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The retinal pigment epithelium (RPE) plays an essential role in the survival and function of the neural retina. RPE uncontrolled proliferation leads to the development of proliferative ocular pathologies, among which proliferative vitreoretinopathy (PVR) is the main cause of retinal surgery failure. Upon the breakdown of the BRB due to trauma or metabolic imbalance the contact of RPE with serum-contained thrombin has been shown to stimulate the proliferation of otherwise quiescent RPE cells. Although the molecular mechanisms involved in this effect are still undetermined, thrombin proteolytic activation of protease-activated G protein coupled receptor-1 (PAR-1) activates PI3K and Akt, known to play an essential role in proliferation. The present study demonstrates that: 1) thrombin stimulates Ser 473 Akt phosphorylation without affecting Thr 308 basal phosphorylation in RPE cells; 2) thrombin-induced Akt stimulation promotes cyclin D1 accumulation through the phosphorylation/ inhibition of GSK-3β, thus preventing Thr 286 cyclin D1 phosphorylation, nuclear export and degradation; 3) Akt signaling requires the upstream activation of PI3K and PLC. Since the pharmacological inhibition of these pathways or the silencing of cyclin expression prevent thrombin-induced RPE cell proliferation, these results contribute relevant evidence for establishing the mechanism involved in the development of proliferative eye diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The development of hepatic fibrosis on a background of chronic liver injury represents a complex disease trait modulated through the interaction of host genetic factors and environmental influences. Early observations that hepatic inflammation and cirrhosis are associated with the presence of microthrombi within the hepatic vasculature and fibrin/fibrinogen deposition were followed by epidemiological studies showing that carriage of the Factor V Leiden (FvL) mutation, protein C deficiency and increased expression of factor VIII are associated with accelerated progression to cirrhosis in a chronic hepatitis C infection. Additional data suggest that these factors may influence fibrogenesis in many forms of chronic liver disease and extra-hepatic fibrotic processes. Drawing evidence both from liver research and studies of fibrogenesis in other organ systems, two hypotheses may explain how activity of the coagulation cascade influences the rate of hepatic fibrogenesis: tissue ischaemia and parenchymal extinction and direct thrombin mediated stellate cell activation via PAR-1 cleavage. Drawing on preclinical and clinical studies we discuss the evidence for a role for coagulation cascade activity in hepatic fibrogenesis and explore the proposed pathogenic mechanisms that lead to stellate cell activation. The corollary of an association between hypercoagulation and increased fibrosis is that interference with the coagulation cascade may reduce hepatic fibrosis. We conclude this article by examining the implications for future therapeutic intervention., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

It is well known that the expression level of secretory group IIA phospholipase A(2) (sPLA(2)-IIA) is elevated in inflammatory diseases and lipopolysaccharide (LPS) up-regulates the expression of sPLA(2)-IIA in human umbilical vein endothelial cells (HUVECs). Recently, lower concentration thrombin could elicit anti-inflammatory responses in HUVECs. Here, the effects of lower concentration thrombin on the expression of sPLA(2)-IIA in LPS-stimulated HUVECs were investigated. Prior treatment of cells with thrombin (25-75 pM) inhibited LPS-induced sPLA(2)-IIA expression by activating its receptor, protease-activated receptor-1 (PAR-1). And pretreatment of cells with either PI3-kinase inhibitor (LY294002) or cholesterol depleting agent (methyl-β-cyclodextrin, MβCD) abolished the inhibitory activity of thrombin against sPLA(2)-IIA expression. Therefore, these results suggest that PAR-1 activation by lower concentration thrombin inhibited LPS mediated expression of sPLA(2)-IIA by PAR-1 and PI3-kinase-dependent manner in lipid raft on the HUVECs., (Copyright © 2011 Wiley-Liss, Inc.)

Thrombin has been shown to increase expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the development of atherosclerosis. However, the precise mechanism of this induction remains unknown. In the present study, we investigated whether the small G protein RhoA, and its effector, Rho-kinase are involved in MCP-1 induction by thrombin in endothelial cells. Y-27632, a specific Rho-kinase inhibitor, potently inhibited MCP-1 induction by thrombin. Y-27632 significantly decreased the chemotactic activity of thrombin-stimulated supernatants of endothelial cells on monocytes. Importantly, fasudil, a specific Rho-kinase inhibitor, attenuated MCP-1 gene expression in the aorta of db/db mice. Y-27632 attenuated thrombin-mediated phosphorylation of p38MAPK and p65, indicating that Rho-kinase mediates thrombin-induced MCP-1 expression through p38MAPK and NF-κB activation. Our findings demonstrate that the Rho/Rho-kinase signaling pathway plays a critical role in thrombin-mediated MCP-1 expression and function, and suggest that Rho/Rho-kinase may be an important target in the development of new therapeutic strategies for atherosclerosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose of Review: Activation of the coagulation system during ischemia/reperfusion injury is an unavoidable event and even further augmented during cardiovascular surgery. Clotting not only leads to disturbance of blood rheology but also enhances the inflammatory response. We aim to highlight the inflammatory properties of the coagulation system and novel potential therapeutic approaches targeting both features., Recent Findings: Heparin, a thrombin inhibitor, is still the drug of choice for preventing coagulation following, for example, cardiovascular surgery. On the contrary, much effort is done to evaluate the utilization of direct thrombin inhibitors to prevent ischemia/reperfusion injury. Furthermore, targeting the inflammatory potential of the coagulation system seems to be very promising. Fibrin(ogen) and its degradation products modulate the inflammatory response, especially by inducing leukocyte migration. Inhibiting these pro-inflammatory effects, for example, by administration of Bβ15-42 was recently shown to be beneficial under various inflammatory conditions., Summary: Ischemia and reperfusion are common activators of coagulation that is also accompanied by inflammation. Therefore, targeting this well orchestrated system might be of therapeutic benefit, as its mode of action is dual: clotting inhibition and anti-inflammation. This novel therapeutic approach might at least be of benefit in the treatment of systemic inflammatory syndromes following, that is, cardiovascular surgery.

Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.

The discovery of circulating angiogenic cells (CAC) with cardiovascular regenerative potential has transformed our understanding of the health and maintenance of cardiovascular tissues. We will describe the influence of acute exercise and exercise training on CAC characteristics and evidence for mechanisms that may be important in the optimization of CAC potential.

Invasion of the decidua by extravillous trophoblasts (EVTs) is accompanied by thrombin generation from decidual cell (DC)-expressed tissue factor (TF). This TF protects against hemorrhage as EVTs breach capillaries and subsequently invade and remodel spiral arteries and arterioles. Pre-eclampsia (P-EC) is the world's leading cause of fetal and maternal morbidity and mortality. It is associated with decidual hemorrhage and maternal thrombophilias, which form excess thrombin from DCs, and with maternal infections and other inflammatory conditions that are associated with excess expression of the proinflammatory cytokines interleukin (IL)-1 β and tumor necrosis factor (TNF) α. In human first-trimester leukocyte-free DCs, (1) thrombin enhances expression of soluble fms-like tyrosine kinase-1 (sFlt-1), a potent inhibitor of angiogenesis; (2) thrombin, IL-1β and TNF-α increase monocyte-recruiting chemokine expression leading to a macrophage excess in the pre-eclamptic decidua. The pathogenesis of P-EC likely stems from shallow EVT invasion leading to impaired decidual vascular remodeling. The resulting reduced uteroplacental blood flow is associated with a hypoxic placenta, which appears to secrete excess sFlt-1 into the maternal plasma. A regulatory role for DCs in vascular remodeling is indicated because impaired decidual vascular remodeling could stem from an aberrant local antiangiogenic milieu elicited by excess sFlt-1 and/or macrophage-inhibited EVT decidual invasion., (© Thieme Medical Publishers.)

Abruption-induced thrombin generation and inflammation/infection induced cytokine production have both been associated with fetal membrane (FM) weakening and preterm premature rupture of the fetal membranes (PPROM). Using our in vitro model system we have demonstrated that thrombin, and separately the cytokines, tumor necrosis factor-alpha (TNFα) and interleukin-1-beta (IL-1β), remodel and weaken full thickness FM. Additionally, we have reported that the anti-oxidant and NFκB inhibitor, alpha-lipoic acid (LA), blocks these thrombin and cytokine induced effects. The purpose of these studies was to determine whether thrombin and cytokines directly weaken the amnion membrane (AM), the major load-bearing component of FM. Isolated AM or full thickness FM fragments from unlabored Cesarean deliveries were incubated with thrombin, TNFα, or IL-1β, for 48 h. Rupture strength (breaking force) of each fragment was thereafter determined using our published methodology. Biochemical evidence of remodeling and apoptosis; immunoreactive Matrix Metalloproteinase 9 (MMP9), Tissue Inhibitor of Matrix Metalloproteinase 3 (TIMP3) and cleaved poly (ADP-ribose) polymerase (C-PARP) levels in tissue extracts, were determined by western blot and densitometry. Thrombin induced a dose-dependent weakening of isolated AM (P < 0.001) coupled with dose dependent increases in PARP cleavage, and reciprocal increases and decreases, respectively, in MMP9 and TIMP3 protein (all P < 0.01). Thrombin receptor activating peptide-6 (TRAP) also weakened isolated AM. Neither TNFα nor IL-1β weakened isolated AM. However, both cytokines weakened AM when it was incubated together with the choriodecidua as part of full thickness FM (P < 0.001). Cytokine-conditioned choriodecidua medium also weakened isolated AM (P < 0.001). Under conditions in which cytokines weakened the AM, the changes in MMP9, TIMP3 and PARP cleavage were consistent with those seen after thrombin incubation. LA blocked the FM weakening and remodeling effects. In summary, thrombin weakens AM directly whereas cytokines weaken AM indirectly by causing the release of soluble intermediates from the choriodecidua., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Atrial fibrillation (AF) is the most common cardiac rhythm disorder and a major risk factor for ischemic stroke. Antithrombotic therapy using aspirin or vitamin K antagonists (VKA) is currently prescribed for prevention for ischemic stroke in patients with AF. A narrow therapeutic range and the need of regular monitoring of its anticoagulatory effect impair effectiveness and safety of VKA, causing a need for alternative anticoagulant drugs. Recently developed anticoagulants include direct thrombin antagonists such as dabigatran or factor Xa inhibitors such as rivaroxaban, apixaban, betrixaban, and edoxaban. Currently, data from a phase III clinical trial are available for dabigatran only, which show the direct thrombin antagonist to be at least noninferior in efficacy to VKA for the prevention of stroke and systemic embolism in patients with AF. This review focuses on current advances in the development of directly acting oral anticoagulant drugs and their potential to replace the VKA class of drugs in patients with AF., (Copyright © 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Unlabelled: We previously identified osteopontin (OPN) as a promoter and thus a potential therapeutic target for hepatocellular carcinoma (HCC) metastasis. The serine protease thrombin interacts with OPN and can modify its biological activity. To explore the role of thrombin alone or in conjunction with OPN in HCC, we studied the correlation of thrombin levels to HCC prognosis in patients with various OPN levels, and evaluated the effects of OPN fragments generated by thrombin cleavage on proliferation and adhesion of HCC cells. We found that the thrombin level was strongly associated with the metastatic potential of HCC cell lines, and that thrombin was remarkably overexpressed in HCC tissue compared with adjacent nontumor tissue. In addition, HCC tissue from patients with recurrent disease displayed much higher thrombin levels, particularly in those with elevated OPN levels. Only HCCs with elevated OPN levels had a significant correlation between high thrombin levels and overall survival (OS; P < 0.01), or time to recurrence (TTR; P < 0.0001) of HCC. Multivariate analysis revealed that thrombin was an independent prognostic indicator. In vitro assays demonstrated that thrombin promotes the proliferation and adhesion of OPN+ HCC cells. Furthermore, thrombin activated the focal adhesion kinase (FAK) pathway of OPN+ HCC cells, which was blocked by the inhibition of integrin β1., Conclusion: Thrombin plays an important role in OPN-mediated aggressive phenotype of HCC through activation of integrin β1-FAK signaling, and is an independent poor prognostic factor for HCC. Thus, thrombin may be a potential therapeutic target to inhibit HCC metastasis in OPN+ patients., (Copyright © 2010 American Association for the Study of Liver Diseases.)

Background: We recently demonstrated in an experimental model the expression of tissue factor (TF) in aortic valves. Thrombin, generated at the end of the TF-initiated coagulation cascade, has been shown to cleave the anti-calcific osteopontin (OSP) liberating the pro-inflammatory OSP N-half., Objectives: We hypothesized that TF might play an important role in calcific aortic valve stenosis (AS) through thrombin generation and hence evaluated the valvular expression of TF and its inhibitor (TF pathway inhibitor), α-thrombin, OSP and its thrombin-cleaved form (OSP N-half)., Methods: Calcified aortic valves were obtained from patients undergoing valve replacement. Protein expression was evaluated by immunostaining and measured using ELISA kits. Transcripts were analyzed by RT-PCR., Results: We included 52 patients (31 men; age 70 ± 10 years; aortic valve area index 0.35 ± 0.13 cm(2)/m(2)). Immunohistochemistry revealed that TF, OSP and α-thrombin expressions were associated with calcifications at the aortic side of the leaflets. There was an overexpression in calcified regions as compared to non-calcified zones of TF (733.3 ± 70.5 pg/mg vs. 429.4 ± 73.2 pg/mg; p<0.0001), OSP (88.9 ± 12.7 ng/mg vs. 15.0 ± 3.3 ng/mg; p<0.0001) and OSP N-half (0.41 ± 0.06 pmol/mg vs. 0.056 ± 0.011 pmol/mg; p<0.0001). Additionally, both TF and α-thrombin expressions were associated with OSP N-half (r=0.52; p<0.0001 and r=0.33; p=0.019, respectively)., Conclusions: Aortic leaflet TF and α-thrombin expressions and their association with the thrombin-cleaved form of OSP, are a new and important feature of AS. We hypothesize that TF may be involved in the mineralization process of aortic valves by enhancing the generation of the pro-inflammatory OSP N-half through thrombin induction. This pathway deserves further studies to address its implication in the aortic valve calcification process., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Monocytes and monocyte-derived microparticles (MMPs) play a major role in acute coronary syndrome (ASC). Activated monocytes (ac-M) and MMPs support thrombin generation via tissue factor (TF). The aim of this study was to evaluate the inhibitory effect of fondaparinux, a selective Xa inhibitor, on thrombin generation supported by activated monocytes and MMPs. Monocytes were purified by elutriation. They were activated by LPS, allowing to obtain both ac-M and MMPs. Thrombin generation was performed using Fluoroscan(®) in these two cell models, in comparison with a cell-free model (TF 5 pM final). Two concentrations of ac-M (0.2 × 10⁶ and 1 × 10⁶/well) and four concentrations of MMPs (40,000; 80,000; 120,000 and 160,000/well) were tested. TGT was evaluated for increasing fondaparinux concentrations (0, 0.1, 0.4, 0.7 and 1.2 μg/ml). Without fondaparinux, 0.2 × 10⁶ ac-M and 160,000 MMPs induced comparable results. Fondaparinux inhibited thrombin generation in the three models. Inhibition was fondaparinux concentration dependent. Rate index was the most sensitive parameter, compared to lag-time, peak and endogenous thrombin potential. The rate index IC(50) were 0.69 ± 0.03 μg/ml for ac-M, 0.20 ± 0.03 μg/ml for MMPs, and 0.22 ± 0.02 μg/ml for cell-free model. Fondaparinux exerted an inhibitory effect at all concentrations, including the lowest (0.1 μg/ml). The extend of inhibition was similar between MMPs and cell-free models, and stronger than ac-M model. We assume that the efficacy of fondaparinux 2.5 mg once daily in ACS patients may be in part attributed to its inhibitory effect on MMPs.

Despite the availability of dual antiplatelet therapy comprised of aspirin and clopidogrel, there is still significant unmet medical need for treating and preventing arterial thrombotic diseases. To achieve further reduction of cardiovascular events without exceeding bleeding tolerability and safety limits, novel antiplatelet strategies might need to trade in antiplatelet efficacy by partial inhibition of an important platelet activation pathway or by differentially targeting pathological versus physiological thrombogenesis pathways. Thrombin, the central enzyme in coagulation and the most potent platelet agonist tested in vitro, is one of the key factors driving the formation of occlusive thrombi. Platelet thrombin receptors, namely protease-activated receptor 1 (PAR-1) and protease-activated receptor 4 (PAR-4), act in concert to elicit robust platelet responses to thrombin. PAR-1 is the high affinity thrombin receptor and represents a novel antithrombotic target. PAR-4 is a low affinity thrombin receptor with less understood function. This review discusses the genetic and pharmacological evidence for PAR-1 target validation and highlights the progresses and challenges in developing oral PAR-1 antagonists, especially SCH 530348 from Merck/Schering-Plough and E-5555 from Eisai Co. Recent patents disclosing several novel chemical series of PAR-1 antagonists from Sanofi-Aventis and Pierre Fabre are also presented.

Background: In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We hypothesized that protracted intravenous infusion of thrombin at steady state will allow to study isolated thrombin effects in vivo., Methods: Thrombin (0.05-0.9U/kg/min) was continuously infused in Sprague Dawley rats over five hours (n=38). The study consisted of three parts: dose escalation (n=21), dose verification (n=5) and a parallel group study to investigate whether thrombin effects can be antagonised by concomitant infusion of lepirudin (n=12)., Results: A thrombin dose of 0.9U/kg/min decreased platelet counts by 70% compared to the control group (median 230×10^9/L vs. 752×10^9/L; p=0.041). In accordance, infusion of 0.9U/kg/min of thrombin decreased fibrinogen level by 75% compared to the control group (56mg/dl vs. 220mg/dl; p=0.046). Cumulative thrombin doses of ≥0.1U/kg/min caused bleedings but not thromboembolic events. Thrombin at doses ≥0.15U/kg/min was lethal in four cases (30%). Platelet counts and fibrinogen levels after thrombin infusion correlated with bleeding events and mortality. Administration of thrombin at cumulative doses of 0.3-0.9U/kg/min was associated with a 3 to 6.5 -fold increase in IL-6 levels (139-306pg/ml vs. 47pg/ml, p<0.05). In contrast, thrombin infusion did not alter other markers of inflammation (IL-10, MCP-1 or TNF-alpha). In addition, lepirudin prevented thrombin- induced thrombocytopenia., Conclusion: Protracted intravenous infusion of thrombin offers a new experimental model, where consumption of fibrinogen and platelets correlates with bleedings and mortality. Infusion of thrombin increased only IL-6 levels but not other cytokines., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cytokine-mediated inflammation and abruption-induced thrombin generation are separately implicated in matrix metalloproteinase (MMP)-mediated weakening of fetal membranes (FM) leading to preterm premature rupture of the fetal membranes (PPROM). At term, FM of both labored vaginal and unlabored Cesarean deliveries exhibit a weak zone overlying the cervix exhibiting ECM remodeling characterized by increased MMP9 protein and activity. We have reproduced these biochemical changes as well as FM weakening in vitro using tumor necrosis factor-alpha (TNF) and interleukin (IL)-1β, inflammatory cytokines implicated in PPROM. Additionally, we have reported that the antioxidant and NFκB inhibitor alpha-lipoic Acid (LA) blocks these TNF-induced effects. We now present the first direct evidence that thrombin also can induce FM weakening in vitro, and LA treatment inhibits this thrombin-induced-weakening. Full thickness FM fragments from unlabored Cesarean deliveries were incubated with increasing doses of thrombin (0-100 u/ml) for 48 h. Fragments were then strength tested (breaking force and work to rupture) using our published methodology. MMP3 and 9 levels in tissue extracts were determined by Western blot and densitometry. To determine the effect of LA, FM fragments were incubated with control medium or 10 u/ml thrombin, with or without 0.25 mM LA. Strength testing and MMP induction were determined. Thrombin induced a dose-dependent decrease in FM strength (42% baseline rupture force and 45% work to rupture) coupled with a dose-dependent increase in MMP3 and 9 expression (all p < 0.001). Treatment of FM with 0.25 mM LA completely inhibited thrombin-induced FM weakening and MMP expression (all p < 0.001). Thrombin treatment of cultured FM induces mechanical weakening and increased MMP3 and 9. Treatment of FM with LA inhibits these thrombin-induced effects. We speculate LA may prove clinically useful in prevention of PPROM associated with abruption., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background and Purpose: Activation of human platelets by thrombin is mediated predominately through two proteinase-activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3-kinase (PI3K) inhibition leads to reversible PAR1-mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated., Experimental Approach: The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC-1. Western blotting and fluo-3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca(2+) mobilization respectively., Key Results: When PAR4 function was inhibited either by the PAR4 antagonist YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin-elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD-3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin-stimulated platelets. The aggregation-reversing activity mainly resulted from inhibition of both PI3K-dependent PKC activation and PAR4-mediated sustained intracellular Ca(2+) rises. Blockade of ADP P2Y(12) receptor with 2-methylthioadenosine 5'-monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K-dependent PKC activation and its ability to reverse PAR1-activating peptide-induced platelet aggregation. Co-administration of 2-methylthioadenosine 5'-monophosphate triethylammonium salt with YD-3 also decreased the stability of thrombin-induced platelet aggregation., Conclusions and Implications: These results suggest that PAR4 acts in parallel with the P2Y(12)/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.

Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca(2+)-permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca(2+) influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca(2+) influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH(2)-terminal kinase, and nuclear factor-κB signaling and required de novo protein synthesis. The Ca(2+) signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca(2+) signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca(2+)-signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH(2), a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own expression.

Thrombogenesis depends on biochemical reactions affected by blood flow dynamics. While mathematical models of mural thrombogenesis provide a means of understanding how blood flow affects thrombus growth, comparisons to experimental data are needed to validate the models and enable prediction of thrombus growth under diverse conditions. In this paper, we present mathematical models of mural thrombogenesis under flow and validation of the models with experimental data collected from a thrombogenic vascular graft segment. The grafts were placed in exteriorized high-flow arteriovenous (AV) shunts in baboons. Radiolabeled platelet deposition onto the thrombogenic segment, a marker of thrombus size, and plasma thrombin-antithrombin (TAT) concentration downstream of the graft, a marker of local thrombin generation, were monitored over time. The mathematical model of mural thrombogenesis consisted of transport-reaction equations in which platelets and thrombin were explicitly considered. We found that the transport-reaction model captured the order of magnitude of TAT sampled levels, while calculated rates of platelet deposition agreed well with radioimaging results. Analysis of experimental and modeling data indicates that, at least during part of thrombus growth progression, thrombin generation is in excess and platelet adhesion rates would be sustained even at lower local thrombin concentrations.

Abstract Endothelin-1 (ET-1) appears to be involved in drug-induced proliferation of gingival fibroblasts. Thrombin induces proliferation of human gingival fibroblasts via protease-activated receptor 1 (PAR1). In this study, using cultured rat gingival fibroblasts, we investigated whether thrombin-induced proliferation of gingival fibroblasts is mediated by ET-1. Thrombin-induced proliferation (0.05-2.5 U/mL). Proliferation was also induced by a PAR1-specific agonist (TFLLR-NH(2,) 0.1-30 microm), but not by a PAR2-specific agonist (SLIGRL-NH(2)). Thrombin (2.5 U/mL) induced an increase in immunoreactive ET-1 expression, which was inhibited by cycloheximide (10 microg/mL), and an increase in preproET-1 mRNA expression, as assessed by reverse transcription polymerase chain reaction. TFLLR-NH(2) increased ET-1 release into the culture medium in both a concentration (0.01-10 microm)- and time (6-24 h)-dependent manner, as assessed by solid phase sandwich enzyme-linked immunosorbent assay. The thrombin (2.5 U/mL)-induced proliferation was inhibited by a PAR1-selective inhibitor, SCH79797 (0.1 microm) and an ET(A) antagonist, BQ-123 (1 microm), but not by an ET(B) antagonist, BQ-788 (1 microm). These findings suggest that thrombin, acting via PAR1, induced proliferation of cultured rat gingival fibroblasts that was mediated by ET-1 acting via ET(A).

We observed that human rhabdomyosarcoma (RMS) cells highly express a tissue factor that promotes thrombin formation, which indirectly and directly affects RMS progression. First, we found that thrombin activates platelets to generate microvesicles (PMV), which transfer to RMS cells' alpha2beta3 integrin and increase their adhesiveness to endothelial cells. Accordingly, RMS cells covered with PMVs showed higher metastatic potential after i.v. injection into immunodeficient mice. Furthermore, PMVs activate mitogen-activated protein kinase (MAPK)p42/44 and AKT to chemoattract RMS cells. We also found that RMS cells express functional protease-activated receptor-1 (PAR1) and PAR3 and respond to thrombin stimulation by MAPKp42/44 and MAPKp38 phosphorylation. To our surprise, thrombin did not affect RMS proliferation or survival; it inhibited the chemotactic and adhesive properties of RMS cells. However, when PAR1-specific agonist thrombin receptor-activating peptide 6 was used, which does not activate PAR3, selective PAR1 stimulation enhanced RMS proliferation. To learn more on the role of PAR1 and PAR3 antagonism in RMS proliferation and metastasis, we knocked down both receptors by using a short hairpin RNA strategy. We found that although thrombin does not affect growth of PAR1(-/-) cells, it stimulated the proliferation of PAR3(-/-) cells. More importantly, PAR3(-/-) cells, in contrast to PAR1(-/-) ones, formed larger tumors in immunodeficient mice. We conclude that thrombin is a novel underappreciated modulator of RMS metastasis and that we have identified a novel role for PAR3 in thrombin signaling., ((c)2010 AACR.)

It is well recognized that the proliferation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of various vascular diseases, including atherosclerosis and hypertension. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42- and 44-kDa isoforms (ERK1/2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS) and thrombin (Thr). However, understanding of the intracellular signal transduction pathways involved is incomplete. This review considers the recent findings in INS and Thr signaling mechanisms that modulate the proliferation of VSMCs with particular emphasis on the ERK1/2 signaling pathway, an important mediator of VSMCs hypertrophy and vascular disease. Moreover, because the ERK1/2 pathway have been acknowledged as an important mediator of VSMCs hypertrophy, ERK1/2 is identified as a key target for novel therapeutic interventions to minimize irreversible tissue damage associated with hypertension and atherosclerosis.

Summary Background: Activated protein C (APC) in complex with endothelial protein C receptor (EPCR) can reverse the barrier-disruptive and cytotoxic effects of proinflammatory cytokines by cleaving protease-activated receptor 1 (PAR-1). Recently, it was reported that the PAR-1-dependent vascular barrier-protective effect of APC is mediated through transactivation of the angiopoietin (Ang)-Tie2 signaling pathway. The antagonist of this pathway, Ang2, is stored in Weibel-Palade bodies within endothelial cells., Objectives: To determine whether the occupancy of EPCR by its ligand can switch the PAR-1-dependent signaling specificity of thrombin through the Ang-Tie2 axis., Methods: We activated endothelial cells with thrombin before and after treating them with the catalytically inactive Ser195-->Ala substitution mutant of protein C. The expression levels of Ang1, Ang2 and Tie2 in response to thrombin were measured by both an enzyme-linked immunosorbent assay and a cell permeability assay in the absence and presence of small interfering RNA and a blocking antibody to Tie2., Results: Thrombin upregulated the expression of both Ang1 and Tie2 but downregulated the expression of Ang2 when EPCR was occupied by its ligand. The Ang1-Tie2-dependent protective effect of thrombin was initiated through protein C inhibiting the rapid mobilization of Ang2 from Weibel-Palade bodies. Interestingly, the protein C mutant also inhibited the thrombin mobilization of P-selectin., Conclusions: These results suggest a physiologic role for the low concentration of thrombin in maintaining the integrity of the EPCR-containing vasculature through the PAR-1-dependent inhibition of Ang2 and P-selectin release from Weibel-Palade bodies.

Platelets play an important role in hemostasis by forming a thrombotic plug that seals the vessel wall and promotes vascular healing. After platelets adhere and aggregate at the wound site, their next step is to generate contractile forces through the coordination of physicochemical interactions between actin, myosin, and alpha(IIb)beta(3) integrin receptors that retract the thrombus' size and strengthen its adhesion to the exposed matrix. Although platelet contractile forces (PCF) are a definitive feature of hemostasis and thrombosis, there are few approaches that can directly measure them. In this study, we describe the development of an approach to measure PCF in microthrombi using a microscopic flexible post force sensor array. Quasi-static measurements and live microscopic imaging of thrombin-activated platelets on the posts were conducted to assay the development of PCF to various hemostatic conditions. Microthrombi were observed to produce forces that monotonically increased with thrombin concentration and activation time, but forces subsided when thrombin was removed. PCF results were statistically similar on arrays of posts printed with fibronectin or fibrinogen. PCF measurements were combined with clot volume measurements to determine that the average force per platelet was 2.1 +/- 0.1 nN after 60 min, which is significantly higher than what has been measured with previous approaches. Overall, the flexible post arrays for PCF measurements are a promising approach for evaluating platelet functionality, platelet physiology and pathology, the impacts of different matrices or agonists on hemostatic responses, and in providing critical information regarding platelet activity that can guide new hemostatic or thrombotic strategies.

The nature of protease-activated receptors (PARs) capable of activating respiratory vagal C-fibres in the mouse was investigated. Infusing thrombin or trypsin via the trachea strongly activated vagal lung C-fibres with action potential discharge, recorded with the extracellular electrode positioned in the vagal sensory ganglion. The intensity of activation was similar to that observed with the TRPV1 agonist, capsaicin. This was mimicked by the PAR1-activating peptide TFLLR-NH(2), whereas the PAR2-activating peptide SLIGRL-NH(2) was without effect. Patch clamp recording on cell bodies of capsaicin-sensitive neurons retrogradely labelled from the lungs revealed that TFLLR-NH(2) consistently evokes a large inward current. RT-PCR revealed all four PARs were expressed in the vagal ganglia. However, when RT-PCR was carried out on individual neurons retrogradely labelled from the lungs it was noted that TRPV1-positive neurons (presumed C-fibre neurons) expressed PAR1 and PAR3, whereas PAR2 and PAR4 were rarely expressed. The C-fibres in mouse lungs isolated from PAR1(-/-) animals responded normally to capsaicin, but failed to respond to trypsin, thrombin, or TFLLR-NH(2). These data show that the PAR most relevant for evoking action potential discharge in vagal C-fibres in mouse lungs is PAR1, and that this is a direct neuronal effect.

Glial cells are abundant in the CNS and play diverse roles in the regulation of neuronal activity, vascular function, and gliotransmitter release, whereas pathologically activated glial cells have been reported to disturb brain function in conjunction with Ca(2+) signaling, however there is no enough explanation for a unique Ca(2+) entry. Transient receptor potential (TRP) superfamily comprises a group of non-selective cation channels that open in response to divergent stimuli in their environment. Although TRP channels are widely distributed in the mammalian brain, their roles remain to be elucidated. Here we provide an overview of the roles of TRP channels in pathophysiological processes, especially focusing on TRPC3 and TRPV4 channels in glial cells. Using rat cortical astrocytes, we found that TRPC3 was upregulated by thrombin via Ca(2+) signaling through TRPC3 itself. Thrombin also upregulated S100B, a marker of reactive astrocytes, and increased cell proliferation, both of which were inhibited by Ca(2+) signaling blockers and specific knockdown of TRPC3 using siRNA, suggesting that TRPC3 contributes to the pathological activation of astrocytes in part through a feed-forward upregulation of its own expression. Moreover, we found that TRPV4 stimulation by its agonist 4alpha-PDD suppressed LPS-induced microglial activation while TRPV4 mRNA was downregulated in LPS-treated cultured rat microglia. These results suggest that TRP channels play pivotal roles in the process of astrocytic and microglial activation.

Host defense and blood coagulation are tightly connected and interacting systems, necessary for the integrity of an organism. Complex mechanisms regulate the intensity of a host response to invading pathogens or other potentially dangerous situations. Under regular conditions, this response is limited in time and located to the site of injury. Sometimes, however, systemic host response is overwhelming and disproportional and causes damage, not cure. Dependent on the genetical predisposition of the host, its current immunocompetence, or the type of injury, the reaction leads to the clinical picture of the different degrees of sepsis. Septic organ dysfunction is caused by intravascular fibrin deposition as a result of coagulation activation, anticoagulant breakdown, and shut down of fibrinolysis. This article describes the major pathophysiologic reactions in these situations and presents www.SepDIC.eu, an online tool on sepsis and associated coagulopathy.

The clinical observation that thrombosis in some patients heralds the onset of malignancy has been recognized for over a century. Thrombin the key terminal enzyme of coagulation also promotes angiogenesis and stimulates tumor-platelet adhesion, adhesion to endothelium, tumor implantation, tumor cell growth and metastasis. The thrombin receptor, a member of the protease-activated receptor family, is expressed on many tumor cell lines and on breast tumor biopsy specimens. In addition to mitogenic effects on fibroblast, smooth muscle cells and endothelial cells, thrombin also exerts direct effects on cancer cells by activation of the cell cycle through downregulation of p27(Kip1) and induction of Skp2, and cyclins D and A. MicroRNA 222, which inhibits p27(Kip1), is upregulated by thrombin. In the transgenic TRAMP mouse model of prostate cancer inhibition of endogenous thrombin by hirudin retards spontaneous tumor growth. Inhibition of thrombin may lead to tumor dormancy and could explain inhibition of tumor growth and metastasis by anticoagulants observed in animal models and a beneficial effect on survival observed in some clinical trials of anticoagulants in cancer patients.

The binding of thrombin to its receptor stimulates inflammatory cytokines including IL-6 and monocyte chemoattractant protein-1 (MCP-1); both are associated with the development of insulin resistance. Because increased adiposity enhanced the expression of coagulation factor VII that stimulates the coagulation pathway in adipose tissue, we tested whether the inhibition of thrombin action ameliorates insulin resistance in obese diabetic (Lpr(-/-):db/db) mice. The 4-wk administration of argatroban, a selective thrombin inhibitor, reduced fasting plasma glucose and ameliorated insulin resistance in these mice. It also reduced adipocyte size and macrophage infiltration into adipose tissue. The aberrant gene expression of MCP-1, IL-6, adiponectin, and factor VII and suppressed insulin receptor substrate-1-Akt signaling in adipose tissue of db/db mice were reversed by argatroban treatment. These results demonstrate that increased adiposity enhances the production of thrombin in adipose tissue by stimulating factor VII expression and suggest that increased thrombin activity in adipose tissue plays an important role in the development of insulin resistance via enhancing MCP-1 production, leading to macrophage infiltration and insulin receptor substrate-1-Akt pathway inactivation.

Objectives: Does thrombin activation seen in patients with abdominal aortic aneurysms (AAA) relate to the thrombus surface area or volume within the aneurysm?, Patients and Methods: A total of 130 patients with AAA were analyzed regarding levels of the complex between activated protein C-protein C inhibitor (APC-PCI) and AAA morphology. Analysis of APC-PCI complex was made using a sandwich immunofluorometric method., Results: Increased APC-PCI concentrations were seen in patients with AAA (0.44 microg/L; P < .001 compared with controls). The correlations of APC-PCI values were r = .13, P = .13 for aneurysm size, r = .08, P = .35 for thrombus surface area, and r = .13, P = .14 for thrombus volume. APC-PCI values elevated to 0.45 microg/L in 10 patients with AAA having no or very little thrombus mass., Conclusion: Disappointingly, no correlation was found between thrombus surface area or volume and levels of the APC-PCI complex. Mechanisms other than the AAA-sac thrombus must be evaluated as cause of thrombin activation in patients with AAA.

In the pathogenesis of sepsis, inflammation and coagulation play a pivotal role. Increasing evidence points to an extensive cross-talk between these two systems, whereby inflammation leads to activation of coagulation, and coagulation also considerably affects inflammatory activity. Molecular pathways that contribute to inflammation-induced activation of coagulation have been precisely identified. Pro-inflammatory cytokines and other mediators are capable of activating the coagulation system and down-regulating important physiologic anticoagulant pathways. Activation of the coagulation system and ensuing thrombin generation is dependent on expression of tissue factor and the simultaneous down-regulation of endothelial-bound anticoagulant mechanisms and endogenous fibrinolysis. Conversely, activated coagulation proteases may affect specific cellular receptors on inflammatory cells and endothelial cells and thereby modulate the inflammatory response.

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders characterized by an increased incidence of thrombo-hemorrhagic complications. The acquired somatic Janus kinase 2 (JAK2) V617F mutation is present in the majority of PV and ET patients. Because aberrant protein Tyr-phosphorylation has been associated with hematopoietic malignancies, the activity of the tyrosine kinases Src and JAK2 was analyzed in resting and thrombin-stimulated platelets from 13 PV and 42 ET patients. JAK2 was found inactive in healthy and pathological resting cells regardless of the V617F mutation. In addition, Src was inactive in all resting platelets, but in the pathological specimens it was present in a preactivated conformation as a consequence of anomalous dephosphorylation of its inhibitory phospho-Tyr527 residue, likely mediated by Src homology-2 domain-containing protein Tyr-phosphatase-2 (SHP-2), whose constitutive activity correlated with its recruitment to Src. Low thrombin concentration triggered a more rapid Src-signaling activation, higher [Ca(2+)](c) increase, and aggregation in pathological platelets compared with controls. Thrombin-induced Src activation preceded JAK2 activation, which occurred simultaneously in normal and pathological platelets. Our results indicate that a constitutive Src kinase preactivation is implicated in platelet hypersensitivity and likely involved, at least partially, in the functional abnormalities of PV and ET platelets.

Intracerebral hemorrhage (ICH) is a devastating neurological disorder with high mortality and poor prognosis, for which virtually no effective drug therapies are available at present. Experimental animal models, based on intrastriatal injection of collagenase or autologous blood, have enabled great advances in elucidation of cellular/molecular events contributing to brain pathogenesis associated with ICH. Many lines of evidence indicate that blood constituents, including hemoglobin-derived products as well as proteases such as thrombin, play important roles in the pathogenic events. Inflammatory reactions involving neutrophils, activated microglia, and production of proinflammatory cytokines also constitute a critical aspect of pathology leading to neurodegeneration and tissue damage. Efforts are continuing to find drugs that potentially alleviate pathological and neurological outcomes of ICH. Various drugs that possess antioxidative, anti-inflammatory or neurotrophic/neuroprotective properties have been demonstrated to produce therapeutic effects on ICH animal models. Drugs already in clinical use such as minocycline, statins, and several nuclear receptor ligands are among the list of effective drugs, but whether they also show therapeutic efficacy in human ICH patients remains unproven. Here, current knowledge of ICH pathogenesis and problems arising with respect to exploration of new drug candidates are discussed.

Vascular smooth muscle cells (VSMC) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMC allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMC proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Signal transduction pathways in eukaryotic cells integrate diverse extracellular signals, and regulate complex biological responses such as growth, differentiation and death. One group of proline-directed Ser/Thr protein kinases, the mitogen-activated protein kinases (MAPKs), plays a central role in these signalling pathways. Much attention has focused in recent years on subfamilies of MAPKs, the extracellular signal regulated kinases (ERKs). Here we overview the work on ERKs 1 to 2, emphasising when possible their biological activities in VSMC proliferation. It is clear from numerous studies including our own, that ERK1/ERK2 pathway has an important role in VSMC proliferation induced by insulin (INS) and thrombin. Despite the physiological and pathophysiological importance of INS and thrombin, possible signal transduction pathways involved in INS and thrombin regulation of VSMC's proliferation remains poorly understood. Thus, this review examines recent findings in signaling mechanisms involved in INS and thrombin- triggered VSMC's proliferation with particular emphasis on ERK1/2 signaling pathways. Future investigations should now focus on the mechanisms of MAPK activation which might therefore represent a new mechanism involved in the antiproliferative effect revealed in this review.

Diverse oncogenic transformations result in the constitutive expression of tissue factor (TF) in cancer cells. The local and systemic activation of the coagulation cascade has long been a recognized hallmark for aggressive cancer, but genetic mouse models and new experimental therapeutics have only recently demonstrated crucial roles for TF initiated cell signaling in the pathogenesis of cancer. On tumor cells, the TF-VIIa binary complex mediates activation of protease activated receptor (PAR) 2 and thereby shapes the tumor microenvironment by inducing an array of proangiogenic and immune modulating cytokines, chemokines, and growth factors. PAR2 also uniquely triggers tumor cell migration by G protein-independent pathways through beta-arrestin scaffolding. Metastatic tumor cells use additional signaling networks of the coagulation cascade by activating PAR1 through thrombin or the ternary TF-VIIa-Xa signaling complex in the vascular and potentially lymphatic system. Selective antagonists of TF-VIIa-PAR2 signaling may be used as antiangiogenic therapy without increasing the risk of bleeding, whereas coagulation and associated signaling pathways on platelets and other host cells may be targeted for therapeutic benefit in advanced cancer and metastatic disease.

Morbidity and mortality in patients with atherothrombotic disease remain high despite the use of antiplatelet therapy with aspirin and an ADP receptor antagonist. Selective inhibition of the principal protease-activated receptor (PAR)-1 for thrombin, the most potent agonist for platelet activation, represents a promising novel strategy to reduce thrombosis and ischaemic events. SCH 530348, a potent thrombin receptor antagonist (TRA) selective for PAR-1, has been evaluated in preclinical studies, demonstrating complete and sustained inhibition of thrombin/TRAP-induced platelet aggregation without a concomitant increase in the risk of bleeding. Phase 2 studies in patients undergoing non-urgent or urgent PCI showed that treatment with SCH 530348 in addition to the standard of care (aspirin plus an ADP receptor antagonist) is not associated with an increased risk of TIMI bleeding and is well tolerated, with a rate of adverse events comparable to standard therapy alone. These studies also demonstrated that the use of SCH 530348 in combination with aspirin and an ADP receptor antagonist may reduce the incidence of major adverse cardiac events, specifically periprocedural myocardial infarction, vs aspirin plus an ADP receptor antagonist alone. On the basis of these encouraging results, 2 ongoing large phase 3 randomized trials are evaluating the efficacy and safety of SCH 530348 in combination with the standard-of-care therapy in approximately 35,000 patients with NSTE ACS or established atherosclerosis.

Platelet stimulation with thrombin induces an elevation in cytoplasmic free Ca(2+) concentration ([Ca(2+)]c) due to Ca(2+) release from intracellular stores and entry from the extracellular medium. Two different intracellular Ca(2+) stores have been described in human platelets: the dense tubular system and the lysosomal-like acidic stores. In the present study, we investigated the contribution of the acidic stores in thrombin-induced platelet aggregation. We have found that platelet aggregation induced by thrombin is reduced in a Ca(2+)-free medium. Discharge of the acidic Ca(2+) stores by treatment with the sarcoendoplasmic Ca(2+)-ATPase (SERCA)3 selective inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone reduced thrombin-evoked platelet aggregation. In the presence of 2,5-di-(tert-butyl)-1,4-hydroquinone, platelet aggregation induced by the protease-activated receptor (PAR)-1 and PAR-4 agonist peptides, SFLLRN and AYPGKF, respectively, was significantly reduced. In cells with depleted acidic stores, activation of GPIb-IX-V by thrombin resulted in reduced or no platelet aggregation in a medium containing 1 mmol/l Caor in a Ca(2+)-free medium, respectively. This finding suggests that Ca(2+) accumulation in the acidic Ca(2+) compartments is required for platelet aggregation induced by activation of the G-coupled PAR-1 and PAR-4 thrombin receptors and, by the occupation of the leucine-rich glycoprotein GPIb-IX-V and provide evidence supporting a functional role of the lysosomal-like acidic Ca(2+) stores in human platelets.

We have developed an integrated approach that combines empirical and computational methodologies to define an individual's thrombin phenotype. We have evaluated the process of thrombin generation in healthy individuals and individuals with defined pathologies in order to develop general criteria relevant to assess an individual's propensity for hemorrhage or thrombosis. Three complementary hypotheses have emerged from our work: (i) compensation by the ensemble of other coagulation proteins in individuals with specific factor deficiencies can 'normalize' an individual's thrombin generation process and represents a rationale for their unexpected phenotype; (ii) individuals with clinically unremarkable factor levels may present thrombin generation profiles typical of individuals with hemostatic complications; and (iii) in some hemostatic disorders a specific pattern of expression of a small ensemble of coagulation factors may be sufficient to explain the overall phenotype.

Thrombin can amplify inflammation induced by other stimuli, either through ischemia (consequent upon thrombosis), indirectly through generation of downstream mediators such as activated protein C, or directly via signals through protease activated receptors (PAR). This paper will summarize recent data from our laboratory indicating that thrombin is required to initiate CCR2-dependent leukocyte recruitment and that it is the principal determinant of the outcome after vascular injury, via PAR-1 activation of a distinct subset of smooth muscle cell progenitors. In both, tissue factor (TF) initiates thrombin generation and the thrombin acts locally, exemplifying that the initiation phase can generate autocrine or paracrine signalling molecules. Thrombin is an important constituent of innate immunity, able to amplify and modify responses to invading pathogens or tissue damage. With novel anti-thrombin therapeutics and agents to target PAR, a new understanding of the importance of thrombin may allow the development of innovative anti-inflammatory strategies.

Activation of NF-kappaB is essential for protease-activated receptor-1 (PAR-1)-mediated ICAM-1 expression in endothelial cells. Here we show that PAR-1 activation induces binding of both p65/RelA and NFATc1 to the NF-kappaB binding site localized in intron-1 of the ICAM-1 gene to initiate transcription in endothelial cells. We discovered the presence of two NF-kappaB binding sites in intron-1 (+70, NF-kappaB site 1; +611, NF-kappaB site 2) of the human ICAM-1 gene. Chromatin immunoprecipitation results showed that thrombin induced binding of p65/RelA and of NFATc1 specifically to intronic NF-kappaB site 1 of the ICAM-1 gene. Electrophoretic mobility shift and supershift assays confirmed the binding of p65/RelA and NFATc1 to the intronic NF-kappaB site 1 in thrombin-stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (-1,385 to +234) construct approximately 25-fold and mutation of intronic NF-kappaB site 1 markedly reduced thrombin-induced reporter expression. Moreover, inhibition of calcineurin, knockdown of either NFATc1 or p65/RelA with siRNA significantly reduced thrombin-induced ICAM-1 expression and polymorphonuclear leukocyte adhesion to endothelial cells. In contrast, NFATc1 knockdown had no effect on TNF-alpha-induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-kappaB site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.

To date, most clinicians and researchers have been using platelet concentrate within the range of 4x to 8x baseline. In this case report a new procedure involving strategic pooling and triple spin was developed and used to concentrate platelets to 23x baseline. The concentrate alone was infused into morselized resorbable collagen sponge, activated with calcium chloride and autologous thrombin, then placed as an autologous graft into the left sinus floor of a healthy 80-year-old woman. The floor of the sinus had approximately 2 mm of bony height. A computed tomography scan taken after 5 months showed that new bone was formed and it was as dense as native bone around the surgical site. From the computed tomography scan bone density analysis using Hounsfield Units revealed that the bone formed was D3 bone (518.3 +/- 224.9 Hounsfield Units) and it was as dense as native bone on the axial, coronal and sagittal slices. Histomorphometric analysis of 2 bone core biopsies taken after 6 months showed that 1 core had 34% bone of which 98% was vital new bone and the other core had 39% bone of which 100% was vital new bone. The height of the bone formed in the sinus floor was 12 mm. It was also characterized by good trabecular pattern and connectivity. The quality of the bone generated was such that 2 endosseous implants were placed and torqued to 30 N cm after the cores were removed.

In addition to its established roles in the haemostatic system, thrombin is an intriguing coagulation protease demonstrating an array of effects on endothelial cells, vascular smooth muscle cells (VSMC), monocytes, and platelets, all of which are involved in the pathophysiology of atherosclerosis. There is mounting evidence that thrombin acts as a powerful modulator of many processes like regulation of vascular tone, permeability, migration and proliferation of VSMC, recruitment of monocytes into the atherosclerotic lesions, induction of diverse pro-inflammatory markers, and all of these are related to the progression of cardiovascular disease. Recent studies in transgenic mice models indicate that the deletion of the natural thrombin inhibitor heparin cofactor II promotes an accelerated atherogenic state. Moreover, the reduction of thrombin activity levels in apolipoprotein E-deficient mice, because of the administration of the direct thrombin inhibitor melagatran, attenuates plaque progression and promotes stability in advanced atherosclerotic lesions. The combined evidence points to thrombin as a pivotal contributor to vascular pathophysiology. Considering the clinical development of selective anticoagulants including direct thrombin inhibitors, it is a relevant moment to review the different thrombin-induced mechanisms that contribute to the initiation, formation, progression, and destabilization of atherosclerotic plaques.

Background: High expression of vascular endothelial growth factor (VEGF) induces subepithelial fibrosis associated with angiogenesis in asthma. Thrombin is recognized as a new candidate mediating airway remodeling. Therefore, this study was designed to determine the potential mechanisms of airway remodeling initiated by activated thrombin in asthma., Methods: Levels of biochemical parameters in induced sputum were examined in 21 asthmatic patients and 11 normal controls., Results: Thrombin activity in induced sputum was significantly higher in asthmatic patients than in normal controls (normal controls: median [range] 1.26 (0.93-2.42) U/mL; asthmatic patients: 3.67 (1.15-10.2) U/mL, p < 0.0001). VEGF level in induced sputum was positively correlated with thrombin activity in all study subjects. Levels of basic fibroblast growth factor (bFGF), which is a major profibrotic factor, were also significantly higher in asthmatic patients than in normal controls. Moreover, thrombin activity was significantly correlated with bFGF level in all study subjects. We also observed a significant correlation between bFGF and procollagen type III peptide level., Conclusion: Increase in VEGF level leads to up-regulation of thrombin activity in asthmatic airways, and this elevated thrombin activity induces elevation of bFGF level. It will become to be a new strategy of asthma therapy to attenuate thrombin activity for the regulation of airway remodeling.