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J. E. Sadler - One of the best experts on this subject based on the ideXlab platform.
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Thrombomodulin structure and function.
Thrombosis and haemostasis, 1997Co-Authors: J. E. SadlerAbstract:Thrombomodulin is protein cofactor expressed on endothelial cell surfaces that modifies the substrate specificity of thrombin, apparently by an allosteric mechanism. The thrombin-Thrombomodulin complex activates protein C, initiating an essential anticoagulant pathway. The cofactor function of membrane-associated Thrombomodulin requires the last three of six tandemly repeated EGF-like domains (numbers 4, 5, and 6), as well as a Ser/Thr-rich spacer between EGF-like domain 6 and the transmembrane domain. The Ser/Thr-rich domain is variably modified with a chondroitin sulfate chain that influences the affinity of thrombin binding and the calcium ion dependence of cofactor function. The structure of EGF-like domain 4 has been determined by NMR spectroscopy, and the structure of a complex between thrombin and a peptide from Thrombomodulin EGF-like domain 5 was determined by X-ray crystallography. These structures are small steps toward an understanding of how Thrombomodulin regulates thrombin.
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Sequences required for Thrombomodulin cofactor activity within the fourth epidermal growth factor-like domain of human Thrombomodulin.
The Journal of biological chemistry, 1993Co-Authors: Steven R. Lentz, Yan Chen, J. E. SadlerAbstract:Abstract Activation of protein C by thrombin is stimulated by the endothelial cell cofactor Thrombomodulin. The structural regions of Thrombomodulin necessary for cofactor activity have been localized to the fourth through sixth epidermal growth factor (EGF)-like domains. The fourth EGF-like domain is unnecessary for high affinity thrombin binding, but is required for cofactor activity. To identify essential sequences within the fourth EGF-like domain, a series of recombinant human Thrombomodulins consisting of EGF-like domains four through six were expressed in human kidney cells. These mutants contain replacements of disulfide loops within the fourth EGF-like domain, thereby conserving overall disulfide bond structure. All of the mutants bound to thrombin with high affinity, and inhibited the fibrinogen-clotting activity of thrombin to a similar extent. Two regions of the fourth EGF-like domain were identified to be essential for cofactor activity: 1) the sequence consisting of amino acids Glu-357, Tyr-358, and Gln-359 shared by the overlapping first and second disulfide loops, and 2) the amino-terminal region of the third disulfide loop containing amino acids Glu-374, Gly-375, and Phe-376. These results suggest that amino acids critical for Thrombomodulin cofactor activity are located near the junction between the two subdomains of the fourth EGF-like domain.
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Structure-function relationships of the thrombin-Thrombomodulin interaction.
Haemostasis, 1993Co-Authors: J. E. Sadler, S R Lentz, J P Sheehan, M TsiangAbstract:Thrombomodulin is an anticoagulant protein cofactor that modulates the substrate specificity of thrombin and promotes the cleavage of protein C. The structure-function relationships of the thrombin-Thrombomodulin interaction have been explored by recombinant DNA and protein chemistry methods. Thrombomodulin binds to thrombin at an anion-binding exosite on the carboxyl-terminal side of the substrate binding cleft. This interaction interferes with the recognition and cleavage of fibrinogen, factor V, and the platelet thrombin receptor. Binding to Thrombomodulin also protects thrombin from inhibition by heparin cofactor II. The major thrombin binding site on Thrombomodulin consists of EGF-like domains 5 and 6. In addition, EGF-like domain 4 is required for Thrombomodulin to accelerate the activation of protein C. Some Thrombomodulin molecules contain a chondroitin sulfate moiety attached to a Ser/Thr-rich domain adjacent to the cell membrane. This modification is not required for the cofactor activity of Thrombomodulin, but appears to contribute to 'direct anticoagulant' activity--the ability of Thrombomodulin to inhibit fibrinogen clotting, factor V activation, and platelet activation. The chondroitin sulfate moiety of Thrombomodulin also can affect the rate of thrombin inhibition by antithrombin III, possibly by competing with heparin for the heparin binding site on thrombin. Detailed understanding of these interactions could lead to new strategies for the treatment of bleeding or thrombotic disorders.
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ligand specificity of human Thrombomodulin equilibrium binding of human thrombin meizothrombin and factor xa to recombinant Thrombomodulin
Journal of Biological Chemistry, 1992Co-Authors: M Tsiang, Steven R. Lentz, J. E. SadlerAbstract:Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human Thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human Thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to Thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to Thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of Thrombomodulin, whereas no significant activation by factor Xa was detected with or without Thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of Thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important Thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell Thrombomodulin.
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inhibition of Thrombomodulin surface expression and protein c activation by the thrombogenic agent homocysteine
Journal of Clinical Investigation, 1991Co-Authors: Steven R. Lentz, J. E. SadlerAbstract:: Elevated levels of plasma homocysteine are associated with both venous and arterial thrombosis. Homocysteine inhibits the function of Thrombomodulin, an anticoagulant glycoprotein on the endothelial surface that serves as a cofactor for the activation of protein C by thrombin. The effects of homocysteine on Thrombomodulin expression and protein C activation were investigated in cultured human umbilical vein endothelial cells and CV-1(18A) cells that express recombinant human Thrombomodulin. Addition of 5 mM homocysteine to endothelial cells produced slight increases in Thrombomodulin mRNA and Thrombomodulin synthesis without affecting cell viability. In both cell types, Thrombomodulin synthesized in the presence of homocysteine remained sensitive to digestion with endoglycosidase H and failed to appear on the cell surface, suggesting impaired transit along the secretory pathway. In a cell-free protein C activation assay, homocysteine irreversibly inactivated both Thrombomodulin and protein C in a process that required free thiol groups and was inhibited by the oxidizing agents diamide or N-ethylmaleimide. By inhibiting both Thrombomodulin surface expression and protein C activation, homocysteine may contribute to the development of thrombosis in patients with cystathionine beta-synthase deficiency.
Steven R. Lentz - One of the best experts on this subject based on the ideXlab platform.
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Targeted Replacement of the Murine Thrombomodulin Gene with Human Thrombomodulin Coding Sequence Results in Decreased Protein C Activation and Enhanced Thrombotic Response to Photochemical Injury.
Blood, 2004Co-Authors: Thomas J. Raife, Rochelle A. Erger, Katina M. Wilson, José A. Fernández, Hyung Suk Kim, John H. Griffin, Jennifer Alternburg, Nobuyo Maeda, Steven R. LentzAbstract:Thrombomodulin is an endothelial transmembrane glycoprotein that regulates hemostasis and inflammation. Deletion of the Thrombomodulin gene in mice results in embryonic lethality. To examine the function of human Thrombomodulin in a murine model, we used gene targeting to replace the entire coding sequence of the intronless murine Thrombomodulin gene with a human Thrombomodulin cDNA. Mice derived from successfully targeted 129/SvEv embryonic stem cells were maintained on either a 129/SvEv background or a mixed (129/B6) background. Mice homozygous for the modified allele ( hthm/hthm ) were viable, and the total amount of Thrombomodulin mRNA (measured by real time PCR) did not differ between hthm/hthm and wild-type ( +/+) mice (p>0.05). In heterozygous ( hthm/+) mice, the ratio of human to murine Thrombomodulin mRNA was similar in lung, heart, and kidney, and expression of murine Thrombomodulin transcripts was 50% of that in wild-type (+/+) mice. Expression of human Thrombomodulin protein was detected in endothelium by immunohistochemistry and in lung lysates by enzyme linked immunoassay. Thrombomodulin cofactor activity, measured in a protein C activation assay with human protein C and thrombin, was decreased in the aorta of hthm/hthm mice (1.1±0.5 nmol/mm) compared with +/+ mice (3.6±0.3 nmol/mm; p hthm/hthm mice (0.7±0.1 nmol/mg protein) than in +/+ mice (2.5±0.5 nmol/mg protein; p hthm/hthm and +/+ mice was even greater when measured with murine protein C and thrombin (0.7±0.6 vs. 5.2±0.9 nmol/mg protein; p Hthm/+ mice had intermediate levels of cofactor activity with either human or murine reagents. Time to thrombotic occlusion of the carotid artery after photochemical injury (rose bengal and green laser) was shorter in hthm/hthm mice than in +/+ mice (10.4±3.0 vs. 34.6±10.6 minutes; p hthm/hthm mice suggests that this animal model may be useful for investigation of human Thrombomodulin function in vivo .
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Expression of Thrombomodulin and consequences of Thrombomodulin deficiency during healing of cutaneous wounds.
The American journal of pathology, 1999Co-Authors: Jeffrey J. Peterson, Thomas J. Raife, Donna J. Lager, Helen Rayburn, G. Patrick Kealey, Robert D. Rosenberg, Steven R. LentzAbstract:Thrombomodulin is a cell surface anticoagulant that is expressed by endothelial cells and epidermal keratinocytes. Using immunohistochemistry, we examined Thrombomodulin expression during healing of partial-thickness wounds in human skin and full-thickness wounds in mouse skin. We also examined Thrombomodulin expression and wound healing in heterozygous Thrombomodulin-deficient mice, compound heterozygous mice that have
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Cellular localization of Thrombomodulin in human epithelium and squamous malignancies.
The American journal of pathology, 1995Co-Authors: Donna J. Lager, Thomas J. Raife, E. J. Callaghan, S. F. Worth, Steven R. LentzAbstract:Thrombomodulin is a cell surface glycoprotein that functions as an anticoagulant. Although initially identified on endothelial cells, Thrombomodulin is also expressed by other vascular cells, by mesothelial cells, and by epidermal keratinocytes. To determine whether Thrombomodulin is expressed by epithelial cells in locations other than skin, we conducted a survey of Thrombomodulin protein and mRNA in human epithelium. Thrombomodulin protein was detected by immunohistochemistry in all samples containing stratified squamous epithelium, including oral mucosa, larynx, esophagus, uterine ectocervix, and vagina. In these tissues, Thrombomodulin staining localized to the suprabasal layer, with minimal staining observed in the basal or superficial layers of epithelium. Thrombomodulin was not detected in cuboidal, simple columnar, or pseudostratified columnar epithelium and was detected variably in transitional epithelium. Thrombomodulin staining was also observed in 21 of 26 cases of invasive squamous cell carcinoma and in several examples of squamous carcinoma-in-situ and squamous metaplasia. Expression of Thrombomodulin mRNA was confirmed by in situ hybridization in both normal and malignant squamous epithelium. Full-length, functionally active Thrombomodulin was demonstrated in cultured squamous epithelial cells. These data demonstrate that Thrombomodulin expression correlates with the squamous phenotype and suggest that hemostasis is regulated by compartmentalization of procoagulant and anti-coagulant epithelial proteins.
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Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation.
The Journal of clinical investigation, 1994Co-Authors: Thomas J. Raife, Yan Chen, Donna J. Lager, Kathi C. Madison, Warren W. Piette, E. J. Howard, M. T. Sturm, Steven R. LentzAbstract:Thrombomodulin is an endothelial cell surface glycoprotein that inhibits the procoagulant activities of thrombin and accelerates activation of the anticoagulant protein C. Because protein C deficiency is associated with cutaneous thrombosis, we investigated the expression of Thrombomodulin in human skin. Thrombomodulin was detected by immunohistochemical staining both in dermal endothelial cells and in epidermal keratinocytes. Within the epidermis, Thrombomodulin staining was limited to keratinocytes of the spinous layer, suggesting that Thrombomodulin is induced when basal keratinocytes begin to terminally differentiate. Thrombomodulin expression also correlated with squamous differentiation in epidermal malignancies; little or no Thrombomodulin staining was seen in five basal cell carcinomas, whereas strong Thrombomodulin staining was observed in each of five squamous cell carcinomas. Human foreskin keratinocytes cultured in medium containing 0.07 mM calcium chloride synthesized functional Thrombomodulin with cofactor activity comparable to Thrombomodulin in human umbilical vein endothelial cells. Stimulation of keratinocyte differentiation with 1.4 mM calcium chloride for 48 h produced 3.5-, 3.2-, and 5.6-fold increases in Thrombomodulin cofactor activity, antigen, and mRNA, respectively. These observations suggest that thrombin is regulated by keratinocyte Thrombomodulin at sites of cutaneous injury, and indicate a potential role for Thrombomodulin in epidermal differentiation.
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Sequences required for Thrombomodulin cofactor activity within the fourth epidermal growth factor-like domain of human Thrombomodulin.
The Journal of biological chemistry, 1993Co-Authors: Steven R. Lentz, Yan Chen, J. E. SadlerAbstract:Abstract Activation of protein C by thrombin is stimulated by the endothelial cell cofactor Thrombomodulin. The structural regions of Thrombomodulin necessary for cofactor activity have been localized to the fourth through sixth epidermal growth factor (EGF)-like domains. The fourth EGF-like domain is unnecessary for high affinity thrombin binding, but is required for cofactor activity. To identify essential sequences within the fourth EGF-like domain, a series of recombinant human Thrombomodulins consisting of EGF-like domains four through six were expressed in human kidney cells. These mutants contain replacements of disulfide loops within the fourth EGF-like domain, thereby conserving overall disulfide bond structure. All of the mutants bound to thrombin with high affinity, and inhibited the fibrinogen-clotting activity of thrombin to a similar extent. Two regions of the fourth EGF-like domain were identified to be essential for cofactor activity: 1) the sequence consisting of amino acids Glu-357, Tyr-358, and Gln-359 shared by the overlapping first and second disulfide loops, and 2) the amino-terminal region of the third disulfide loop containing amino acids Glu-374, Gly-375, and Phe-376. These results suggest that amino acids critical for Thrombomodulin cofactor activity are located near the junction between the two subdomains of the fourth EGF-like domain.
Yuichi Kanmura - One of the best experts on this subject based on the ideXlab platform.
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Efficacy of recombinant human soluble Thrombomodulin in disseminated intravascular coagulation
Critical Care, 2010Co-Authors: Tomotsugu Yasuda, N Kiyonaga, T Ohryorji, M Nakahara, Naoko Okayama, T Kikuchi, T Imabayashi, Yasuyuki Kakihana, Yuichi KanmuraAbstract:Soluble Thrombomodulin is a promising therapeutic natural anticoagulant that is comparable with antithrombin, tissue factor pathway inhibitor and activated protein C. Recently, a recombinant human soluble Thrombomodulin, composed of the active, extracellular domain of Thrombomodulin, has become commercially available for patients with disseminated intravascular coagulation (DIC). However, its effect on adhesion molecule or plasminogen activator inhibitor 1 (PAI-1) in patients with DIC is not clear. To elucidate the possible effect of a soluble Thrombomodulin on endothelial cell and neutrophil interaction in DIC, we investigated the efficacy of a recombinant human soluble Thrombomodulin against not only soluble fibrin (SF) for the sensitive marker of DIC, but also soluble E-selectin (sES) and PAI-1 in patients with DIC.
Alain Beretz - One of the best experts on this subject based on the ideXlab platform.
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Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing Thrombomodulin and decreasing tissue factor activities.
British Journal of Pharmacology, 1993Co-Authors: Gisele Haan-archipoff, Alain Beretz, K Bartha, C Brisson, C De La Salle, C Froget-léon, C Klein-soyer, Jean-pierre CazenaveAbstract:1. The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were investigated on the expression of tissue factor and Thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2. Forskolin (10(-6) to 10(-4) M), PGE1 (10(-7) to 10(-5) M) and db cyclic AMP (10(-4) to 10(-3) M) caused a concentration-dependent decrease of cytokine-induced tissue factor activity. 3. Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive Thrombomodulin activity and reversed the decrease of this activity caused by interleukin-1 (IL-1). 4. IBMX (10(-4) M) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and Thrombomodulin activities. 5. Forskolin (10(-4) M) decreased the IL-1-induced tissue factor mRNA and increased the Thrombomodulin mRNA level. IL-1 did not change the Thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6. Dibutyryl cyclic GMP (10(-4) M to 10(-3) M) did not influence tissue factor or Thrombomodulin activity. 7. Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and Thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium.
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Thrombin regulates tissue factor and Thrombomodulin mRNA levels and activities in human saphenous vein endothelial cells by distinct mechanisms.
Journal of Biological Chemistry, 1993Co-Authors: K Bartha, Gisele Haan-archipoff, C Brisson, C De La Salle, Jean-pierre Cazenave, Francois Lanza, Alain BeretzAbstract:The effects of thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK)-inhibited thrombin, and thrombin receptor agonist peptide, SFLLRNPNDKYEPF (SFLL, a portion of the receptor unmasked after thrombin cleavage), on the expression of tissue factor (TF) and Thrombomodulin by human saphenous vein endothelial cells (HSVECs) in culture were studied. Unstimulated cells contained very low amounts of TF mRNA as measured by the reverse transcriptase-PCR method. Thrombin treatment increased TF mRNA to 8.0 +/- 1.9 (n = 3) times the control level. The increase was detectable within 2 h and declined to near basal level by 6 h. Induction of TF mRNA was not blocked by cycloheximide, treatment with cycloheximide alone also increased TF mRNA levels, and thrombin in combination with cycloheximide further enhanced the accumulation of TF mRNA. Thrombin caused a 14.5 +/- 1.5-fold (n = 5) increase in TF activity on the surface of HSVECs and a 20.5 +/- 1.4-fold (mean +/- S.D., n = 2) increase in the extracellular matrix. The thrombin-induced effects on TF synthesis could be fully reproduced by the thrombin receptor agonist peptide, SFLL, whereas PPACK-inhibited thrombin did not influence TF expression. Thrombin increased Thrombomodulin mRNA to 190 +/- 39% (n = 5) of control levels, whereas PPACK-inhibited thrombin or SFLL did not influence Thrombomodulin mRNA levels. In contrast, surface-bound Thrombomodulin cofactor activity and Thrombomodulin antigen in the cell lysates did not change over 24 h of incubation with thrombin. However, thrombin caused a 2-fold increase in Thrombomodulin antigen released into the conditioned medium, and immunoelectron microscopy of HSVECs also demonstrated the presence of Thrombomodulin vesicles close to the luminal cell surface in thrombin-treated cultures. The Western blot pattern Thrombomodulin in the conditioned medium of untreated and thrombin-treated cells was found to be similar, and soluble Thrombomodulin occurred mainly as fragments of the cell-associated form. We conclude that the transcriptional control by thrombin causes an increase in both TF and Thrombomodulin mRNA. The increase in TF mRNA levels is also paralleled by an increase in surface expression, is dependent on the proteolytic activity of thrombin, and is mediated by the same receptor as the recently cloned thrombin receptor in platelets. Up-regulation of Thrombomodulin mRNA levels by thrombin is distinct from this pathway and is associated with unchanged expression on the cell surface.
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Use of annexin-V to demonstrate the role of phosphatidylserine exposure in the maintenance of haemostatic balance by endothelial cells.
The Biochemical Journal, 1992Co-Authors: C Ravanat, Alain Beretz, Jean-pierre Cazenave, G Archipoff, G Freund, Jf FreyssinetAbstract:Annexin-V (PAP-I, lipocortin-V) acts as a potent anticoagulant in vitro by binding to negatively charged phospholipids with higher affinity than vitamin K-dependent proteins, with a Kd in the 10(-10) M range. The purpose of the present study was to use annexin-V as a probe to assess the catalytic potential of phospholipids in pro- and anti-coagulant reactions in purified systems and at the surface of endothelial cells in culture after stimulation. Procoagulant tissue factor and anticoagulant Thrombomodulin activities were compared by using specific two-stage amidolytic assays performed with purified proteins. Procoagulant activity was estimated by the generation of Factor Xa by the Factor VII(a)-tissue factor complex. Anticoagulant activity was estimated by the generation of activated protein C by either the thrombin-Thrombomodulin complex or Factor Xa. Annexin-V induced a decrease of 70% of Thrombomodulin activity when Thrombomodulin (5.4-214 nM) was reconstituted into phosphatidylcholine/phosphatidylserine (1:1, mol/mol) vesicles at 37.5 or 75 microM-phospholipid concentration, the apparent Ki being 0.5 microM at 75 microM-lipid. The saturating concentration of annexin-V was dependent on phospholipid concentration, but was independent of the phospholipid/Thrombomodulin ratio. By contrast, when Thrombomodulin was not reconstituted in vesicles, annexin-V had no effect. At 2 microM, annexin-V totally inhibited the generation of activated protein C by Factor Xa in the presence of 75 microM-lipid, the saturating inhibitory concentration being dependent on phospholipid concentration. At 0.1 microM, annexin-V totally inhibited tissue-factor activity present in crude brain thromboplastin. In the absence of stimulation, human endothelial cells in culture expressed significant Thrombomodulin activity and no detectable tissue-factor activity. Basal Thrombomodulin activity was only slightly inhibited (less than 15%) by 0.5 microM-annexin-V. Phorbol myristate acetate (PMA) induced the expression of tissue-factor activity and decreased Thrombomodulin activity at the endothelial-cell surface. Annexin-V, at a concentration of 16 microM, caused an 80% decrease of tissue-factor activity induced by PMA at 10 ng/ml, whereas it inhibited Thrombomodulin activity by only 15% on the same stimulated cells. Our results confirm that annexin-V inhibits, in vitro, procoagulant tissue-factor activity and anticoagulant activities (activation of protein C by the thrombin-Thrombomodulin complex and by Factor Xa), through phospholipid-dependent mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)
Jean-pierre Cazenave - One of the best experts on this subject based on the ideXlab platform.
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Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing Thrombomodulin and decreasing tissue factor activities.
British Journal of Pharmacology, 1993Co-Authors: Gisele Haan-archipoff, Alain Beretz, K Bartha, C Brisson, C De La Salle, C Froget-léon, C Klein-soyer, Jean-pierre CazenaveAbstract:1. The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were investigated on the expression of tissue factor and Thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2. Forskolin (10(-6) to 10(-4) M), PGE1 (10(-7) to 10(-5) M) and db cyclic AMP (10(-4) to 10(-3) M) caused a concentration-dependent decrease of cytokine-induced tissue factor activity. 3. Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive Thrombomodulin activity and reversed the decrease of this activity caused by interleukin-1 (IL-1). 4. IBMX (10(-4) M) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and Thrombomodulin activities. 5. Forskolin (10(-4) M) decreased the IL-1-induced tissue factor mRNA and increased the Thrombomodulin mRNA level. IL-1 did not change the Thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6. Dibutyryl cyclic GMP (10(-4) M to 10(-3) M) did not influence tissue factor or Thrombomodulin activity. 7. Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and Thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium.
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Thrombin regulates tissue factor and Thrombomodulin mRNA levels and activities in human saphenous vein endothelial cells by distinct mechanisms.
Journal of Biological Chemistry, 1993Co-Authors: K Bartha, Gisele Haan-archipoff, C Brisson, C De La Salle, Jean-pierre Cazenave, Francois Lanza, Alain BeretzAbstract:The effects of thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK)-inhibited thrombin, and thrombin receptor agonist peptide, SFLLRNPNDKYEPF (SFLL, a portion of the receptor unmasked after thrombin cleavage), on the expression of tissue factor (TF) and Thrombomodulin by human saphenous vein endothelial cells (HSVECs) in culture were studied. Unstimulated cells contained very low amounts of TF mRNA as measured by the reverse transcriptase-PCR method. Thrombin treatment increased TF mRNA to 8.0 +/- 1.9 (n = 3) times the control level. The increase was detectable within 2 h and declined to near basal level by 6 h. Induction of TF mRNA was not blocked by cycloheximide, treatment with cycloheximide alone also increased TF mRNA levels, and thrombin in combination with cycloheximide further enhanced the accumulation of TF mRNA. Thrombin caused a 14.5 +/- 1.5-fold (n = 5) increase in TF activity on the surface of HSVECs and a 20.5 +/- 1.4-fold (mean +/- S.D., n = 2) increase in the extracellular matrix. The thrombin-induced effects on TF synthesis could be fully reproduced by the thrombin receptor agonist peptide, SFLL, whereas PPACK-inhibited thrombin did not influence TF expression. Thrombin increased Thrombomodulin mRNA to 190 +/- 39% (n = 5) of control levels, whereas PPACK-inhibited thrombin or SFLL did not influence Thrombomodulin mRNA levels. In contrast, surface-bound Thrombomodulin cofactor activity and Thrombomodulin antigen in the cell lysates did not change over 24 h of incubation with thrombin. However, thrombin caused a 2-fold increase in Thrombomodulin antigen released into the conditioned medium, and immunoelectron microscopy of HSVECs also demonstrated the presence of Thrombomodulin vesicles close to the luminal cell surface in thrombin-treated cultures. The Western blot pattern Thrombomodulin in the conditioned medium of untreated and thrombin-treated cells was found to be similar, and soluble Thrombomodulin occurred mainly as fragments of the cell-associated form. We conclude that the transcriptional control by thrombin causes an increase in both TF and Thrombomodulin mRNA. The increase in TF mRNA levels is also paralleled by an increase in surface expression, is dependent on the proteolytic activity of thrombin, and is mediated by the same receptor as the recently cloned thrombin receptor in platelets. Up-regulation of Thrombomodulin mRNA levels by thrombin is distinct from this pathway and is associated with unchanged expression on the cell surface.
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Use of annexin-V to demonstrate the role of phosphatidylserine exposure in the maintenance of haemostatic balance by endothelial cells.
The Biochemical Journal, 1992Co-Authors: C Ravanat, Alain Beretz, Jean-pierre Cazenave, G Archipoff, G Freund, Jf FreyssinetAbstract:Annexin-V (PAP-I, lipocortin-V) acts as a potent anticoagulant in vitro by binding to negatively charged phospholipids with higher affinity than vitamin K-dependent proteins, with a Kd in the 10(-10) M range. The purpose of the present study was to use annexin-V as a probe to assess the catalytic potential of phospholipids in pro- and anti-coagulant reactions in purified systems and at the surface of endothelial cells in culture after stimulation. Procoagulant tissue factor and anticoagulant Thrombomodulin activities were compared by using specific two-stage amidolytic assays performed with purified proteins. Procoagulant activity was estimated by the generation of Factor Xa by the Factor VII(a)-tissue factor complex. Anticoagulant activity was estimated by the generation of activated protein C by either the thrombin-Thrombomodulin complex or Factor Xa. Annexin-V induced a decrease of 70% of Thrombomodulin activity when Thrombomodulin (5.4-214 nM) was reconstituted into phosphatidylcholine/phosphatidylserine (1:1, mol/mol) vesicles at 37.5 or 75 microM-phospholipid concentration, the apparent Ki being 0.5 microM at 75 microM-lipid. The saturating concentration of annexin-V was dependent on phospholipid concentration, but was independent of the phospholipid/Thrombomodulin ratio. By contrast, when Thrombomodulin was not reconstituted in vesicles, annexin-V had no effect. At 2 microM, annexin-V totally inhibited the generation of activated protein C by Factor Xa in the presence of 75 microM-lipid, the saturating inhibitory concentration being dependent on phospholipid concentration. At 0.1 microM, annexin-V totally inhibited tissue-factor activity present in crude brain thromboplastin. In the absence of stimulation, human endothelial cells in culture expressed significant Thrombomodulin activity and no detectable tissue-factor activity. Basal Thrombomodulin activity was only slightly inhibited (less than 15%) by 0.5 microM-annexin-V. Phorbol myristate acetate (PMA) induced the expression of tissue-factor activity and decreased Thrombomodulin activity at the endothelial-cell surface. Annexin-V, at a concentration of 16 microM, caused an 80% decrease of tissue-factor activity induced by PMA at 10 ng/ml, whereas it inhibited Thrombomodulin activity by only 15% on the same stimulated cells. Our results confirm that annexin-V inhibits, in vitro, procoagulant tissue-factor activity and anticoagulant activities (activation of protein C by the thrombin-Thrombomodulin complex and by Factor Xa), through phospholipid-dependent mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)
