Protease-activated Receptor-1 Signaling Research Articles

Cathepsin G Recruits Osteoclast Precursors via Proteolytic Activation of Protease-Activated Receptor-1

Metastatic breast cancer shows extreme tropism for the bone microenvironment, leading to the establishment of osteolytic metastases. Perpetuation of tumor-induced osteolysis requires a continuous supply of osteoclast precursors migrating into the bone microenvironment that can subsequently differentiate into mature osteoclasts and resorb bone. Thus, identification and subsequent targeting of chemoattractants of osteoclast precursors that are up-regulated at the tumor-bone interface represents a potential avenue to interrupt osteolysis. We report that cathepsin G, a serine protease, plays a vital role in the bone microenvironment by modulating tumor-stromal interaction in a manner that favors tumor establishment and regulates chemotaxis of monocytes, a subset of which has the potential to differentiate into osteoclasts. Our data show that cathepsin G-induced chemotaxis of monocytes is mediated by proteolytic activation of protease-activated receptor-1 (PAR-1). Attenuation of PAR-1 activation abrogates cathepsin G-mediated induction of monocyte chemotaxis. We also show that in vivo inhibition of cathepsin G reduces the number of CD11b(+) osteoclast precursors and mature osteoclasts at the tumor-bone interface. Together, these data suggest that therapeutic targeting of both PAR-1 signaling in osteoclast precursors as well as cathepsin G at the tumor-bone interface has the potential to reduce osteolysis by inhibiting the recruitment, differentiation, and activation of osteoclast precursors.

The complex role of fibrin in acute lung injury

in the last decade there have been many advances in our understanding of the pathogenesis of acute lung injury (ALI), but there have been frustratingly few successful therapeutic interventions. In fact, the only treatment proven to reduce mortality is low tidal volume lung-protective mechanical

Differential Effects of Serine Proteases on the Migration of Normal and Tumor Cells: Implications for Tumor Microenvironment

The supporting role of proteases in tumor progression and invasion is well known; however, the use of proteases as therapeutic agents has also been demonstrated. In this article, the authors report on the differential effects of exogenous serine proteases on the motility of tumor and normal cells. The treatment of normal and tumor cells with a single dose of pancreatic serine proteases, trypsin (TR) and chymotrypsin (CH), leads to a concentration-dependent response by cells, first accelerating and then slowing mobility. Tumor cells are 10 to 20 times more sensitive to exogenous TR/CH, suggesting that a single dose of proteases may cause discordant movements of normal and tumor cells within the tumor environment. The inhibitory effects of TR on cell motility are contradicted by thrombin (TH), particularly in the regulation of normal cells' migration. The purpose of this investigation was to ascertain the role of protease-activated receptors (PARs) in terms of normal and tumor cell motility. Duplicate treatments with proteases resulted in diminished mobility of both normal and tumor cells. Repeated application of TR and TH in 1-hour treatment intervals initially desensitizes cell surface PARs. However, cell surface PARs reappear regardless of subsequent protease treatments in both normal and tumor cells. The resensitization process is retarded in tumor cells when compared with normal cells. This is evidenced by lower expression of PARs as well as by their relocalization at the tumor cell surfaces. Under these conditions, normal cells remain responsive to exogenous proteases in terms of cell motility. Exogenous proteases do not modulate motility of repeatedly stimulated tumor cells, and consequently, the migration of tumor cells appears disconnected from the PAR signaling pathways. The use of activating peptides in lieu of the cognate proteases for a given PAR system indicated that proteases may act through additional targets not regulated by PAR signaling. We hypothesize that the divergent migration patterns of normal and tumor cells due to exposure to proteases is in part mediated by PARs. Thus, treatment with exogenous proteases may cause rearrangement of the tumor and stromal cells within the tumor microenvironment. Such topographical effects may lead to the inhibition of tumor progression and metastasis development.

Inhibition of the ADP/P2Y12 Pathway Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice

Clopidogrel, a potent anti-thrombotic drug widely used for the secondary prevention of myocardial infarction and ischemic stroke, inhibits the ADP/P2Y12 pathway by inducing irreversible alteration of the platelet receptor P2Y12, ultimately uncoupling ADP from adenylyl cyclase inhibition. Clinical trials have demonstrated additional benefits of dual antiplatelet therapy, such as combined treatment with clopidogrel and aspirin, in the prevention of ischemic cardiovascular disease. We previously established that protease-activated receptor (PAR) signaling is necessary for platelet activation by thrombin and plays an important role in mouse models of hemostasis and thrombosis. Indeed a PAR antagonist is currently being used as an anti-thrombotic agent in clinical trials.In order to evaluate the possible interaction between the thrombin/PAR and ADP/P2Y12 pathways, we orally administered clopidogrel (30mg/kg) or vehicle to PAR-4 null, heterozygous and wild type littermates, and evaluated arterial thrombus formation and bleeding during surgical challenge. In a FeCl3-induced carotid thrombosis model, PAR-4 deficient mice treated with clopidogrel were completely protected from thrombosis compared to their wild type treated littermates. Interestingly, clopidogrel treated PAR-4 heterozygotes showed an intermediate level of protection compared to treated wild type and null mice that was similar to vehicle-treated PAR-4 deficient mice. Additionally, we scored bleeding during the surgical procedure while blinded to genotype and treatment. The bleeding score was increased in clopidogrel treated mice compared to vehicle treated mice and was exacerbated with increasing haplotype deficiency of Par-4.To better define the relationship between these two activation pathways, we generated mice lacking both PAR-4 and P2Y12. All genetic combinations were born at the expected Mendelian distribution. Females lacking both receptors carried offspring to term, but signs of bleeding were observed during parturition. Preliminary data from PAR-4/P2Y12 double null mice, in both the tail bleeding assay and in a FeCl3-induced carotid thrombosis model, is in agreement with the results obtained in clopidogrel treated PAR-4 deficient mice. This study provides useful information to evaluate the efficacy and safety of dual antiplatelet therapy inhibiting the thrombin/PAR and ADP/P2Y12 pathways and explores the relationship between these two critical pro-thrombotic pathways in an in vivo setting.

Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

The ATP-gated P2X1 Receptor Plays a Pivotal Role in Activation of Aspirin-treated Platelets by Thrombin and Epinephrine

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.

Protein C supports platelet binding and activation under flow: role of glycoprotein Ib and apolipoprotein E receptor 2.

Activated protein C (APC) regulates thrombin generation and inhibits apoptosis. Endothelial protein C receptor (EPCR)-bound protein C is activated by thrombomodulin-bound thrombin. APC inactivates coagulation factors (F)Va/VIIIa and generates cytoprotective signaling downstream of protease-activated receptor-1 (PAR-1). Binding of APC to EPCR both modifies and induces PAR-1 signaling, but it is unknown if protein C interacts with cells in an alternative manner. To determine whether platelets possess receptors for protein C that can generate intracellular signals. Immobilized protein C or APC supported platelet adhesion, lamellipodia formation and elevation of intracellular Ca(2+). Adhesion of platelets to protein C or APC was inhibited by soluble recombinant apolipoprotein E receptor 2' (ApoER2') and by receptor-associated protein (RAP), an inhibitor of the low-density lipoprotein receptor family. Under shear, surface-bound protein C supported platelet adhesion and aggregation in a glycoprotein (GP)Ibalpha-dependent manner, and adhesion of platelets to immobilized protein C was abrogated by the addition of soluble forms of ApoER2' or RAP. APC bound to purified recombinant ApoER2' or GPIbalpha. Our data demonstrate that activation of platelets with rapid intracellular signaling caused by binding to immobilized protein C or APC occurs via mechanisms that require ApoER2 and GPIbalpha and that APC directly binds to purified ectodomains of the receptors ApoER2 and GPIbalpha. These findings imply that protein C and APC may directly promote cell signaling in other cells by binding to ApoER2 and/or GPIbalpha.

LIGR, a protease‐activated receptor‐2‐derived peptide, enhances skin pigmentation without inducing inflammatory processes

The protease-activated receptor-2 (PAR-2) is a seven transmembrane G-protein-coupled receptor that could be activated by serine protease cleavage or by synthetic peptide agonists. We showed earlier that activation of PAR-2 with Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), a known PAR-2 activating peptide, induces keratinocyte phagocytosis and increases skin pigmentation, indicating that PAR-2 regulates pigmentation by controlling phagocytosis of melanosomes. Here, we show that Leu-Ile-Gly-Arg-NH(2) (LIGR) can also induce skin pigmentation. Both SLIGRL and LIGR increased melanin deposition in vitro and in vivo, and visibly darkened human skins grafted onto severe combined immuno-deficient (SCID) mice. Both SLIGRL and LIGR stimulated Rho-GTP activation resulting in keratinocyte phagocytosis. Interestingly, LIGR activates only a subset of the PAR-2 signaling pathways, and unlike SLIGRL, it does not induce inflammatory processes. LIGR did not affect many PAR-2 signaling pathways, including [Ca(2+)] mobilization, cAMP induction, the induction of cyclooxgenase-2 (COX-2) expression and the secretion of prostaglandin E2, interleukin-6 and -8. PAR-2 siRNA inhibited LIGR-induced phagocytosis, indicating that LIGR signals via PAR-2. Our data suggest that LIGR is a more specific regulator of PAR-2-induced pigmentation relative to SLIGRL. Therefore, enhancing skin pigmentation by topical applications of LIGR may result in a desired tanned-like skin color, without enhancing inflammatory processes, and without the need of UV exposure.

Persistent transactivation of EGFR and ErbB2/HER2 by protease-activated receptor-1 promotes breast carcinoma cell invasion

Hyperactivation of ErbB signaling is implicated in metastatic breast cancer. However, the mechanisms that cause dysregulated ErbB signaling and promote breast carcinoma cell invasion remain poorly understood. One pathway leading to ErbB activation that remains unexplored in breast carcinoma cell invasion involves transactivation by G-protein-coupled receptors (GPCRs). Protease-activated receptor-1 (PAR1), a GPCR activated by extracellular proteases, is overexpressed in invasive breast cancer. PAR1 is also proposed to function in breast cancer invasion and metastasis, but how PAR1 contributes to these processes is not known. In this study, we report that proteolytic activation of PAR1 by thrombin induces persistent transactivation of EGFR and ErbB2/HER2 in invasive breast carcinoma, but not in normal mammary epithelial cells. PAR1-stimulated EGFR and ErbB2 transactivation leads to prolonged extracellular signal-regulated kinase-1 and -2 signaling and promotes breast carcinoma cell invasion. We also show that PAR1 signaling through Galpha(i/o) and metalloprotease activity is critical for ErbB transactivation and cellular invasion. Finally, we demonstrate that PAR1 expression in invasive breast carcinoma is essential for tumor growth in vivo assessed by mammary fat pad xenografts. These studies reveal a critical role for PAR1, a receptor activated by tumor-generated proteases, in hyperactivation of ErbB signaling that promotes breast carcinoma cell invasion.

Tissue Factor and Protease-Activated Receptor Signaling in Cancer

The activation of the coagulation cascade in the tumor microenvironment is a key feature of advanced malignancies. On tumor cells, tissue factor (TF) plays a central role to initiate cross-talk through the release of procoagulant microparticles or through direct, protease-activated receptor (PAR)-mediated cell signaling that leads to the production of soluble cytokines and angiogenic growth factors. In addition, the hemostatic system in the host compartment sustains crucial circuits that promote metastasis and support tumor growth and angiogenesis. Experimental tumor and genetic models have defined specific pathways that are supported by tumor cell and host TF and have identified potential therapeutic modalities to specifically interrupt TF signaling in tumor biology without impairment of hemostatic functions.

The contribution of PARs to inflammation and immunity to fungi

During inflammation, host- and microbial-derived proteases trigger the activation of protease-activated receptors (PARs), a family of G-protein-coupled receptors. We report here that activation of Toll-like receptors (TLRs) by fungi unmasks an essential and divergent role for PAR1 and PAR2 in downstream signaling and inflammation. TLRs activated PARs and triggered distinct signal transduction pathways involved in inflammation and immunity to Candida albicans and Aspergillus fumigatus. Inflammation was promoted by PAR1 and PAR2 activation in response to Candida and by PAR2 inhibition in response to Aspergillus. This occurred by TLR regulation of PAR signaling, with TLR2 promoting PAR1 activity, and TLR4 suppressing PAR2 activity. Thus, tissue injury and pathogens induce signals that are integrated at the level of distinct TLR/PAR-dependent pathways, the exploitation or subversion of which contributes to divergence in microbial promotion of inflammatory response.

Dendritic cell PAR1–S1P3 signalling couples coagulation and inflammation

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.

Proteinase-activated Receptor-2 Induces Cyclooxygenase-2 Expression through β-Catenin and Cyclic AMP-response Element-binding Protein

Chronic inflammation of mucosae is associated with an increased cancer risk. Tumorigenesis in these tissues is associated with the activity of some proteinases, cyclooxygenase-2 (COX-2), and beta-catenin. Serine proteinases participate in both inflammation and tumorigenesis through the activation of proteinase-activated receptor-2 (PAR(2)), which up-regulates COX-2 by an unknown mechanism. We sought to determine whether beta-catenin participated in PAR(2)-induced COX-2 expression and through what cellular mechanism. In A549 epithelial cells, we showed that PAR(2) activation increased COX-2 expression through the beta-catenin/T cell factor transcription pathway. This effect was dependent upon ERK1/2 MAPK, which inhibited the beta-catenin-regulating protein, glycogen synthase kinase-3beta, and induced the activity of the cAMP-response element-binding protein (CREB). Knockdown of CREB by small interfering RNA revealed that PAR(2)-induced beta-catenin transcriptional activity and COX-2 expression were CREB-dependent. A co-immunoprecipitation assay revealed a physical interaction between CREB and beta-catenin. Thus, PAR(2) up-regulated COX-2 expression via an ERK1/2-mediated activation of the beta-catenin/Tcf-4 and CREB pathways. These findings reveal new cellular mechanisms by which serine proteinases may participate in tumor development and are particularly relevant to cancers associated with chronic mucosal inflammation, where serine proteinases are abundant and COX-2 overexpression is a common feature.

Thrombin and protease-activated receptors (PARs) in atherothrombosis

Thrombin is a multifunctional serine protease generated at the site of vascular injury that transforms fibrinogen into fibrin, activates blood platelets and elicits multiple effects on a variety of cell types including endothelial cells, vascular smooth muscle cells (VSMC), monocytes, T lymphocytes and fibroblasts. Cellular effects of thrombin are mediated by protease-activated receptors (PARs), members of the G protein-coupled receptors that carry their own ligand which remains cryptic until unmasked by proteolytic cleavage. Thrombin signalling in platelets contributes to haemostasis and thrombosis. In normal arteries PARs are mainly expressed in endothelial cells, while their expression in VSMC is limited. Endothelial PARs participate in the regulation of vascular tone, vascular permeability and endothelial secretory activity while in VSMC they mediate contraction, migration, proliferation, hypertrophy and production of extracellular matrix. PARs contribute to the pro-inflammatory phenotype observed in endothelial dysfunction and their up-regulation in VSMC seems to be a key element in the pathogenesis of atherosclerosis and restenosis. In the last years a myriad of studies have emphasized the critical role of PAR signalling in thrombin mediated effects in haemostasis, inflammation, cancer and embryonic development. Lately, PARs have become a therapeutic target to inhibit platelet aggregation and thrombosis. Early data from a clinical trial (TRA-PCI) to evaluate safety and efficacy of a potent new oral thrombin receptor antagonist (TRA) have promisingly indicated that overall TRA treatment reduces adverse event rates without an increase in bleeding risk. In this paper we review cellular responses triggered by thrombin and their implication in vascular pathophysiology.

Activated protein C–cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin

Activated protein C (APC) signals in endothelial cells ex vivo through protease activated receptor-1 (PAR1). However, it is controversial whether PAR1 can mediate APC's protective effects in sepsis because the inflammatory response results in thrombin generation and thrombin proteolytically activates PAR1 much more efficiently than APC. Here we show that APC can induce powerful barrier protective responses in an endothelial cell monolayer in the presence of thrombin. Using cell surface immunoassays with conformation sensitive monoclonal anti-PAR1 antibodies we analyzed cleavage of endogenous PAR1 on the endothelial cell surface by APC in the absence and presence of thrombin. Incubation with APC caused efficient PAR1 cleavage and upon coincubation with thrombin APC supported additional PAR1 cleavage. Thrombin-cleaved PAR1 rapidly disappeared from the cell surface whereas, unexpectedly, the APC-cleaved PAR1 remained and could be detected on the cell surface, even when thrombin at concentrations of up to 1 nM was also present. Our findings demonstrate for the first time directly that APC can generate a distinct PAR1 population on endothelial cells in the presence of thrombin. The data suggest that different trafficking of activated PAR1 might explain how PAR1 signaling by APC can be relevant when thrombin is present.

A small interfering RNA targeting proteinase‐activated receptor‐2 is effective in suppression of tumor growth in a Panc1 xenograft model

Proteinase-activated receptor-2 (PAR-2), which is a G protein-coupled receptor, is activated in inflammatory processes and cell proliferation. We previously demonstrated that an anti-PAR-2 antibody suppresses proliferation of human pancreatic cells in vitro. However, there have been no studies of PAR-2 signaling pathways in vivo. The aim of this study was to determine whether blockade of PAR-2 by RNA interference influences pancreatic tumor growth. We originally constructed small interfering RNAs (siRNAs) targeting human PAR-2, and performed cell proliferation assays of Panc1 human pancreatic cancer cell line with these siRNAs. Intratumoral treatment with these PAR-2 siRNAs and atelocollagen was also performed in a xenograft model with nude mice and Panc1 cells. siRNAs against human PAR-2 inhibited proliferation of Panc1 cells, whereas control scramble siRNAs had no effect on proliferation. The PAR-2 siRNAs dramatically suppressed tumor growth in the xenograft model. PAR-2-specific siRNA inhibited growth of human pancreatic cancer cells both in vitro and in vivo. Blockade of PAR-2 signaling by siRNA may be a novel strategy to treat pancreatic cancer.

Glutamic Acid 192 of Activated Protein C Restricts the Specificity of PAR-1 Cleavage in Endothelial Cells.

It is known that residue 192 (chymotrypsin numbering) plays a key role in determining the P3 binding specificity of coagulation proteases. This residue is a Glu in both thrombin and activated protein C. Previous studies have indicated that the substrates containing an acidic residue at the P3 position are not recognized efficiently by either thrombin or APC in the absence of a cofactor. Protease activated receptor 1 (PAR-1), a common substrate for both thrombin and APC contains an acidic Asp at the P3 position. In this study we examined the ability of Glu-192 to Gln (E192Q) substitution mutants of both thrombin and APC to recognize and cleave PAR-1 in human umbilical vein endothelial cells (HUVEC) transfected with a PAR-1 cleavage reporter plasmid in which the exodomain of the receptor has been fused to a cDNA fragment encoding the soluble alkaline phosphatase. Thrombin E192Q cleaved PAR-1 with a catalytic efficiency that was approximately 5-fold higher than that of wild-type thrombin. On the other hand, the activity of APC E192Q toward PAR-1 was improved approximately 100-fold. Thus, unlike an approximately 1000-fold lower activity for APC in cleaving PAR-1 relative to thrombin, the activity of APC E192Q toward PAR-1 was only 10-fold lower than that of thrombin. These results suggest that the inhibitory interaction of Glu-192 of APC with P3-Asp of PAR-1 is responsible for its poor activity toward PAR-1. It is known that the cleavage of PAR-1 by thrombin in endothelial cells elicits a proinflammatory response. However, the cleavage of the same receptor by APC in complex with endothelial protein C receptor (EPCR) invokes a protective antiinflammatory response. The mechanism of the paradoxical effect of PAR-1 signaling by the two proteases is not known. We used these mutants to investigate the possibility that the level of PAR-1 activation by either thrombin or APC dictates the type of the response in endothelial cells. Furthermore, we used these mutants in TNF-a-stimulated endothelial cell permeability and apoptosis assays to understand the mechanism by which EPCR enables APC to activate PAR-1 in endothelial cells. We discovered that the dose of receptor activation is not responsible for the paradoxical effect of PAR-1 signaling by APC and thrombin in endothelial cells. Furthermore, the interaction of APC with EPCR is not associated with an improvement in the catalytic efficiency of the protease toward PAR-1. The possible EPCR and PAR-1 dependent recognition and signaling mechanism of APC and thrombin is discussed.

Tissue Factor Pathway Inhibitor Anticoagulant and Anti-PAR Signaling Activity Requires GPI-Anchor Attachment to the Cell Surface in Chinese Hamster Ovary (CHO) Cells.

Introduction: TFPI is an endothelial associated protein that prevents intravascular thrombosis. It produces active site directed inhibition of the TF-fVIIa catalytic complex. TF-fVIIa activates blood coagulation via cleavage of fIX and fX. TF-fVIIa also initiates inflammation via activation of protease activated receptors (PARs). TFPI is attached to the endothelial surface through a GPI-anchor that localizes it to lipid raft/caveolae microdomains. We examined how GPI-anchor mediated localization of TFPI to these microdomains alters its ability to inhibit TF-fVIIa mediated coagulation and signaling events in vitro and in vivo.Methods: CHO cells were transfected with TF to create CHO-TF cells. CHO-TF cells were transfected with chimeric forms of TFPI containing either a GPI-anchor or a transmembrane (TM) attachment sequence at the C-terminus to create CHO-TF/TFPI-GPI and CHO-TF/TFPI-TM cells. Clones expressing equal amounts of TFPI-GPI and TFPI-TM were selected using flow cytometry. TF-fVIIa activity on the surface of these cells was compared to that of CHO-TF cells in three assays. TF-fVIIa coagulation and PAR signaling activity were examined in vitro using fXa activation assays and ERK1/2 phosphorylation assays, respectively. A TF-mediated tumor metastasis assay was used to assess TF activity in vivo.Results: GPI-anchored TFPI displays significantly more anti-TF-fVIIa activity than TM-anchored TFPI in the in vitro assays. In the anticoagulant activity assays, when compared to CHO-TF cells, the amount of fXa generated on the cell surface for 30 min was reduced by 64% in the CHO-TF/TFPI-GPI cells but only 8% in the CHO-TF/TFPI-TM cells (CHO-TF cells generated fXa at 6.3 pmol/min; CHO-TF/TFPI-GPI at 2.3 pmol/min; CHO-TF/TFPI-TM at 5.8 pmol/min). The signaling assays were performed using flow cytometry. When compared to CHO-TF cells, ERK1/2 phosphorylation was reduced 73% in CHO-TF/TFPI-GPI cells while no effect was observed in CHO-TF/TFPI-TM cells (MFI for CHO-TF cells at 3.17±2.04; CHO-TF/TFPI-GPI at 0.86±1.9; CHO-TF/TFPI-TM at 3.5±0.95). To assess TFPI inhibitory activity in vivo, cell lines were injected into the tail vein of SCID mice and the lungs examined for tumors at 10 days. TF-fVIIa activity is required for tumor formation in this assay. Consistent with the in vitro results, the CHO-TF/TFPI-GPI cells had greatly decreased tumor burden while CHO-TF/TFPI-TM had much less effect. Interestingly, transfection of CHO-TF cells with wild type TFPI that is secreted by CHO cells had no effect on tumor burden indicating that cell surface association of TFPI is required for anti-TF-fVIIa activity in this assay. These CHO cell results contrast with our previously published data where we found no difference in the anticoagulant activity of TFPI-GPI and TFPI-TM on the surface of HEK293 cells (Thromb. Hemost. 2003, 89:65). Since CHO cells express abundant caveolin-1 while HEK293 cells express very little, we considered that caveolin-1 may be responsible for the differential activity observed. In support of this hypothesis we found that TFPI and caveolin-1 co-immunoprecipitate from human placental lysates.Conclusions: A CHO cell model was used to examine the inhibition of TF-fVIIa by TFPI-GPI or TFPI-TM. GPI-anchor attachment of TFPI to the cell surface appears to be critical for its anti-TF activity. Localization to caveolae microdomains and/or a direct interaction with a protein complex containing caveolin-1 may be required for full TFPI activity in vivo.

Protease-Activated Receptor-1 Contributes to Cardiac Remodeling and Hypertrophy

Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1(-/-) mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

Protective Signaling Pathways of Activated Protein C in Endothelial Cells

Activated protein C (APC) has been reported to improve survival in patients with severe sepsis.1 Protein C (PC) is physiologically activated on the endothelial cell surface by the key procoagulant enzyme thrombin, and APC downregulates thrombin formation in a negative feedback loop.2 This anticoagulant effect of APC is unlikely to explain its benefit in systemic inflammation because other anticoagulants did not improve survival in septic patients. How the protective effects of APC in systemic inflammation are mediated has thus received a considerable amount of attention in recent years. See ATVB 2007;2634–2641. In vitro studies have shown that incubation of cultured endothelial cells with APC leads to a number of potentially antiinflammatory cellular changes. These include downregulation of the expression of adhesion molecules, enhanced barrier function in cell monolayers, and effects on gene expression that result in decreased susceptibility to apoptosis. Most of these cellular responses have been shown to require the binding of APC to endothelial cell protein C receptor (EPCR) and cleavage-mediated activation of protease activated receptor-1 (PAR1).3–6 EPCR may not only enhance activation of PAR1 by APC but also modify downstream responses through other mechanisms.7–9 EPCR is a transmembrane receptor with a very short cytoplasmic domain and an extracellular domain that binds both PC and APC. EPCR recruits and positions PC/APC on the cell surface, and EPCR binding enhances activation of PC. PAR1 is the prototypical thrombin receptor and belongs to a family of 7-transmembrane G protein–coupled receptors. Enzymatic cleavage of PAR1 exposes a new extracellular N terminus that acts as a tethered activation ligand. Importantly, in mouse models of stroke and endotoxemia EPCR-dependent PAR1 signaling is required for protective effects of APC.5,10 However, it is unknown which cellular effects of APC lead to protection in vivo, and it remains possible that mechanisms …