Aspirin Inhibits the Inflammatory Response of Protease-Activated Receptor 1 in Pregnancy Neutrophils: Implications for Treating Women with Preeclampsia.

Neutrophils expressing cyclooxygenase-2 (COX-2) extensively infiltrate maternal blood vessels in preeclampsia, associated with vascular inflammation. Because pregnancy neutrophils also express protease-activated receptor 1 (PAR-1, F2R thrombin receptor), which they do not in non-pregnant subjects, they can be activated by proteases. We tested the hypothesis that aspirin at a dose sufficient to inhibit COX-2 would reduce inflammatory responses in preeclampsia neutrophils. Neutrophils were isolated from normal pregnant and preeclamptic women at approximately 30 weeks' gestation. Normal pregnancy neutrophils were treated with elastase, a protease elevated in preeclampsia, or elastase plus aspirin to inhibit COX-2, or elastase plus pinane thromboxane, a biologically active structural analog of thromboxane and a thromboxane synthase inhibitor. Preeclamptic pregnancy neutrophils were treated with the same doses of aspirin or pinane thromboxane. Confocal microscopy with immunofluorescence staining was used to determine the cellular localization of the p65 subunit of nuclear factor-kappa B (NF-κB) and media concentrations of thromboxane were measured to evaluate the inflammatory response. In untreated neutrophils of normal pregnant women, p65 was localized to the cytosol. Upon stimulation with elastase, p65 translocated from the cytosol to the nucleus coincident with increased thromboxane production. When neutrophils were co-treated with aspirin or pinane thromboxane, elastase was not able to cause nuclear translocation of p65 or increase thromboxane. In untreated neutrophils of preeclamptic women, the p65 subunit was present in the nucleus and thromboxane production was elevated, but when preeclamptic neutrophils were treated with aspirin or pinane thromboxane, p65 was cleared from the nucleus and returned to the cytosol along with decreased thromboxane production. These findings suggest that COX-2 is a downstream mediator of PAR-1 and demonstrate that PAR-1- mediated inflammation can be inhibited by aspirin. Given the extensive and ubiquitous expression of PAR-1 and COX-2 in preeclamptic women, consideration should be given to treating women with preeclampsia using a dose of aspirin sufficient to inhibit COX-2.

Factor Xa Inhibition with Apixaban Does Not Influence Cardiac Remodelling in Rats with Heart Failure After Myocardial Infarction.

BACKGROUND: Heart failure (HF) is considered to be a prothrombotic condition and it has been suggested that coagulation factors contribute to maladaptive cardiac remodelling via activation of the protease-activated receptor 1 (PAR1). We tested the hypothesis that anticoagulation with the factor Xa (FXa) inhibitor apixaban would ameliorate cardiac remodelling in rats with HF after myocardial infarction (MI). METHODS AND RESULTS: Male Sprague-Dawley rats were either subjected to permanent ligation of the left ascending coronary artery (MI) or sham surgery. The MI and sham animals were randomly allocated to treatment with placebo or apixaban in the chow (150 mg/kg/day), starting 2 weeks after surgery. Cardiac function was assessed using echocardiography and histological and molecular markers of cardiac hypertrophy were assessed in the left ventricle (LV). Apixaban resulted in a fivefold increase in anti-FXa activity compared with vehicle, but no overt bleeding was observed and haematocrit levels remained similar in apixaban- and vehicle-treated groups. After 10 weeks of treatment, LV ejection fraction was 42 ± 3% in the MI group treated with apixaban and 37 ± 2 in the vehicle-treated MI group (p > 0.05). Both vehicle- and apixaban-treated MI groups also displayed similar degrees of LV dilatation, LV hypertrophy and interstitial fibrosis. Histological and molecular markers for pathological remodelling were also comparable between groups, as was the activity of signalling pathways downstream of the PAR1 receptor. CONCLUSION: FXa inhibition with apixaban does not influence pathological cardiac remodelling after MI. These data do not support the use of FXa inhibitor in HF patients with the aim to amend the severity of HF. Graphical Abstract.

Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking.

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.

Signaling mechanism of protease activated receptor 1-induced proliferation of astrocytes: stabilization of hypoxia inducible factor-1α triggers glucose metabolism and accumulation of cyclin D1.

Thrombin was found to stimulate astrocytes proliferation. In this study, we want to clarify whether thrombin-activated protease-activated receptor will affect the glucose metabolism signaling pathways to accelerate the proliferation of astrocytes. In addition, we study if thrombin has effects on cell cycle transition to promote astrocytes proliferation. We firstly observed that thrombin activated protease-activated receptor 1 (PAR-1) inducing the increases of intracellular Ca(2+) and ROS production, which contribute to the astrocytes' proliferation. We further confirmed that ROS stabilized HIF-1α, the latter subsequently accelerated glucose uptake in astrocytes. On the other hand, we demonstrated that thrombin triggered PI3K/Akt/cyclin D1 signal transduction, which may promote the cell cycle transition to enhance astrocytes proliferation. As a result, we discovered three signaling pathways mainly accounting for cell proliferation induced by thrombin: (1) thrombin-stimulated ERK, JNK/ROS/HIF-1α and (2) PI3K/Akt/ROS/HIF-1α pathways to increase expression of hexokinase 2 which mediated glucose metabolism in astrocytes, and (3) thrombin stimulates PAR-1/PI3K/Akt/cyclin D1 to promote the cell cycle transition and finally to increase cell proliferation.

Active proteases in nephrotic plasma lead to a podocin-dependent phosphorylation of VASP in podocytes via protease activated receptor-1.

Focal segmental glomerulosclerosis (FSGS) is associated with glomerular podocyte injury. Podocytes undergo dramatic changes in their actin structure, with little mechanistic insight to date into the human disease. Post-transplantation recurrence of FSGS is the archetypal form of the disease caused by unknown circulating plasma 'factors'. There is increasing indication that plasma protease activity could be central to this disease. Using clinical plasma exchange material, collected from patients in relapse and remission stages of disease, the effects of FSGS plasma on human conditionally immortalized podocytes (ciPods) were studied. We show that vasodilator stimulated phosphoprotein (VASP) is phosphorylated in response to relapse plasma from ten consecutively tested patients, and not in response to paired remission plasma or non-FSGS controls. The phosphorylation signal is absent in human podocytes carrying a pathological podocin mutation. To test for a plasma ligand, inhibition of proteases in relapse plasma leads to the loss of VASP phosphorylation. By the use of siRNA technology, we show that proteases in the plasma signal predominantly via protease activated receptor-1 (PAR1) to VASP. Mechanistically, FSGS plasma increases podocyte motility, which is dependent on VASP phosphorylation. These data suggest a specific biomarker for disease activity, as well as revealing a novel and highly specific receptor-mediated signalling pathway to the actin cytoskeleton.

Proteinase-activated receptors 1 and 2 exert opposite effects on renal renin release.

Proteinase-activated receptors (PARs) 1 to 4 are highly expressed in the kidney and are involved in the regulation of renal hemodynamics and tubular function. Since intravascular infusion of the proteinase thrombin, which activates PARs, has been shown to decrease plasma renin activity in rats, we investigated the effects of the respective PAR subtypes on renin release using the isolated perfused mouse kidney model. Thrombin dose-dependently reduced perfusate flow and inhibited renin secretion rates (RSRs) that had been prestimulated by the ß-adrenoreceptor agonist isoproterenol. The suppression of RSRs was prevented by the selective PAR1 inhibitor SCH79797, and direct activation of PAR1 by TFLLR mimicked the effects of thrombin on RSRs and vascular tone. Moreover, TFLLR suppressed the stimulations of RSRs in response to the loop diuretic bumetanide, to prostaglandin E(2), or to a decrease in renal perfusion pressure but not in response to a reduction in extracellular calcium. The PAR2-activating peptide SLIGRL concentration dependently increased RSR and perfusate flow. The stimulation of RSRs by SLIGRL was markedly attenuated by N(G)-nitro-L-arginine methyl ester, suggesting an NO-dependent mechanism. Activation of PAR4 by AYPGKF did not modulate RSRs or perfusate flow. PAR1 and PAR2 immunoreactivity were detected in the juxtaglomerular region and were colocalized with renin immunoreactivity. Our data provide evidence that PAR1 activation inhibits renal renin secretion and induces renal vasoconstriction, whereas PAR2 activation stimulates renin release and induces vasodilation mainly via the release of NO.

PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease-activated receptor-1 but not protease-activated receptor-4 in human platelets.

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.

[Activated protein C, a protein at the crossroads between coagulation and inflammation]. / La protéine C activée: une protéine à l'interface de l'inflammation et de la coagulation.

Sepsis is defined as a systemic response to infection, characterized by an intense inflammatory response linked to coagulation activation and fibrinolysis inhibition, two processes which are intimately associated. In a field where mortality remains very high, administration of activated protein C, a physiological coagulation inhibitor with cytoprotective properties, has demonstrated its effectiveness and was able to reduce mortality. Protein C belongs to a system that involves plasma proteins and endothelial cell receptors. In addition to well documented effects on coagulation and fibrinolysis, activated protein C exhibits anti-inflammatory, anti-apoptotic but also anti-histone activities. Indeed, a recent study focusing on the cytoprotective effects of activated protein C showed that extracellular histones are released during severe sepsis and may participate in the pathophysiology of severe sepsis. These histones appear to be new targets of activated protein C.

Statins prevent tissue factor induction by protease-activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro.

BACKGROUND: Protease-activated receptors (PARs) are G-protein-coupled receptors that function in hemostasis and thrombosis, as well as in the inflammatory and proliferative responses triggered by tissue injury. We have previously shown that PAR1 or PAR2 occupancy by specific PAR-agonist peptides (PAR-APs) induces tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs), where TF regulation by PAR1 (but not by PAR2) requires intact endothelial caveolin-enriched membrane microdomains in which PAR1 and caveolin-1 associate. OBJECTIVES: The aim of this study was to determine the effects of cholesterol-lowering agents (statins) and cholesterol-loading lipoprotein on PAR1-AP-mediated and PAR2-AP-mediated TF induction in HUVECs. RESULTS: Statins completely prevented TF induction by PAR-APs in an isoprenoid-independent manner, induced the delocalization of PAR1 from caveolin-enriched membrane microdomains without affecting PAR1 mRNA, and decreased PAR2 mRNA and protein levels. Statins also prevented PAR-AP-mediated extracellular signal-related kinase 1/2 activation, which is crucial for TF induction. The redistribution of PAR1 is accompanied by the relocation of the membrane microdomain-associated G-protein α, caveolin-1, and Src, which we previously showed to play a key role in signal transduction and TF induction. Conversely, cholesterol loading potently amplified PAR1-AP-induced TF, probably as a result of the increased abundance of PAR1 and the Src and G-protein α signaling molecules in the caveolin-1-enriched fraction, without affecting PAR1 mRNA. CONCLUSIONS: As PARs have important functions in hemostasis, cancer, thrombosis, and inflammatory processes, our findings that statins prevent TF induction by PAR-APs altering the membrane localization of PAR1 and the expression of PAR2 suggest that they may provide health benefits other than reducing atherosclerosis.

Phosphatidylserine exposure and other apoptotic-like events in Bernard-Soulier syndrome platelets.

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.

Signal transduction by protease-activated receptors.

The family of G protein-coupled receptors (GPCRs) constitutes the largest class of signalling receptors in the human genome, controlling vast physiological responses and are the target of many drugs. After activation, GPCRs are rapidly desensitized by phosphorylation and beta-arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and beta-arrestin-dependent pathway and then recycled back to the cell surface or sorted to lysosomes for degradation. Given the vast number and diversity of GPCRs, different mechanisms are likely to exist to precisely regulate the magnitude, duration and spatial aspects of receptor signalling. The G protein-coupled protease-activated receptors (PARs) provide elegant examples of GPCRs that are regulated by distinct desensitization and endocytic sorting mechanisms, processes that are critically important for the spatial and temporal fidelity of PAR signalling. PARs are irreversibly activated through proteolytic cleavage and transmit cellular responses to extracellular proteases. Activated PAR(1) internalizes through a clathrin- and dynamin-dependent pathway independent of beta-arrestins. Interestingly, PAR(1) is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, beta-arrestins mediate activated PAR(2) internalization and function as scaffolds that promote signalling from endocytic vesicles. Moreover, activated PAR(2) is modified with ubiquitin, which facilitates lysosomal degradation. Activated PARs also adopt distinct active conformations that signal to diverse effectors and are likely regulated by different mechanisms. Thus, the identification of the molecular machinery important for PAR signal regulation will enable the development of new strategies to manipulate receptor signalling and will provide novel targets for the development of drugs.

Thrombin activation of proteinase-activated receptor 1 potentiates the myofilament Ca2+ sensitivity and induces vasoconstriction in porcine pulmonary arteries.

BACKGROUND AND PURPOSE: Thrombus formation is commonly associated with pulmonary arterial hypertension (PAH). Thrombin may thus play an important role in the pathogenesis and pathophysiology of PAH. Hence, we investigated the contractile effects of thrombin and its mechanism in pulmonary artery. EXPERIMENTAL APPROACH: The cytosolic Ca(2+) concentrations ([Ca(2+)](i)), 20 kDa myosin light chain (MLC20) phosphorylation and tension development were evaluated using the isolated porcine pulmonary artery. KEY RESULTS: Thrombin induced a sustained contraction in endothelium-denuded strips obtained from different sites of a pulmonary artery, ranging from the main pulmonary artery to the intrapulmonary artery. In the presence of endothelium, thrombin induced a transient relaxation. The contractile effect of thrombin was abolished by either a protease inhibitor or a proteinase-activated receptor 1 (PAR(1)) antagonist, while it was mimicked by PAR(1)-activating peptide (PAR(1)AP), but not PAR(4)AP. The thrombin-induced contraction was associated with a small elevation of [Ca(2+)](i) and an increase in MLC20 phosphorylation. Thrombin and PAR(1)AP induced a greater increase in tension for a given [Ca(2+)](i) elevation than that obtained with high K(+)-depolarization. They also induced a contraction at a fixed Ca(2+) concentration in alpha-toxin-permeabilized preparations. CONCLUSIONS AND IMPLICATIONS: The present study revealed a unique property of the pulmonary artery. In contrast to normal arteries of the systemic circulation, thrombin induces a sustained contraction in the normal pulmonary artery, by activating PAR(1) and thereby increasing the sensitivity of the myofilament to Ca(2+). This responsiveness of the pulmonary artery to thrombin may therefore contribute to the pathogenesis and pathophysiology of PAH.

Extracellular osmolarity modulates G protein-coupled receptor-dependent ATP release from 1321N1 astrocytoma cells.

We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755-763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca(2+) mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.

Opposite effects of two thiazolidinediones, ciglitazone and troglitazone, on proteinase-activated receptor-1-triggered prostaglandin E(2) release.

Thiazolidinediones, known as peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists, may modify prostaglandin formation and exert gastroprotective effects. Since activation of proteinase-activated receptor-1 (PAR1) reveals endogenous prostanoid-dependent gastroprotection, we investigated if two thiazolidinediones, ciglitazone and troglitazone, modulate the prostaglandin E(2) (PGE(2)) release caused by activation of PAR1 in normal rat gastric mucosal epithelial RGM1 cells. Ciglitazone dramatically facilitated the PAR1-triggered PGE(2) production and cyclooxygenase-2 (COX-2) upregulation, although it had no effect by itself. In contrast, troglitazone suppressed the PAR1-triggered PGE(2) production and COX-2 upregulation. Either effect of ciglitazone and troglitazone was resistant to GW9662, a PPARgamma antagonist. The facilitation of the PGE(2) release by ciglitazone was blocked by inhibitors of MEK, p38 MAP kinase (p38MAPK) and PI3-kinase (PI3K), but not JNK. Nonetheless, ciglitazone failed to enhance the PAR1-triggered phosphorylation of ERK and p38MAPK. In conclusion, ciglitazone and troglitazone, exert opposite effects on the PAR1-triggered PGE(2) production and COX-2 upregulation by targeting molecules other than PPARgamma.

Differential effects of quercetin, apigenin and genistein on signalling pathways of protease-activated receptors PAR(1) and PAR(4) in platelets.

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 novel oral thrombin receptor antagonist.

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.

Activated protein C and acute kidney injury: Selective targeting of PAR-1.

Protein C is a plasma serine protease that when activated plays a central role in modulating the function of the vascular endothelium and its interface with the innate immune system. Activated protein C (APC) has a dual mechanism of action via the feedback inhibition of thrombin generation, and as an agonist of protease activated receptor-1 (PAR-1). Through different cofactor interactions, this dual mechanism of antithrombotic and cytoprotective activity results in the ability of APC to modulate endothelial dysfunction by blocking cytokine signaling, functional cell adhesion expression, vascular permeability, apoptosis, and modulating leukocyte migration and adhesion. Deficiency in protein C, which occurs during systemic inflammatory activation, is highly associated with organ dysfunction. APC has shown efficacy in a number of preclinical models of thrombosis and ischemia, and the recombinant human APC drotrecogin alfa (activated), reduces mortality in patients with high-risk severe sepsis. The ability of APC to suppress pro-inflammatory pathways and enhance cellular survival suggests that APC plays a key role in the adaptive response to protect the vessel wall from insult and to enhance endothelial, cellular, and organ survival. The focus of this review will be to summarize the emerging data suggesting the potential therapeutic benefit of APC and related members of the pathway in the prevention and treatment of acute kidney injury.

[Characterization of endothelial progenitor cells and putative strategies to improve their expansion]. / Caractérisation des progéniteurs endothéliaux et stratégies d'expansion in vitro.

Injection of endothelial progenitor cells (EPC) expanded ex vivo has been shown to increase neovascularization in preclinical models of ischemia and in adult patients, but the precise origin and identity of the cell population responsible for these clinical benefits are controversial. Given the potential usefulness of EPC as a cell therapy product, their thorough characterization is of major importance. This review describes the two cell populations currently called EPC and the means to find differential phenotypic markers. We have shown that BMP2/4 are specific markers of late EPC and play a key role in EPC commitment and outgrowth during neovascularization. Several authors have attempted to expand EPC ex vivo in order to obtain a homogeneous cell therapy product. One possible mean of expanding EPC ex vivo is to activate the thrombin receptor PAR-1 with the specific peptide SFLLRN. Indeed, PAR-1 activation increases angiogenic properties of EPC through activation of SDF-1, angiopoietin and IL-8 pathways. This review summarizes the characterization of EPC and different methods of ex vivo expansion.

Activated protein C protects against myocardial ischemia/ reperfusion injury via inhibition of apoptosis and inflammation.

OBJECTIVE: In spite of major advances in reperfusion therapy for patients presenting with acute coronary syndrome, long-term morbidity is still substantial. A limitation of initial treatment of myocardial ischemia is the lack of prevention of ischemia/reperfusion (I/R) injury. Activated protein C (APC), a crucial mediator in the coagulation process, plays a prominent role in the crosstalk between coagulation and inflammation and provides cytoprotective effects via inhibition of apoptosis and inflammation in several human and animal studies. METHODS AND RESULTS: APC was administered in an animal model for myocardial I/R. APC largely inhibited early myocardial I/R injury after varying reperfusion times, an effect that was absent on administration of heparin, a nonspecific anticoagulant agent. The protective effects of APC were absent in case of absence or blockade of protease activated receptor-1 (PAR-1), indicating a critical role for PAR-1 in this process. Furthermore, we showed a strong antiapoptotic effect of APC in the early phase of reperfusion combined with an antiinflammatory effect at an early stage (IL-6), as well as at a later stage (leukocyte infiltration). CONCLUSIONS: APC exerts strong protective effects on early myocardial I/R injury, primarily via inhibition of apoptosis and inflammation, which are regulated via PAR-1.