Effector Cell Protease Receptor-1 Research Articles

Chromenes - A Novel Class of Heterocyclic Compounds: Recent Advancements and Future Directions.

Chromenes are an important class of oxygen-containing heterocyclic compounds with intriguing biological activity, a simple structure with mild adverse effects. Chromenes are abundantly found in nature in the form of alkaloids, tocopherols, flavone, and anthocyanins. The Chromene nucleus is an important moiety for the discovery of new drug candidates. Chromene derivatives have shown various pharmacological activities like antiviral, anticancer, anti-inflammatory, antitumour, antimicrobial, antiproliferative, anticholinesterase, EPR-1 (Effector cell Protease Receptor-1) antagonist and MAO (Mono-Amine Oxidase) inhibitors. In SAR (Structure Activity Relationship) studies with chromene nucleus, it was found that 4-aryl moiety, 3-cyano group, and 2-amino group are essential for the cytotoxic activity. Substitution at the 7th position with electron donating group enhances the pharmacological activity whereas the electron withdrawing group decreases the pharmacological activity. Structural modifications at the chromene ring, middle aliphatic portion, and terminal aromatic ring yielded more potential 5-HT1A (5-Hydroxytryptamine 1A) receptor affinity and antidiabetic activity. Chromenes with cyclic secondary amine and 4-hydroxy phenyl substituents yielded potent antimicrobial compounds. This review summarizes the importance of chromenes in rational drug design and the development of novel molecules with a variety of pharmacological activities.

Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant

Reactivation of survivin expression is involved in carcinogenesis and angiogenesis in colon cancer. Previous in vitro studies showed that mutation of the cysteine residue at position 84 (Cys84Ala) of survivin generates a dominant-negative mutant that triggers mitotic catastrophe and apoptosis. We investigated the therapeutic effect of the adeno-associated virus (AAV)-mediated survivin mutant (Cys84Ala) on colon cancer. Survivin mutant (Cys84Ala) (Sur-Mut(Cys84Ala)) was cloned into the AAV expression vector pAM/CAG-WPRE.poly(A) to generate recombinant AAV-Sur-Mut(Cys84Ala) virus. Cell proliferation, apoptosis, mitotic catastrophe, and tumor growth were measured in vitro and in vivo. Transduction of colon cancer cells with rAAV-Sur-Mut(Cys84Ala) inhibited cell proliferation and induced apoptosis and mitotic catastrophe in vitro. rAAV-Sur-Mut(Cys84Ala) sensitized colon cancer cells to chemotherapeutic drugs. Furthermore, expression of survivin mutant mediated by AAV inhibited tumorigenesis in colon cancer cells. Intratumoral injection of rAAV-Sur-Mut(Cys84Ala) significantly induced apoptosis and mitotic catastrophe and inhibited angiogenesis and tumor growth in a colon cancer xenograft model in vivo. No obvious cytotoxicity to other tissues was observed. More importantly, rAAV-Sur-Mut(Cys84Ala) expression strongly enhanced the antitumor activity of 5-Fluorouracil (5-FU), resulting in regression of established tumors. Our results showed that rAAV-Sur-Mut(Cys84Ala) induced apoptosis and mitotic catastrophe and inhibited tumor angiogenesis and tumor growth. Thus, use of AAV-mediated survivin mutant (Cys84Ala) is a promising strategy in colon cancer gene therapy.

Downregulation of survivin expression by induction of the effector cell protease receptor-1 reduces tumor growth potential and results in an increased sensitivity to anticancer agents in human colon cancer

Survivin, a novel inhibitor of apoptosis, is expressed in cancer cells and not in normal adult tissues, and is recognised as a potential target in anticancer therapy. The induction of a natural antisense of survivin, effector cell protease receptor-1 (EPR-1), in a human colon cancer cell line resulted in a downregulation of survivin expression, with a similar decrease in cell proliferation, an increase in apoptosis and an increase in the sensitivity to anticancer agents. In addition, subcutaneous (s.c.) tumours from EPR-1 transfectants showed a significant reduction in size compared with parental cells, and this antitumour efficacy was further enhanced in combination with anticancer agents. These findings suggest that regulation of survivin by the induction of EPR-1 cDNA may have significant potential as a therapy for human colon cancer.

Factor Xa-Evoked Relaxation in Rat Aorta: Involvement of PAR-2

Protease-activated receptor-2 (PAR-2) and/or effector cell protease receptor-1 (EPR-1) may mediate the direct cellular actions of coagulation factor Xa in some cultured cell lines. The present study examined if factor Xa could actually evoke relaxation through either of these receptor systems in isolated rat aorta. Factor Xa at 8.5–85 nM, like the PAR-2-activators trypsin and SLIGRL-NH2, produced nitric oxide-dependent relaxation in the precontracted aortic rings. PAR-2 desensitization abolished relaxation responses to factor Xa as well as trypsin in the rings. The factor Xa interepidermal growth factor synthetic peptide L83FTRKL88(G)-NH2, known to block factor Xa binding to EPR-1, failed to inhibit factor Xa-evoked relaxation in the preparations. Our findings provide evidence that factor Xa evokes relaxation by activating PAR-2, but independently of EPR-1, in the rat aorta. The factor Xa-PAR-2 pathway might thus contribute to the severe hypotension during sepsis, in which multiple coagulation factors including factor X would become activated and PAR-2 would be induced.

Isolation and mapping the pig homologs survivin (BIRC5) and effector cell protease receptor 1 (EPR1) genes

Isolation and mapping the pig homologs survivin (BIRC5) and effector cell protease receptor 1 (EPR1) genes

Disturbed expression of the anti-apoptosis gene, Survivin, and EPR-1 in hematological malignancies

Survivin is a newly discovered inhibitor of the apoptosis protein, IAP, expressed during development and in human cancers. The effector cell protease receptor-1 (EPR-1) gene is oriented in the opposite direction on the same DNA double strand. Thus, the Survivin and EPR-1 (Survivin/EPR-1) genes exist in a head-to-head configuration. It is not clear whether mutual expression of the Survivin/EPR-1 genes occurs in both normal cells and cancer cells. Here, we investigated the mutual expression of the Survivin/EPR-1 genes in 12 normal peripheral blood (PB) specimens, seven normal bone marrow (BM) specimens, five lymph node (LN) specimens, and seven leukemic cell lines, and 27 patients with malignant lymphoma (ML), four with acute lymphocytic leukemia (ALL), three with acute myelocytic leukemia (AML), and four with chronic myelocytic leukemia in blastic crisis (CML-BC). Using Northern blot analysis, small amounts of EPR-1 mRNA were detected in normal PB, normal BM and LN specimens, but no Survivin mRNA was detected. However, Survivin mRNA was detected in two of the 12 normal PB, six of the seven normal BM and one of the five LN specimens using reverse transcription and polymerase chain reaction (RT-PCR). Expression of both the Survivin and EPR-1 genes was detected in six of the seven cell line samples by Northern blot, and in all of them by RT-PCR. Mutual expression of the Survivin and EPR-1 genes was detected in three of the four CML-BC samples, 15 of the 27 ML samples, two of the four ALL samples, and all three AML samples using the RT-PCR method. No EPR-1 expression with or without Survivin expression was clearly detected in eight of the nine diffuse large B-cell lymphoma (DLB) specimens, two of the six follicular center lymphoma specimens, one of the four specimens of nodular sclerosis of Hodgkin's lymphoma, two of the four ALL specimens or one of the four CML-BC specimens. The data presented here show that disrupted expression of the Survivin/EPR-1 genes occurred in many kinds of hematologically malignant cells. This may be of biological importance.

The elusive factor Xa receptor: failure to detect transcripts that correspond to the published sequence of EPR-1

The coagulation protease factor Xa induces cellular responses implicated in cardiovascular and inflammatory disease. Effector-cell protease receptor 1 (EPR-1) is a functionally characterized receptor of factor Xa, and the EPR-1complementary DNA (cDNA) was published. Remarkably, the cDNA encoding an inhibitor of apoptosis, survivin, is reportedly identical to that ofEPR-1 except for a few nucleotide differences and its orientation opposite to EPR-1. To isolate the EPR-1cDNA and gene, we surveyed gene databases for expressed sequence tags (ESTs) that could be derived from EPR-1. All ESTs with strong homology to EPR-1/survivin were derived from survivinand could not encode EPR-1. By polymerase chain reaction and Southern blot hybridization, EPR-1 was not detectable in the human or murine genome, but survivin was. Our data suggest that EPR-1 is either highly cell-specific or the published EPR-1 cDNA includes sequences from clones derived from survivin messenger RNA. The means by which factor Xa mediates its cellular effects requires further evaluation.

The elusive factor Xa receptor: failure to detect transcripts that correspond to the published sequence of EPR-1

AbstractThe coagulation protease factor Xa induces cellular responses implicated in cardiovascular and inflammatory disease. Effector-cell protease receptor 1 (EPR-1) is a functionally characterized receptor of factor Xa, and the EPR-1complementary DNA (cDNA) was published. Remarkably, the cDNA encoding an inhibitor of apoptosis, survivin, is reportedly identical to that ofEPR-1 except for a few nucleotide differences and its orientation opposite to EPR-1. To isolate the EPR-1cDNA and gene, we surveyed gene databases for expressed sequence tags (ESTs) that could be derived from EPR-1. All ESTs with strong homology to EPR-1/survivin were derived from survivinand could not encode EPR-1. By polymerase chain reaction and Southern blot hybridization, EPR-1 was not detectable in the human or murine genome, but survivin was. Our data suggest that EPR-1 is either highly cell-specific or the published EPR-1 cDNA includes sequences from clones derived from survivin messenger RNA. The means by which factor Xa mediates its cellular effects requires further evaluation.

Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti-effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells.

Factor Xa Induces Cytokine Production and Expression of Adhesion Molecules by Human Umbilical Vein Endothelial Cells

Proinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to factor Xa (5-80 nM) concentration dependently stimulated the production of IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the expression of E-selectin, ICAM-1, and VCAM-1, which was accompanied by polymorphonuclear leukocyte adhesion. The effects of factor Xa were blocked by antithrombin III, but not by the thrombin-specific inhibitor hirudin, suggesting that factor Xa elicits these responses directly and not via thrombin. IL-1alpha and TNF-alpha were not implicated, since neither the IL-1 receptor antagonist nor a TNF-neutralizing Ab could suppress the factor Xa responses. Active site-inhibited factor Xa and factor Xa depleted from gamma-carboxyglutamic acid residues were completely inactive. The effector cell protease receptor-1 (EPR-1) seems not to be involved since anti-EPR-1 Abs failed to inhibit cytokine production. Moreover, neither the factor X peptide Leu83-Leu88, representing the inter-epidermal growth factor sequence in factor Xa that mediates ligand binding to EPR-1, nor the peptide AG1, corresponding to the EPR-1 sequence Ser123-Pro137 implicated in factor Xa binding, inhibited the factor Xa-induced cytokine production. In conclusion, these findings indicate that factor Xa evokes a proinflammatory response in endothelial cells, which requires both its catalytic and gamma-carboxyglutamic acid-containing domain. The receptor system involved in these responses induced by factor Xa remains to be established.

Structural basis for chemical inhibition of human blood coagulation factor Xa.

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1-44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-A resolution. The racemic mixture of inhibitor FX-2212, (2RS)-(3'-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1-45] factor Xa and Des[1-44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.

Induction of Apoptosis and Inhibition of Cell Proliferation bysurvivin Gene Targeting

Survivin is a new IAP apoptosis inhibitor expressed during development and in human cancer in vivo. The coding strand of the survivin gene was extensively complementary to that of effector cell protease receptor-1 (EPR-1), prompting the present investigation on the origin and functional relationship of these two transcripts. Southern blots of genomic DNA were consistent with the presence of multiple, evolutionarily conserved, EPR-1/Survivin-related genes. By pulsed field gel electrophoresis and single- and two-color fluorescence in situ hybridization, these were contained within a contiguous physical interval of 75-130 kilobases (kb) on chromosome 17q25. In Northern blots, a single strand-specific probe identified a 1.3-kb EPR-1 mRNA broadly distributed in normal adult and fetal tissues, structurally distinct from the 1.9-kb Survivin transcript expressed in transformed cell lines. Transient co-transfection of an EPR-1 cDNA potentially acting as a Survivin antisense with a lacZ reporter plasmid resulted in loss of viability of HeLa cells. In contrast, co-transfection of an antisense cDNA of intercellular adhesion molecule-1 or a sense-oriented Survivin cDNA was without effect. In stably transfected HeLa cells, ZnSO4 induction of an EPR-1 mRNA under the control of a metallothionein promoter suppressed the expression of endogenous survivin. This resulted in (i) increased apoptosis as detected by analysis of DNA content and in situ internucleosomal DNA fragmentation and (ii) inhibition of cell proliferation as compared with induced vector control transfectants. These findings suggest the existence of a potential EPR-1/survivin gene cluster and identify survivin as a new target for disrupting cell viability pathways in cancer.

Hypotension and inflammatory cytokine gene expression triggered by factor Xa-nitric oxide signaling.

The signaling pathway initiated by factor Xa on vascular endothelial cells was investigated. Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1-2.5 microG/ml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective. A factor Xa inter-epidermal growth factor synthetic peptide L (83)FTRKL(88) (G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective. Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) but not by D-NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55-3.1 ng/ml) in a reaction inhibited by L-NAME and by the inter-epidermal growth factor peptide Leu(83)-Leu(88) but unaffected by hirudin. Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, unaffected by subsequent addition of tissue factor pathway inhibitor. These data suggest that factor Xa-induced NO release modulates endothelial cell-dependent vasorelaxation and cytokine gene expression. This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulation in vivo.

Effector protease receptor 1 mediates the mitogenic activity of factor Xa for vascular smooth muscle cells in vitro and in vivo.

The binding of 125I-factor Xa to human aortic smooth muscle cell (SMC) monolayers was studied. At 4 degreesC, 125I-factor Xa bound to a single class of binding sites with a dissociation constant value of 3.6+/-0.7 nM and a binding site density of 11,720+/-1,240 sites/cell (n = 9). 125I-factor Xa binding was not affected by factor X, thrombin, or by DX9065, a direct inhibitor of factor Xa, but was inhibited by factor Xa (IC50 = 5.4+/-0.2 nM; n = 9) and by antibodies specific for the effector cell protease receptor 1 (EPR-1), a well-known receptor of factor Xa on various cell types. A factor X peptide duplicating the inter-EGF sequence Leu83-Leu88-(Gly) blocked the binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 = 110+/-21 nM). Factor Xa increased phosphoinositide turnover in SMCs and when added to SMCs in culture was a potent mitogen. These effects were inhibited by DX9065 and by antibodies directed against EPR-1 and PDGF. Increased expression of EPR-1 was identified immunohistochemically on SMCs growing in culture and in SMCs from the rabbit carotid artery after vascular injury. When applied locally to air-injured rabbit carotid arteries, antibodies directed against EPR-1 (100 mug/ artery) strongly reduced myointimal proliferation 14 d after vascular injury (65-71% inhibition, P < 0.01). DX9065 (10 mg/kg, subcutaneous) inhibited myointimal proliferation significantly (43% inhibition, P < 0.05). These findings indicate that SMCs express functional high affinity receptors for factor Xa related to EPR-1, which may be of importance in the regulation of homeostasis of the vascular wall and after vascular injury.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

We evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP's anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme's active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP's antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Factor X‐dependent, thrombin‐generating activities on a neuroblastoma cell and their disappearance upon differentiation

Some tumor cells induce platelet aggregation in the bloodstream, which has been implicated in tumor metastasis. In this study, we investigated the mechanism of platelet aggregation induced by a human neuroblastoma cell line, GOTO. It was revealed that GOTO cells had tissue factor on their surface and converted factor X (FX) to FXa with the aid of factor VIIa. The produced FXa formed prothrombinase complex on the cells and activated prothrombin. From experiments on activity inhibition by specific monoclonal as well as polyclonal antibodies, it was concluded that factor V did not constitute this prothrombinase complex. Another cofactor known to constitute prothrombinase complex on some cells, effector cell protease receptor-1 (EPR-1), was not expressed on GOTO cells, suggesting that the cofactor composing FXa-dependent prothrombinase activity on GOTO cells is not factor V or EPR-1 but, rather, is an unknown molecule. Upon the culturing in the presence of 5-bromo-2′-deoxyuridine for 4 days, GOTO cells differentiated into Schwann-like cells, and both FXase and prothrombinase activities were greatly diminished. Flow cytometric analyses revealed that the decrease of FXase activity should be attributed to the decrease of tissue factor expression on GOTO cells. Because these activities greatly diminished upon cellular differentiation, the expression of both cofactor molecules may be related to the malignant and metastatic nature of the tumor cells. J. Cell. Physiol. 173:406–414, 1997. © 1997 Wiley-Liss, Inc.

Factor X-dependent, thrombin-generating activities on a neuroblastoma cell and their disappearance upon differentiation.

Some tumor cells induce platelet aggregation in the bloodstream, which has been implicated in tumor metastasis. In this study, we investigated the mechanism of platelet aggregation induced by a human neuroblastoma cell line, GOTO. It was revealed that GOTO cells had tissue factor on their surface and converted factor X (FX) to FXa with the aid of factor VIIa. The produced FXa formed prothrombinase complex on the cells and activated prothrombin. From experiments on activity inhibition by specific monoclonal as well as polyclonal antibodies, it was concluded that factor V did not constitute this prothrombinase complex. Another cofactor known to constitute prothrombinase complex on some cells, effector cell protease receptor-1 (EPR-1), was not expressed on GOTO cells, suggesting that the cofactor composing FXa-dependent prothrombinase activity on GOTO cells is not factor V or EPR-1 but, rather, is an unknown molecule. Upon the culturing in the presence of 5-bromo-2'-deoxyuridine for 4 days, GOTO cells differentiated into Schwann-like cells, and both FXase and prothrombinase activities were greatly diminished. Flow cytometric analyses revealed that the decrease of FXase activity should be attributed to the decrease of tissue factor expression on GOTO cells. Because these activities greatly diminished upon cellular differentiation, the expression of both cofactor molecules may be related to the malignant and metastatic nature of the tumor cells.

Human umbilical vein endothelial cells express high affinity receptors for factor Xa.

The binding of [125I]-factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7 degrees C, [125I]-factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 +/- 0.8 nM and a binding site density of 57,460 +/- 5,200 sites/cell (n = 3). Association and dissociation kinetics were of a pseudo-first order and gave association and dissociation rate constant values of 0.15 x 10(6) M-1 s-1 and 4.0 x 10(-4) s-1, respectively. [125I]-factor Xa binding was inhibited by factor Xa but was not affected by factor X, thrombin or monoclonal antibodies against factor V, antithrombin-III or tissue factor pathway inhibitor (TFPI) but was inhibited by an antibody specific for the effector cell protease receptor-1 (EPR-1), a well-known receptor of factor Xa on various cell types. [125I]-factor Xa binding to HUVEC was not affected by various inhibitors of factor Xa such as DX 9065, pentasaccharide-antithrombin-III or TFPI. Factor Xa increased intracellular free calcium levels and phosphoinositide turnover in endothelial cells and, when added to HUVEC in culture, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. HUVEC-bound factor Xa promoted prothrombin activation in the presence of factor Va only. This effect was inhibited by both indirect and direct inhibitors of factor Xa. These findings indicate that HUVEC express functional high affinity receptors for factor Xa, related to EPR-1, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall.

Human umbilical vein endothelial cells express high affinity receptors for factor Xa

The binding of [125I]-factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7°C, [125I]-factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 ± 0.8 nM and a binding site density of 57,460 ± 5,200 sites/cell (n = 3). Association and dissociation kinetics were of a pseudo-first order and gave association and dissociation rate constant values of 0.15 × 106 M-1 s-1 and 4.0 × 10-4 s-1, respectively. [125I]-factor Xa binding was inhibited by factor Xa but was not affected by factor X, thrombin or monoclonal antibodies against factor V, antithrombin-III or tissue factor pathway inhibitor (TFPI) but was inhibited by an antibody specific for the effector cell protease receptor-1 (EPR-1), a well-known receptor of factor Xa on various cell types. [125I]-factor Xa binding to HUVEC was not affected by various inhibitors of factor Xa such as DX 9065, pentasaccharide-antithrombin-III or TFPI. Factor Xa increased intracellular free calcium levels and phosphoinositide turnover in endothelial cells and, when added to HUVEC in culture, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. HUVEC-bound factor Xa promoted prothrombin activation in the presence of factor Va only. This effect was inhibited by both indirect and direct inhibitors of factor Xa. These findings indicate that HUVEC express functional high affinity receptors for factor Xa, related to EPR-1, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall. J. Cell. Physiol. 172:36–43, 1997. © 1997 Wiley-Liss, Inc.

Factor Xa as an interface between coagulation and inflammation. Molecular mimicry of factor Xa association with effector cell protease receptor-1 induces acute inflammation in vivo.

Coagulation proteases were tested in a rat model of acute inflammation. Subplantar injection of Factor Xa (10-30 microg) produced a time- and dose-dependent edema in the rat paw, and potentiated carrageenin-induced edema. In contrast, the homologous protease Factor IXa was ineffective. This inflammatory response was recapitulated by the Factor Xa sequence L83FTRKL88(G), which mediates ligand binding to effector cell protease receptor-1 (EPR-1), while a control scrambled peptide did not induce edema in vivo. Conversely, injection of the EPR-1-derived peptide S123PGKPGNQNSKNEPP137 (corresponding to the receptor binding site for Factor Xa) inhibited carrageenin-induced rat paw edema, while the adjacent EPR-1 sequence P136PKKRERERSSHCYP150 was without effect. EPR-1-Factor Xa-induced inflammation was characterized by fast onset and prominent perivascular accumulation of activated and degranulated mast cells, was inhibited by the histamine/serotonin antagonists cyproheptadine and methysergide, but was unaffected by the thrombin-specific inhibitor, Hirulog. These findings suggest that through its interaction with EPR-1, Factor Xa may function as a mediator of acute inflammation in vivo. This pathway may amplify both coagulation and inflammatory cascades, thus contributing to the pathogenesis of tissue injury in vivo.