB 4 ) and its receptor leukotriene B 4 receptor 1 (BLT1) are proposed as novel therapeutic targets for ICH therapy. After the onset of ICH, the LTB 4 content in the brain transiently elevated. Microglia are considered as the source of LTB 4 production. Thrombin, a blood constituent, activated the BV-2 microglia and increased the LTB 4 secretion from the BV-2 cells. Microglia-released LTB 4 promoted its own microglial activation and neutrophil-like differentiated HL-60 cell migration activity. LTB 4 receptors comprised of two types: BLT1 and BLT2, with BLT1 known to be a high-affinity receptor associated with chemotaxis. BLT1 knockout mice showed decreased neutrophil invasion, attenuating sensorimotor dysfunction after ICH. Furthermore, therapeutic administration of ONO-4057, an orally active LTB 4 receptor antagonist, attenuated neutrophil invasion, microglial activation, axonal fragmentation, and sensorimotor deficits induced by ICH. These results suggest that LTB 4 and its receptor BLT1 can be potential promising therapeutic targets that prevent tissue damages following ICH.

Introduction: Thrombin inhibitor and anti-Xa are now widely used in clinical practice. However, the difference between thrombin inhibitor and anti-Xa in prevention of thrombosis is still to be elucidated., Materials and Methods: Computer simulator implementing the function of platelet, coagulation, fibrinolysis and blood flow was developed. The function of thrombin is defined as to activated platelet at the rate of 0.01 s -1 and to produce fibrin at the rate of 0.1 s -1 in control. The effect of thrombin inhibitor was settled to reduce the rate of platelet activation and fibrin generation changed from 10 to 100% as compared to the control. The local thrombin generation rate on activated platelet was settled as 1.0 s -1 as a control. The effect of anti-Xa was settled to reduce to thrombin generation rate on activated platelet from 10% to 100% as compared to the control. The sizes of thrombi formed at site of endothelial injury in the presence and absence of thrombin inhibitor and anti-Xa were compared., Results and Conclusions: The size of thrombi formed by 30-s perfusion of blood at site of endothelial injury reduced both in the presence of thrombin inhibitor and anti-Xa. There was significant positive relationship between thrombin inhibitor effect and the size of formed thrombi with R value of 0.96. (p < 0.0001) However, the sizes of thrombi were not influence by anti-Xa until it decreased 30% or less as compared to control. There was no significant relationship between anti-Xa effect and the size of formed thrombi. (R = 0.39, p = 0.09) Our results suggest the different dose-dependent effects of thrombin inhibitor and anti-Xa on thrombus formation at least in specific conditions. Computer simulation may help to predict quantitative antithrombotic effects of various antithrombotic agents., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ancient organisms have a combined coagulation and immune system, and although links between inflammation and hemostasis exist in mammals, they are indirect and slower to act. Here we investigated direct links between mammalian immune and coagulation systems by examining cytokine proproteins for potential thrombin protease consensus sites. We found that interleukin (IL)-1α is directly activated by thrombin. Thrombin cleaved pro-IL-1α at a site perfectly conserved across disparate species, indicating functional importance. Surface pro-IL-1α on macrophages and activated platelets was cleaved and activated by thrombin, while tissue factor, a potent thrombin activator, colocalized with pro-IL-1α in the epidermis. Mice bearing a mutation in the IL-1α thrombin cleavage site (R114Q) exhibited defects in efficient wound healing and rapid thrombopoiesis after acute platelet loss. Thrombin-cleaved IL-1α was detected in humans during sepsis, pointing to the relevance of this pathway for normal physiology and the pathogenesis of inflammatory and thrombotic diseases., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Venom can affect any part of the body reachable via the bloodstream. Toxins which specifically act upon the coagulation cascade do so either by anticoagulant or procoagulant mechanisms. Here we investigated the coagulotoxic effects of six species within the medically important pit viper genus Protobothrops (Habu) from the Chinese mainland and Japanese islands, a genus known to produce hemorrhagic shock in envenomed patients. Differential coagulotoxicity was revealed: P. jerdonii and P. mangshanensis produced an overall net anticoagulant effect through the pseudo-procoagulant clotting of fibrinogen; P. flavoviridis and P. tokarensis exhibit a strong anticoagulant activity through the destructive cleavage of fibrinogen; and while P. elegans and P. mucrosquamatus both cleaved the A-alpha and B-beta chains of fibrinogen they did not exhibit strong anticoagulant activity. These variations in coagulant properties were congruent with phylogeny, with the closest relatives exhibiting similar venom effects in their action upon fibrinogen. Ancestral state reconstruction indicated that anticoagulation mediated by pseudo-procoagulant cleavage of fibrinogen is the basal state, while anticoagulation produced by destructive cleavage of fibrinogen is the derived state within this genus. This is the first in depth study of its kind highlighting extreme enzymatic variability, functional diversification and clotting diversification within one genus surrounding one target site, governed by variability in co-factor dependency. The documentation that the same net overall function, anticoagulation, is mediated by differential underlying mechanics suggests limited antivenom cross-reactivity, although this must be tested in future work. These results add to the body of knowledge necessary to inform clinical management of the envenomed patient., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Activation of the blood coagulation cascade leads to fibrin deposition and platelet activation that are required for hemostasis. However, aberrant activation of coagulation can lead to thrombosis. Thrombi can cause tissue ischemia, and fibrin degradation products and activated platelets can enhance inflammation. In addition, coagulation proteases activate cells by cleavage of PARs (protease-activated receptors), including PAR1 and PAR2. Direct oral anticoagulants have recently been developed to specifically inhibit the coagulation proteases FXa (factor Xa) and thrombin. Administration of these inhibitors to wild-type mice can be used to determine the roles of FXa and thrombin in different inflammatory diseases. These results can be compared with the phenotypes of mice with deficiencies of either Par1 (F2r) or Par2 (F2rl1). However, inhibition of coagulation proteases will have effects beyond reducing PAR signaling, and a deficiency of PARs will abolish signaling from all proteases that activate these receptors. We will summarize studies that examine the roles of coagulation proteases, particularly FXa and thrombin, and PARs in different mouse models of inflammatory disease. Targeting FXa and thrombin or PARs may reduce inflammatory diseases in humans.

Background: Thrombin activates hepatic stellate cells via protease-activated receptor-1. The role of Factor Xa (FXa) in hepatic fibrosis has not been elucidated. We aimed to evaluate the impact of FXa and thrombin in vitro on stellate cells and their respective inhibition in vivo using a rodent model of hepatic fibrosis., Methods: HSC-LX2 cells were incubated with FXa and/or thrombin in cell culture, stained for αSMA and relative gene expression and gel contraction calculated. C57BL/6 J mice were administered thioacetamide (TAA) for 8 weeks with Rivaroxaban (n = 15) or Dabigatran (n = 15). Control animals received TAA alone (n = 15). Fibrosis was scored and quantified using digital image analysis and hepatic tissue hydroxyproline estimated., Results: Stellate cells treated with FXa and thrombin demonstrated upregulation of procollagen, TGF-beta, αSMA and significant cell contraction (43.48%+/- 4.12) compared to culturing with FXa or thrombin alone (26.90%+/- 8.90, p = 0.02; 13.1%+/- 9.84, p < 0.001). Mean fibrosis score, percentage area of fibrosis and hepatic hydroxyproline content (2.46 vs 4.08, p = 0.008; 2.02% vs 3.76%, p = 0.012; 276.0 vs 651.3, p = 0.0001) were significantly reduced in mice treated with the FXa inhibitor compared to control mice. FXa inhibition was significantly more effective than thrombin inhibition in reducing percentage area of fibrosis and hepatic hydroxyproline content (2.02% vs 3.70%,p = 0.031; 276.0 vs 413.1,p = 0.001)., Conclusions: FXa promotes stellate cell contractility and activation. Early inhibition of coagulation using a FXa inhibitor significantly reduces TAA induced murine liver fibrosis and may be a viable treatment for liver fibrosis in patients.

Background: Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets., Methods and Results: Recombinant Escherichia coli-expressed exopolyphosphatase deletion mutant PPX&#95;Δ12 labeled with fluorescent Alexa488 dye was used as a polyphosphate probe in flow cytometry. PPX&#95;Δ12-Alexa488-signal dose-dependently increased with long-chain polyphosphate binding to platelets. In contrast, short-chain polyphosphate that is found in the supernatant of activated platelets, did not bind to the platelet surface. Both exopolyphosphatase treatment and polyphosphate pre-incubation abolished PPX&#95;Δ12-Alexa488 binding to polyphosphate on platelets. Stimulation of platelets with thrombin receptor agonist Trap6, and P2Y 12 receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX&#95;Δ12-Alexa488 signal in a dose-dependent manner., Conclusion: This study indicates that long-chain polyphosphate binds to platelet plasma membranes and presents a promising diagnostic assay to measure this interaction on human platelets in platelet-rich plasma. Future investigations will aim to determine if polyphosphate can be used as a novel biomarker of thrombosis. © 2016 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)

Transient amnesia is a common consequence of minimal traumatic brain injury (mTBI). However, while recent findings have addressed the mechanisms involved in its onset, the processes contributing to its recovery have not yet been addressed. Recently, we have found that thrombin is detected at high concentrations in the brain of mice after exposure to mTBI and that in such settings amnesia is rescued by either inhibiting thrombin activity or by blockade of PAR1. Here, we report that mice spontaneously recover from amnesia after two weeks from mTBI exposure. At this time point, long term potentiation was equally evoked in injured vs. control animals with thrombin concentration in the brain being normalized at this stage. These findings, which refer to the specific aspect of memory retrieval upon mTBI, together with our previous work, hint to a strong correlation between cognitive defects in the context of mTBI and thrombin concentrations in the brain. This may suggest that a possible scavenging of thrombin in the brain at early phases following mTBI may improve memory function.

Background and Aims: Thrombin, the active enzyme of the coagulation system, plays a critical role in the pathogenesis of atherosclerosis. Vascular repair promoted by stromal cell-derived factor-1 is a protective process in atherosclerosis. Consumption of low amount of alcohol is believed to reduce the risk of atherosclerotic cardiovascular disease but the mechanism is unclear. This study evaluated whether alcohol can modulate the expression of stromal cell-derived factor-1 and the pro-atherosclerotic activity of thrombin., Methods: Hepatocytes, monocytes, vascular endothelial and vascular smooth muscle cells were pre-treated with increasing concentrations of ethanol before stimulation with thrombin. The expression of cytokines, chemokines, cell adhesion molecules and epigenetic factors, including histone deacetylases and sirtuins, was evaluated., Results: Thrombin stimulation significantly enhanced the expression of pro-inflammatory cytokines, chemokines and cell adhesion molecules, but significantly decreased the expression of stromal cell-derived factor-1. Pre-treatment of cells with a low dose of ethanol significantly decreased thrombin-induced production of pro-inflammatory cytokines and chemokines, and significantly increased the production of stromal cell-derived factor-1 compared to cells treated with thrombin alone. Ethanol significantly counteracted the decreased expression of histone deacetylases and sirtuins induced by thrombin. Inhibition of histone deacetylase-2 with trichostatin A or with specific siRNA abolished the stimulatory activity of low-dose ethanol on stromal cell-derived factor-1., Conclusions: Low-dose of ethanol attenuates the inflammatory response and counteracts the reduced expression of stromal cell-derived factor-1 induced by thrombin via an epigenetic mechanism, providing a potential explanation for the protective activity of low dose of alcohol in atherosclerosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Adequate platelet reactivity is required for maintaining hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated to regulate platelet function and thrombosis ex vivo, supporting a key role for 12-LOX in the regulation of in vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Here, we studied the effect of the first highly selective 12-LOX inhibitor, ML355, on in vivo thrombosis and hemostasis., Approach and Results: ML355 dose-dependently inhibited human platelet aggregation and 12-LOX oxylipin production, as confirmed by mass spectrometry. Interestingly, the antiplatelet effects of ML355 were reversed after exposure to high concentrations of thrombin in vitro. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen were attenuated in whole blood treated with ML355 comparable to aspirin. Oral administration of ML355 in mice showed reasonable plasma drug levels by pharmacokinetic assessment. ML355 treatment impaired thrombus growth and vessel occlusion in FeCl 3 -induced mesenteric and laser-induced cremaster arteriole thrombosis models in mice. Importantly, hemostatic plug formation and bleeding after treatment with ML355 was minimal in mice in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model., Conclusions: Our data strongly support 12-LOX as a key determinant of platelet reactivity in vivo, and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy., (© 2017 American Heart Association, Inc.)

Thrombin is a multifunctional enzyme that plays an important role in blood coagulation, cell growth, and metastasis. Depending upon the binding of sodium ions, thrombin presents significantly different enzymatic activities. In the environment with sodium ions, thrombin is highly active in cleaving the coagulated substrates and this is referred to as the "fast" form; in the environment without sodium ions, thrombin turns catalytically less active and is in the "slow" form. Although many experimental studies over the last two decades have attempted to reveal the structural and kinetic differences between these two forms, it remains vague and disputed how the functional switch between the "fast" and "slow" forms is mediated by Na + cations. In this work, we employ microsecond-scale all-atom molecular dynamics simulations to investigate the differences in the structural ensembles in sodium-bound/unbound and potassium-bound/unbound thrombin. Our calculations indicate that the regulatory regions, including the 60s, γ loops, and exosite I and II, are primarily affected by both the bound and unbound cations. Conformational free energy surfaces, estimated from principal component analysis, further reveal the existence of multiple conformational states. The binding of a cation introduces changes in the distribution of these states. Through comparisons with potassium-binding, the binding of sodium ions appears to shift the population toward conformational states that might be catalytically favorable. Our study of thrombin in the presence of sodium/potassium ions suggests Na + -mediated generalized allostery is the mechanism of thrombin's functional switch between the "fast" and "slow" forms.

Background: Plasma transfusion remains the mainstay hemostatic therapy during liver transplantation (LT) in most countries. However, a large volume is required for plasma to achieve clinically relevant factor increases. Prothrombin complex concentrate (PCC) is a low-volume alternative to plasma in warfarin reversal, but its efficacy has not been well studied in LT., Methods: Blood samples were collected from 28 LT patients at baseline (T0) and 30 minutes after graft reperfusion (T1). Factor X and antithrombin levels were measured. Ex vivo effects of PCC (0.2 and 0.4 IU/mL) and 10% volume replacement with normal plasma were compared in LT and warfarin plasma by measuring lag time, thrombin peak, and endogenous thrombin potential (ETP) using thrombin generation (TG) assay., Results: Coagulation status was worsened at T1 as international normalized ratio increased from 1.7 to 3.0, and factor X was decreased from 49% to 28%. TG measurements showed normal lag time and ETP at T0 and T1, but low-normal peak at T0, and below-normal peak at T1. Both doses of PCC increased peak and ETP, while 10% volume plasma had minimal effects on TG. Thrombin inhibition appears to be very slow after adding 0.4 IU/mL of PCC in LT plasma due to low antithrombin. The same doses of PCC and plasma were insufficient for warfarin reversal., Conclusions: Reduced TG in LT can be more effectively restored by using PCC rather than plasma. The required doses of PCC for LT patients seem to be lower than warfarin reversal due to slow thrombin inhibition.

Heparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.

Objective: Progestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte-endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis., Study Design: Liquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both., Results: Thrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (p<.02, n=3). Compared to pre-DMPA endometria (n=5), stromal cells in post-DMPA endometria (n=5) displayed stronger CSPG4 immunostaining. In HEEC cultures (n=3), total tube-formed mesh area was significantly higher in rh-CSPG4 versus control (p<.05). However, thrombin disrupted HEEC tube formation by a concentration- and time-dependent reduction of angiogenic parameters (p<.05), whereas CSPG4 co-treatment did not reverse these thrombin-mediated effects., Conclusion: These results suggest that disruption of HEEC tube formation by thrombin induces aberrant angiogenesis and abnormal uterine bleeding in DMPA users., Implications: Mass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin-only contraceptive-induced abnormal uterine bleeding., (Copyright © 2017. Published by Elsevier Inc.)

Atrial fibrillation (AF) is associated with increased risk of thromboembolic complications. One of the markers of the increased risk of hypercoagulable state is platelet hyperreactivity. The aim of the study was to assess impact of arrhythmia on platelet reactivity., Methods: The study included 36 (mean age 48,3; range 21-60) male patients with lone atrial fibrillation, with exclusion of concomitant diseases known to trigger hypercoagulable state. The AF patients underwent cardioversion to restore sinus rhythm and were subsequently under observation for 1month. Echocardiography, ECG and blood collection was performed before cardioversion (T0) and 4weeks after successful cardioversion (T1). During the study period patients have been contacted and examined every week and 24h ECG monitoring was performed. Platelet reactivity was assessed based on changes of CD62 and CD42b expression on platelet surface after stimulation with thrombin. Also changes in MPV were assessed., Results: In all patients sinus rhythm was maintained at the end of the study period, however in 14 patients recurrences of AF were observed, confirmed by 24h ECG monitoring (atrial fibrillation recurrence group - AFR) and 22 patients maintained sinus rhythm throughout the whole study period (SR group). Mean fluorescence intensity (MFI) of CD62 on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (48.04±22.42 vs 41.47±16.03; p<0.01). Also MFI of CD42b on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (22.16±10.82 vs 12.06±5.99; p<0.0001). Platelets reactivity estimated by CD 62 expression in SR group decreased significantly after 4weeks observation (58.01±15.26 vs 46.57±13.44; p<0.001) opposite to AFR group 35.66±21.87 vs 34.54±16.4; p-ns). Moreover there were significant differences between basal reactivity during AF between SR and AFR groups (58.01±15.26 vs 35.66±21.87; p-0.01). MFI of CD42b on thrombin stimulated platelets decreased significantly both in AFR and SR groups (22.05±11.36 vs 13.8±6.03; p<0.001 and 21.87±14.18 vs 10.04±5.09; p<0005). MPV decreased significantly 4weeks after electrical cardioversion as compared to T0 (8.81±0.19 vs 8.42±0.14; p<0.0001)., Conclusion: The changes of platelet reactivity to thrombin observed after restoration of sinus rhythm in patients prove that arrhythmia intrinsically leads to increased reactivity of platelets., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

Multicellular interactions of platelets, leukocytes, and the blood vessel wall support coagulation and precipitate arterial and venous thrombosis. High levels of angiotensin II cause arterial hypertension by a complex vascular inflammatory pathway that requires leukocyte recruitment and reactive oxygen species production and is followed by vascular dysfunction. We delineate a previously undescribed, proinflammatory coagulation-vascular circuit that is a major regulator of vascular tone, blood pressure, and endothelial function. In mice with angiotensin II-induced hypertension, tissue factor was up-regulated, as was thrombin-dependent endothelial cell vascular cellular adhesion molecule 1 expression and integrin α M β 2 - and platelet-dependent leukocyte adhesion to arterial vessels. The resulting vascular inflammation and dysfunction was mediated by activation of thrombin-driven factor XI (FXI) feedback, independent of factor XII. The FXI receptor glycoprotein Ibα on platelets was required for this thrombin feedback activation in angiotensin II-infused mice. Inhibition of FXI synthesis with an antisense oligonucleotide was sufficient to prevent thrombin propagation on platelets, vascular leukocyte infiltration, angiotensin II-induced endothelial dysfunction, and arterial hypertension in mice and rats. Antisense oligonucleotide against FXI also reduced the increased blood pressure and attenuated vascular and kidney dysfunction in rats with established arterial hypertension. Further, platelet-localized thrombin generation was amplified in an FXI-dependent manner in patients with uncontrolled arterial hypertension, suggesting that platelet-localized thrombin generation may serve as an inflammatory marker of high blood pressure. Our results outline a coagulation-inflammation circuit that promotes vascular dysfunction, and highlight the possible utility of FXI-targeted anticoagulants in treating hypertension, beyond their application as antithrombotic agents in cardiovascular disease., (Copyright © 2017, American Association for the Advancement of Science.)

Context: Studies have shown that thrombin activation played a central role in cell injuries associated with intracerebral hemorrhage (ICH)., Objective: Here, our study investigated the cytotoxicity of thrombin on neurons, and determined the involvement of JNK pathways in thrombin-induced neuronal apoptosis., Materials and Methods: Primary cultured neurons were treated with different doses of thrombin. Some neurons were given either SP600125 or vehicle. LDH release assay and flow cytometry were used to measure neuronal apoptosis caused by thrombin. The activation of JNK and capases-3 were measured by Western blot., Results: Our results showed large doses of thrombin that increased the LDH release, the level of cleaved caspase-3 and apoptosis rate of neurons. JNK was activated by thrombin in a time-dependent manner. Administration of SP600125 protects neurons from thrombin-induced apoptosis., Conclusion: These data indicate that the activation of JNK is crucial for thrombin-induced neuronal apoptosis, and inhibition of JNK may be a potential therapeutic target for ICH.

Background: Postoperative spinal epidural hematoma (POSEH) is different from spontaneous or post-spinal procedure hematoma because of the application of suction drains. However, it appeared that suction drains were not effective for prevention of POSEH in previous studies. The purpose of this study was to test our hypothesis that POSEH can be caused by hypercoagulability., Methods: This was an experimental study. One hundred fifty milliliters of blood was donated from each of the 12 consecutive patients who underwent spine surgery and infused into 3 saline bags of 50 mL each. One of the 3 bags in each set contained 5,000 units of thrombin. All of them were connected to 120 ± 30 mmHg vacuum suctions: drainage was started 8 minutes after connection to the vacuum system for 12 normal blood bags (BV8) and 12 thrombin-containing blood bags (TBV8) and 15 minutes after connection for the remaining 12 normal blood bags (BV15). The amount of initial and remaining hematoma at 20 minutes, 120 minutes, and 24 hours after vacuum application were measured by their weight (g). The primary endpoint was the difference between BV8 and TBV8. The secondary end point was the difference between BV8 and BV15., Results: The remaining hematoma in TBV8 was significantly greater than that in BV8 at all measurement points: 46.3 ± 12.4 vs. 17.0 ± 1.3 ( p = 0.000) at 20 minutes; 33.0 ± 8.2 vs. 16.3 ± 1.2 ( p = 0.000) at 120 minutes; and 26.1 ± 4.0 vs. 15.8 ± 1.6 ( p = 0.000) at 24 hours after vacuum application. The remaining hematoma of BV15 was significantly greater than that of BV8 at all measurement points: 30.0 ± 12.0 vs. 17.0 ± 1.3 ( p = 0.002) at 20 minutes; 24.2 ± 7.6 vs. 16.3 ± 1.2 at 120 minutes ( p = 0.002); and 22.2 ± 6.6 vs. 15.8 ± 1.6 ( p = 0.004) at 24 hours after vacuum application., Conclusions: With a suction drain in place, the amount of remaining hematoma could be affected by coagulability. Thrombin-containing local hemostatics and the length of time elapsed before the commencement of suction resulted in hypercoagulability, indicating these two factors could be causes of POSEH., Competing Interests: No potential conflict of interest relevant to this article was reported.

Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0 %, in contrast to values of all variants were maintained at above 80 %. The thrombin-AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12 %, whereas that of tested variants was 37 %-56 %. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin-TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin-TM affinity-dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM-mediated anticoagulation.

Introduction: For a long time, vitamin K antagonists (VKA) have been the preferred drugs for the anticoagulation management of both atrial fibrillation and venous thromboembolism. Hereby, the major purpose is to attenuate the onset of thrombosis without affecting hemostasis. Areas covered: Nowadays, non-vitamin K anticoagulants (NOAC), a new class of oral anticoagulants is available for the above-mentioned indications. NOAC are at least as effective and safer with regard to intracranial bleedings compared to VKA, but major bleedings still occur. For this reason, the search for safer anticoagulants is still ongoing. Expert commentary: There are several unmet needs in NOAC management, including selection of optimal drug and dose, uncertainty on specific conditions and lack of drug persistence. There remains to be an important need for safer anticoagulants; 'upstream' anticoagulants including inhibitors of factor XIa may provide additional benefit related to fewer bleeding complications.

Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-α. Slightly lower levels of ADAMTS13 secretion were found with all treatments. This may explain the thrombogenicity of patients with inflammation where extremely high VWF levels and slightly lower ADAMTS13 levels are present., Competing Interests: The authors have declared that no competing interest exists.

Objective: Venous thromboembolism frequently results in thrombi formation near or within the pocket of a venous valve due to recirculating hemodynamics, which has been largely attributed to hypoxia-induced tissue factor (TF) expression. Numerical models are now capable of assessing the spatiotemporal behavior of the TF-initiated coagulation cascade under nonuniform hemodynamics. The aim of this study was to use such a numerical simulation to analyze the degree and location of thrombin formation with respect to TF position in the presence of disturbed flow induced by an open venous valve., Methods: Thrombin formation was simulated using a computational model that captures the hemodynamics, kinetics, and chemical transport of 22 biochemical species. Disturbed flow is described by the presence of a valve in the equilibrium phase of the valve cycle with leaflets in a fully open position. Three different positions of TF downstream of the valve opening were investigated., Results: The critical amount of TF required to initiate a thrombotic response is reduced by up to 80% when it is positioned underneath the recirculating regions near the valve opening. In addition, because of the increased surface area of the open valve cusp in conjunction with recirculating hemodynamics, it was observed that thrombin is generated inside the valve pocket even when the exposed region of TF is downstream of the valve., Conclusions: The presence of prothrombotic surface reactions in conjunction with recirculating hemodynamics provides an additional mechanism for thrombus formation in venous valves that does not require direct damage or dysfunction to the valve itself., (Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Transforming growth factor β (TGFβ) regulates cell proliferation, differentiation, migration, apoptosis, and extracellular matrix production. It also plays a pivotal role in the pathogenesis of gingival overgrowth. Thrombin is a key player in tissue repair, remodeling, and fibrosis after an injury, and it exerts profibrotic effects by activating protease-activated receptors. Connective tissue growth factor (CTGF or CCN2) modulates cell adhesion, migration, proliferation, matrix production, and wound healing. It is overexpressed in many fibrotic disorders, including gingival overgrowth, and it is positively associated with the degree of fibrosis in gingival overgrowth. In human gingival fibroblasts, we previously found that TGFβ1 induced CCN2 protein synthesis through c-jun N-terminal kinase and Smad3 activation. Thrombin stimulates CCN2 synthesis through protease-activated receptor 1 and c-jun N-terminal kinase signaling. Curcumin inhibited TGFβ1- and thrombin-induced CCN2 synthesis. In this study, we demonstrated that thrombin and protease-activated receptor 1 agonist SFLLRN induced latent TGFβ1 activation and Smad3 phosphorylation in human gingival fibroblasts. Pretreatment with a TGFβ-neutralizing antibody, TGFβ type I receptor inhibitor SB431542, and Smad3 inhibitor SIS3 inhibited approximately 86%, 94%, and 100% of thrombin-induced CCN2 synthesis, respectively. Furthermore, blocking integrin subunits αv and β1 with antibodies effectively inhibited SFLLRN-induced Smad3 phosphorylation and CCN2 synthesis and increased activated TGFβ1 levels; however, similar effects were not observed for integrins αvβ3 and αvβ5. These results suggest that protease-activated receptor 1-induced CCN2 synthesis in human gingival fibroblasts is mediated through integrin αvβ1-induced latent TGFβ1 activation and subsequent TGFβ1 signaling. Moreover, curcumin dose dependently decreased thrombin-induced activated TGFβ1 levels. Curcumin-inhibited thrombin-induced CCN2 synthesis in human gingival fibroblasts is caused by the suppression of latent TGFβ1 activation., (© International & American Associations for Dental Research 2016.)

In recent years, injuries induced by explosive blast have got more and more attention owing to weapon development and frequent terrorist activities. Tear, bleeding and edema of tissues and organs are the main manifestations of blast shock wave damage. Vascular endothelial barrier is the main defense of tissues and organs' integrity. This article aims to discuss possible mechanisms of endothelial barrier damage induced by explosive blast and main manifestations of blood brain barrier, bloodeair barrier, and intestinal vascular barrier impairments. In addition, the main regulatory factors of vascular permeability are also summarized so as to provide theoretical basis for prevention and cure of vascular endothelial barrier damage resulting from explosive blast.

Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts., (Copyright © 2016 the American Physiological Society.)

Platelet aggregation is an essential response to tissue injury and is associated with activation of pro-oxidant enzymes, such as cyclooxygenase, and is also a highly energetic process. The two central energetic pathways in the cell, glycolysis and mitochondrial oxidative phosphorylation, are susceptible to damage by reactive lipid species. Interestingly, how platelet metabolism is affected by the oxidative stress associated with aggregation is largely unexplored. To address this issue, we examined the response of human platelets to 4-hydroxynonenal (4-HNE), a reactive lipid species which is generated during thrombus formation and during oxidative stress. Elevated plasma 4-HNE has been associated with renal failure, septic shock and cardiopulmonary bypass surgery. In this study, we found that 4-HNE decreased thrombin stimulated platelet aggregation by approximately 60%. The metabolomics analysis demonstrated that underlying our previous observation of a stimulation of platelet energetics by thrombin glycolysis and TCA (Tricarboxylic acid) metabolites were increased. Next, we assessed the effect of both 4-HNE and alkyne HNE (A-HNE) on bioenergetics and targeted metabolomics, and found a stimulatory effect on glycolysis, associated with inhibition of bioenergetic parameters. In the presence of HNE and thrombin glycolysis was further stimulated but the levels of the TCA metabolites were markedly suppressed. Identification of proteins modified by A-HNE followed by click chemistry and mass spectrometry revealed essential targets in platelet activation including proteins involved in metabolism, adhesion, cytoskeletal reorganization, aggregation, vesicular transport, protein folding, antioxidant proteins, and small GTPases. In summary, the biological effects of 4-HNE can be more effectively explained in platelets by the integrated effects of the modification of an electrophile responsive proteome rather than the isolated effects of candidate proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In the sixties, the clotting cascade was proposed, which describes the coagulation process as a sequence of enzymatic events initiated by two different pathways, the intrinsic and the extrinsic pathways, converging on a common pathway, to generate a multifunctional enzyme, thrombin, whose main function is to convert fibrinogen into fibrin, a protein that polymerizes spontaneously to form the building block of a hemostatic clot. Later, it was proposed a cell-based model of the hemostasis according to that coagulation does not occur as a consequence of linear sequential enzyme activation pathways, but rather via a network of simultaneous interactions between plasmatic and transmembrane proteins, as well as several cellular types, that allow the formation of highly efficient enzymatic complexes that lead to thrombin generation. In this review, we summarize these two approaches highlighting the functions of thrombin within the hemostasis and the inhibition mechanisms that regulate the blood coagulation. Moreover, we described different tests that are used to assess the function of the coagulation system, such as: activated partial thromboplastin time, prothrombin time, thrombin time, reptilase time, ecarin clotting time, and the use of chromogenic substrates to evaluate individual coagulation factors. Finally, because of thrombin generation is a fundamental part of the blood coagulation and, an estimation of how well a particular individual can generate thrombin may correlate with either a risk of bleeding or thrombosis, we also include the existing methods to evaluate the potential of thrombin generation in an individual.

Background: Platelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated., Objective: To investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation., Methods and Results: Highly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins., Conclusions: Both GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Thrombin-mediated proteolysis is a major determinant of metastasis, but is not universally important for primary tumor growth. Here, we report that colorectal adenocarcinoma represents one important exception whereby thrombin-mediated functions support both primary tumor growth and metastasis. In contrast with studies of multiple nongastrointestinal cancers, we found that the growth of primary tumors formed by murine and human colon cancer cells was reduced in mice by genetic or pharmacologic reduction of circulating prothrombin. Reduced prothrombin expression was associated with lower mitotic indices and invasion of surrounding tissue. Mechanistic investigations revealed that thrombin-driven colonic adenocarcinoma growth relied upon at least two targets of thrombin-mediated proteolysis, protease-activated receptor-1 (PAR-1) expressed by stromal cells and the extracellular matrix protein, fibrinogen. Colonic adenocarcinoma growth was reduced in PAR-1-deficient mice, implicating stromal cell-associated PAR-1 as one thrombin target important for tumor outgrowth. Furthermore, tumor growth was dramatically impeded in fibrinogen-deficient mice, offering the first direct evidence of a critical functional role for fibrinogen in malignant tumor growth. Tumors harvested from fibrinogen-deficient mice displayed a relative reduction in cell proliferative indices, as well as increased tumor necrosis and decreased tumor vascular density. Collectively, our findings established a functional role for thrombin and its targets PAR-1 and fibrinogen in the pathogenesis of colonic adenocarcinoma, supporting tumor growth as well as local invasion and metastasis., (©2015 American Association for Cancer Research.)

Human ceruloplasmin (CP) is a multifunctional copper-binding protein produced in the liver. CP oxidizes Fe(2+) to Fe(3+), decreasing the concentration of Fe(2+) available for generating harmful oxidant species. CP is also a potent inhibitor of leukocyte myeloperoxidase (MPO) (Kd=130nM), a major source of oxidants in vivo. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting flexible joints and characterized by activation of both inflammatory and coagulation processes. Indeed, the levels of CP, MPO, and thrombin are markedly increased in the synovial fluid of RA patients. Here we show that thrombin cleaves CP in vitro at (481)Arg-Ser(482) and (887)Lys-Val(888) bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the MPO inhibitory function of CP is abrogated. Analysis of the synovial fluid of 24 RA patients reveals that CP is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with thrombin in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. Using independent biophysical techniques, we show that thrombin has intrinsic affinity for CP (Kd=60-270nM), independent of proteolysis, and inhibits CP ferroxidase activity (KI=220±20nM). Mapping of thrombin binding sites with specific exosite-directed ligands (i.e., hirugen, fibrinogen γ'-peptide) and thrombin analogues having the exosites variably compromised (i.e., prothrombin, prethrombin-2, βT-thrombin) reveals that the positively charged exosite-II of thrombin binds to the negatively charged upper region of CP, while the protease active site and exosite-I remain accessible. These results suggest that thrombin can exacerbate inflammation in RA by impairing the MPO inhibitory function of CP via proteolysis and by competitively inhibiting CP ferroxidase activity. Notably, local administration of hirudin, a highly potent and specifc thrombin inhibitor, reduces the concentration of active MPO in the synovial fluid of RA patients and has a beneficial effect on the clinical symptoms of the disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Traumatic-induced coagulopathy (TIC) is a hemostatic disorder that is associated with significant bleeding, transfusion requirements, morbidity and mortality. A disorder similar or analogous to TIC was reported around 70 years ago in patients with shock, hemorrhage, burns, cardiac arrest or undergoing major surgery, and the condition was referred to as a "severe bleeding tendency," "defibrination syndrome," "consumptive disorder," and later by surgeons treating US Vietnam combat casualties as a "diffuse oozing coagulopathy." In 1982, Moore's group termed it the "bloody vicious cycle," others "the lethal triad," and in 2003 Brohi and colleagues introduced "acute traumatic coagulopathy" (ATC). Since that time, early TIC has been cloaked in many names and acronyms, including a "fibrinolytic form of disseminated intravascular coagulopathy (DIC)." A global consensus on naming is urgently required to avoid confusion. In our view, TIC is a dynamic entity that evolves over time and no single hypothesis adequately explains the different manifestations of the coagulopathy. However, early TIC is not DIC because an increased thrombin-generating potential in vitro does not imply a clinically relevant thrombotic state in vivo as early TIC is characterized by excessive bleeding, not thrombosis. DIC with its diffuse anatomopathologic fibrin deposition appears to be a latter phase progression of TIC associated with unchecked inflammation and multiple organ dysfunction.

Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the extrinsic coagulation cascade, and its expression is reported to be relatively stable. Various pathophysiologic agents have been shown to influence TFPI activity by regulating its expression or by modifying the protein. It is not clear how TFPI activity is regulated in normal physiology or in injury. Because thrombin and TFPI are locally elaborated in pleural injury, we sought to determine if thrombin could regulate TFPI in human pleural mesothelial cells (HPMCs). Thrombin significantly decreased TFPI mRNA and protein levels by > 70%. Thrombin-mediated down-regulation of TFPI promoted factor X activation by HPMCs. The ability of thrombin to significantly decrease TFPI mRNA and protein levels was maintained at nanomolar concentrations. Protease-activated receptor (PAR)-1, a mediator of thrombin signaling, is detectable in the mesothelium in human and murine pleural injury. PAR-1 silencing blocked thrombin-mediated decrements of TFPI in HPMCs. Thrombin activates PI3K/Akt and nuclear factor κB (NF-κB) signaling in HPMCs. Inhibition of PI3K (by PX-866) and NF-κB (by SN50) prevented thrombin-mediated TFPI mRNA and protein down-regulation. These are the first studies to demonstrate that thrombin decreases TFPI expression in HPMCs. Our findings demonstrate a novel mechanism by which thrombin regulates TFPI expression in HPMCs and promotes an unrestricted procoagulant response, and suggest that interactions between PI3K and NF-κB signaling pathways are linked in HPMCs and control TFPI expression. These findings raise the possibility that targeting this pathway could limit the ability of the mesothelium to support extravascular fibrin deposition and organization associated with pleural injury.

The role of thrombin in vascular physiology and pathophysiology continues to impact our understanding of many cellular processes and systems including the function of platelets, endothelial cells, smooth muscle cells, leukocytes and the regulation of the coagulation cascade. Recent acute coronary syndrome clinical trial results that have compared the use of parenteral or oral anticoagulants versus or in combination with anti-platelet agents have forced a reexamination of the importance of thrombin activity in influencing patient outcomes, particularly in the area of secondary prevention. The debate of the need to include oral anticoagulation as a concomitant or replacement therapy to an antiplatelet regimen as a means to improve patient outcomes requires further examination and larger prospective clinical trials., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aim: Thrombus formation is a dynamic process regulated by flow, blood cells, and plasma proteins. The present study was performed to investigate the characteristics of human coronary thrombus in ST-segment elevation myocardial infarction (STEMI)., Methods and Results: Patients admitted with ST-elevation myocardial infarction, in which thrombectomy was performed, were included (n = 86). Intracoronary thrombi and blood from the culprit coronary site and the systemic circulation were obtained during percutaneous coronary intervention (PCI). Thrombi were categorized by onset-of-pain-to-PCI elapsed time in thrombus of <3 (T3) and more than 6 h of evolution (T6). Clinical, morphological, and proteomic variables were investigated. While T3 were mainly composed by platelets and fibrin(ogen), T6 were characterized by a reduced platelet content, increased leucocytes infiltration (including monocytes, neutrophils, T-cells, and B-cells), and appearance of undifferentiated progenitor cells. Significant differences between T3 and T6 were found in the cell cytoskeleton-associated proteome (beta-actin and tropomyosin 3 and 4). By discovery proteomics, we have identified profilin-1 (Pfn-1) in the coronary thrombi and detected higher levels in T3 than in T6. While plasma Pfn-1 levels were low in T3 patients, levels significantly increased in both coronary and peripheral circulation in T6 patients indicating release. In vitro platelet aggregation studies showed that platelets secrete Pfn-1 upon complete activation., Conclusion: Coronary thrombi show rapid dynamic changes both in structure and cell composition as a function of elapsed onset-of-pain-to-PCI time. Aged ischaemic thrombi were more likely to have reduced Pfn-1 content releasing Pfn-1 to the circulation. Onset-of-pain-to-PCI elapsed time in STEMI patients and hence age of occlusive thrombus can be profiled by Pfn-1 levels found in the peripheral circulation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Metabolic acidosis has been implicated in the development of coagulopathy, although the specific mechanisms have not been well characterized. We sought to explore whether resuscitation of injured patients with a balanced crystalloid solution affects coagulation, as measured by endogenous thrombin potential (ETP) and thromboelastography (TEG). We performed an exploratory analysis of a subset of subjects enrolled in a randomized trial comparing the effect of resuscitation with isotonic saline versus Plasma-Lyte A (PLA) on acidosis and electrolyte abnormalities. We collected plasma at admission and 6 h later for subsequent ETP and TEG analysis and compared subjects receiving isotonic saline to those receiving PLA. Among 18 evaluated subjects, baseline characteristics, including ETP and TEG parameters, were similar between the two arms. At 6 h, subjects receiving isotonic saline were more acidemic. At 6 h, there were no differences in ETP parameters between groups; however, TEG results showed the time from initial clot formation to an amplitude of 20 mm (K) was shorter (3.8 ± 2.1 vs. 7.2 ± 2.8 s) and the rapidity of fibrin build-up and cross-linking (α angle) was significantly greater (41 ± 8 vs. 24 ± 15 deg) for the PLA group than in the isotonic saline group. Relative to PLA, isotonic saline does not alter thrombin generation, but isotonic saline and PLA may differentially impact clotting factor availability. The shorter time to reach prespecified clot amplitude and the increased rate of fibrin generation imply faster amplification of clotting factors with PLA without effect on latency time or clot strength.

Purpose: We previously reported that extracellular matrix composition (fibrin versus collagen) modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study, we investigate the role of thrombin and cell contractility in mediating these differences in cell behavior., Methods: To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs were cultured in serum-free media containing platelet-derived growth factor (PDGF), with or without thrombin, the Rho kinase inhibitor Y-27632, and/or the myosin II inhibitor blebbistatin. We used 3-dimensional and 4-dimensional imaging to assess cell mechanical behavior, connectivity and cytoskeletal organization., Results: Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also induced Rho kinase-dependent clustering of cells plated on top of compliant collagen matrices, but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when Rho kinase was inhibited. In nested matrices, cells always migrated independently through collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but cells continued to migrate collectively., Conclusions: The results suggest that while thrombin-induced actomyosin contraction can induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is not required for clustering or collective migration of corneal fibroblasts interacting with fibin., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Animal studies testing whether therapeutic hypothermia is neuroprotective after intracerebral hemorrhage (ICH) have been inconclusive. In rodents, ICH is often produced in the striatum by infusing collagenase, which causes prolonged hemorrhaging from multiple vessels. Our previous data shows that this bleeding (hematoma) is worsened by systemic hypothermia given soon after collagenase infusion. In this study we hypothesized that localized brain hypothermia would also aggravate bleeding in this model (0.2 U of collagenase in 1.2 μL of saline). We also evaluated cooling after intrastriatal thrombin infusion (1 U in 30 μL of saline)-a simplified model of ICH thought to cause bleeding. Focal hypothermia was achieved by flushing cold water through an implanted cooling device attached to the skull underneath the temporalis muscle of adult rats. Previous work and data at this time shows this method cools the striatum to ∼33°C, whereas the body remains normothermic. In comparison to normothermic groups, cooling significantly worsened bleeding when instituted at 6 hours (∼94 vs. 42 μL, p=0.018) and 12 hours (79 vs. 61 μL, p=0.042) post-ICH (24-hour survival), but not after a 24-hour delay (36-hour survival). Rats were cooled until euthanasia when hematoma size was determined by a hemoglobin-based spectrophotometry assay. Cooling did not influence cerebral blood volume after just saline or thrombin infusion. The latter is explained by the fact that thrombin did not cause bleeding beyond that caused by saline infusion. In summary, local hypothermia significantly aggravates bleeding many hours after collagenase infusion suggesting that bleeding may have confounded earlier studies with hypothermia. Furthermore, these findings serve as a cautionary note on using cooling even many hours after cerebral bleeding.

Context: Although it is recognized that thrombin plays a key role in airway remodeling during chronic asthma. In a previous study, we have proved that thrombin promotes airway remodeling via PAR-1 in OVA-allergic rats, but little is known about intracellular signaling pathway involved in the event., Objective: In this study, we intend to explore the impact of pERK1/2 signaling pathway on the process of thrombin-induced airway remodeling in OVA-allergic rats., Materials and Methods: A rat model of chronic asthma was set up by systemic sensitization and repeated challenge to OVA. The doses of thrombin, recombinant hirudin, PAR-1 inhibitor ER-112780-06, and pERK1/2 inhibitor PD98059 varied for different groups. The expression of pERK1/2 was analyzed by western blot and RT-PCR. Secretion of TGF-β1 and IL-6 was detected by ELISA., Results: The expression of pERK1/2 was higher in the airway of asthmatic rats than those of normal rats, and was significantly increased by thrombin treatment but decreased by thrombin-inhibitor treatment. Airway remodeling was enhanced by thrombin but weakened by pERK1/2 inhibitor. Expression of growth factors and IL-6 in asthmatic rats was significantly increased by thrombin treatment and decreased by thrombin-inhibitor treatment and pERK1/2 inhibitor treatment., Conclusion: These results suggest that ERK1/2 signaling pathway may play an important role in the process of thrombin-promoting airway remodeling in OVA-allergic rats, and pERK1/2 inhibitor effectively inhibits the process.

Hodgkin's lymphoma is believed to spread in an orderly fashion within the lymphatic compartment. In a minority of cases, after reaching the spleen, the neoplasm disseminates, reminiscent of metastasis. In the spleen, the Hodgkin-Reed-Sternberg tumor cells come across platelets in the blood vessels and mainly in the splenic red pulp. Based on this knowledge, we investigated the possibility of platelets inducing cell adhesion in Hodgkin's lymphoma cell lines. We showed that L428 and KMH-2 cells strongly adhere to thrombin-activated platelets. Cell adhesion to platelets is partially dependent on CD15 antigens (Lewis(X)), mainly sialyl-CD15, and P-selectin. KMH-2, as compared to L428 cells, showed increased binding due to its differential high expression of the sialyl-CD15. As a consequence of incubation with platelets, KMH-2 cells also produced increased amounts of tumor necrosis factors α (TNFα) followed by enhanced binding to human vascular endothelial cells (HUVEC). Incubation of both cell lines with activated platelets also induced activation of AP-1 transcription complex. Our findings are consistent with the concept that platelets play a critical role in the dissemination of HRS cells in HL, predominantly in the spleen, by increasing cell adhesion and thus promoting their proliferative and migratory properties beyond the lymphatic system.

Despite years of research and efforts to translate stroke research to clinical therapy, ischaemic stroke remains a major cause of death, disability, and diminished quality of life. Primary and secondary preventive measures combined with improved quality of care have made significant progress. However, no novel drug for ischaemic stroke therapy has been approved in the past decade. Numerous studies have shown beneficial effects of activated protein C (APC) in rodent stroke models. In addition to its natural anticoagulant functions, APC conveys multiple direct cytoprotective effects on many different cell types that involve multiple receptors including protease activated receptor (PAR) 1, PAR3, and the endothelial protein C receptor (EPCR). Application of molecular engineered APC variants with altered selectivity profiles to rodent stroke models demonstrated that the beneficial effects of APC primarily require its cytoprotective activities but not its anticoagulant activities. Extensive basic, preclinical, and clinical research provided a compelling rationale based on strong evidence for translation of APC therapy that has led to the clinical development of the cytoprotective-selective APC variant, 3K3A-APC, for ischaemic stroke. Recent identification of non-canonical PAR1 and PAR3 activation by APC that give rise to novel tethered-ligands capable of inducing biased cytoprotective signalling as opposed to the canonical signalling provides a mechanistic explanation for how APC-mediated PAR activation can selectively induce cytoprotective signalling pathways. Collectively, these paradigm-shifting discoveries provide detailed insights into the receptor targets and the molecular mechanisms for neuroprotection by cytoprotective-selective 3K3A-APC, which is currently a biologic drug in clinical trials for ischaemic stroke.

Acute coronary syndrome (ACS) is a medical emergency. Patients who survive the initial event remain at risk of recurrent cardiovascular events. In most cases, ACS is triggered by thrombosis after rupture of an atherosclerotic plaque. Key to thrombus formation at this site is the generation of thrombin, which not only converts fibrinogen to fibrin but also serves as a potent platelet agonist and induces platelet aggregation at the site of vascular injury. Although dual antiplatelet therapy is more effective for the prevention of recurrent events than aspirin alone after ACS, there remains an approximately 10 % risk of recurrent ischaemic events at one year. Recent studies have evaluated whether the addition of an anticoagulant to antiplatelet therapy reduces the risk of recurrent ischaemia after an ACS event. Rivaroxaban, an oral factor Xa inhibitor, attenuates thrombin generation. When used in conjunction with dual antiplatelet therapy in patients with stabilised ACS, rivaroxaban 2.5 mg twice daily significantly reduced the risk of the composite endpoint of cardiovascular death, myocardial infarction and stroke compared with placebo. Although it increased the risk of bleeding, rivaroxaban was associated with a reduction in mortality; a finding that supports the use of a dual-pathway approach that combines anticoagulant and antiplatelet therapy. This review explores the pathophysiology of ACS to provide perspective on the results of recent clinical trials with novel oral anticoagulants for ACS and to identify their potential role in this setting.