D Dupouy

Centre Hospitalier Universitaire de Toulouse, Tolosa de Llenguadoc, Midi-Pyrénées, France

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Publications (35)217.01 Total impact

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    ABSTRACT: Fragile atherosclerotic plaques are rich in apoptotic smooth muscle cells (SMCs) and macrophages, generating microparticules (MPs) which accumulate locally and may be released in blood in case of mechanical or spontaneous plaque disruption. Besides being highly procoagulant, this material may interact with downstream endothelium. Using a model of mouse aorta vaso-reactivity, we have investigated the effects of apoptotic MPs prepared in vitro from Fas-ligand sensitive SMCs. Short-term preincubation of aorta rings with the MPs dose-dependently reduced the vasodilatory response to acetylcholine dependent on the endothelium. This effect was prevented by the addition of abxicimab or eptifibatide, indicating a role for a beta3 integrin in this process. We further investigated its mechanism using cultured endothelial cells. The MPs were found to bind to the cells and to inhibit the production and the release of nitric oxide (NO) in response to bradykinin. This phenomenom was redox sensitive, independent of the generation of activated coagulation proteases, and was abrogated when the MPs were pretreated by trypsin. The metabolic effects of MPs were prevented by the addition of eptifibatide. Taken together, these results suggest a potential, platelet-independent, mechanism for the improvement of microvascular perfusion observed with beta3-integrin antagonists.
    Thrombosis and Haemostasis 11/2005; 94(4):853-8. · 5.76 Impact Factor
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    ABSTRACT: As apoptosis of neo-intimal SMCs is a feature of advanced atherosclerotic plaques, the procoagulant properties of SMCs of synthetic phenotype undergoing apoptosis were investigated. SMCs isolated from rat aorta obtained 10 days after balloon injury, previously found to up-regulate Tissue Factor (TF) and Tissue Factor Inhibitor (TFPI) and to release large amounts of TFPI (Ghrib et al. Thromb Haemost 2002;87:1043-50), were sensitive to the apoptosis induced by Fas-ligand. During this process, surface TF activity rose by a factor 10 over 6 hours, in parallel with a proportional increase in prothrombinase, while TF protein expressed at the membrane significantly decreased. The microparticles (MPs) produced during SMC death bore intact and functional TF, but the release of TFPI did not change, so that the balance shifted to a procoagulant state during apoptosis. Shed MPs enhanced thrombus formation in flowing whole blood over collagen coated-glass slides. Apoptotic SMCs in atherosclerotic plaques represent a reservoir of highly thrombogenic material, released into the blood stream in case of spontaneous or mechanical plaque disruption.
    Thrombosis and Haemostasis 10/2003; 90(3):511-8. · 5.76 Impact Factor
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    ABSTRACT: Tissue factor (TF) and its specific inhibitor TF pathway inhibitor (TFPI) are produced by vascular smooth muscle cells (SMCs) in vitro and are increased in vivo in atherosclerotic compared to normal vessels. Besides local regulation of the hemostatic balance, this may be related to non-hemostatic TF/protease dependent functions such as SMC proliferation, adhesion and migration. The aim of the study was to compare the expression of both proteins between the contractile (normal adult) and synthetic (neo-intimal) SMC phenotypes. Primary cultures of SMCs isolated from rat thoracic aorta before and 10 days after balloon injury displayed stable characteristics of the contractile and synthetic phenotype, respectively. Synthetic SMCs expressed more TF mRNA than contractile SMCs, but released excess TF in the conditioned medium, so that the cell-associated TF activity measured by a factor Xa generating assay remained similar in the two subtypes. Accordingly, cell surface thrombogenicity measured under blood flow conditions was also similar. The production and release of functional TFPI was enhanced by a factor 3 to 6 (p < 0.01) in synthetic SMCs. A difference in the quantitative expression of TF and TFPI is a new distinctive feature of SMC phenotypes. Matrix-associated TFPI derived from synthetic SMCs may serve as an anchorage for their migration and regulate protease-activated processes during neo-intima formation.
    Thrombosis and Haemostasis 06/2002; 87(6):1051-6. · 5.76 Impact Factor
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    ABSTRACT: To determine whether polymorphonuclear leukocytes (PMN) modulate the production of tissue factor (TF) by monocytes, PBMC were incubated with increasing concentrations of PMN. PMN did not express any procoagulant activity. After 20-h cocultures, PMN enhanced or inhibited the TF production of PBMC, and this effect depended on the PMN/PBMC ratio. When the ratio increased from 1/1000 to 1/5, without or with LPS, the TF activity of PBMC increased to peak at 2.5-fold the baseline value (p < 0.01). The TF Ag and TF mRNA also increased. This potentiating effect was mediated by reactive oxygen species (ROS) released by PMN during the coculture; it did not require direct cell contact between PMN and PBMC, it was enhanced when PMN were stimulated by fMLP (a chemotactic peptide), and it was inhibited by two antioxidants, N-acetyl cysteine and pyrrolidine dithiocarbamate. In contrast, when the PMN/PBMC ratio was further increased from 1/2 to 2/1, the PBMC TF activity, Ag, and mRNA decreased and were inhibited compared with those of PBMC cultured alone (p < 0.01). This inhibitory effect required direct cell contact between PMN and PBMC, and it was not due to a PMN-mediated cytotoxicity. To confirm the role of ROS, H2O2 enhanced then inhibited the TF activity of PBMC in a dose-dependent manner, similarly to PMN. Thus, PMN may play an important role in the pathogenesis of thrombosis and atherosclerosis by exerting concentration-dependent regulatory effects on the TF production by PBMC via the release of ROS.
    The Journal of Immunology 05/2000; 164(7):3822-8. · 5.52 Impact Factor
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    ABSTRACT: The antithrombotic beta-D-xyloside, naroparcil, has previously been shown to induce a dose-related increase of circulating glycosaminoglycans (GAGs) together with an antithrombin activity (anti-IIa) via heparin cofactor II (HCII) in the rabbit. In order to go further in the mechanisms, the relationship between the antithrombotic activity, the HCII-mediated anti-IIa activity and the plasma GAG content was investigated. We showed that the in vitro specific activity on the inhibition of thrombin by HCII of the plasma GAG extract from naroparcil-treated rabbits was increased by a factor of 60 when compared to controls. In addition, the fractionation of the plasma GAG extract by affinity chromatography on immobilized HCII led to a more potent material whereas the low-affinity fraction was shown to be inactive in thrombin inhibition by HCII. The qualitative analysis of GAGs showed the presence of the deltaDi-4S DS disaccharide, undetectable in control, which accounted for 22% in the unfractionated GAG extract and for 60% in the high affinity fraction. In vitro experiments using immuno-depleted plasma in antithrombin III (ATIII), HCII or both, indicated that the anti-IIa activity of the plasma GAG extract from naroparcil-treated rabbits was mainly due to HCII potentialisation. The unfractionated GAG extract and the high affinity fraction were shown to be antithrombotic in a Wessler-based model in the rat, giving ED80 values of 610 UA/kg and 56 UA/kg respectively whereas the low-affinity fraction was devoid of any antithrombotic activity. These results show that the antithrombotic activity of naroparcil is dependent on modification in the plasma GAG profile which inactivates thrombin via the HCII.
    Thrombosis and Haemostasis 07/1999; 81(6):945-50. · 5.76 Impact Factor
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    Y Cadroy, D Dupouy, B Boneu
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    ABSTRACT: Monocytes express tissue factor (TF) upon stimulation by inflammatory agents. Dietary administration of fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) results in an impairment of TF expression by monocytes. EPA and DHA are metabolized differently from arachidonic acid (AA), the major fatty acid present in cell membranes. We examined the effects of AA on the TF expression of isolated human PBMC, and we determined whether EPA and DHA modulated this phenomenon differently. Nonstimulated PBMC had a low TF-dependent procoagulant activity. When PBMC were incubated with increasing concentrations of AA, the TF-dependent procoagulant activity increased in a dose-dependent manner to 190% at 7.5 microM. Indomethacin, a cyclo-oxygenase inhibitor, totally abolished the stimulating effect of AA, whereas specific pharmacologic inhibitors of cyclo-oxygenase-2 or of 5-lipoxygenase had no inhibitory effect. A thromboxane (TX)A2/endoperoxides receptor antagonist and a TX synthase inhibitor blocked the potentiating effect of AA. Purified PGG2 and carbocyclic TXA2, a TXA2 agonist, enhanced the procoagulant activity of PBMC in a dose-dependent manner whereas, in contrast, PGE2 inhibited it. Finally, contrary to AA, EPA or DHA did not increase TXB2 production or TF expression by PBMC. The TF-dependent procoagulant activity of isolated PBMC was increased by AA through the production of cyclo-oxygenase-1 metabolites; the combined action of PGG2 and TXA2, which potentiated it, was greater than that of PGE2, which inhibited it. Dietary n-3 fatty acids exert part of their beneficial effect by modulating this procoagulant activity differently from AA.
    The Journal of Immunology 07/1998; 160(12):6145-50. · 5.52 Impact Factor
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    ABSTRACT: We investigated the role of the thrombomodulin (TM/protein C/protein S anticoagulant pathway in modulating the thrombogenic properties of the endothelium. Endothelial cells (ECs) were placed in parallel-plate flow chambers and exposed to nonanticoagulated human blood at a venous wall shear rate (50 s-1). Fibrin deposition on resting ECs treated with a control IgG1 was negligible. In contrast, a significant amount of fibrin deposited when TM expression was specifically suppressed by > 95% by preincubating ECs with an anti-TM IgG1. Similarly, fibrin deposited on interleukin 1-stimulated ECs, but the fibrin deposition was further increased threefold with anti-TM IgG1. Comparable results were found when ECs were perfused at 650 s-1. When TM surface activity was enhanced by 150% by treating ECs with active phorbol ester (4-phorbol 12-myristate 13-acetate; PMA), the deposition of fibrin was 30% lower than on ECs not pretreated with PMA. Finally, fibrin deposition on stimulated ECs was significantly higher in 11 untreated patients with well-characterized deficiencies of protein C or S or heterozygous factor V Leiden mutation than in 11 healthy individuals, and it was significantly correlated to basal plasma levels of thrombin-antithrombin complexes. Thus, this study underlines the central role of the TM/protein C/protein S pathway in modulating the thrombogenic status of resting and stimulated ECs and indicates that basal coagulation system markers may be helpful in monitoring patients presenting a disorder of this anticoagulant pathway.
    Arteriosclerosis Thrombosis and Vascular Biology 04/1997; 17(3):520-7. · 6.34 Impact Factor
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    ABSTRACT: We examined the ability of unfractionated heparin to modulate the procoagulant activities of stimulated endothelial cells (EC). Confluent human venous umbilical EC were incubated with heparin before or after stimulation, then rinsed extensively to eliminate any heparin in the solution. EC, stimulated for 4 h with endotoxin and interleukin 1 beta, expressed tissue factor and prothrombinase activities. When EC were treated with heparin (6 and 60 micrograms/ml) during the last 10 min of the stimulation period, EC-related procoagulant activities were inhibited in a dose-dependent manner (80-90% inhibition at 60 micrograms/ml). The inhibition was antithrombin-dependent and it disappeared after heparin removal in less than 15 min at 37 degrees C but persisted at 4 degrees C. When EC were treated with heparin (60 micrograms/ml) for 24 h then extensively washed before stimulation, the anticoagulant effect was more modest (50% inhibition). The effect was antithrombin-dependent. Inhibition was maximum after 18-24 h of pretreatment of EC with heparin and was stable for at least 7 h. The cell surface displayed a "heparin-like" activity: treatment by heparin doubled the rate of thrombin-antithrombin complex formation and this effect was heparinase sensitive and chondroitinase ABC insensitive. Thus, heparin modulates the procoagulant properties of stimulated EC according to two distinct mechanisms. The first one is rapid and transient, probably related to the presence of heparin molecules bound at the membrane surface. The second is delayed and persistent, and our results suggest that it is mediated by an increase in the membrane heparan sulfate molecules.
    Thrombosis and Haemostasis 02/1996; 75(1):190-5. · 5.76 Impact Factor
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    ABSTRACT: We have compared the anticoagulant and the antithrombotic effects of unfractionated heparin (Calciparine) and low molecular weight heparin (Fraxiparine) in an experimental human venous thrombosis model. One single subcutaneous injection of Calciparine or Fraxiparine was administered to healthy male volunteers at one month interval in a randomised and cross-over design. Ten subjects received doses used in man for preventing venous thrombosis (5,000 IU and 3,075 IU, respectively), and seven other subjects received curative doses (12,500 IU and 6,150 IU, respectively). Thrombus formation was measured 3 h and 8 h after drug administration. Non-anticoagulated human blood was drawn for 5 min directly from an antecubital vein over confluent cultured endothelial cells positioned in a parallel-plate perfusion chamber. The cells were previously stimulated for 4 h with lipopolysaccharides (10 micrograms/ml) and interleukin 1 beta (50 U/ml), resulting in optimal expression of biological active tissue factor. The wall shear rate at the cell surface was 50 s-1 and mimicked venous blood flow conditions. Immunologically quantified fibrin deposition on the stimulated cells was reduced only by curative doses of Calciparine and Fraxiparine at 3 h (3.4 +/- 0.8 versus 1.0 +/- 0.2 micrograms/cm/ and 2.6 +/- 0.8 versus 1.0 +/- 0.1 micrograms/cm2, respectively, p < or = 0.05). The influence of Calciparine and Fraxiparine on the formation of thrombin and fibrin was determined by measuring the plasma levels of thrombin-antithrombin III complexes and fibrinopeptide A (FPA) in blood samples collected distally to the perfusion chamber. The generation of these markers was significantly inhibited (50-83%) by both prophylactic and curative doses of Calciparine and Fraxiparine (p < or = 0.05). However, Fraxiparine still significantly inhibited the thrombin and fibrin generation at 8 h (p < or = 0.05), whereas Calciparine did not. The antithrombotic effects of both heparins were correlated with their plasma activities as measured by the antifactor Xa or the antithrombin assays. Thus, it appears in this model that Calciparine and Fraxiparine produce comparable antithrombotic effects at clinically comparable doses. However Fraxiparine has a longer-lasting anticoagulant activity than Calciparine. These results are in good agreement with clinical observations in man, and thus in favour of our model of human venous thrombogenesis for further studies of antithrombotic molecules.
    Thrombosis and Haemostasis 11/1995; 74(5):1286-92. · 5.76 Impact Factor
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    ABSTRACT: We have evaluated the relationship between the level of tissue factor (TF) expression by stimulated endothelial cells and thrombus formation under blood flow conditions. Cultures of human umbilical venous endothelial cells (HUVECs) were treated in order to express different levels of TF activity. They were stimulated for 4 h with either I) lipopolysaccharides (LPS, 10 micrograms/ml), II) recombinant interleukin 1 beta (IL1 beta, 50 UI/ml) or III) simultaneously with LPS and IL1 beta (LPS+IL1 beta). TF activity was low on confluent HUVECs or on the corresponding extracellular-matrix (ECM prepared by exposure of HUVECs to 0.1 N NH4OH). In contrast, it was high when HUVECs were stimulated with LPS or IL1 beta, and significantly higher (p < 0.05) with LPS+IL1 beta. The TF activity associated with the stimulated ECM was 2-fold higher (p < 0.05) than that expressed on the luminal surface of the stimulated HUVECs, irrespective of the agonist or combination of agonists used. These surfaces were exposed to non-anticoagulated human blood at a venous (50 s-1) and an arterial (650 s-1) wall shear rate in parallel-plate perfusion chambers for 5 min. Thrombus formation was morphologically quantified by measuring the deposition of platelets and fibrin. Fibrin deposition was also immunologically quantified. Fibrin deposition was related to the level of TF expression. Non-stimulated HUVECs and corresponding ECMs were not thrombogenic. The luminal surface of HUVECs stimulated with LPS or IL1 beta alone expressed low levels of TF activity and was a poor inducer of platelet deposition and fibrin deposition (< 15%) at 50 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
    Thrombosis and Haemostasis 09/1995; 74(2):778-83. · 5.76 Impact Factor
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    ABSTRACT: We have investigated the influence of blood flow on thrombin generation, fibrin formation, and fibrin deposition on procoagulant and nonprocoagulant surfaces. Nonanticoagulated human blood was drawn for 5 minutes directly from an antecubital vein over stimulated endothelial cells expressing tissue factor and over human type III collagen fibrils, positioned in parallel-plate perfusion chambers. The shear rates at these surfaces were 50, 650, and 2,600 s-1. Deposition of platelets and fibrin was measured by morphometry. Thrombin and fibrin formation was determined by measuring prothrombin fragments 1 + 2 (F 1 + 2), thrombin-antithrombin III complexes, (T-AT) and fibrinopeptide A (FPA) in blood effluent from the perfusion chamber at the end of the 5-minute perfusion period. On procoagulant endothelial cells, the thrombi were primarily composed of fibrin. The fibrin deposition (81%, 21%, and 2% at 50, 650, and 2,600 s-1, respectively) and plasma levels of F 1 + 2, T-AT and FPA were shear rate dependent and highest at 50 s-1. There was a positive correlation between F 1 + 2 and T-AT and the fibrin deposition (P < .01). In contrast, the collagen surface triggered primarily thrombi that were composed of platelets. The platelet thrombi and plasma levels of F 1 + 2 and T-AT were also dependent on the shear rate, but highest at 650 and 2,600 s-1. F 1 + 2 and T-AT reached the same level as observed with procoagulant endothelial cells at the higher shear rates. There was a positive correlation between F 1 + 2 and T-AT and the platelet thrombus formation (P < .05), confirming the predominant role of platelets in thrombin generation. Thus, thrombin formation is strongly influenced by the blood flow, and this effect depends on the composition of the thrombogenic surface.
    Blood 11/1994; 84(7):2206-13. · 9.78 Impact Factor
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    ABSTRACT: Clot-bound thrombin proteolyses fibrinogen and amplifies the coagulation cascade at its close vicinity, thereby ensuring the growth of fibrin-rich thrombus. The present study compares the ability of various glycosaminoglycans (GAGs) to inhibit these 2 properties. Unfractionated heparin (UH), 3 low molecular weight heparins (LMWHs) with increasing antifactor Xa/antifactor IIa ratio, the synthetic pentasaccharide (PS), devoid of antifactor IIa activity, and dermatan sulfate (DS), a catalyst of thrombin inhibition by heparin cofactor II, were selected on the basis of their different properties. Proteolysis of fibrinogen by clot-bound thrombin was evaluated by measuring fibrinopeptide A (FPA) generation after an incubation of standardized washed clots in plasma for 120 min in absence or in presence of increasing concentrations of heparins or of DS. The results were compared to those obtained when free alpha-thrombin (0.4 nM) was added to plasma in the same experimental conditions. On the basis of equivalent antithrombin units, UH and LMWHs gave identical results. To inhibit by 70% fibrinogen proteolysis induced by clot-bound thrombin (IC 70), 5- to 9-fold higher concentrations of UH or of LMWHs were required in comparison with those required to inhibit free thrombin. For DS, only a 1.3 times higher concentration was required. PS (final concentration 1 anti Xa U.ml-1) was devoid of any inhibitory effect. The amplification of the coagulation cascade induced by clot-bound thrombin was evaluated by measuring the shortening of whole blood clotting time (WBCT) resulting from the incubation of washed clots in native blood. In absence of GAG, clot-bound thrombin reduced WBCT from 18 +/- 2 min to 9 +/- 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)
    Thrombosis and Haemostasis 06/1994; 71(5):576-80. · 5.76 Impact Factor
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    ABSTRACT: Two groups of 23 and 84 patients with hip fracture received intramuscularly 100 and 300 mg dermatan sulfate (MF701) b.i.d., respectively, for the prophylaxis of deep vein thrombosis. Median duration of treatment was 17 and 16 days, respectively. Four blood samples were collected from each patient while under treatment. Plasma levels of dermatan sulfate were determined by a chromogenic substrate assay. A one-compartment model for multiple doses was employed to estimate the pharmacokinetic parameters. Fitting was applied to mean plasma concentrations calculated for each sampling time and weighted according to the number of samples available at each time. Thrombin clotting time was measured on the same plasma samples. Antithrombotic efficacy was assessed by bilateral venography. Plasma levels of dermatan sulfate increased gradually throughout the treatment, indicating a marked accumulation process. Time to reach steady-state was 14 or 9 days with 100 or 300 mg b.i.d., respectively. This was due to an apparent prolonged terminal half-life (68 or 43 h), which actually reflected slow absorption from the injection sites. The clinical efficacy of MF701 in preventing DVT was found to be dependent on the plasma concentration of the drug and also, but less significantly, on the prolongation of thrombin clotting time. Dermatan sulfate plasma levels greater than 9 micrograms/ml are advisable to optimize efficacy in hip fracture patients.
    Thrombosis and Haemostasis 05/1994; 71(5):553-7. · 5.76 Impact Factor
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    ABSTRACT: The relationship between the antithrombotic activity of dermatan sulfate (DS) in vivo and its catalytic effect on the inhibition of thrombin by heparin cofactor II (HC II) in vitro was investigated. DS was depolymerized by Smith degradation and the fragments obtained were separated by gel filtration. The fragment of minimal size with full catalytic activity was a hexadecasaccharide, which was further fractionated by affinity for immobilized HC II. Only a small proportion by weight (6.7%) was recovered in the high-affinity fraction, which had about 10 times more catalytic activity than the unfractionated oligosaccharide; the change in activity was primarily caused by the removal of inert materials, recovered in the low-affinity fraction. 1H-NMR spectra indicated strengthening of the signal given by Ido A (2S04) in the high-affinity fraction compared with that of the low-affinity fraction. The anticoagulant activity of the high-affinity fraction was exclusively HC II-dependent. The antithrombotic potency was evaluated in rabbits using the Wessler-thromboplastin model. Half-maximal prevention of thrombosis was obtained after injection of 250 micrograms/kg DS, of 500 micrograms/kg hexadecasaccharide, or of 60 micrograms/kg of its high-affinity fraction. The low-affinity fraction was ineffective at the highest dose tested (1,200 micrograms/kg) and did not potentiate the effect of the high-affinity fraction. These results show that the antithrombotic effect of DS is essentially dependent on HC II binding and activation and that HC II is therefore a suitable target for antithrombotic drugs.
    Blood 05/1993; 81(7):1771-7. · 9.78 Impact Factor
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    ABSTRACT: We have investigated the pharmacological properties of an O-acetylated butyryl derivative of the low molecular weight heparin CY 216 (C4-CY 216). In a purified system the ability of C4-CY 216 to catalyze thrombin and factor Xa inhibition was comparable to that of CY 216. The antithrombin and antifactor Xa catalytic efficiencies of C4-CY 216 were reduced 217 and 12 times respectively when albumin (10 mg ml-1) was added to the reagents, while those of CY 216 were essentially unchanged. In plasma, the antifactor Xa specific activity of C4-CY 216 was close to that of CY 216 but the antithrombin specific activity was 2 times lower. After bolus and continuous intravenous injection to rabbits, the clearances of the two activities of C4-CY 216 were on average half the corresponding values of CY 216. After subcutaneous injection, the bioavailability of C4-CY 216 was comparable to that of CY 216. C4-CY 216 was as potent as CY 216 in preventing venous thrombosis in the thromboplastin-Wessler model and the duration of the antithrombotic effect was longer than that of the parent compound. The chemical alteration of CY 216 did not enhance the prohaemorrhagic effect in the rat tail transection model. Therefore, the new concept of heparin derivative having a low clearance and long lasting effects that we have recently reported for unfractionated heparin may also be applied to a low molecular weight heparin.
    Thrombosis and Haemostasis 04/1992; 67(3):346-51. · 5.76 Impact Factor
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    ABSTRACT: This article reports on the pharmacologic properties of an O-acylated butyryl derivative (C4-UH) of unfractionated heparin (UH). In a purified system, the ability of C4-UH to catalyze the inhibition of thrombin and of factor Xa in the presence of antithrombin III was similar to that of UH. Addition of albumin (10 mg/ml) to the reagents reduced the antithrombin and antifactor Xa catalytic potency of C4-UH 68-fold and 20-fold, respectively, and did not alter those of UH. As judged from the prolongation of the activated partial thromboplastin time and the thrombin clotting time, the anticoagulant activities of C4-UH were two times weaker than those of UH. After calibration against UH, the antifactor Xa-specific and antithrombin-specific activities were two and 6.6 times lower, respectively. After bolus intravenous injection into rabbits, the apparent clearances of C4-UH were reduced 2.4 (antifactor Xa activity) and 3.2 times (antithrombin activity) in comparison with those of UH. This property accounted for the higher plasma concentrations generated during a constant infusion of the same dose. In the Wessler thromboplastin model, the minimum doses providing the maximum antithrombotic effect after bolus injection were equivalent for both compounds when expressed as antifactor Xa units; the duration of the antithrombotic effect of this derivative was prolonged, whereas the hemorrhagic potential was unaffected. This study opens a new concept for heparin derivatives having lower clearances and long-lasting effects. These properties could be linked to nonspecific binding of C4-UH to plasma proteins, thereby reducing the amount of free compound available to interact with antithrombin III.
    Journal of Laboratory and Clinical Medicine 03/1992; 119(2):189-96. · 2.62 Impact Factor
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    ABSTRACT: Antibody inhibitors against human thrombin are rare and have remained poorly characterized. We report the case of a 40-yr-old patient who developed a potent thrombin inhibitor revealed by mild bleeding symptoms and marked prolongation of most laboratory clotting times. After two years of evolution, he died from cerebral hemorrhage. The inhibitor, a polyclonal IgG, was associated with hematological and immunological criteria of autoimmune disorder. Antithrombin IgG was isolated from the patient's plasma by protein A- and thrombin-affinity chromatography. Fab fragments inhibited amidolytic activity of alpha thrombin, and thrombin-thrombomodulin catalyzed protein C activation with a Ki of approximately 10(-8) M in a noncompetitive manner. Alpha to gamma conversion of thrombin resulted in a moderate loss of affinity for the inhibitor. Upon complex formation of thrombin with staphylocoagulase or alpha 2-macroglobulin (alpha 2M), inhibition was decreased by two orders of magnitude and acquired an apparent competitive character. In Western blot experiments, the antibody reacted with active alpha-thrombin, did not react with chloromethylketone-inhibited thrombin and reacted with a lower affinity with iPr2P-thrombin. The inhibitor did not block thrombin binding to benzamidine-, heparin-, or fibrin-Sepharose, but displaced proflavin from its complex with thrombin. Taken together, these results indicate that the patient's autoantibody recognized a conformational structure which includes, at least in part, the apolar binding site adjacent to the catalytic site of thrombin.
    Journal of Clinical Investigation 08/1991; 88(1):290-6. · 12.81 Impact Factor
  • P Sié, D Dupouy, F Dol, B Boneu
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    ABSTRACT: 1. Routine clotting assays and tests used for monitoring heparin therapy are not suitable for measuring plasma DS in most clinical conditions. 2. Sensitive and specific assays have been developed recently for this purpose. They are based on different biological properties of DS. 3. As DS is heterogeneous, significant differences between the results obtained with different assays are expected. 4. Ex vivo measurements of DS do not reflect an actual concentration because, due to complex pharmacokinetics, the spectrum of molecular species found in a patient's plasma may be different from that present in the material administered. 5. DSs from different sources may exhibit wide variations in structure and biological properties. Ex vivo assays must be calibrated with the DS preparation administered to the patient. Information obtained with one DS is not reliable for other DSs. 6. The relationship between ex vivo activities of DS and antithrombotic effects in man is unknown. Simultaneous assessment of DS concentration and of activation of coagulation in models such as hemodialysis will be very welcome.
    Seminars in Thrombosis and Hemostasis 02/1991; 17 Suppl 2:181-5. · 4.22 Impact Factor
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    ABSTRACT: Dermatan sulfate (DS), a catalyst of the thrombin-heparin cofactor II interaction, has antithrombotic activity and is devoid of significant hemorrhagic risk in several animal models. We investigated the pharmacodynamic and pharmacokinetic properties of DS in humans. DS was injected in single bolus intravenous injections of four increasing doses (0.5, 1, 1.5, 2 mg/kg) to six healthy volunteers. The resulting anticoagulant activities were assessed by the activated partial thromboplastin time (APTT) and the thrombin clotting time (TCT). There were dose-dependent prolongations of the APTT and TCT, and the anticoagulant activities disappeared in less than three hours. The pharmacokinetic parameters were calculated from the plasma concentrations of DS measured with a new chromogenic assay. The volume of distribution was approximately 1.8 times greater than the theoretical plasma volume and was independent of dose. In contrast, the clearance decreased with dose and the terminal half-life ranged from 0.45 +/- 0.08 hours at 0.5 mg/kg to 0.72 +/- 0.11 hours (mean +/- SD) at 2 mg/kg. The bioavailabilities of subcutaneous (SC) and intramuscular (IM) administration relative to those of intravenous administration were determined in 12 other volunteers. The respective bioavailabilities were 24.7% +/- 12.9% and 12.4% +/- 9.2% for SC and IM administration. There was no detectable change in the APTT and the TCT when the volunteers were injected with 1.5 mg/kg SC or IM. In addition, the pharmacokinetic parameters derived from plasma concentrations of DS showed considerable interindividual variations by the two later routes of administration. Peak concentrations were noted 2.7 +/- 1.3 hours after SC injection and 4.3 +/- 4.9 hours after IM injection. The average peak concentrations were 0.7 +/- 0.3 and 0.4 +/- 0.2 mg/L after SC and IM injections, respectively. The half-lives of DS were 7.9 +/- 6.5 hours (SC) and 6.3 +/- 7.4 hours (IM). No adverse reaction to DS was recorded during this study.
    Blood 11/1989; 74(5):1577-82. · 9.78 Impact Factor
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    ABSTRACT: The neutralization of the anticoagulant, anti-thrombin, and bleeding effects of dermatan sulfate (DS), a potential antithrombotic agent, was investigated. Protamine sulfate (PS) and hexadimethrine bromide (Polybrene), which reverse the anticoagulant effect of heparin, also neutralized DS in vitro. In human plasma, polybrene was approximately 3 times more active on a weight basis than PS for neutralizing DS (1.5 micrograms polybrene inhibits 1 microgram DS). Intravenous administration of polybrene to rabbits pretreated with DS in a 1:1 weight ratio immediately neutralized 90% of DS and this effect was stable with time. In contrast, PS in a weight ratio of 6:1 (PS to DS) only neutralized 50% of DS injected. When plasma DS concentrations were maintained by continuous infusion between 3 and 15 micrograms/ml, a bolus of polybrene 0.25 mg/kg induced an immediate drop of about 4 micrograms/ml but initial values of DS were recovered within 20 min. PS was again much less effective than polybrene for neutralizing DS. The bleeding effect of DS and its correction by polybrene was studied by using the rat tail transection model. Very large doses of DS (greater than 10 mg/kg) were required to get a modest prolongation of bleeding time. The injection of equivalent doses of polybrene in animals pretreated by DS induced a strong bleeding effect associated with a drop in platelet and leukocyte counts. Animal models are thus inappropriate for investigating the correction of DS-induced bleeding, because high doses of both DS and neutralizing agents are required in these models. Our results indicate that, provided the doses of neutralizing agents remain below their established levels of toxicity in man, DS could if necessary be neutralized completely by polybrene and partially by PS.
    Thrombosis Research 05/1989; 54(1):63-74. · 3.13 Impact Factor