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Aims: Atrial fibrillation (AF) produces a hypercoagulable state. Stimulation of protease-activated receptors by coagulation factors provokes pro-fibrotic, pro-hypertrophic, and pro-inflammatory responses in a variety of tissues. We studied the effects of thrombin on atrial fibroblasts and tested the hypothesis that hypercoagulability contributes to the development of a substrate for AF., Methods and Results: In isolated rat atrial fibroblasts, thrombin enhanced the phosphorylation of the pro-fibrotic signalling molecules Akt and Erk and increased the expression of transforming growth factor β1 (2.7-fold) and the pro-inflammatory factor monocyte chemoattractant protein-1 (6.1-fold). Thrombin also increased the incorporation of 3 H-proline, suggesting enhanced collagen synthesis by fibroblasts (2.5-fold). All effects could be attenuated by the thrombin inhibitor dabigatran. In transgenic mice with a pro-coagulant phenotype (TM pro/pro ), the inducibility of AF episodes lasting >1 s was higher (7 out of 12 vs. 1 out of 10 in wild type) and duration of AF episodes was longer compared with wild type mice (maximum episode duration 42.8 ± 68.4 vs. 0.23 ± 0.39 s). In six goats with persistent AF treated with nadroparin, targeting Factor Xa-mediated thrombin generation, the complexity of the AF substrate was less pronounced than in control animals (LA maximal activation time differences 23.3 ± 3.1 ms in control vs. 15.7 ± 2.1 ms in nadroparin, P < 0.05). In the treated animals, AF-induced α-smooth muscle actin expression was lower and endomysial fibrosis was less pronounced., Conclusion: The hypercoagulable state during AF causes pro-fibrotic and pro-inflammatory responses in adult atrial fibroblasts. Hypercoagulability promotes the development of a substrate for AF in transgenic mice and in goats with persistent AF. In AF goats, nadroparin attenuates atrial fibrosis and the complexity of the AF substrate. Inhibition of coagulation may not only prevent strokes but also inhibit the development of a substrate for AF., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Objective: Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms., Approach and Results: We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells., Conclusions: Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists., (© 2016 American Heart Association, Inc.)

Thrombin's role in the nervous system is not well understood. Under conditions of blood-brain barrier compromise (e.g., neurosurgery or stroke), thrombin can result in neuroapoptosis and the formation of glial scars. Despite this, preconditioning with thrombin has been found to be neuroprotective in models of cerebral ischemia and intracerebral hemorrhage. We investigated the effects of physiologically relevant concentrations of thrombin on cortical neurons using two culture-based assays. We examined thrombin's effect on neurites by quantitative analysis of fluorescently labeled neurons. To characterize thrombin's effects on neuron survival, we spectrophotometrically measured changes in enzymatic activity. Using receptor agonists and thrombin inhibitors, we separately examined the role of thrombin and its receptor in neuroprotection. We found that low concentrations of thrombin (1 nM) enhances neurite growth and branching, neuron viability, and protects against excitotoxic damage. In contrast, higher concentrations of thrombin (100 nM) are potentially detrimental to neuronal health as evidenced by inhibition of neurite growth. Lower concentrations of thrombin resulted in equivalent neuroprotection as the antifibrinolytic, aprotinin, and the direct thrombin inhibitor, argatroban. Interestingly, exogenous application of the species-specific thrombin inhibitor, antithrombin III, was detrimental to neuronal health; suggesting that some endogenous thrombin is necessary for optimal neuron health in our culture system. Activation of the thrombin receptor, protease-activated receptor-1 (PAR-1), via micromolar concentrations of the thrombin receptor agonist peptide, TRAP, did not adversely affect neuronal viability. An optimal concentration of thrombin exists to enhance neuronal health. Neurotoxic effects of thrombin do not involve activation of PAR receptors and thus separate pharmacologic manipulation of thrombin's receptor in the setting of direct thrombin inhibitors could be a potential neuroprotective strategy.

Background: Human platelet activation and aggregation is a complex process. To date, many therapies have been developed targeting proteins that mediate this process to prevent unwanted activation. However, the current standard of care for acute coronary syndromes still has limitations, including bleeding risk., Objective: To evaluate the protease-activated receptor 4 (PAR4) anionic cluster as a viable antiplatelet target by using a polyclonal antibody (CAN12)., Methods: We used western blotting, aggregation and secretion ex vivo to evaluate the ability of CAN12 to interact with PAR4 and inhibit platelet activation. The effects of CAN12 in vivo were evaluated with the Rose Bengal arterial thrombosis model and two models of hemostasis., Results: CAN12 was able to interact with human PAR4 and delay PAR4 cleavage. In addition, CAN12 inhibited thrombin-induced human platelet aggregation and secretion in a dose-dependent manner. The specificity of CAN12 was agonist-dependent. In vivo, we determined that CAN12 was able to inhibit arterial thrombosis, and, using two independent methods, we found that CAN12 did not influence hemostasis., Conclusion: Targeting the extracellular anionic cluster on PAR4 is a viable novel strategy as an antiplatelet therapy., (© 2014 International Society on Thrombosis and Haemostasis.)

Objective: Statins decrease mortality in patients with vascular disorders, and evidence for the pleiotropic effects of statins is accumulating. Statins enhance endothelial NO synthase (eNOS) expression, thereby attenuating platelet activation and thrombus formation. Our goal was to determine whether statins have eNOS-independent effects on platelet activation., Methods and Results: Wild-type and eNOS-deficient mice were given a 14-day course of oral atorvastatin, and platelet activation was evaluated in vitro and in vivo. Whereas in wild-type mice atorvastatin inhibited platelet activation in vitro in response to numerous agonists, in eNOS-deficient mice, atorvastatin inhibited only thrombin-induced and protease-activated receptor 4 agonist peptide-induced platelet activation. Consistent with an eNOS-independent effect, atorvastatin inhibited platelet activation in vivo in both wild-type and eNOS-deficient mice., Conclusions: Atorvastatin inhibits platelet activation via eNOS-dependent and eNOS-independent mechanisms with the latter restricted to protease-activated receptor 4-induced activation downstream to the receptor.

The unifying basis of acute coronary syndrome (ACS) is the complication of a vulnerable coronary plaque, an event primarily mediated by platelet activation. Three major pathways are predominantly involved in this process: thromboxane A(2) via the thromboxane A(2) receptor, adenosine diphosphate via the P2Y(12) receptor, and thrombin via the protease-activated receptor (PAR)-1, with the latter being the most potent platelet activator. Despite the effective inhibition of the first two pathways with aspirin and an expanding family of P2Y(12) inhibitors, respectively, the recurrence of ischemic events in patients with ACS remains high. There is also a growing concern regarding the safety profile in terms of bleeding with more powerful antiplatelet agents, which has tempered expectations of newly developed compounds. PAR-1 inhibitors are a novel class of antiplatelet agents that inhibit thrombin-mediated platelet activation. Preliminary data indicate that these compounds have the potential to improve ischemic prognosis without increasing the bleeding risk. In this chapter we will discuss the rationale for developing this novel class of antiplatelet agents and specifically, the two compounds in most advanced clinical development, vorapaxar and atopaxar., (Copyright © 2012 S. Karger AG, Basel.)

Background: Platelet aggregation varies among individuals; and genetic factors may alter platelet activation through G-protein-coupled receptors, thus influencing results of point-of-care platelet aggregometry in whole blood. We tested the hypothesis that the C825T polymorphism of the gene GNB3 encoding the G-protein β-3 subunit and the platelet GPIIIa Pl(A1)/(A2) polymorphism of the glycoprotein IIIa influence platelet aggregation., Methods: Evoked [thrombin receptor activating peptide (TRAP), ADP, TXA(2) agonist U46619, epinephrine, and collagen] platelet aggregation in whole blood was measured using impedance aggregometry (Multiplate) in 143 healthy individuals (age: 40.2 years ±11.7 SD). Genotypes were determined using pyrosequencing and restriction analysis. Data were analyzed by linear one-way analysis of variance and Student's t-test, linear and multiple regression, and the χ(2)-test, as appropriate., Results: Homozygous carriers of the GNB3 825C-allele showed significantly (P≤0.022) increased maximum aggregation for EC(75) dosages compared with CT and TT genotypes [e.g. ADP: CC 150±36 vs. TT 126±33 aggregation unit (AU); thrombin receptor activating peptide: CC 175±46 vs. TT 150±38 AU; U46619: CC 164±33 vs. 149±32 AU; epinephrine: CC 66±41 vs. TT 48±34 AU]. In contrast, genotypes of glycoprotein IIb/IIIa PI(A)-polymorphism had no effect. Regression analysis revealed the GNB3 C825T polymorphism as an independent factor for enhanced platelet aggregation, besides factors such as female sex and blood cell values., Conclusion: In human whole blood, the GNB3 825CC genotype is associated with enhanced platelet aggregation.

Background: Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca(2+) signaling must be tightly controlled. Thrombin-induced Ca(2+) signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca(2+) signal, although the mechanisms by which this occurs and its physiological relevance are still unclear., Objectives: To investigate the mechanisms by which PKC inhibitors enhance thrombin-induced Ca(2+) signaling, and to determine the importance of this pathway in platelet activation., Methods: Cytosolic Ca(2+) signaling was monitored in fura-2-loaded human platelets. Phosphatidylserine (PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry., Results: PKC inhibition by bisindolylmaleimide-I (BIM-I) enhanced α-thrombin-induced Ca(2+) signaling in a concentration-dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or γ-thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM-I had little effect on α-thrombin-induced signaling following treatment with the PAR1 antagonist, SCH-79797. BIM-I enhanced Ca(2+) release from intracellular stores and Ca(2+) entry, as assessed by Mn(2+) quench. However, the plasma membrane Ca(2+) ATPase inhibitor, 5(6)-carboxyeosin, did not prevent the effect of BIM-I. PKC inhibition strongly enhanced α-thrombin-induced PS exposure, which was reversed by blockade of PAR1., Conclusions: Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca(2+) release and Ca(2+) entry, which leads to reduced platelet PS exposure., (© 2011 International Society on Thrombosis and Haemostasis.)

In the Bernard-Soulier syndrome (BSS), the giant platelets are said to have increased phosphatidylserine (PS) surface exposure in the resting state and shortened survival in the circulation. When normal platelets are activated, they undergo many biochemical and morphological changes, some of which are apoptotic. Herein, we investigated apoptotic-like events in BSS platelets upon activation, specifically, PS exposure, microparticle (MP) formation, cell shrinkage, and loss of mitochondrial inner membrane potential (DeltaPsi(m)). Platelets from two unrelated BSS patients were examined in whole blood; agonists used were collagen, thrombin, PAR1- or PAR4-activating peptides (APs), or combinations of collagen with thrombin, and the PAR-APs. Flow cytometry was used to measure PS exposure (annexin A5 binding), platelet-derived MPs (forward scatter; events <0.75 microm size), and DeltaPsi(m) (TMRM fluorescence). PS exposure was increased on resting and activated BSS platelets, and this was independent of the platelet size. MP formation by BSS platelets was generally enhanced. Cell shrinkage occurred on activation to form smaller, PS-exposing platelets in BSS and controls. A proportion of PS-exposing BSS and control platelets exhibited DeltaPsi(m) loss, but unlike controls, there was also loss of DeltaPsi(m) in the BSS platelets not exposing PS. Thus, BSS platelets undergo apoptotic-like events upon activation, with PS exposure and MP formation being enhanced. These events may play a role in the shortened survival in BSS, as well as affecting thrombin generation., ((c) 2010 Wiley-Liss, Inc.)

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.

Background and Purpose: The modulation by flavonoids of platelet responses induced by thrombin has been little investigated, and the antiplatelet activity, as well as possible inhibitory mechanisms of these compounds on thrombin signalling, has not yet been elucidated. We explored whether flavonoids affect platelet signalling pathways triggered by thrombin and by the selective activation of its protease-activated receptors (PARs) 1 and 4, and analysed the antagonism of these polyphenols at thrombin receptors., Experimental Approach: We investigated the effect of a range of polyphenolic compounds on platelet aggregation, 5-HT secretion, intracellular calcium mobilization, protein kinase activity and tyrosine phosphorylation, triggered by thrombin and PAR agonist peptides (PAR-APs). The ability of these flavonoids to bind to thrombin receptors was investigated by competitive radioligand binding assays using (125)I-thrombin., Key Results: Quercetin, apigenin and genistein impaired platelet aggregation, as well as 5-HT release and calcium mobilization, induced by thrombin and PAR-APs. Quercetin and apigenin were inhibitors of protein kinases, but genistein exhibited a minimal ability to suppress platelet phosphorylation. Binding assays did not establish any kind of interaction between thrombin receptors and any of the flavonoids tested., Conclusions and Implications: Quercetin, apigenin and genistein did not inhibit thrombin responses by interacting with thrombin receptors, but by interfering with intracellular signalling. While inhibition by genistein may be a consequence of affecting calcium mobilization, subsequent platelet secretion and aggregation, for quercetin and apigenin, inhibition of kinase activation may also be involved in the impairment of platelet responses.

SCH 530348, a synthetic tricyclic 3-phenylpyridine, is an orally active fourth generation himbacine-based antagonist of the protease-activated receptor (PAR)-1, the primary receptor for thrombin on platelets in humans. SCH 530348 is the first in a new class of compounds that inhibit thrombin-mediated platelet aggregation without affecting the enzymatic activity of thrombin on fibrinogen. Preclinical and initial clinical studies have demonstrated this compound to be a highly potent inhibitor of thrombin-induced platelet activation, to have excellent oral bioavailability and to have a favorable safety profile. These data suggest that this compound has the potential to reduce the risk of ischemic events without significantly increasing the rate of bleeding. Two large Phase III clinical outcome trials are currently underway to evaluate the safety and efficacy of SCH 530348 for the management of acute coronary syndromes and the secondary prevention of atherothrombotic events.

The present review focuses on the roles of thromboxane A2 (TxA2) in arterial thrombosis, atherogenesis, vascular stent-related ischemic events and renal proteinuria. Particular emphasis is laid on therapeutic interventions targeting the TxA2 (TP) receptors and TxA2 synthase (TS), including dual TP-receptor antagonists and TS inhibitors. Their significant inhibitory efficacies on arterial thrombogenesis, atherogenesis, restenosis after stent placement, vasoconstriction and proteinuria indicate novel and improved treatments for cardiovascular and selected renal diseases. New therapeutic interventions of the TxA2 pathway may also be beneficial for patients with poor biological antiplatelet drug response, for example, to aspirin and/or clopidogrel. These new TP/TS agents offer novel improved treatments to efficiently and simultaneously interfere with thrombogenesis and atherogenesis, and to enlarge the existing panel of platelet inhibitors for efficient prophylaxis and treatment of arterial thrombosis and renal proteinuria.

Thrombin is now recognized as an important factor in many cancers. Here, we examined the expression and role of the recently discovered thrombin receptor PAR4, in human colon cancer cells. PAR4 mRNA was found in 10 out of 14 (71%) human colon cancer cell lines tested but not in epithelial cells isolated from normal human colon. This finding is in line with immunostaining results of PAR4 in human colon tumors and its absence in normal human colonic mucosa. Investigation of the functional significance of the aberrant expression of PAR4 in colon cancer cells revealed (i) a prompt increase in intracellular calcium concentration on challenge with PAR4-specific agonist AP4 (100 microM) and (ii) marked mitogenic response (2.5-fold increase in cell number) in a dose-dependent manner on treatment with AP4 (0.1-300 microM). Analysis of the signaling pathways downstream of PAR4 activation in HT29 cells showed (i) a sustained phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2) and (ii) the involvement of epidermal growth factor receptor B-2 (ErbB-2) but not of epidermal growth factor receptor in PAR4-induced mitogen-activated protein kinase activation. Tyrphostin AG1478, the ErbB inhibitor, reversed the action of AP4 on ERK1/2 and ErbB-2 phosphorylation and HT29 cell growth. Finally, the Src inhibitor PP2 abrogated ErbB-2 and ERK phosphorylation and HT29 cell proliferation, suggesting the essential role of Src activity in PAR4-induced phosphorylation of ErbB-2. These data highlight the role of PAR4 as a new important player in the control of colon tumors and underline the critical role of ErbB-2 transactivation.

A synthetic peptide representing the receptor-binding domain of human thrombin (TP508) promotes angiogenesis and accelerates wound healing in animal models. However, the mechanisms underlying the therapeutic effects of TP508 have not been clearly defined. In this study, we set out to determine whether TP508 could stimulate stem cell proliferation. Adipose tissue-derived stem cells (ASCs) were incubated with TP508 (5 microg/ml) and cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation. Our data showed that TP508 treatment significantly stimulated BrdU incorporation in ASCs (p < 0.01). The increased BrdU incorporation induced by TP508 was abolished by the PI3 kinase (PI3K) inhibitor LY294002 at 50 microM. Western blot analysis of ASCs revealed increased phosphorylation of Akt in response to TP508 when compared to unstimulated controls. These results indicate that TP508 exerts proliferative effects on ASCs via the PI3K/Akt pathway.

Second messenger-mediated inside-out activation of integrin alphaIIbbeta3 is a key step in platelet aggregation. We recently showed strongly impaired but not absent alphaIIbbeta3-mediated aggregation of CalDAG-GEFI-deficient platelets activated with various agonists. Here we further evaluated the roles of CalDAG-GEFI and protein kinase C (PKC) for alphaIIbbeta3 activation in platelets activated with a PAR4 receptor-specific agonist, GYPGKF (PAR4p). Compared with wild-type controls, platelets treated with the PKC inhibitor Ro31-8220 or CalDAG-GEFI-deficient platelets showed a marked defect in aggregation at low (< 1mM PAR4p) but not high PAR4p concentrations. Blocking of PKC function in CalDAG-GEFI-deficient platelets, how-ever, strongly decreased aggregation at all PAR4p concentrations, demonstrating that CalDAG-GEFI and PKC represent separate, but synergizing, pathways important for alphaIIbbeta3 activation. PAR4p-induced aggregation in the absence of CalDAG-GEFI required cosignaling through the Galphai-coupled receptor for ADP, P2Y12. Independent roles for CalDAG-GEFI and PKC/Galphai signaling were also observed for PAR4p-induced activation of the small GTPase Rap1, with CalDAG-GEFI mediating the rapid but reversible activation of this small GTPase. In summary, our study identifies CalDAG-GEFI and PKC as independent pathways leading to Rap1 and alphaIIbbeta3 activation in mouse platelets activated through the PAR4 receptor.

Objectives: We sought to test the hypothesis that cigarette smoking adversely alters protease-activated receptor type 1 (PAR-1)-mediated vascular effects in vivo in humans., Background: Distinct from its role in the coagulation cascade, thrombin exerts its major cellular and cardiovascular actions via PAR-1. The activation of PAR-1 causes endothelium-dependent arterial vasodilation and the release of endogenous fibrinolytic factors., Methods: Forearm blood flow was measured with venous occlusion plethysmography in 12 cigarette smokers and 12 age- and gender-matched nonsmokers during intrabrachial infusions of PAR-1-activating-peptide (SFLLRN; 5 to 50 nmol/min), bradykinin (100 to 1,000 pmol/min), and sodium nitroprusside (2 to 8 mug/min). Plasma tissue plasminogen activator (t-PA) and plasminogen-activator inhibitor 1 antigen and activity concentrations were measured throughout the experiment., Results: All agonists caused dose-dependent increases in forearm blood flow (p < 0.0001 for all). Although bradykinin and sodium nitroprusside caused similar vasodilation, SFLLRN-induced vasodilation was attenuated in smokers (p = 0.04). Smokers had modest reductions in bradykinin-induced active t-PA release (reduced by 37%, p = 0.03) and had a marked impairment of SFLLRN-induced t-PA antigen (p = 0.02) and activity (p = 0.006) release, with a 96% reduction in overall net t-PA antigen release. The use of SFLLRN also caused similar (p = NS) increases in inactive plasminogen-activator inhibitor 1 in both smokers and nonsmokers (p

We have performed intrastriatal injection of thrombin and searched for distant effects in the cell body region. In striatum, thrombin produced a slight loss of striatal neurons as demonstrated by neural nuclei immunostaining - a non-specific neuronal marker - and the expression of glutamic acid decarboxylase 67 mRNA, a specific marker for striatal GABAergic interneurons, the most abundant phenotype in this brain area. Interestingly, striatal neuropil contained many boutons immunostained for synaptic vesicle protein 2 and synaptophysin which colocalize with tyrosine hydroxylase (TH), suggesting a degenerative process with pre-synaptic accumulation of synaptic vesicles. When we studied the effects on substantia nigra, we found the disappearance of dopaminergic neurons, shown by loss of TH immunoreactivity, loss of expression of TH and dopamine transporter mRNAs, and disappearance of FluoroGold-labelled nigral neurons. The degeneration of substantia nigra dopaminergic neurons was produced through up-regulation of cFos mRNA, apoptosis and accumulation of alpha-synuclein shown by colocalization experiments. Thrombin effects could be mediated by protease-activated receptor 4 activation, as protease-activated receptor 4-activating peptide mimicked thrombin effects. Our results point out the possible relationship between synapse elimination and retrograde degeneration in the nigral dopaminergic system.

Proteinase-activated receptor-1 (PAR(1)) and PAR(2) mediate contraction in the guinea-pig gallbladder. To investigate and compare the effects mediated by PARs in the human gallbladder with those in the guinea-pig gallbladder, we measured contractions of isolated human and guinea-pig gallbladder strips caused by PAR agonists. Results in human were similar to those in guinea-pig gallbladder. The PAR(1) agonists, thrombin, TFLLR-NH2 and SFLLRN-NH2, as well as the PAR(2) agonists, trypsin, SLIGKV-NH2 and SLIGRL-NH2, caused contraction in both human and guinea-pig gallbladders. These indicate the existence of PAR(1) and PAR(2) mediating gallbladder contraction. Furthermore, the existence of PAR(1) and PAR(2) in the human gallbladder was confirmed by reverse transcription-polymerase chain reaction. In contrast, FSLLR-NH2, a PAR(1) control peptide, and VKGILS-NH2, a PAR(2) control peptide, as well as three PAR(4) agonists, GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2, did not cause any contraction or relaxation. The contractile responses to TFLLR-NH2, SFLLRN-NH2 and trypsin in both human and guinea-pig gallbladders were insensitive to atropine and tetrodotoxin, suggesting direct effects. These results demonstrate that, similar to the guinea-pig gallbladder, both PAR(1) and PAR(2) but not PAR(4) mediate muscle contraction in the human gallbladder. PAR(1) and PAR(2) may play important roles in the control of both human and guinea-pig gallbladder motility.

High concentrations of thrombin (Thr) have been linked to neuronal damage in cerebral ischemia and traumatic brain injury. In the present study we found that Thr markedly enhanced swelling-activated efflux of (3)H-glutamate from cultured astrocytes exposed to hyposmotic medium. Thr (0.5-5 U/mL) elicited small (3)H-glutamate efflux under isosmotic conditions and increased the hyposmotic glutamate efflux by 5- to 10-fold, the maximum effect being observed at 15% osmolarity reduction. These Thr effects involve its protease activity and are fully mimicked by SFFLRN, the synthetic peptide activating protease-activated receptor-1. Thr potentiation of (3)H-glutamate efflux was largely dependent on a Thr-elicited increases in cytosolic Ca(2+) (Ca(2+) (i)) concentration ([Ca(2+)](i)). Preventing Ca(2+) (i) rise by treatment with EGTA-AM or with the phospholipase C blocker U73122 reduced the Thr-increased glutamate efflux by 68%. The protein kinase C blockers Go6976 or chelerythrine reduced the Thr effect by 19%-22%, while Ca/calmodulin blocker W7 caused a 63% inhibition. In addition to this Ca(2+)-sensitive pathway, Thr effect on glutamate efflux also involved activation of phosphoinositide-3 kinase (PI3K), since it was reduced by the PI3K inhibitor wortmannin (51% inhibition). Treating cells with EGTA-AM plus wortmannin essentially abolished Thr-dependent glutamate efflux. Thr-activated glutamate release was potently inhibited by the blockers of the volume-sensitive anion permeability pathway, NPPB (IC(50) 15.8 microM), DCPIB (IC(50) 4.2 microM), and tamoxifen (IC(50) 6.6 microM. These results suggest that Thr may contribute to the excitotoxic neuronal injury by elevating extracellular glutamate release from glial cells. Therefore, this work may aid in search of neuroprotective strategies for treating cerebral ischemia and brain trauma., ((c) 2007 Wiley-Liss, Inc.)

Thrombin is a pivotal enzyme formed in the coagulation cascade and an important and potent platelet activator. The two protease-activated thrombin receptors on human platelets are denoted PAR1 and PAR4. The physiological relevance of PAR4 is still unclear, as both aggregation and secretion can be accomplished by PAR1 activation alone. In the present study we have investigated the role of PARs in platelet activation, blood coagulation, clot elasticity and fibrinolysis. Flow cytometry, free oscillation rheometry and thrombin generation measurements were used to analyze blood or platelet-rich plasma from healthy individuals. Maximum PAR1 activation with the peptide SFLLRN gave fewer fibrinogen-binding platelets with lower mean fluorescent intensity than maximum PAR4 activation with AYPGKF. Inhibition of any of the receptors prolonged clotting times. However, PAR1 is more important for fibrinolysis; inhibition of this receptor prolonged all the steps in the fibrinolytic process. Clot elasticity decreased significantly when the PAR4 receptor was inhibited. In the thrombin generation measurements, PAR4 inhibition delayed the thrombin generation start and peak, but did not affect the total amount of thrombin generated. PAR1 inhibition had no significant impact on thrombin generation. We found that PAR4 is most likely activated by low concentrations of thrombin during the initial phase of thrombin generation and is of importance to the clotting time. Furthermore, we suggest that the PAR4 receptor may have a physiological role in the stabilisation of the coagulum.

Serine proteinases, like trypsin, can play a hormone-like role by triggering signal transduction pathways in target cells. In many respects these hormone-like actions of proteinases can now be understood in terms of the pharmacodynamics of the G protein-coupled 'receptor' responsible for the cellular actions of thrombin (proteinase-activated receptor-1, or PAR1). PAR1, like the other three members of this receptor family (PAR2, PAR3 and PAR4), has a unique mechanism of activation involving the proteolytic unmasking of an N-terminally tethered sequence that can activate the receptor. The selective activation of each PAR by short synthetic peptides representing these sequences has demonstrated that PAR1, PAR2 and PAR4 play important roles in regulating physiological responses ranging from vasoregulation and cell growth to inflammation and nociception. We hypothesise that the tissue kallikreins may regulate signal transduction via the PARs. Although PARs can account for many of their biological actions, kallikreins may also cause effects by mechanisms not involving the PARs. For instance, trypsin activates the insulin receptor and thrombin can act via a mechanism involving its non-catalytic domains. Based on the data we summarise, we propose that the kallikreins, like thrombin and trypsin, must now be considered as important 'hormonal' regulators of tissue function.

Background: Thromboxane A2 (TXA2) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA2 generation., Results: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane-bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR-C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF- and thrombin-induced TXA2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA2 phosphorylation and TXA2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists., Conclusions: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA2 generation, which is potentiated by the P2Y12 receptor-mediated signaling pathway but not the P2Y1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.

A series of synthetic 2-morpholinochromones have been evaluated as inhibitors of platelet phosphodiesterase, PDE3A. While previous assertions about the anti-PDE3 activity of this class have been confirmed, in some cases the reported anti-platelet activities clearly derive from a non-PDE3 regulated mechanism. The potential utility of 2-morpholinochromones either as PDE3 inhibitors and/or anti-thrombotic agents thus remains only poorly examined.

We have previously reported that the contractile response to thrombin and trypsin was enhanced in the pregnant rat myometrium. We herein determined whether or not sex hormones contribute to this enhancement and the expression of protease-activated receptors (PARs). The nonpregnant rats received daily injections of either 17beta-estradiol or progesterone, and then the contractile response of the myometrium was examined ex vivo. Treatment with either 17beta-estradiol or progesterone had almost no significant enhancing effect on the high K(+)- or oxytocin-induced contraction. On the other hand, both 17beta-estradiol and progesterone dose-dependently enhanced the contractile response to trypsin. A maximal enhancement was obtained at 25 and 40 mg kg weight(-1) day(-1) for 17beta-estradiol and progesterone, respectively. The extent of the enhancement of the trypsin-induced contraction seen in the sex hormone-treated rats in the present study was comparable to that reported in the pregnant rats. However, the contractile response to thrombin and PAR1/PAR2-AP, SFLLRNP was not enhanced either by progesterone or 17beta-estradiol. PAR2-AP and PAR4-AP failed to induce contraction under any conditions. PAR1 mRNA was scarcely detected in the control myometrium by an RT-PCR analysis, while it slightly increased only in the progesterone-treated rats. Neither PAR2 nor PAR4 mRNA was detected. We thus conclude that the responsiveness to trypsin, but not thrombin, is controlled by sex hormones. A novel type of receptor, other than PAR1, PAR2 or PAR4, is suggested to mediate the trypsin-induced contraction as in the case of the pregnant rat myometrium.

Background: Circulating PLTs have a low activation state and high responsiveness, which ensures adequate hemostatic activity at sites of vessel wall damage. PLTs collected for transfusion purposes preferably have retained these properties to restore impaired hemostasis with thrombocytopenia., Study Design and Methods: We determined activation properties and coagulant activity of PLT-plasma preparations that were pooled or collected from single donors via apheresis., Results: In comparison to freshly isolated PLTs, both apheresis and pooled PLTs exhibited slow exposure of CD62 upon storage, followed by surface appearance of procoagulant phosphatidylserine (PS) but not activated integrin alpha IIb beta 3. During storage, thrombin- and ADP-induced Ca2+ signal generation consistently decreased in apheresis and pooled PLTs, which was accompanied by lower agonist-induced CD62 exposure and alpha IIb beta 3 activation. In flowing whole blood, stored apheresis PLTs showed lower collagen-induced Ca2+ responses and strikingly diminished participation in thrombus formation. Both apheresis and pooled PLT-plasma concentrates exhibited high tissue factor-triggered thrombin generation, which was insensitive to PLT inhibition and attributable to PS-exposing microparticles., Conclusion: PLTs stored in plasma develop surface activation markers but, simultaneously, show markedly decreased responsiveness toward physiologic agonists. The plasma contains high coagulant activity, which is no longer PLT (activation)-dependent.

Thrombin signaling in the endothelium is linked to multiple phenotypic changes, including alterations in permeability, vasomotor tone, and leukocyte trafficking. The thrombin signal is transduced, at least in part, at the level of gene transcription. In this review, we focus on the role of thrombin signaling and transcriptional networks in mediating downstream gene expression and endothelial phenotype. In addition, we report the results of DNA microarrays in control and thrombin-treated endothelial cells. We conclude that (1) thrombin induces the upregulation and downregulation of multiple genes in the endothelium, (2) thrombin-mediated gene expression involves a multitude of transcription factors, and (3) future breakthroughs in the field will depend on a better understanding of the spatial and temporal dynamics of these transcriptional networks.

Objective: Protease-activated receptors (PARs) mediate DNA synthesis in endothelial cells when activated by serine proteases. However, despite the existence of heterogeneity among endothelial cells from each tissue, the responses to PAR-1, PAR-2, and PAR-4 activation are poorly defined and compared between endothelial cells from different sites. The aim of this study was to investigate whether PAR-mediated DNA synthesis differed in various endothelial cell types., Methods: We examined the incorporation of BrdU by human pulmonary artery endothelial cells (HPAECs), human aortic endothelial cells (HAECs), and human umbilical vein endothelial cells (HUVECs)., Results: When the endothelial cells were treated with the selective PAR-1-activating peptide, SFLLRN, HAECs showed the highest BrdU incorporation rate (182 +/- 28%). In contrast, treatment with the PAR-2-activating peptide, SLIGKV, resulted in the highest BrdU incorporation rate (173 +/- 37%) in HPAECs, when pretreated with TNF-alpha. The PAR-4-activating peptide, GYPGQV, induced DNA synthesis in HPAECs and HAECs, but not in HUVECs., Conclusion: These findings suggest that each PAR preferentially targets an endothelial cell type, and thus plays a distinct role in diverse physiological or pathological conditions., (Copyright 2004 S. Karger AG, Basel)

Introduction: Platelets play an important role in the natural history of ischemic stroke, and are known to be activated in the acute phase. Although aspirin reduces risks of myocardial infarction, stroke and cardiovascular death, the extent of platelet action and the effect of aspirin on platelet function in patients recovering from stroke remain unclear., Methods: We studied 120 individuals divided into three equal groups: aspirin-free patients after ischemic stroke, post-stroke patients receiving aspirin (81-650 mg/daily), and aspirin-free subjects with multiple risk factors for vascular disease. Conventional platelet aggregation induced by 5 microM ADP and 5 microM epinephrine, cartridge-based analyzers (Ultegra, and PFA-100) readings, and expression of CD31, CD41a, CD42b, GPIIb/IIIa activity, CD51/CD61, CD62p, CD63, CD107a, CD154, CD165, formation of platelet-monocyte aggregates, intact (SPAN12), and cleaved (WEDE15) PAR-1 thrombin receptors by flow cytometry were analyzed., Results: There were no differences between aspirin-free post-stroke patients and aspirin-free controls. Although aggregation was slightly higher, 12 out of the 14 receptor analyses, were surprisingly lower in the post-stroke cohort. Aspirin-treated patients exhibited highly significant inhibition of epinephrine-induced aggregation (p=0.0001), prolongation of the closure time (p=0.03), and reduction of the aspirin reactive units (p=0.02) measured by the Ultegra device. In addition, surface platelet expression of thrombospondin (p=0.001), GPIIb/IIIa activity (p=0.04), P-selectin (p=0.03), CD40-ligand (p=0.04), CD165 (p=0.02), the formation of the platelet-monocyte aggregates (p=0.01), and intact epitope of PAR-1 thrombin receptor (p=0.03) were significantly lower in the aspirin-treated group., Conclusions: Platelets are not activated in aspirin-free patients after ischemic stroke. Platelet function is significantly inhibited in those treated with aspirin when compared with healthy subjects with risk factors for vascular disease. Bleeding complications and hemorrhagic transformations after aggressive antiplatelet regimens could be related to the decreased or normal baseline platelet characteristics in such patients. Further analysis of platelet heterogeneity and its clinical significance remains to be determined in randomized trials., (Copyright 2004 Elsevier Ltd.)

X-Ray crystal structures of four different fluorine-substituted phenylalanines (two mono- and two tri-substitutions) were analyzed to investigate the effect of fluorine atom on the association pattern of benzene rings. Although respective structures showed similar molecular packing in such a way that the layers of hydrophobic benzene rings and hydrophilic amino/carboxyl groups were alternately running along a crystallographic axis, the association patterns of benzene rings were different depending on the substitution position and number of fluorine atoms. The general features could be that the partially displaced face-to-face interactions are increased with increase in the number of fluorine atoms, whereas the edge-to-face interactions are decreased. The C-H bond next to a fluorine-substituted carbon atom could serve as a donor of an intermolecular C-H.F hydrogen bond.

Protease-activated receptor-2 (PAR-2) has been demonstrated to be highly expressed in the gastrointestinal tract. In the present study, we investigated the effects of PAR-2 stimulation on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line, in comparison with the PAR-1 stimulation. PAR-2 stimulation by agonist peptide SLIGKV concentration-dependently induced the increase in [Ca2+]i and the proliferation of DLD-1 whereas the inverse peptide LSIGKV did not. Trypin (10(-9) M), an agonist protease for PAR-2, also enhanced the proliferation of DLD-1. The proliferative response of DLD-1 to PAR-2 stimulation was associated with the transient phosphorylation of MEK and MAP kinase, but not p38 MAP kinase and JNK. Inhibition of MEK by PD98059 (50 microM) completely inhibited the proliferation-stimulating effects as well as the phosphorylation of MAP kinase induced by PAR-2 agonist peptide (100 microM) and trypsin (10(-9) M). The prolonged treatment with PAR-2 agonist peptide for more than one hour was required for the enhanced proliferative response, suggesting the existence of unknown long-lasting cooperative signaling with MAP kinase cascade. PAR-1 stimulation by the agonist peptide SFLLRN (100 microM) or thrombin (10(-8) M) produced Ca2+ signaling, however, the stimulation neither produced the cell proliferative response nor the activation of MEK-MAP kinase cascade. These results indicated that Ca2+ signaling induced by PARs activation was not enough for inducing the cell proliferation in DLD-1 cells and that stimulation of PAR-2 can induce the activation of MEK-MAP kinase cascade, leading to the growth promoting response.

A new generation of indole-based peptide mimetics, bearing a basic amine at the C-terminus, was developed by the agency of two complementary, multistep, trityl resin-based approaches. Thus, we obtained several high-affinity thrombin receptor (PAR-1) ligands, such as 32 and 34. Compounds 32 and 34 were found to bind to PAR-1 with excellent affinity (IC(50)=25 and 35 nM, respectively) and to effectively block platelet aggregation induced by SFLLRN-NH(2) (TRAP-6) and alpha-thrombin.

Investigations determined the mechanism(s) by which Arg-Pro-Pro-Gly-Phe (RPPGF) inhibits thrombin-induced platelet activation. High concentrations of RPPGF inhibit thrombin-induced coagulant activity. RPPGF binds to the active site of thrombin by forming a parallel beta-strand with Ser214-Gly216 and interacts with His57, Asp189, and Ser195 of the catalytic triad. RPPGF competitively inhibits alpha-thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with a Ki = 1.75 +/- 0.03 mM. Other mechanisms were sought to explain why RPPGF inhibits thrombin activation of platelets at concentrations below that which inhibits its active site. Soluble RPPGF blocks biotinylated NATLDPRSFLLR of the thrombin cleavage site on protease-activated receptor (PAR)1 from binding to the peptide RPPGC (IC50 = 20 microM). The soluble recombinant extracellular domain of PAR1 (rPAR1EC) blocks biotinylated RPPGF binding to rPAR1EC (IC50 = 50 microM) bound to microtiter plates, but rPAR1EC deletion mutants missing the sequence LDPR or PRSF do not. RPPGF and related forms prevent the thrombin-like enzyme thrombocytin from proteolyzing rPAR1EC at concentrations that do not block thrombocytin's active site. These studies indicate that RPPGF is a bifunctional inhibitor of thrombin: it binds to PAR1 to prevent thrombin cleavage at Arg41 and interacts with the active site of alpha-thrombin.

Functions of thrombin as a modulator of inflammation and tissue repair are mediated by the proteinase-activated receptor (PAR) family. Some of these effects may be induced by activation of mast cells. To characterize the degranulation of rat peritoneal mast cells in response to PAR agonists, the effects of thrombin, trypsin and peptide agonists of PARs (PAR-AP, proteinase-activated receptor-activating peptides) on secretion were investigated. The release of beta-hexosaminidase by thrombin (0.01-1 microM) was concentration-dependent and mediated via PAR(1), as evidenced by cathepsin G (100 microM)-induced inactivation of PAR(1) and thrombin-stimulated PAR(1) desensitization. Trypsin (1 microM) accelerated histamine secretion. The PAR(1)-AP, TRAP (SFFLRN, 1-100 microM) and the PAR(2)-AP SLIGRL (5-100 microM) caused the release of histamine, and beta-hexosaminidase from inflammatory mast cells were obtained from a model of acute peritonitis in rats. Relative to the response to compound 48/80, the thrombin- and TRAP-induced release of beta-hexosaminidase was higher in inflammatory mast cells than in the control. This suggests that additional exposure of PAR(1) on mast cells to PAR agonists or an increase in PARs sensitivity to PAR agonists probably occurred during acute inflammation.

Aspectrin-based skeleton uniformly underlies and supports the plasma membrane of the resting platelet, but remodels and centralizes in the activated platelet. alpha-Adducin, a phosphoprotein that forms a ternary complex with F-actin and spectrin, is dephosphorylated and mostly bound to spectrin in the membrane skeleton of the resting platelet at sites where actin filaments attach to the ends of spectrin molecules. Platelets activated through protease-activated receptor 1, FcgammaRIIA, or by treatment with PMA phosphorylate adducin at Ser726. Phosphoadducin releases from the membrane skeleton concomitant with its dissociation from spectrin and actin. Inhibition of PKC blunts adducin phosphorylation and release from spectrin and actin, preventing the centralization of spectrin that normally follows cell activation. We conclude that adducin targets actin filament ends to spectrin to complete the assembly of the resting membrane skeleton. Dissociation of phosphoadducin releases spectrin from actin, facilitating centralization of spectrin, and leads to the exposure of barbed actin filament ends that may then participate in converting the resting platelet's disc shape into its active form.

The traditional view on the role of serine proteases in tumor biology has changed with the recent discovery of a family of protease-activated receptors (PARs). In this study we explored the expression and functional role of the thrombin receptor PAR-1 in human colon cancer cells. Reverse transcriptase-polymerase chain reaction analysis showed that PAR-1 mRNAs are present in 11 of 14 human colon cancer cell lines tested but not in normal human colonic epithelial cells. This is in line with the immunolocalization of PAR-1 in human colon tumors and its absence in normal human colonic mucosa. The functional significance of the aberrant expression of PAR-1 in colon cancer cells was then investigated. We found that 1) a prompt increase in intracellular calcium concentration was observed on thrombin (10 nmol/L) or PAR-1 agonist AP1 (100 micro mol/L) challenge of HT29 cells; 2) HT29 quiescent cells treated with thrombin (0.01 to 20 nmol/L) or AP1 (1 to 300 micro mol/L) exhibited dramatic mitogenic responses (3.5-fold increase in cell number). Proliferative effects of thrombin or AP1 were also observed in other colon cancer cell lines expressing PAR-1. This effect was reversed by the MEK inhibitor PD98059 in consonance with the ability of thrombin or AP1 to induce phosphorylation of p42/p44 extracellular-regulated protein kinases. 3) PAR-1 activation by thrombin or AP1 led to a two-fold increase in cell motility of wounded HT29-D4. Our results demonstrate for the first time the aberrant expression of the functional thrombin receptor PAR-1 in colon cancers and its important involvement in cell proliferation and motility. Thrombin should now be considered as a growth factor for human colon cancer.

Thrombin-induced platelet activation involves cleavage of protease-activated receptors (PARs) 1 and 4, and interaction, via glycoprotein (Gp)Ibalpha, with the platelet GpIb/IX/V complex. This study investigated inhibition of platelet activation by thrombin inhibitors with different modes of action: two reversible direct thrombin inhibitors, melagatran and inogatran; hirudin, a tightly binding direct thrombin inhibitor; and two indirect thrombin inhibitors, heparin and dalteparin. Up-regulation of P-selectin (CD62P) and PAR-1 cleavage was measured in human whole blood, by flow cytometry. The thrombin concentration that induced 50% of maximum (EC50 ) PAR-1 cleavage was 0.028 nmol/l, while that of platelet activation (CD62P) was over two-fold higher (0.64 nmol/l). The EC50 of a PAR-1-independent component, defined as a further activating effect of thrombin on top of the maximum PAR-1-activating peptide (AP) effect, was 3.2 nmol/l. All anticoagulants were concentration-dependent inhibitors of thrombin-induced platelet activation and PAR-1 cleavage, but none inhibited PAR-1-AP or PAR-4-AP induced activation. Melagatran and inogatran were more potent inhibitors of CD62P up-regulation than of PAR-1 cleavage; conversely, hirudin, heparin and dalteparin were more potent inhibitors of PAR-1 cleavage.Thus, reversible direct thrombin inhibitors, such as melagatran, are potent inhibitors of thrombin-induced platelet activation, acting mainly by inhibition of a PAR-1-independent component.

It has been shown that human airway trypsin-like protease (HAT) is localized in human bronchial epithelial cells (HBEC), and trypsin activates protease-activated receptor-2 (PAR-2). Activation of PAR-2 activates G-protein followed by an increase of intracellular free Ca2+, [Ca2+]in. This study was undertaken to clarify whether HAT can activate PAR-2 in HBEC or not. RT-PCR showed that HAT mRNA is expressed in HBEC, and PAR-2 mRNA is the most strongly expressed of the known PARs in HBEC. Both PAR-2 agonist peptide (PAR-2 AP) and HAT increased [Ca2+]in in HBEC in a biphasic fashion; a prompt, sharp increase (peak I) and a sustained low plateau (peak II). PAR-2 AP over 100-200 microM and HAT over 200-300 mU/ml (0.08-0.12 microM) induced both peak I and II, and PAR-2 AP below 100 microM and HAT below 200 mU/ml induced only peak II. Both PAR-2 AP-induced and HAT-induced peak I were induced by Ca2+ mobilization from intracellular stores, because they appeared even in Ca2+-free medium. Both PAR-2 AP-induced and HAT-induced peak II were induced by an influx of extracellular Ca2+, because they were abolished in Ca2+-free medium. The Ca2+ response to HAT was desensitized by exposure of HBEC to PAR-2 AP. These results indicate that HBEC have a functional PAR-2, and HAT regulates cellular functions of HBEC via activation of PAR-2.

Purpose: Platelet-endothelial cell adhesion is an important pathologic response to vessel injury or inflammation. On binding to its endothelial or platelet G protein-linked seven-transmembrane domain receptor, protease-activated receptor-1 (PAR1), thrombin releases a 41-amino acid peptide (TR(1-41)). We examined the effect of TR(1-41) on platelet activation and on platelet-endothelial cell adhesion., Methods: A monolayer of confluent human saphenous vein endothelial cells was incubated with washed human platelets. Platelets were stimulated with either TR(1-41), TR(21-41), scrambled TR(1-41), adenosine diphosphate (ADP)-epinephrine (EPI), thrombin, or thrombin receptor activating peptide (TRAP). Platelet activation was identified with flow cytometry. The magnitude of platelet-endothelial cell adhesion was determined with a laser scanning cytometer that scanned the monolayer of endothelial cells and identified fluorescently bound platelets., Results: Maximal thrombin stimulation (0.1 U/mL) induced a threefold increase in platelets bound to endothelial cells compared with buffer alone. Stimulation with TR(1-41) (20 mmol/L) tripled the number of platelets bound to endothelial cells compared with thrombin. Scrambled sequence of TR(1-41) (20 mmol/L) and TR(21-41) (20 mmol/L), neither of which induces platelet activation, had minimal effect on platelet adhesion. Both TRAP (20 mmol/L) and ADP-EPI (20 mmol/L) induced less platelet-endothelial cell adhesion than did thrombin. TR(1-41)-induced platelet-endothelial cell adhesion was partially blocked by glycoprotein (GP)IIb-IIIa-specific monoclonal antibody, 10E5 (10 mg/mL)., Conclusions: TR(1-41), the cleaved peptide of PAR1, is a more potent stimulant of platelet-endothelial cell adhesion than is thrombin, TRAP, or ADP-EPI, and this adhesion is at least in part mediated by the platelet GPIIb-IIIa receptor.

We have analysed the role of N-linked glycosylation in regulating human proteinase-activated receptor-2 (hPAR(2)) expression and function. Epitope-tagged wild-type hPAR(2) (wt-hPAR(2)) or hPAR(2) that lacked glycosylation sequons (following site-directed mutagenesis) in either the N-terminus [hPAR(2)N30A (Asn(30)-->Ala)], extracellular loop 2 [ECL2; hPAR(2)N222Q (Asn(222)-->Gln) or hPAR(2)N222A (Asn(222)-->Ala)] or both (hPAR(2)N30A,N222A or hPAR(2)N30A,N222Q) were expressed in the Chinese-hamster ovary (CHO) fibroblast cell line, Pro5. Western blot analysis of wt-hPAR(2) showed mature wt-hPAR(2) to have a molecular mass of 55-100 kDa, and 33-48 kDa following N -glycosidase F deglycosylation. FACS analysis and immunocytochemistry of the wt-hPAR(2) and PAR(2) mutant cell lines revealed that removal of both glycosylation sequons decreases (50% of wt-hPAR(2)) cell surface expression. Western blot analysis indicated that both N-linked sites are glycosylated. In functional studies, hPAR(2)N30A displayed a selective and significant increase in sensitivity towards tryptase. Interestingly, hPAR(2)N222A displayed a loss in sensitivity towards all PAR(2) agonists tested. However, further analysis revealed receptor sensitivity to alanine mutations in this domain, as the more conservative substitution hPAR(2)N222Q displayed no change in response to PAR(2) agonists. hPAR(2)N30A,N222Q displayed increased sensitivity towards tryptase, but a loss in sensitivity towards trypsin and the synthetic peptide SLIGRL-NH(2), although this loss in sensitivity towards trypsin and SLIGRL-NH(2) was secondary to changes in cell-surface expression. Finally, expression of sialic-acid-deficient wt-hPAR(2) in the CHO Lec2 glycosylation-deficient mutant cell line, showed a 40 kDa loss in molecular mass, in addition to a marked and selective increase in sensitivity towards tryptase. We conclude that hPAR(2) N-linked glycosylation and sialylation regulates receptor expression and/or signalling.

Inflammation is a process culminated by cellular components and soluble mediators act in concert to evolve a sustainable process to impart distress in vascularized tissue. The role of thrombin receptors in neutrophil degranulation is not fully understood. Thrombin-mediated signaling has been shown to occur at least in part by a family of G protein-coupled protease-activated receptors (PARs). Protease-activated receptors-1 (PAR1) is the prototype for understanding on the fundamental signaling mechanism and related characterization. The inference of thrombin receptors and various synthetic ligand formulations on neutrophil degranulation ascertained an enormous disparity in the response by human donors. Based on the previous reports and supposition in perpetuity, it is hypothesized that thrombin through its receptor, and its subtypes, may very improbably have any functional role in PMN degranulation.

Activation of rat proteinase-activated receptor 2 (PAR2) by trypsin involves the unmasking of the tethered sequence S(37)LIGRL(42) that either tethered or on its own as a free peptide, activates PAR2. We aimed to determine whether different peptide sequences acting either as trypsin-revealed tethered ligands or as soluble peptides had the same relative activities for triggering the receptor. A comparison was also made between the different soluble and tethered receptor activating sequences in receptor constructs with extracellular loop 2 (ECL2) residues E(232)E(233) (PAR2SR/EE) mutated to R(232)R(233) (PAR2SR/RR). Using site-directed mutagenesis, we prepared PAR2 constructs with trypsin-revealed tethered ligand sequences corresponding to the synthetic receptor-activating peptides (PAR2APs): SLIGRL-NH(2) (SR-NH(2)), SLIGAL-NH(2) (SA-NH(2)), and SLIGEL-NH(2) (SE-NH(2)). Kirsten virus-transformed rat kidney cells stably expressing 1) wild-type PAR2 with site-mutated tethered ligands (PAR2SA/EE and PAR2SE/EE); 2) wild-type PAR2 with ECL2 mutated to R(232)R(233) (PAR2SR/RR); and 3) PAR2 constructs with both the RR mutation in ECL2 and a mutation in the tethered ligand (PAR2SA/RR and PAR2SE/RR) were assessed for receptor-mediated calcium signaling and cell growth inhibition, upon activation either by trypsin or the above-mentioned PAR2APs. Trypsin exerted equivalent and full agonist activity on the PAR2 constructs, causing a maximum response between 20 to 80 nM. In contrast, the PAR2APs as free peptide agonists showed marked potency differences in all wild-type receptors with mutated tethered ligands (SR-NH(2) >> SA-NH(2) >> SE-NH(2)) and in all ECL2 RR mutated constructs (SE-NH(2) > SR-NH(2) >> SA-NH(2)). We conclude that for receptor activation, the trypsin-revealed PAR2 tethered ligand sequence interacts differently for receptor activation than does the same peptide sequence as a free peptide.

Protease-activated receptors (PARs) are G-protein-coupled, seven transmembrane domain receptors that act as cellular enzyme sensors. These receptors are activated by the proteolytic cleavage at the amino terminus, enabling interaction between the newly formed "tethered ligand" and the second extracellular loop of the receptor to confer cellular signalling. PARs can also be activated by small peptides that mimic the tethered ligand. In the respiratory tract, PARs may be regulated by endogenous proteases, such as airway trypsin and mast cell tryptase, as well as exogenous proteases, including inhaled aeroallergens such as those from house dust mite faecal pellets. Immunoreactive PARs have been identified in multiple cell types of the respiratory tract, and PAR activation has been reported to stimulate cellular mitogenesis and to promote tissue inflammation. Activation of PARs concurrently stimulates the release of bronchorelaxant and anti-inflammatory mediators, which may serve to induce cytoprotection and to minimise tissue trauma associated with severe chronic airways inflammation. Furthermore, airway inflammatory responses are associated with increased epithelial PAR expression and elevated concentrations of PAR-activating, and PAR-inactivating, proteases in the extracellular space. On this basis, PARs are likely to play a regulatory role in airway homeostasis, and may participate in respiratory inflammatory disorders, such as asthma and chronic obstructive pulmonary disease. Further studies focussing on the effects of newly developed PAR agonists and antagonists in appropriate models of airway inflammation will permit better insight into the role of PARs in respiratory pathophysiology and their potential as therapeutic targets.

To develop new therapeutic strategies we examined the expression and signaltransduction of G protein-coupled receptors in a human transitional cell carcinoma cell line. The receptors for lysophosphatidic acid (LPA) and thrombin potently stimulate cell migration. Pretreatment with PTX completely inhibited cell motility induced by LPA. In the model of chemically induced bladder carcinoma in rats the effects of intravesical instillation of PTX or phosphate buffered saline was examined. The incidence of G2-G3 cells in cytology was significantly reduced in rats treated with PTX. To ascertain the side effects of intravesical instillation of PTX a Phase I study was initiated. 15 patients were instilled with PTX at 5 dose levels (14-72 micrograms/100 ml) 3 times a week. Instillation of PTX up to 72 micrograms was without local or systemic side effects. PTX is a substance which potently inhibits tumor cell motility and progression. Intravesical treatment was well tolerated and therefore, the influence of PTX on local tumor should be evaluated in a Phase II study.

Protease-activated receptor-2 (PAR-2) plays a role in inflammatory reactions in airway physiology. Proteases cleaving the extracellular NH(2) terminus of receptors activate or inactivate PAR, thus possessing a therapeutic potential. Using RT-PCR and immunocytochemistry, we show PAR-2 in human airway epithelial cell lines human bronchial epithelial (HBE) and A549. Functional expression of PAR-2 was confirmed by Ca(2+) imaging studies using the receptor agonist protease trypsin. The effect was abolished by soybean trypsin inhibitor and mimicked by the specific PAR-2 peptide agonist SLIGKV. Amplitude and duration of PAR-2-elicited Ca(2+) response in HBE and A549 cells depend on concentration and time of agonist superfusion. The response is partially pertussis toxin (PTX) insensitive, abolished by the phospholipase C inhibitor U-73122, and diminished by the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Cathepsin G altered neither the resting Ca(2+) level nor PAR-2-elicited Ca(2+) response. Thermolysin, a prototypic bacterial metalloprotease, induced a dose-dependent Ca(2+) response in HBE, but not A549, cells. In both cell lines, thermolysin abolished the response to a subsequent trypsin challenge but not to SLIGKV. Thus different epithelial cell types express different PAR-2 with identical responses to physiological stimuli (trypsin, SLIGKV) but different sensitivity to modifying proteases, such as thermolysin.

Proteinase-activated receptors (PARs) are a novel class of G protein-coupled receptors that respond to signals through endogenous proteinases. PAR activation involves enzymatic cleavage of the extracellular NH(2)-terminal domain and unmasking of a new NH(2) terminus, which serves as an anchored ligand to activate the receptor. At least four PAR subtypes have been identified. In the present study, we used the in vitro perfused hydronephrotic rat kidney to examine the effects of activating PAR-2 on the afferent arteriole. The synthetic peptide SLIGRL-NH(2), which corresponds to the exposed ligand sequence and selectively activates PAR-2, did not alter basal afferent arteriolar diameter but caused a concentration-dependent vasodilation (3-30 microM) of arterioles preconstricted by angiotensin II (0.1 nM). A modified peptide sequence (LSIGRL-NH(2), inactive at PAR-2) had no effect. This vasodilation was characterized by an initial transient component followed by a smaller sustained response. A similar pattern of vasodilation was seen when SLIGRL-NH(2) was administered to isolated perfused normal rat kidney. The sustained component of the PAR-2-induced afferent arteriolar vasodilation was eliminated by nitric oxide (NO) synthase inhibition (100 microM nitro-L-arginine methyl ester). In contrast, the transient vasodilation persisted under these conditions. This transient response was not observed when afferent arterioles were preconstricted with elevated KCl, suggesting involvement of an endothelium-derived hyperpolarizing factor. Finally, RT-PCR revealed the presence of PAR-2 mRNA in isolated afferent arterioles. These findings indicate that PAR-2 is expressed in the afferent arteriole and that its activation elicits afferent arteriolar vasodilation by NO-dependent and NO-independent mechanisms.

Activation of protease-activated receptor (PAR)-2 has been proposed to be protective in myocardial ischemia/reperfusion (I/R) injury, an effect possibly related to an action on the coronary vasculature. Therefore, we investigated the effects of PAR2 activation on coronary tone in isolated perfused rat hearts and elucidated the mechanisms of any observed effects. Although having a negligible effect on ventricular contractility, the PAR2 activating peptide SLIGRL produced an endothelium-dependent coronary vasodilatation (ED(50)=3.5 nmol). Following I/R injury, the response to SLIGRL was selectively preserved, whereas the dilator response to acetylcholine was converted to constriction. Trypsin also produced a vasodilator dose-response curve that was biphasic in nature (ED(50-1)=0.36 U, ED(50-2)=38.71 U). Desensitization of PAR2 receptors indicated that the high potency phase was mediated by PAR2. Removal of the endothelium but not treatment with L-NAME (300 micromol/L), indomethacin (5 micromol/L), or oxyhemoglobin (10 micromol/L) inhibited the response to SLIGRL and trypsin. Treatment with the K(+)-channel blockers TEA (10 mmol/L), charybdotoxin (20 nmol/L)/apamin (100 nmol/L), or elevated potassium (20 mmol/L) significantly suppressed responses. Similarly, inhibition of lipoxygenase with nordihydroguaiaretic acid (1 micromol/L), eicosatetraynoic acid (1 micromol/L), or baicalein (10 micromol/L), desensitization of C-fibers using capsaicin (1 micromol/L, 20 minutes), or blockade of vanilloid (VR1) receptors using capsazepine (3 micromol/L) inhibited the responses. This study shows, for the first time, that PAR2 activation causes endothelium-dependent coronary vasodilation that is preserved after I/R injury and is not mediated by NO or prostanoids, but involves the release of an endothelium-derived hyperpolarizing factor (EDHF), possibly a lipoxygenase-derived eicosanoid, and activation of VR1 receptors on sensory C-fibers.

Classical ligands bind to the extracellular surface of their cognate receptors and activate signaling pathways without crossing the plasma membrane barrier. We selectively targeted the intracellular receptor-G protein interface by using cell-penetrating membrane-tethered peptides. Attachment of a palmitate group to peptides derived from the third intracellular loop of protease-activated receptors-1 and -2 and melanocortin-4 receptors yields agonists and/or antagonists of receptor-G protein signaling. These lipidated peptides--which we have termed pepducins--require the presence of their cognate receptor for activity and are highly selective for receptor type. Mutational analysis of both intact receptor and pepducins demonstrates that the cell-penetrating agonists do not activate G proteins by the same mechanism as the intact receptor third intracellular loop but instead require the C-tail of the receptor. Construction of such peptide-lipid conjugates constitutes a new molecular strategy for the development of therapeutics targeted to the receptor-effector interface.

Progestogens are used clinically for contraception, to control excessive menstrual bleeding and to oppose oestrogen in hormone replacement therapy. The use of intrauterine levonorgestrel (LNG) is however, associated with endometrial atrophy and decidualisation of the stroma. In this study, we aimed to identify genes whose expression is modulated by LNG either alone or in combination with progesterone. Thus endometrial stromal cells were stimulated with progesterone, LNG or LNG and progesterone. Poly-A RNA was isolated and used to probe expression arrays. The expression of a number of genes was altered on exposure to LNG or LNG and progesterone. Alteration of expression patterns was confirmed using semi-quantitative RT-PCR and western blot analysis. In particular, the protease activated receptor-1 (PAR-1) gene that encodes a receptor for thrombin was down regulated. This is the first demonstration that PAR-1 is down regulated by the progestogen LNG in human endometrium. Alteration in the expression levels of this receptor may affect both growth and haemostatic activity within the endometrium and may account for the observed morphological effects seen in users of intrauterine LNG delivery devices.

Protease-activated receptors (PARs) are newly identified members of the superfamily of G-protein-coupled receptors that initiate cell signaling by the proteolytic activity of extracellular serine proteases. Certain proteases are believed to be involved in development and repair processes and most likely regulate multiple functions of the CNS by activating PARs. Three members of this family (PAR-1, PAR-3, and PAR-4) are considered thrombin receptors, whereas PAR-2 is activated by trypsin. In the present study, using reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and Ca(2+) mobilization studies, we demonstrate that PAR-1, PAR-2, PAR-3, and PAR-4 are functionally co-expressed in cultured rat astrocytes. Short-term stimulation of astrocytes with thrombin, trypsin, and peptides corresponding to the tethered ligand domains of PAR-1, PAR-2, PAR-3, and PAR-4 induced a transient rise of [Ca(2+)](i) in cultured astrocytes. In studying calcium signaling, based on receptor desensitization, and using an antagonist of thrombin receptor PAR-1, we provide evidence that the thrombin-induced [Ca(2+)](i) response in astrocytes in addition to PAR-1 stimulation, involves also stimulation of PAR-3 and PAR-4. Trypsin, in addition to PAR-2, can also activate PAR-1 and PAR-4. Furthermore we find that activation of PAR-1, and PAR-2 induces proliferation of astrocytes while PAR-4 activation exerts toxic effects. This study is the first to show that (1) cultured astrocytes functionally express PAR-3 and PAR-4 together with PAR-1 and PAR-2; (2) PAR-3-activating peptide (TFRGAP) is effective in eliciting Ca(2+) signaling; and (3) activation of different PARs leads to distinct downstream effects., (Copyright 2002 Wiley-Liss, Inc.)