RESUMO
Heart transplant recipients show platelet hyperaggregability, which may be related to the incidence of graft vasculopathy. We investigated whether trapidil can inhibit the aggregation of platelets from these patients. Platelet count, mean platelet volume (MPV), and adenosine diphosphate (ADP)-induced platelet aggregation were determined in 18 heart transplant recipients and 12 healthy subjects. Additionally, platelet-rich plasma from the patients was incubated with trapidil or with saline, prior to measuring ADP-induced aggregation. The MPV was significantly greater in patients compared to controls (9.4+/-1.1 vs 8.5+/-0.7 fL; P=.01), and ADP-induced platelet aggregation was significantly increased in patients compared to controls (81.2%+/-13.1% vs 69.6%+/-16.2%; P=.04, respectively). The trapidil-treated samples showed significantly decreased platelet aggregation compared to the control samples (24.2%+/-12.6% vs 66.7%+/-11.7%; P<.001). Platelets from heart transplant recipients showed an increased MPV and increased ADP-induced aggregation. Trapidil effectively reduced the ADP-induced aggregation ex vivo.
Assuntos
Transplante de Coração/fisiologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Trapidil/farmacologia , Difosfato de Adenosina/farmacologia , Adulto , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Valores de ReferênciaRESUMO
During the transformation of fibrinogen to fibrin, excess fibrinogen suppresses further polymerization of fibrin, thereby enabling the nascent fibrin to be transported in a soluble form in blood. The question of possible complex formation between fibrin and fibrinogen was addressed by analyzing fibrin/fibrinogen (1:20, mol/mol) mixtures in the presence of calcium ions in stable linear sucrose density gradients by ultracentrifugation at 37 degrees C. During the period of ultracentrifugation in independent experiments, 40% of desAA-fibrin and 30% of desAABB-fibrin, respectively, precipitated without the participation of fibrinogen. The desAABB-fibrin, recovered in the gradient fractions, appeared as high-molecular-weight polymers (22 S), whereas the recovered desAA-fibrin exhibited only a slight increase in molecular weight (9 S) compared to fibrinogen (8 S). In contrast to this finding, both types of fibrin were totally recovered in gradient fractions provided that fibrinogen was present in the gradient at a uniform concentration of 2 mg/ml. In addition, the presence of fibrinogen but not human serum albumin reduced the size of desAABB-fibrin polymers (17 S). However, stable fibrin-fibrinogen complexes could not be demonstrated, since cosedimentation of differently labelled desAABB-fibrin and fibrinogen was not detectable. These studies suggest a specific but weak interaction of the solubilizing fibrinogen with the soluble fibrin polymers as demonstrated by a rapid exchange of both macromolecules.
Assuntos
Fibrina/metabolismo , Fibrinogênio/metabolismo , Centrifugação com Gradiente de Concentração , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinopeptídeo A/metabolismo , Fibrinopeptídeo B/metabolismo , Humanos , Polímeros/metabolismo , UltracentrifugaçãoRESUMO
Thrombin preferentially cleaves fibrinopeptides A (FPA) from fibrinogen resulting in the formation of desAA-fibrin from which most of the fibrinopeptides B (FPB) are then released with an enhanced rate. Kinetics of fibrinopeptide release from normal and dysfunctional fibrinogens were investigated in order to further characterize the mechanism of accelerated FPB release during desAA-fibrin polymerization. Dysfunctional fibrinogens London I and Ashford, exhibiting primary polymerization abnormalities (i.e., an abnormality present when all fibrinopeptides have been cleaved), which in the case of fibrinogen London I is believed to be caused by a defect in the D-domain, were shown to exhibit a decreased rate of FPB release compared with normal fibrinogen. While Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was shown to decrease the rate of FPB release from normal fibrinogen by a factor of 5, normal fragment D1, although inhibiting clot formation of normal fibrinogen, did not influence the acceleration of FPB release. On the other hand, the presence of fragment D1 did not enhance FPB release from fibrinogen London I, suggesting that interaction of D-domains in functional isolation with desAA-fibrin E-domains is not sufficient to enhance FPB release. Although clot formation was inhibited by the concentrations of fragment D1 used, the formation of small desAA-fibrin oligomers was hardly affected. Thus, small fibrin polymers, but not desAA-fibrin monomers, act as optimal substrates for the release of FPB by thrombin.
Assuntos
Fibrina/antagonistas & inibidores , Fibrinogênio/metabolismo , Fibrinogênios Anormais , Fibrinopeptídeo B/metabolismo , Trombina/metabolismo , Transtornos da Coagulação Sanguínea , Fibrina/metabolismo , Humanos , Cinética , Nefelometria e Turbidimetria , PolímerosRESUMO
Rabbits were injected with a platelet antiserum to examine the role of thrombocytopenia of the precipitation of soluble fibrin by endotoxin.dotoxin. Platelet antiserum removed more than 98% of the circulating platelets, and the resulting thrombocytopenia with platelet counts below 10,000 per mul persisted during the entire 10 hr-period of the experiment. Leukocyte counts were not significantly influenced by the platelet antiserum. Since the rabbits were treated with high doses of heparin, activation of intravascular coagulation by the antiserum did not occur. Precipitation of soluble fibrin was achieved by injection of endotoxin into rabbits with ancrod-induced circulating soluble fibrin. Thrombocytopenia did not prevent the occurrence of glomerular microclots after ancrod and endotoxin administration. On the contrary, if endotoxin was injected into antiserum- and heparin-treated rabbits with circulating soluble fibrin, glomerular microclot formation occurred even in a higher percentage than in control rabbits treated with absorbed platelet antiserum. This investigation indicates that platelet antiserum-induced thrombocytopenia does not protect against precipitation of soluble fibrin by en
Assuntos
Plaquetas , Coagulação Intravascular Disseminada/sangue , Endotoxinas/farmacologia , Fibrina/metabolismo , Ancrod/farmacologia , Animais , Plaquetas/imunologia , Precipitação Química , Coagulação Intravascular Disseminada/induzido quimicamente , Feminino , Heparina/farmacologia , Soros Imunes , Contagem de Leucócitos , Masculino , Coelhos , Fenômeno de Shwartzman/sangue , TrombocitopeniaRESUMO
Most of the knowledge acquired on platelet function and biochemistry has been obtained from platelets prepared from blood anticoagulated with sodium citrate. Using washed platelets from human blood (PNB) to which no anticoagulants were added, we report on responses not observed with platelets prepared from citrate-anticoagulated blood. Native blood was passed rapidly (within 5 min of venepuncture) through a Sephadex G-25/G-50 column to remove divalent ions and thus prevent coagulation. Platelets were separated from the gel-filtered blood by differential centrifugation. Responses of PNB to thrombin, collagen, calcium ionophore, ristocetin, release of 14C-5hydroxytryptamine and beta-thromboglobulin, and generation of thromboxane A2 were similar to those observed for citrated platelets. Comparison of PNB with thrombin-treated platelets, which demonstrate an increase of platelet factor 3 activity, a reduction of the adenylate energy charge and an impairment of clot retraction, indicated the absence of platelet activation. Unlike citrated platelets, however, aggregation of PNB in response to ADP was irreversible in the presence of Ca2+ and fibrinogen, even at concentrations as low as 0.2 microM ADP, with aggregation taking up to 25 times longer to reach the same extent of aggregation as for citrated platelets. PNB did not aggregate to epinephrine even in the presence of Ca2+ and fibrinogen. Sodium citrate impaired ADP-induced aggregation and clot retraction of PNB. Thus citrate affects platelet function and may cause changes resulting in the unphysiological behaviour and responses of platelets.
Assuntos
Anticoagulantes , Citratos/farmacologia , Agregação Plaquetária , Difosfato de Adenosina/farmacologia , Calcimicina/farmacologia , Cálcio/farmacologia , Retração do Coágulo , Epinefrina/farmacologia , Humanos , Cinética , Agregação Plaquetária/efeitos dos fármacos , Fator Plaquetário 3/análise , Ristocetina/farmacologiaRESUMO
The present study is concerned with the formation of fibrin-fibrinogen associations in the presence of FXIIIa while fibrinolysis was inhibited by aprotinin and EACA. SDS agarose-polyacrylamide gel electrophoresis on 2.5% gels under non-reducing conditions and ultracentrifugation of the associations on urea-sucrose density gradients showed the formation of soluble cross-linked high molecular weight (HMW) fibrin-fibrinogen polymers with an estimated molecular size up to 10 times that of fibrinogen. After incubation of a mixture of 131I-fibrinogen (4 mg/ml) and 125I-desAA-fibrin (0.2 mg/ml) with FXIIIa (2 U/ml) for 1 h at 37 degrees C, about 5% of the fibrinogen and 80% of the fibrin was incorporated into the generated soluble HMW polymers. The detection of soluble crosslinked fibrin-fibrinogen polymers could be a useful diagnostic criterion for imminent DIC.
Assuntos
Fibrina/metabolismo , Fibrinogênio/metabolismo , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/diagnóstico , Humanos , Técnicas In Vitro , Peso Molecular , Polímeros , Conformação Proteica , SolubilidadeRESUMO
The interaction of radiolabeled factor VII (FVII) and factor VIIa (FVIIa) with endotoxin-stimulated endothelial cells (EC), known to express tissue factor (TF), and unstimulated EC was studied. FVII/FVIIa binding to EC-monolayers was saturable within 4.5-6 h, reversible, temperature and calcium dependent on both, endotoxin-stimulated and on unstimulated EC. Upon 2 h of incubation on EC, FVII was partially converted to FVIIa in the absence of protease inhibitors. The affinity of this binding was Kd = 45.4 +/- 18.7 nM with a calculated number of binding sites Bmax = 3.75 +/- 0.31 x 10(6) molecules/cell. In addition to unlabeled FVII and FVIIa, other vitamin K-dependent proteins reduced binding of [125I]-FVII/FVIIa to about 60-70%, and this type of common binding site for vitamin K-dependent proteins revealed a Kd = 32.2 +/- 5.6 nM and a Bmax = 3.03 +/- 0.14 x 10(6) molecules/cell. Moreover, in the presence of 1 microM prothrombin to suppress common binding sites, only on endotoxin-stimulated EC additional inhibition of FVII/FVIIa binding was achieved by anti-TF antibodies. The characteristics of the FVII/FVIIa-TF interaction with a Kd = 17.2 +/- 5.2 nM and a Bmax = 342,000 +/- 1,100 binding sites/cell revealed a similar saturation kinetics in radioligand binding and in functional factor X activation within 90-120 min. These data indicate the presence of at least two independent binding sites for FVII/FVIIa on stimulated EC of which about 10% are TF specific.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Endotélio Vascular/metabolismo , Fator VII/metabolismo , Fator VIIa/metabolismo , Sítios de Ligação , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotoxinas/farmacologia , Fator Xa/biossíntese , Humanos , Cinética , Lipídeo A/farmacologia , Tromboplastina/metabolismoRESUMO
16 non-pregnant female and 19 pregnant rabbits were injected with purified rabbit 125I-fibrinogen in order to study the catabolism of fibrinogen before and after delivery. The half-life time (T 1/2) of the clottable 125I-radioactivity in pregnant rabbits during the last third of gestation was 27.3 +/- 5.3 hr in comparison to 54.4 +/- 3.7 hr in non-pregnant rabbits. After delivery, T 1/2 returned to normal values of 47.9 +/- 10.9 hr. The fractional catabolic rate (FCR) of the clotabble 125I-radioactivity was 67.1% X d-1 +/- 8.6 before and 41.6% X d-1 after delivery whereas FCR in non-pregnant rabbits amounted to 44.7% X d-1 +/- 4.8. These figures demonstrate a pronounced increase in fibrinogen catabolism during the last third of gestation in pregnant rabbits and a normalization immediately after delivery. Although in pregnant rabbits the elimination of fibrinogen from the circulating blood was pronouncedly increased, the plasma fibrinogen concentration did not change. Only after delivery did the fibrinogen concentration increase when the fibrinogen catabolism had already normalized.
Assuntos
Fibrinogênio/metabolismo , Animais , Feminino , Meia-Vida , Gravidez , Terceiro Trimestre da Gravidez , Prenhez , CoelhosRESUMO
The binding properties of purified bovine fibrinogen to bovine aortic endothelial cells have been examined in a tissue culture system. Endothelial cells bound 125I-fibrinogen in a calcium dependent fashion. Removal of calcium by EDTA instantaneously detached most of the cell-associated fibrinogen. Binding of fibrinogen to the endothelial cells was not saturable with time and dosage. Competition studies and displacement experiments did not indicate the involvement of a specific receptor site for the fibrinogen-endothelial cell-interaction. Bovine serum albumin provided in the physiological ratio of 20:1 to fibrinogen competed as effectively as unlabelled fibrinogen for 125I-fibrinogen binding to the endothelial cells. And furthermore, internalization of cell-associated tracer into the endothelial cells that could be demonstrated in the presence of serum free medium did not occur in the presence of albumin. These data suggest that fibrinogen binding to intact bovine endothelial cells is unspecific and presumably negligible in the presence of physiological albumin concentrations.
Assuntos
Aorta/metabolismo , Fibrinogênio/metabolismo , Animais , Ligação Competitiva , Cálcio/farmacologia , Bovinos , Contagem de Células , Células Cultivadas , Endotélio/metabolismo , Glucosamina/farmacologia , Heparina/farmacologia , Cinética , Soroalbumina Bovina/metabolismoRESUMO
A quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20 degrees C and 37 degrees C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20 degrees C as well as at 37 degrees C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20 degrees C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20 degrees C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37 degrees C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37 degrees C (95%) but only 58% of the desAABB-fibrin were bound at 20 degrees C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patient's plasma.
Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Fibrina/análise , Fibrinogênio/análise , Temperatura Corporal , Cromatografia de Afinidade/métodos , Interações Medicamentosas , Fibrina/metabolismo , Fibrinogênio/metabolismo , Fibrinopeptídeo A , Fibrinopeptídeo B , Humanos , SolubilidadeRESUMO
The lysis of fibrin clots on the surface of cultured human omental tissue microvascular endothelial cells (HOTMEC) and cultured human umbilical vein endothelial cells (HUVEC) was studied. Fibrin clots were made by mixing fibrinogen, plasminogen and thrombin on the surface of both cell types. Clot lysis was seen only on the surface of HOTMEC, which were found to synthesize about 100-fold more tissue plasminogen activator (tPA) antigen than HUVEC. Clot lysis of HOTMEC could be blocked by anti-tPA IgG but was not affected by the incorporation of exogenous plasminogen activator (PAI) into the clot in concentrations (75 arbitrary units) exceeding the tPA activity (21 +/- 2.5 IU) of the cells. Thus, it is likely that tPA secreted by HOTMEC is protected from inhibition by PAI in the presence of fibrin and endothelial cells. The stimulation of EC to release an excess of tPA over PAI, in contrast to the secretion of an excess of PAI over tPA found in unstimulated cells in the absence of fibrin, is obviously no prerequisite for the initiation of fibrinolysis on the surface of HOTMEC. As thrombin was used for clot formation, its influence on tPA and PAI synthesis of both cell types was investigated. In contrast to HOTMEC, which were not affected by alpha-thrombin, HUVEC revealed a dose-dependent increase in tPA and PAI synthesis upon incubation with the enzyme. This increase in tPA production by HUVEC was not sufficient to lyse the clots within 48 hours. Furthermore, HUVEC behaved differently towards thrombin as these cells in contrast to HOTMEC revealed the typical shape change reaction upon incubation with the enzyme.
Assuntos
Endotélio Vascular/fisiologia , Trombose , Capilares/citologia , Células Cultivadas , Endotélio Vascular/citologia , Fibrina/biossíntese , Glicoproteínas/análise , Humanos , Proteínas de Membrana/biossíntese , Plasminogênio/metabolismo , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio , Trombina/farmacologia , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
The regulation of tissue-plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) synthesis was studied in cultured human mesothelial cells derived from omentum (HOMC). Heparin (100 U/ml) as well as pentosan polysulfate (300 micrograms/ml) stimulated tPA synthesis by HOMC 2.9-4.5-fold. Heparin-induced tPA production was dose- and time-dependent and was inhibited by cycloheximide. tPA production by HOMC was also stimulated by phorbol 12-myristate 13-acetate (2.6-fold), fibrin clots (1.9-fold), or batroxobin (1.9-fold). Heparin and pentosan polysulfate did not stimulate PAI-1 production by HOMC, while phorbol 12-myristate 13-acetate (100 nM) increased the concentration of PAI-1 in the conditioned medium by 2.6-fold over 24 h. The interaction of heparin with HOMC was studied by direct binding experiments. Dose-dependent specific binding of biotinylated heparin to HOMC was saturable at about 10 micrograms/ml, the KD was estimated to about 0.15 microM. Biotinylated heparin bound rapidly to HOMC and reached a plateau within 60 min. Unlabeled heparin as well as pentosan polysulfate inhibited binding of biotinylated heparin in a dose-dependent fashion. These data demonstrate that heparin interacts with HOMC, and increases the fibrinolytic capacity in these cells by selectively increasing the production of tPA.
Assuntos
Epitélio/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Heparina/farmacologia , Inativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/biossíntese , Biotina , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Indução Enzimática/efeitos dos fármacos , Epitélio/enzimologia , Epitélio/metabolismo , Heparina/metabolismo , Antagonistas de Heparina/farmacologia , Humanos , OmentoRESUMO
In 1994, shortly after a heat-treated prothrombin complex concentrate (PCC) had been withdrawn from the German market due to transmission of hepatitis B, the license of another brand was withdrawn, due to 3 acute fatalities associated with the use of this product. We report on the clinical data of altogether 5 patients, who died during a 3 month period in Germany after having received this brand of PCC. All patients had surgery, acquired deficiencies of coagulation factors, and underlying diseases predisposing for thrombosis or disseminated intravascular coagulation. PCC was administered for the prevention of bleeding. In three patients, a drug interaction of PCC with aprotinin may also have played a role. Several points, however, are suspicious of a major causative effect of the respective product, (a) the close temporal correlation between administration of the drug and the subsequent clinical as well as laboratory deterioration, (b) the accumulation of these adverse events in a short period of time, when the use and market share of this brand increased due to the shortage of other products, and (c) laboratory abnormalities of this brand which have been consistently observed in several in vitro studies.
Assuntos
Fator IX/efeitos adversos , Complicações Pós-Operatórias/prevenção & controle , Protrombina/efeitos adversos , Tromboembolia/etiologia , Adulto , Aprotinina/efeitos adversos , Aprotinina/uso terapêutico , Pré-Escolar , Interações Medicamentosas , Fator IX/uso terapêutico , Evolução Fatal , Feminino , Hemostáticos/efeitos adversos , Hemostáticos/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Protrombina/uso terapêuticoRESUMO
Intact vascular endothelium provides several anticoagulant mechanisms for the maintenance of blood fluidity and the prevention of thrombosis. High-affinity binding of proteolytic active thrombin to thrombomodulin at the cell surface effectively facilitates the activation of the potent anticoagulant protein C (PC). Rapid inactivation of cell-bound thrombin by antithrombin III (ATIII) accelerated by heparin-like structures represents another anticoagulant mechanism. In the present investigation the interference of these two events has been studied. Inhibition of thrombin bound to cultured bovine aortic endothelial cells (BAEC) by ATIII and the effect of the inhibitor on the activation of PC has been studied using purified components of bovine origin. Exposure of thrombin (45 nM) with prewashed confluent BAEC-monolayers for 10 min resulted in the binding of 12% thrombin. The subsequent incubation with various concentrations (0.3-2.4 microM) of ATIII revealed no acceleration of the inhibition of thrombin by ATIII at the endothelial cell surface when compared with the uncatalyzed fluid phase reaction. However, compared with the uncatalyzed fluid phase reaction. However, heparin added to the reaction mixture substantially increased the inactivation of cell-bound thrombin. Modified ATIII that did not possess heparin cofactor activity presented a comparable inactivation pattern for endothelial cell bound-thrombin as native ATIII indicating that heparin-like structures did not accelerate the interaction. When PC (32 nM) and ATIII (1.8 microM) competed for thrombin bound to BAEC, activation of PC was demonstrated within the initial 6 min of the incubation amounting to 62% of the activated PC formation in the absence of ATIII.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antitrombina III/fisiologia , Endotélio/citologia , Proteína C/metabolismo , Trombina/antagonistas & inibidores , Animais , Coagulação Sanguínea , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Heparina/farmacologia , Ligação Proteica , Trombina/metabolismoRESUMO
In the absence of its cofactor thrombomodulin (TM) thrombin is only a poor activator of the anticoagulant serine protease protein C (PC). The TM-dependence of PC-activation has been restricted to a series of molecular structures of the PC molecule including high-affinity calcium binding sites and single amino acid residues. However, thrombin induced activation of a PC derivative altered in all these critical positions is markedly enhanced by TM indicating that additional structures of the PC molecule are involved in determining the TM specificity. Based on the hypothesis that such an additional regulatory element should be located near the thrombin cleavage site and should include negatively charged amino acids to ascertain calcium binding, we studied whether Glu and Asp in positions P7 and P6 relative to the thrombin cleavage site together with Asp in P3 are involved in formation of such a regulatory element. Three PC derivatives containing the neutral counterpart of the negatively charged amino acids in positions P3; P3 and P6; and P3, P6, and P7, respectively, were generated using site-directed mutagenesis. Compared to rPC-wt the initial rates of PC activation of all three mutants were increased 4.0-fold for thrombin/TM and 4.0-, 5.3-fold for activation by thrombin alone. However, compared to the PC derivative neutralized exclusively in P3, additional changes in P6 and P7 showed no increase in the thrombin activation kinetics and calcium binding properties were identical in all of the three mutants.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ácido Aspártico/fisiologia , Cálcio/fisiologia , Ácido Glutâmico/fisiologia , Proteína C/metabolismo , Trombina/farmacologia , Trombomodulina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Transformada , Células Cultivadas , Fenômenos Químicos , Físico-Química , Endotélio Vascular/citologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína C/química , Proteína C/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The use of recombinant (r) hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor IIa assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin-spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 micrograms/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin-spiked blood samples obtained from 50 healthy blood donors. CV-values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 micrograms/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.
Assuntos
Ponte Cardiopulmonar , Fibrinolíticos/uso terapêutico , Terapia com Hirudina , Ponte de Artéria Coronária , Endopeptidases , Fibrinolíticos/administração & dosagem , Próteses Valvulares Cardíacas , Heparina/efeitos adversos , Hirudinas/administração & dosagem , Humanos , Monitorização Intraoperatória , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Valores de Referência , Análise de Regressão , Reprodutibilidade dos Testes , Trombocitopenia/induzido quimicamente , Trombocitopenia/prevenção & controle , Venenos de Víboras , Tempo de Coagulação do Sangue TotalRESUMO
CD40 is a type I member of the tumour necrosis factor (TNF) receptor superfamily of proteins, and is present on a wide variety of cells including vascular endothelial cells. Ligation of this receptor on endothelial cells is known to increase expression of inflammatory adhesion molecules. We have recently demonstrated that platelets express the ligand of CD40 (CD154) within seconds of exposure to agonist, and interact with endothelial cells to participate directly in the induction of an inflammatory response. Here we show that activated platelets induce tissue factor (TF) expression on endothelial cells in a CD40/CD154-dependent manner, and that the magnitude of this response can equal that induced by TNFe. Moreover, CD40 ligation on endothelial cells downregulates the expression of thrombomodulin. We also show that CD40-mediated TF expression is less sensitive to inhibition with the oxidative radical scavenger pyrrolidine dithiocarbamate than is that mediated by TNFalpha, indicating that CD40 has a distinct signalling pathway. Tissue factor is a cell membrane protein which functions as the main trigger of the extrinsic pathway of blood coagulation, and its expression on endothelial cells is implicated in wound healing and angiogenesis. Since platelets are among the first cells involved in haemostasis following tissue injury, our data showing that ligation of CD40 by CD154 induces a procoagulant phenotype on vascular endothelial cells suggests that platelets may play an important role in the induction of wound healing.
Assuntos
Antígenos CD40/fisiologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Glicoproteínas de Membrana/fisiologia , Ativação Plaquetária , Tromboplastina/biossíntese , Plaquetas/metabolismo , Ligante de CD40 , Células Cultivadas , Endotélio Vascular/citologia , Sequestradores de Radicais Livres , Hemostasia/fisiologia , Humanos , Ligantes , Fenótipo , Pirrolidinas/farmacologia , Tiocarbamatos/farmacologia , Trombomodulina/biossíntese , Trombomodulina/genética , Tromboplastina/genética , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais , Cicatrização/fisiologiaRESUMO
A multicenter study of a chromogenic substrate method for photometric determination of prothrombin time was conducted in order to evaluate its clinical application. Seven laboratories participated in the study using a total of 742 plasma samples from 417 patients on oral anticoagulant therapy, 261 healthy subjects and 64 patients with different diseases especially of the liver as well as 30 patients with hereditary deficiency of coagulation factors II, V, VII, X. The chromogenic PT method was compared to a standardized coagulometric PT assay which uses the same sensitive human placenta thromboplastin calibrated against international reference preparations. A high correlation of the prothrombin ratio values of the chromogenic and the coagulometric assay was obtained in 402 plasma samples (r = 0.940; y = 1.02x - 0.1). The study showed that the chromogenic PT reagent is sensitive to deficiency of the coagulation factors of the extrinsic pathway but not affected by heparin up to 1 IU/ml because of the heparin antagonist added. The precision (coefficient of variation) of the photometric method ranged between 0.6 and 3% (intraassay CV) and between 1.4 and 5.8 (interassay CV). The International Sensitivity Index (ISI) obtained for the used lot was 1.09. The therapeutical range in percentage activity for patients in a stable phase of an anticoagulant therapy was found to be from 15 to 27 percent of normal. The results of the clinical evaluation proved the good comparability of the new chromogenic PT test with coagulometric methods, its high factor sensitivity, good reproducibility and easy performance.
Assuntos
Tempo de Protrombina , Anticoagulantes/uso terapêutico , Transtornos da Coagulação Sanguínea/sangue , Testes de Coagulação Sanguínea , Compostos Cromogênicos/normas , Estudos de Avaliação como Assunto , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Heparina/sangue , Humanos , Hepatopatias/sangue , FotometriaRESUMO
Tissue factor pathway inhibitor (TFPI) is an important regulator in the extrinsic blood coagulation pathway. Although the regulatory biochemical role of TFPI is evident, the clinical significance of this proteinase inhibitor remains to be elucidated. The definition of a clinical TFPI deficiency seems to be more complex than that of other coagulation inhibitors because the activity and concentration of circulating TFPI can not be considered a true measure of in vivo levels. Its determination in plasma samples by immunological methods or functional assays has been shown to be inadequate in the detection of a clinical deficiency. Therefore, we screened genomic DNA samples of blood donors and thrombotic patients for alterations in the TFPI gene to assess the influence of a modified TFPI in venous thromboembolic diseases. We detected a single nucleotide substitution in exon 7 (536C-->T) leading to a proline to leucine exchange at amino acid position 151 of the protein ([P151L]TFPI) and found the prevalence of heterozygous carriers in German unrelated blood donors to be 0.2% (n = 5120). Four unrelated persons out of 14 probands carrying the genetic variation could be linked to venous thrombosis. For calculation of a potential risk for venous thrombosis for carriers of the mutation we investigated healthy blood donors about thrombotic events. 7 out of 308 blood donors were found to have a history of venous thrombosis, one of them carried the TFPI mutation. Statistical calculation showed a significant relative risk for venous thrombosis for individuals with the trait (odds ratio, 9.3; confidence interval, 1.8-48.6; p <0.01).
Assuntos
Lipoproteínas/genética , Mutação Puntual , Trombose Venosa/genética , Sequência de Aminoácidos , Éxons , Heterozigoto , Homozigoto , Humanos , Dados de Sequência Molecular , Fatores de RiscoRESUMO
There are currently three established low-density lipoprotein (LDL) apheresis systems: immunoadsorption, heparin-induced extracorporeal LDL precipitation (HELP), and dextran sulfate. We treated the same patient with all three systems and compared the lipid reductions achieved. A total of 135 consecutive treatments were studied, 57 with immunoadsorption, followed by 30 with HELP and 48 with dextran sulfate adsorption. The mean plasma volume (mean +/- SD) treated was 4.9 +/- 0.05, 3.08 +/- 0.091, and 3.39 +/- 0.71 L, respectively. The LDL-cholesterol (LDL-C) reduction was 75.5% +/- 7.4%, 61.6% +/- 5.1%, and 57.1% +/- 12.4%, respectively (P < .001 for immunoadsorption vHELP and dextran sulfate). The mean removal efficiency (mass removed/plasma volume treated) for LDL-C was 1.0 +/- 0.12, 1.42 +/- 0.25, and 1.15 +/- 0.21 g/L, respectively (P < .001 for HELP v immunoadsorption and dextran sulfate). The mean LDL-C plasma concentration before apheresis was 199 +/- 23.9, 201 +/- 25.7, and 186 +/- 28 mg/dL, respectively (P < .001 for dextran sulfate adsorption v immunoadsorption and HELP). Among the three LDL apheresis systems, immunoadsorption caused the greatest percent reduction in LDL-C, while HELP eliminated LDL-C from the plasma most efficiently. Dextran sulfate was similar to HELP in terms of LDL-C reduction, and its removal efficiency was similar to immunoadsorption. Dextran sulfate was also associated with the lowest pretreatment plasma LDL-C concentration.