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Biomedical subjects

G Elgue

Publications and source records attributed to G Elgue.

At least 19 recordsLinked to original sources
PubMed2001

Coagulation and complement activation.

UNLABELLED: The purpose of this investigation was to assess the effect of heparin coating of a new stent construction (Stent Graft, Jomed Implantate GmbH, Germany) on platelet and coagulation activity. METHODS: Stent grafts with an ePTFE membrane interfoliated between two stents were deployed in tubings to form Chandler loops. Fresh human blood with a low concentration of heparin was rotated for 1 h, then collected and used for measurements of platelet number, thrombin-antithrombin complex (TAT), CD11b, C3a and C5b-9. There were five study groups: Group 1, conventional unmodified stents (n = 8); Group 2, untreated stent grafts (n = 8); Group 3, heparin-coated stents and untreated membrane (n = 7); Group 4, heparin-coated stents and membrane (n = 8); Group 5, heparin-coated PVC tubings with no stents (n = 8). RESULTS: There was a significant drop in platelet count, increase in TAT-values and CD11b expression in Groups 1-3 but not in Group 4 compared to Group 5. Examination by scanning electron microscopy revealed extensive activation on non-modified stents but almost no deposition of thrombotic material on heparin-modified stent grafts. CONCLUSIONS: With unmodified stents and membrane there were signs of significant activation of platelets and coagulation. In contrast, the heparin-coated stent graft induced much less alterations, indicating improved blood compatibility.

Blood Coagulation↗
PubMed2001

Contact between a polymer and whole blood: sequence of events leading to thrombin generation.

The mechanism by which thrombin is generated on a polymer surface in an extracorporeal circuit is not yet fully understood. To address this question we have developed an in vitro chamber model in which whole blood containing heparin (1 IU/mL) comes in contact with a commonly used biomaterial, polyvinyl chloride (PVC). Incubation of blood in the chamber for 60 minutes at 37 degrees C resulted in the binding of platelets to the material surface and the generation of thrombin-antithrombin complexes. Corn trypsin inhibitor, a specific inhibitor of factor XIIa, inhibited this thrombin-antithrombin complex generation in blood in contact with PVC, which is not considered an efficient activator of factor XII. The addition of the glycoprotein IIb/IIIa inhibitor Ro44-9883 abrogated platelet binding and aggregation and resulted in decreased generation of thrombin-antithrombin complexes. Thrombin-antithrombin generation was also negligible in platelet-rich plasma but could be partially restored in the presence of erythrocytes. Taken together, these data are compatible with a model in which thrombin generation is triggered by factor XII. The response to contact with PVC appears to begin with a low-grade generation of thrombin that involves both erythrocytes and leukocytes and that activates platelets, followed by the activation of a platelet-dependent amplification loop that produces most of the thrombin.

Acetates↗
PubMed2001

A new in vitro model for the study of pig-to-human vascular hyperacute rejection.

Studies on vascular hyperacute xenograft rejection (HAR) are usually conducted in vitro on cultured endothelial cells (EC) exposed to human serum, in complex whole organ perfusion models using heparinized blood or in vivo models. Here we describe a new model allowing perfusion of pig vessels with human whole blood without anticoagulants. Segments of the porcine iliac artery were connected to circular polyvinyl chloride (PVC) tubing, whose inner surface was conjugated with immobilized heparin. The vessels were perfused with 7 to 8 ml of fresh, non-anticoagulated human blood by rocking of the tubing device for 5, 15 or 60 min in an incubator at 37 degrees C. Human iliac arteries (n = 4) were perfused with fresh human ABO-compatible blood as controls. Perfusion of human vessels resulted in changes in the blood and plasma parameters similar to those in the PVC control loop. Overall, perfusion of the porcine vessels generated high levels of C3a, sC5b-9 and thrombin-anti-thrombin (TAT). Platelet consumption was near total (97.2 +/- 1.2%; "high" responders) in six of 13 vessels perfused and only moderate (55.8 +/- 9.9%; "low" responders) in the remaining seven vessels. The "high" responder vessel group showed a significantly higher platelet reduction, neutrophil loss and monocyte consumption and higher C3a and TAT factor at 60 min compared with the human vessels. The "low" responder porcine vessel group also generated significantly higher TAT levels at 60 min compared with the human vessels, but lower levels compared with the "high" responder porcine vessel group. Immunohistochemical examination of perfused porcine vessels revealed binding of human IgM, IgG, IgA, C1q, C3, fibrin and platelets at 5 min. The binding of these proteins was even stronger at 15 and 60 min, and at 60 min C9 could also be detected. Addition of soluble complement receptor 1 (sCR1) to the blood resulted in a significant reduction in C3a and sC5b-9 (P = 0.046 and P = 0.046, respectively). However, sCR1 did not reduce C1q, C3c or C5 staining, but did abolish C9 binding to the endothelium. In conclusion, in vitro perfusion of porcine vessel segments with non-heparinized, fresh human blood triggered events characterizing HAR. The small quantity of blood and xenogenic tissue that is needed makes this model ideal for investigations of the mechanisms and treatments of rejections associated with xenogeneic pig-to-human xenotransplantation.

ABO Blood-Group System↗
PubMed2000

Damage to porcine islets of Langerhans after exposure to human blood in vitro, or after intraportal transplantation to cynomologus monkeys: protective effects of sCR1 and heparin.

BACKGROUND: Porcine islets offer an attractive alternative to human islets in clinical islet transplantation. The preferred method of islet transplantation is intra-portal injection into the liver. We have recently shown, both in vitro with human islets and in vivo with porcine islets, that islets exposed to allogeneic blood trigger an injurious inflammatory reaction characterized by activation of both coagulation and the complement systems. We have now tested whether a similar reaction is triggered when xenogeneic porcine islets are exposed to human blood in vitro and after intraportal transplantation into primates. Furthermore, we investigated the effect of inhibiting the complement and coagulation systems. METHOD: Islets isolated from adult and fetal porcine pancreas were perfused with fresh human blood in surface heparinized PVC tubings for 5-60 min. Blood cell counts and parameters related to coagulation and the complement system were analyzed, and islets were retrieved after the perifusion was examined by immunohistochemical method. Heparin and soluble complement receptor 1 (sCR1; TP10, 100 microg/ml) were added to the system in some experiments. Furthermore, adult porcine islets were transplanted intraportally into untreated and sCR1- (40 mg/kg BW i.v.) treated cynomolgus monkeys, and plasma insulin concentration was monitored during 60 min after transplantation. RESULTS: Porcine islets perifused with human blood triggered an immediate inflammatory reaction, characterized by a rapid consumption and activation of platelets, consumption of neutrophils and monocytes, activation of the coagulation and complement systems, and release of large amounts of insulin. Islet morphologic analysis revealed damaged islets embedded in clots and infiltrated with CD11+ leukocytes. C3a and C5b-9 was deposited on the islet surface, but human immunoglobulin was not. Complement inhibition with sCR1 reduced insulin release significantly. Intraportal islet transplantation into untreated cynomolgus monkeys resulted in a marked and rapid increase in plasma insulin concentration indicative of islet damage. Pretreatment of the monkeys with sCR1 resulted in significantly less insulin release than in untreated control monkeys. CONCLUSION: Exposure of isolated xenogeneic islets of Langerhans to blood, both in vitro and in vivo, resulted in acute islet damage. Complement and platelets seem to have a central role in the reactions described. Strategies to efficiently inhibit these reactions will be crucial for clinical intraportal islet xenotransplantation to be successful.

Animals↗
PubMed1999

Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation?

The remarkable difference in success rates between clinical pancreas transplantation and islet transplantation is poorly understood. Despite the same histocompatibility barrier and similar immunosuppressive treatments in both transplantation procedures, human intraportal islet transplantation has a much inferior success rate than does vascularized pancreas transplantation. Thus far, little attention has been directed to the possibility that islets transplanted into the blood stream may elicit an injurious incompatibility reaction. We have tested this hypothesis in vitro with human islets and in vivo with porcine islets. Human islets were exposed to nonanticoagulated human ABO-compatible blood in surface-heparinized polyvinyl chloride tubing loops. Heparin and/or the soluble complement receptor 1 (sCR1) TP10 were tested as additives. Adult porcine islets were transplanted intraportally into pigs, and the liver was recovered after 60 min for immunohistochemical staining. Human islets induced a rapid consumption and activation of platelets. Neutrophils and monocytes were also consumed, and the coagulation and complement systems were activated. Upon histological examination, islets were found to be embedded in clots and infiltrated with CD11+ leukocytes. Furthermore, the cellular morphology was disrupted. When heparin and sCR1 were added to the blood, these events were avoided. Porcine islets retrieved in liver biopsies after intraportal islet allotransplantation showed a morphology similar to that of human islets perifused in vitro. Thus, exposure of isolated islets of Langerhans to allogenic blood resulted in significant damage to the islets, a finding that could explain the unsatisfactory clinical results obtained with intraportal islet transplantation. Because administration of heparin in combination with a soluble complement receptor abrogated these events, such treatment would presumably improve the outcome of clinical islet transplantation by reducing both initial islet loss and subsequent specific immune responses.

Adult↗
PubMed1999

Titanium is a highly thrombogenic biomaterial: possible implications for osteogenesis.

Titanium has superior osteointegrating properties compared to other biomaterials. The mechanism for this is unknown. During the initial phase of bone implantation the biomaterial comes into direct contact with whole blood. In this study we use a newly developed in vitro chamber model to compare different commonly used biomaterials in contact with whole blood. These materials were selected with respect to their different osteointegrating properties in order to correlate these properties with the response to whole blood. In the presence of 3 IU/ml of heparin only titanium induced macroscopic clotting. This was reflected by the generation of thrombin-antithrombin which was much increased in blood in contact with titanium compared with steel and PVC. The coagulation activation caused by titanium was triggered by the intrinsic pathway because the generation of FXIIa-AT/C1 esterase inhibitor paralleled that of thrombin-antithrombin, and both thrombin-antithrombin complex and FXIIa-AT/C1 esterase inhibitor generation were abrogated by corn trypsin inhibitor, which is a specific inhibitor of FXIIa. The binding of platelets was increased on the titanium surface compared to the other biomaterial surfaces and the state of platelet activation was much more pronounced as reflected by the levels of beta-thromboglobulin and PDGF. This study indicates that titanium is unsuitable as a biomaterial in devices which are in direct contact with blood for a prolonged period. Furthermore, PDGF and other alpha-granule proteins e.g. TGF-beta, are known to be potent promotors of osteogenesis which suggests that the pronounced thrombogenic properties of titanium might contribute to the good osteointegrating properties.

Blood Coagulation↗
PubMed1999

Phosphorylation of coagulation factor XI by a casein kinase released by activated human platelets increases its susceptibility to activation by factor XIIa and thrombin.

Previous studies suggest that activated platelets facilitate the cleavage of factor XI by both factor XIIa and thrombin. Extracellular phosphorylation is a mechanism by which the function of plasma proteins can be regulated. Phosphorylation is mediated by a casein kinase which is released by activated platelets concomitant with large amounts of ATP and Ca2+. The purpose of this study was to investigate if factor XI is phosphorylated by a platelet casein kinase and whether phosphorylation may affect its activation properties. It was shown that supernatants from platelets which contain platelet casein kinase phosphorylated factor XI. By Western blot analysis it was shown that phosphorylation of factor XI substantially increased its susceptibility to cleavage by factor XIIa, and, to a lesser extent, by thrombin. The generated factor XIa was functionally active in that it cleaved the chromogenic substrate S2366, and in that factor XIa-antithrombin and thrombin-antithrombin complexes were generated when phosphorylated factor XI was added to blood plasma. The present study indicates that platelet-mediated phosphorylation of factor XI enhances the cleavage of factor XI into XIa and that the generated XIa possesses functional activity. Phosphorylation of factor XI might be an essential regulatory mechanism by which platelets mediate amplification of the coagulation cascade.

Blood Platelets↗
PubMed1998

Compstatin inhibits complement and cellular activation in whole blood in two models of extracorporeal circulation.

Recently, a C3-binding cyclic synthetic peptide (Compstatin) has been identified that binds to complement component C3 and inhibits complement activation. Here we have examined the influence of Compstatin on complement activation and its indirect effects on cellular responses in whole blood in two models for extracorporeal circulation. Compstatin effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/ C3 fragments to the polymer surface. As a result of the inhibition of complement activation, the activation of polymorphonuclear leukocytes (PMNs; as assessed by the expression of CD11b) and the binding of these cells (CD16(+)) to the polymer surface were almost completely lost. In contrast, blood cell counts were not affected. Using surface plasmon resonance technology, we have confirmed that Compstatin exerts its inhibitory effect on complement activation by binding to native C3. These data show that complement activation, leading to activation and binding of PMNs to the biomaterial surface, can be abolished by the addition of Compstatin. The properties of Compstatin make Compstatin a promising drug for use in extracorporeal circuits to avoid bioincompatibility reactions, eg, during cardiopulmonary bypass, but also a favorable precursor peptide for the development of an anticomplement drug for oral use.

Amino Acid Sequence↗
PubMed1998

Studies of adsorption, activation, and inhibition of factor XII on immobilized heparin.

The aim of the present investigation was to clarify whether immobilized heparin does, as previously suggested, prevent triggering of the plasma contact activation system. Purified FXII in the absence or presence of antithrombin and/or C1 esterase inhibitor as well as plasma was exposed for 1 to 600 seconds to a surface modified by end-point immobilization of heparin. With purified reagents, a process including surface adsorption and activation of FXII occurred within 1 second. In the presence of antithrombin, the resulting surface-bound alpha-FXIIa was inhibited within that time. Likewise, the adsorption of native FXII from plasma was a rapid process. However, the inhibition of surface-bound alpha-FXIIa was slightly slower than with purified components. Nevertheless, neither beta-FXIIa nor FXIa were found in the plasma phase. Exposure of a surface prepared from heparin molecules, lacking antithrombin binding properties, to plasma resulted in surface-bound alpha-FXIIa within 1 second. In the liquid phase, beta-FXIIa was detected after 2.5 seconds and, 12 seconds later, FXIIa and FXIa in complex with the C1 esterase inhibitor appeared. Addition of heparin to plasma prior to surface exposure did not prevent activation of surface-bound FXII, nor did it increase the beta-FXIIa inhibition rate and prevent FXI activation in plasma, although beta-FXIIa and FXIa-AT complex formation occurred. It is concluded that surface-immobilized heparin, unlike heparin in solution, effectively inhibits the initial contact activation enzymes by an antithrombin-mediated mechanism, thereby suppressing the triggering of the intrinsic plasma coagulation pathway.

Adsorption↗
PubMed1997

Inhibition of the plasma contact activation system of immobilized heparin: relation to surface density of functional antithrombin binding sites.

End-point immobilization of heparin to artificial materials gives rise to a surface that prevents triggering of the plasma contact activation system and, presumably as a result thereof, generally has thrombo-resistant properties. The present investigation was undertaken to determine what density of immobilized heparin molecules expressing functionally intact antithrombin binding sites is required to achieve these blood compatible properties. Six different heparin surfaces were prepared on polyethylene tubing and studied in contact with human plasma. The content of bound heparin was the same on all surfaces while the densities of antithrombin binding sites ranged from 1 to 28 pmol/cm2. The surfaces expressing 4 pmol/cm2 or more of specific antithrombin binding sites generated no measurable enzymatic activity in contact with plasma, either on the exposed surfaces or in the plasma phases. Below this level, the degree of activation gradually increased with decreasing densities, and in parallel the thrombo-resistant properties deteriorated. Addition of heparin to the plasma phase reduced the capacity of the heparin surfaces to bind antithrombin, leading to a diminished ability of the surfaces to prevent contact activation. This finding supports the hypothesis that antithrombin is the critical coagulation inhibitor for the suppression of contact activation on end-point immobilized heparin.

Anticoagulants↗
PubMed1997

Inhibition of complement activation by soluble recombinant CR1 under conditions resembling those in a cardiopulmonary circuit: reduced up-regulation of CD11b and complete abrogation of binding of PMNs to the biomaterial surface.

The influence of soluble recombinant CR1 (sCR1) on complement activation, and its indirect effects on the coagulation system and cellular responses were assessed in two models for the study of blood/surface and blood/air interactions, as are encountered in e.g. cardiopulmonary bypass circuits. The concentrations of C3a and sC5b-9 and the amount of bound C3/C3 fragments were analyzed as indicators of complement activation. Thrombin-antithrombin complexes, the platelet count, surface-ATP, beta-thromboglobulin, and the expression of CD11b on leukocytes were the parameters analyzed to reflect coagulation and cellular responses. In addition, immunochemical analyses of the phenotypes of surface-bound leukocytes and platelets were performed. Recombinant sCR1, at doses ranging between 0.1-0.25 mg/ml, was found to completely inhibit the generation of sC5b-9, and of C3a by two thirds; the binding of C3 and/or C3 fragments to the surface was almost entirely abolished. As a result of the inhibition of complement activation, the expression of CD11b on PMNs, and the binding of these cells to the biomaterial surface was almost completely lost. In contrast, the thrombin-antithrombin complexes, the platelet count, and the adherence of platelets to the surface, as reflected by the ATP binding and the release of beta-thromboglobulin, were not affected. These data show that complement activation, in association with extra-corporeal treatment, causes activation and binding of PMNs to the biomaterial and that these effects can be completely abolished by the addition of soluble recombinant sCR1.

Biocompatible Materials↗
PubMed1995

Control of contact activation on end-point immobilized heparin: the role of antithrombin and the specific antithrombin-binding sequence.

The uptake and activation of FXII from blood plasma was studied in small-diameter polyethylene tubing, surface-modified by end-point immobilization of heparin. Two preparations of heparin were used to modify the contact-activating properties of the plastic tubing: unfractionated, functionally active heparin and low-affinity heparin, lacking the specific antithrombin-binding sequence and virtually devoid of anticoagulant activity. The uptakes of FXII on the two heparin surfaces were similar. No activated FXII could be demonstrated on the unfractionated heparin surface, whereas on the low-affinity heparin surface nearly all FXII underwent spontaneous activation. The suppression of FXII activation on the unfractionated heparin surface was investigated by using plasma depleted of antithrombin, complement C1 esterase inhibitor, or both. The removal of antithrombin resulted in extensive activation of FXII, whereas the depletion of C1 esterase inhibitor had only a minor effect. Experiments with recalcified plasma showed rapid clot formation during exposure to the low-affinity heparin surface. After depletion of antithrombin, but not complement C1 esterase inhibitor, the recalcified plasma clotted in contact with the unfractionated heparin surface as well. We conclude that antithrombin and the antithrombin-binding sequence in the surface-immobilized heparin are essential for the prevention of surface activation of FXII and triggering of the intrinsic coagulation system.

Animals↗
PubMed1994

Coagulation inhibition capacities of low-molecular mass and unfractionated heparin, as determined by thrombin generation.

The common unfractionated heparin preparations (UFH) accelerate inhibition of most of the enzymes in the coagulation cascade, while low-molecular mass heparin (LMMH) mainly accelerates inhibition of activated coagulation factor X (FXa). The present study addresses the question of whether LMMH may be a weaker anticoagulant than UFH when the two preparations are added to plasma with equal FXa inhibitory activities. Normal and coagulation factor VIII (FVIII)-deficient plasma was used. Thrombin generation was determined by assaying the formation of the thrombin-antithrombin complexes (TAT) and of fibrinopeptide A (FPA), two parameters that showed a strong positive correlation. At a heparin concentration of 0.5 or 1.0 FXa-inhibiting IU/ml, the formation of TAT and FPA was substantial and always much more increased with LMMH than with UFH. At 4.0 FXa-inhibiting IU/ml, no FPA was generated, but traces of thrombin were. In recalcified FVIII-deficient plasma (one of the batches containing FVIII antibodies), more TAT was formed with 0.1 FXa-inhibiting IU/ml LMMH than with UFH with the same FXa-inhibiting activity. It is concluded that LMMH is a weaker anticoagulant than UFH, partly because of a poor thrombin inhibition capacity which facilitates acceleration of coagulation by FVIII activation and partly because of a poor inhibition of enzymes preceding the prothrombinase stage, both mechanisms leading to increased enzymatic activity above the prothrombin stage. As judged from the higher degree of thrombin generation with LMMH than with UFH, there is no support for the assumption that LMMH is as good an antithrombotic agent as UFH is, without reducing the haemostatic capacity as much as UFH does.

Antithrombin III↗
PubMed1994

The effect of plasma antithrombin concentration on thrombin generation and fibrin gel structure.

Congenital deficiency of antithrombin (AT) is associated with thrombotic events and AT consumption occurs in some severe disorders and after treatment with heparin. The aim of this study was to investigate whether variations in the level of plasma AT modify thrombin generation and the fibrin formation process after the intrinsic coagulation mechanism is triggered. Normal plasma was depleted of AT by immunoadsorption on CNBr-Sepharose coupled with the anti-AT-IgG fraction of antiserum. The AT-depleted plasma was reconstituted with AT (between 0.3 and 1.5 AT units per ml). Thrombin generation was measured as the development of thrombin-antithrombin complexes (TAT). The lag phase preceding fibrin formation depended on the concentration of AT. The short lag phase was seen in completely AT-depleted plasma and the long in plasma with 1.5 AT units per ml. TAT generation, determined in parallel consecutive samples, showed that the rate at which thrombin was generated was inverse to the AT concentration in plasma. The network structure of hydrated fibrin gels in the clotted plasma was studied by measuring the wavelength dependence of gel turbidity. The mass/length ratio value, -i.e. the thickness of fiber strands and porosity of the gel increased with increasing AT concentrations. It is concluded that plasma AT regulates the rate of prothrombin-thrombin conversion, the clotting time and the consequently network structure of the fibrin gel.

Antithrombin III↗
PubMed1993

On the mechanism of coagulation inhibition on surfaces with end point immobilized heparin.

A well established technique to improve blood compatibility of artificial materials for use in the circulation is to coat the surface with heparin. The present report describes the antithrombin mediated inhibition of thrombin and factor Xa by surfaces modified with end point immobilized heparin. The reaction was followed by conventional chromogenic substrate based enzyme assays as well as by immunological measurement of the enzyme inhibitor (thrombin-antithrombin) complex formation. Both enzymes were rapidly inactivated by heparin surfaces after selective presaturation with antithrombin on the immobilized high affinity heparin molecules. The thrombin inhibitory capacity was enhanced when both high and low affinity heparin were preadsorbed with inhibitor. The main part of the thrombin-antithrombin complex formed remained bound to the surface, however, without functionally blocking the activity of the high affinity sequence of the immobilized heparin. Aliquots of recalcified plasma were slowly rotated in loops of heparinized tubing to investigate whether the main thromboresistant function of the surface was exerted at the level of thrombin or by inactivation of preceding enzymes. After 1 h no visible clotting occurred and only trace amounts of thrombin (0.07 IU/ml), measured as thrombin-antithrombin complexes, had been formed. In non-heparinized loops and in the test tube plasma clotted after 20 min. The thrombin generation when clotting occurred was in the order of 10 IU/ml. It is concluded that the immobilized heparin mediates inhibition of the coagulation cascade prior to prothrombin activation.

Adsorption↗
PubMed1993

Effect of surface-immobilized heparin on the activation of adsorbed factor XII.

Two different heparin surfaces, structurally closely related and of similar negative charge characteristics, were compared with regard to adsorption and activation of coagulation Factor XII (FXII). One surface was prepared by immobilization of unfractioned heparin, which yielded a surface containing both heparin molecules with high and with low affinity for antithrombin (unfractioned [UF] heparin surface). The other surface consisted of a fraction of heparin molecules with low affinity for antithrombin (LA heparin surface) and essentially devoid of antithrombin-binding as well as anticoagulant activity. Both surfaces adsorbed FXII from plasma to a similar extent, and essentially the same quantities of bound factor could be recovered from the surfaces. The two heparin surfaces, however, differed markedly with regard to activation of the adsorbed FXII. On the LA heparin surface, a major portion of the surface-bound FXII was recovered in its enzymatically active form (FXIIa), but only trace amounts of the FXII taken up by the UF heparin surface had undergone activation. When FXII-deficient plasma was used instead of normal plasma, no surface-associated enzyme activity could be recovered on either surface. The presence of free standard heparin or low molecular weight heparin in the plasma exposed to the LA heparin surface did not prevent conversion of FXII to FXIIa.

Adsorption↗