RESUMEN
Thrombolysis with tissue plasminogen activator (tPA) has been a disappointment and has now been replaced by an endovascular procedure whenever possible. Nevertheless, thrombolysis remains the only means by which circulation in a thrombosed artery can be restored rapidly. In contrast to tPA monotherapy, endogenous fibrinolysis uses both tPA and urokinase plasminogen activator (uPA), whose native form is a proenzyme, prouPA. This combination is remarkably effective as evidenced by the fibrin degradation product, D-dimer, which is invariably present in plasma. The two activators have complementary mechanisms of plasminogen activation and are synergistic in combination. Since tPA initiates fibrinolysis when released from the vessel wall and prouPA is in the blood, they induce fibrinolysis sequentially. It was postulated that this may be more effective and fibrin-specific. The hypothesis was tested in a model of clot lysis in plasma in which a clot was first exposed to tPA for 5 min, washed and incubated with prouPA. Lysis was compared with that of clots incubated with both activators simultaneously. The sequential combination was almost twice as effective and caused less fibrinogenolysis than the simultaneous combination (p < 0.0001) despite having significantly less tPA, as a result of the wash. A mechanism is described by which this phenomenon can be explained. The findings are believed to have significant therapeutic implications.
Asunto(s)
Fibrina/metabolismo , Fibrinólisis/efectos de los fármacos , Activadores Plasminogénicos/farmacología , Sinergismo Farmacológico , Productos de Degradación de Fibrina-Fibrinógeno , Humanos , Modelos Biológicos , Activador de Tejido Plasminógeno/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiologíaRESUMEN
Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard 125I-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (less than 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA. Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.
Asunto(s)
Activadores Plasminogénicos/farmacología , Activador de Tejido Plasminógeno/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Carboxipeptidasa B , Carboxipeptidasas/farmacología , Sinergismo Farmacológico , Fibrinolisina/farmacología , Fibrinólisis , Humanos , Lisina/sangre , Activadores Plasminogénicos/antagonistas & inhibidores , Inactivadores Plasminogénicos , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
Plasmin generation by equimolar concentrations of tissue plasminogen activator (t-PA), pro-urokinase (pro-UK), and urokinase (UK), and a twofold higher concentration of a plasmin-resistant mutant rpro-UK (Ala-158-pro-UK) was measured on a microtiter plate reader. The promoting effects on this reaction of equimolar concentrations of fibrinogen, soluble fibrin (Desafib), CNBr fragment FCB-2 (an analogue of fragment D), or purified fragment E-2 were compared. Plasmin generation by t-PA was moderately promoted by fibrinogen, substantially promoted by Desafib and FCB-2, but not at all promoted by fragment E-2. By contrast, plasmin generation by pro-UK or by Ala-158-pro-UK was not promoted either by fibrinogen, Desafib, or FCB-2, but was significantly promoted by fragment E-2. Plasmin generation by UK was not significantly promoted by any of the fibrin(ogen) preparations. Treatment of fragment E-2 by carboxypeptidase-B (CPB), eliminated its promotion of pro-UK and Ala-158-pro-UK-induced plasmin generation. Pretreatment of FCB-2 with plasmin slightly potentiated its promotion of t-PA activity. This effect of plasmin pretreatment of FCB-2 was reversed by CPB treatment. Plasmin pretreatment of FCB-2 did not induce any promotion of activity in pro-UK or Ala-158-pro-UK. The findings show that the intrinsic activity of pro-UK and the activity of t-PA are promoted by different regions of the fibrin(ogen) molecule. The latter is stimulated primarily by a determinant in the fragment D region, which is available in intact fibrin. By contrast, plasminogen activation by the intrinsic activity of pro-UK was stimulated exclusively by fragment E-2, which is unavailable in intact fibrin. The findings are believed relevant to fibrinolysis and support the concept that t-PA and pro-UK are complementary, sequential, and synergistic in their actions.
Asunto(s)
Precursores Enzimáticos/farmacología , Productos de Degradación de Fibrina-Fibrinógeno/farmacología , Fibrinolisina/biosíntesis , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Carboxipeptidasa B , Carboxipeptidasas/farmacología , Sinergismo Farmacológico , Electroforesis en Gel de Poliacrilamida , Fibrinolisina/farmacologíaRESUMEN
Two plasmin-resistant mutant forms of pro-urokinase (pro-UK) constructed by site-directed mutagenesis of Lys158 to Val158 and Met158 were used to evaluate the intrinsic enzymatic and fibrinolytic properties of pro-UK as distinct from those of its two-chain UK (TC-UK) derivative. Both mutants, while resistant to plasmin activation, were as sensitive as pro-UK to degradation by thrombin. Since thrombin cleaves a peptide bond only two residues from the activation site, the integrity of this loop was maintained in the two mutants. The amidolytic and plasminogen-activating activities of the mutants averaged 0.14 and 0.12% that of TC-UK, respectively. The fibrin plate activities were 2,400 IU/ml and 700 IU/mg for the Met158 and Val158 mutants or about 1.5% that of TC-UK. These findings attest to a discrete but low intrinsic activity for pro-UK and suggest that the higher values reported in the literature may be related to UK contaminants or plasmin-induced TC-UK generation during the assay. Clot lysis by the mutants required doses greater than 100-fold higher than those of pro-UK to induce a comparable effect. From this it appears that pro-UK activation is a major determinant of the rate of clot lysis occurring with pro-UK. Clot lysis by the mutants was potentiated by plasmin pretreatment of the fibrin and by the addition of small amounts of TC-UK or tissue plasminogen activator (t-PA). Combinations of t-PA and the mutants were synergistic in their fibrinolytic effects. These findings mirror those previously obtained with pro-UK. We concluded that the previously described potentiation of pro-UK-induced clot lysis by UK or t-PA is mediated primarily by pro-UK itself rather than by a promotion of its activation.
Asunto(s)
Fibrinolisina/farmacología , Fibrinólisis , Lisina , Activadores Plasminogénicos/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Resistencia a Medicamentos , Fibrinólisis/efectos de los fármacos , Humanos , Metionina , Datos de Secuencia Molecular , Mutación , Activadores Plasminogénicos/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Activador de Tejido Plasminógeno/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/genética , ValinaRESUMEN
A single-chain 55,000-mol wt form of urokinase (UK), similar to that previously isolated from urine, was purified from a transformed kidney cell culture medium and characterized; and its fibrinolytic properties were evaluated. The preparation immunoprecipitated with UK antiserum, had a low intrinsic amidolytic activity that was 0.1% of its active derivative, and resisted diisopropyl fluorophosphate treatment and inactivation by plasma inhibitors. The single-chain UK was therefore designated pro-UK. In the presence of plasmin and during clot lysis, activation by conversion to two-chain, 55,000-mol wt UK (TC-UK) was demonstrated. This did not occur during blood clotting nor on incubation with purified thrombin. Clot lysis in plasma consistently occurred in 2-5 h with 50-100 IU per ml of pro-UK, whereas comparable lysis was inconsistently achieved by 500-1,000 IU of UK. Pro-UK, in sharp contrast to UK, caused no fibrinogen degradation at fibrinolytic concentrations. In the absence of a clot, pro-UK in plasma was stable for more than 2 d. When a clot was added after incubation (37 degrees C) for 50 h, activation to full lytic activity took place. The findings in vivo were comparable but the rapid clearance of pro-UK required that it be given by a constant infusion despite its plasma stability. In rabbits, a UK-resistant species, pro-UK was significantly (P less than 0.001) more efficacious than TC-UK but neither induced significant fibrinogen degradation. In dogs, a more sensitive species, the high specificity of thrombolysis by pro-UK contrasted with the defibrinogenation and uncontrollable bleeding that accompanied thrombolysis by UK. It was concluded that clot lysis by pro-UK is more effective and specific than UK. The advantage of pro-UK is in the limitation of its activation to the site of a clot. This can be explained by an activation mechanism that is dependent, under physiological conditions, on fibrin-stabilized plasmin.
Asunto(s)
Precursores Enzimáticos/metabolismo , Fibrina/metabolismo , Fibrinólisis , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Perros , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos/aislamiento & purificación , Fibrinógeno/análisis , Humanos , Cinética , Peso Molecular , Oxidación-Reducción , Conejos , Especificidad de la Especie , Activador de Plasminógeno de Tipo Uroquinasa/aislamiento & purificaciónRESUMEN
BACKGROUND: Prourokinase (prouPA) is unstable in plasma at therapeutic concentrations. A mutant form, M5, made more stable by reducing its intrinsic activity was therefore developed. Activation to two-chain M5 (tcM5) induced a higher catalytic activity than two-chain urokinase plasminogen activator (tcuPA), implicating an active site functional difference. Consistent with this, an unusual tcM5 complex with plasma C1-inhibitor was recently described in dog and human plasma. The effect of C1-inhibitor on fibrinolysis and fibrinogenolysis by M5 is the subject of this study. METHODS AND RESULTS: Zymograms of tcM5 and tcuPA incubated in plasma revealed prominent tcM5-C1-inhibitor complexes, which formed within 5 min. The inhibition rate by purified human C1-inhibitor (250 microg mL(-1)) was about 7-fold faster for tcM5 than it was for tcuPA (10 microg mL(-1)). The effect of the inhibitor on the stability of M5 and prouPA was determined by incubating them in plasma at high concentrations (10-20 microg mL(-1)) +/- C1-inhibitor supplementation. Above 10 microg mL(-1), depletion of all plasma plasminogen occurred, indicating plasmin generation and tcM5/tcuPA formation. With supplemental C1-inhibitor, M5 stability was restored but not prouPA stability. Clot lysis by M5 +/- supplemental C1-inhibitor showed no attenuation of the rate of fibrinolysis, whereas fibrinogenolysis was prevented by C1-inhibitor. Moreover, because of higher dose-tolerance, the rate of fibrin-specific lysis reached that achievable by non-specific fibrinolysis without inhibitor. CONCLUSIONS: Plasma C1-inhibitor stabilized M5 in its proenzyme configuration in plasma by inhibiting tcM5 and thereby non-specific plasminogen activation. At the same time, fibrin-specific plasminogen activation remained unimpaired. This unusual dissociation of effects has significant implications for improving the safety and efficacy of fibrinolysis.
Asunto(s)
Proteínas Inactivadoras del Complemento 1/farmacología , Fibrina/metabolismo , Plasminógeno/efectos de los fármacos , Serpinas/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Proteína Inhibidora del Complemento C1 , Electroforesis en Gel de Poliacrilamida , Fibrinólisis , Humanos , Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
BACKGROUND: A single site mutant (M5) of prourokinase (proUK) was developed to make proUK less vulnerable to spontaneous activation in plasma. This was a problem that seriously compromised proUK in clinical trials, as it precluded proUK-mediated fibrinolysis at therapeutic concentrations. METHODS AND RESULTS: After completing dose-finding studies, 12 anesthetized dogs with femoral artery thrombosis were given either M5 (2.0 mg kg(-1)) or tissue plasminogen activator (t-PA) (1.4 mg kg(-1)) by i.v. infusion over 60 min (20% administered as a bolus). Two pairs of standardized injuries were inflicted at which hemostasis was completed prior to drug administration. Blood loss was quantified by measuring the hemoglobin in blood absorbed from these sites. Thrombolysis was evaluated at 90 min and was comparably effective by both activators. Rethrombosis developed in one t-PA dog. The principal difference found was that blood loss was 10-fold higher with t-PA (mean approximately 40 mL) than with M5 (mean approximately 4 mL) (P = 0.026) and occurred at more multiple sites (mean 2.7 vs. 1.2). This effect was postulated to be related to differences in the mechanism of plasminogen activation by t-PA and M5 in which the latter is promoted by degraded rather than intact (hemostatic) fibrin. In addition, two-chain M5 was efficiently inactivated by plasma C1 inactivator, an exceptional property which helped contain its non-specific proteolytic effect. CONCLUSIONS: Intravascular thrombolysis by M5 was accompanied by significantly less bleeding from hemostatic sites than by t-PA. This was attributed to the proUK paradigm of fibrinolysis being retained at therapeutic concentrations by the mutation.
Asunto(s)
Hemorragia/inducido químicamente , Hemostasis/efectos de los fármacos , Mutación , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/administración & dosificación , Activador de Plasminógeno de Tipo Uroquinasa/administración & dosificación , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Modelos Animales de Enfermedad , Perros , Estabilidad de Enzimas/genética , Arteria Femoral , Fibrinólisis/efectos de los fármacos , Fibrinólisis/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Terapia Trombolítica/efectos adversos , Trombosis/tratamiento farmacológicoRESUMEN
Pro-urokinase is a single chain, precursor form of two chain, 54,000 Mr urokinase. Although originally isolated from urine, pro-urokinase is also found in blood, where it is believed to participate in natural fibrinolysis alongside tissue plasminogen activator (t-PA). These two plasminogen activators share the property of inducing fibrin-selective plasminogen activation, but many of their other properties, including their modes of action, are dissimilar. A comparison of some of the clinically relevant properties of pro-urokinase and t-PA is provided. A multicenter, dose-finding clinical trial of native pro-urokinase is underway in the United States and in West Germany. At the time of this writing, 110 patients with angiographically proved acute coronary thrombosis have been treated. The findings from one center are summarized in some detail and the overall experience is reviewed. Preliminary evidence for a potentiating effect on pro-urokinase-induced thrombolysis by urokinase is presented. The findings suggest that a bolus of urokinase (200,000 IU) at the outset increases the reperfusion rate from 60 to greater than 80% and shortens the lysis time from about 50 to 30 minutes. A modest (19%) but significant (p less than 0.05) decrease in fibrinogen accompanied thrombolysis by urokinase/pro-urokinase. Nonetheless, significant bleeding complications in the multicenter study have, to date, not been encountered and it is suggested that this may be related to pro-urokinase's sensitivity to inactivation by thrombin and lack of potentiation by heparin.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Fibrinolíticos/uso terapéutico , Activadores Plasminogénicos/uso terapéutico , Activador de Tejido Plasminógeno/uso terapéutico , Activador de Plasminógeno de Tipo Uroquinasa/uso terapéutico , Enfermedad Aguda , Ensayos Clínicos como Asunto , Trombosis Coronaria/tratamiento farmacológico , Sinergismo Farmacológico , Quimioterapia Combinada , Fibrinólisis/efectos de los fármacos , Humanos , Proteínas Recombinantes/uso terapéutico , Factores de TiempoRESUMEN
OBJECTIVES: The present study was designed to test the efficacy and safety of a sequential combination of recombinant tissue-type plasminogen activator (rt-PA) and pro-urokinase in patients with acute myocardial infarction. BACKGROUND: Efforts continue to identify a thrombolytic regimen that induces rapid, complete and sustained coronary artery patency in acute myocardial infarction. The two endogenous plasminogen activators rt-PA and pro-urokinase have been shown experimentally to induce fibrinolysis by sequential and complementary mechanisms. As a result, certain combinations of these activators have been found to be synergistic in vitro and in vivo. METHODS: In a multicenter observational study with core facilities for angiographic and laboratory analysis, 101 patients with acute myocardial infarction were enrolled and given a low dose bolus of rt-PA (5 to 10 mg) followed by a 90-min infusion of pro-urokinase (40 mg/h). All patients received intravenous heparin and oral aspirin. Coronary angiography was performed in all patients at 90 min. RESULTS: Angiography at 90 min showed the infarct-related artery to be patent (Thrombolysis in Myocardial Infarction [TIMI] grade 2 or 3 flow) in 77% of patients, and 60% achieved TIMI grade 3 flow. At one center, angiography was repeated at 24 h to detect a possible reocclusion. All 28 patients with a patent infarct-related artery at 90 min had patency at 24 h (82% achieved TIMI grade 3 flow). Treatment was well tolerated, with bleeding complications essentially confined to arterial puncture site hematomas. There was only one in-hospital death. CONCLUSIONS: A sequential combination of low dose rt-PA and reduced-dose pro-urokinase produced a high TIMI 3 patency rate, was well tolerated and was associated with a low reocclusion rate.
Asunto(s)
Precursores Enzimáticos/uso terapéutico , Fibrinolíticos/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/uso terapéutico , Activador de Plasminógeno de Tipo Uroquinasa/uso terapéutico , Adulto , Anciano , Factores de Confusión Epidemiológicos , Angiografía Coronaria , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/diagnóstico por imagen , Proteínas Recombinantes/uso terapéutico , Terapia Trombolítica/efectos adversos , Factores de Tiempo , Resultado del Tratamiento , Grado de Desobstrucción Vascular/efectos de los fármacosRESUMEN
The measurement of high-density lipoprotein (HDL) for the purpose of assessing the risk of cardiovascular disease in the individual subject was evaluated. Three laboratory methods were compared, two electrophoretic and one heparin-manganese precipitation, and the HDL results were expressed both as a percent and as an absolute concentration. In phase 1 of the study, the optimal method and the best cut point were identified. In phase 2, these were applied to a larger population who were assigned, on the basis of clinical criteria, to a coronary heart disease and to a control group. The overall probability of correct classification of an individual by his HDL result was calculated. When HDL was expressed as a percent and determined by gel electrophoresis, 82.6% of control subjects and 83.0% of patients with coronary heart disease were classified correctly using the optimal cut point of 23.5%.
Asunto(s)
Enfermedades Cardiovasculares/sangre , Enfermedad Coronaria/sangre , Lipoproteínas HDL/sangre , Adulto , Anciano , Precipitación Química , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Persona de Mediana Edad , Riesgo , Estadística como AsuntoRESUMEN
Measurement of fibrin degradation products (FDP) was evaluated in 152 consecutive patients referred because diagnosis of deep vein thrombosis (DVT) was suspected. All patients underwent impedance plethysmography (IPG), and venograms were obtained in 59. Sensitivity and specificity of FDP measurement, using venography as the standard, was 88% and 66%, respectively. Sensitivity and specificity of IPG was 77% and 79%, respectively. Specificity of the two tests combined, when both yielded abnormal results, was 93%. Patients were classified as (1) normal, (2) abnormal, or (3) equivocal on the basis of IPG. A significant difference in mean FDP concentration was found between groups 1 and 2 and groups 2 and 3. An overall correlation was found between degree of abnormality on IPG and FDP concentration. A quantitative determination of FDP is a practical and reliable screening test for DVT and, when used in conjunction with IPG, improves both sensitivity and specificity of the latter.
Asunto(s)
Productos de Degradación de Fibrina-Fibrinógeno/análisis , Pletismografía de Impedancia , Tromboflebitis/diagnóstico , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , FlebografíaRESUMEN
Two hours before surgery and every 12 hours thereafter 5,000 units of heparin sodium was administered subcutaneously to 100 general surgical patients. Hemostasis was evaluated by a template bleeding time and an activated partial thromboplastin time (PTT). The latter was sensitive to 0.05 units/ml of heparin and gave a straight-line response up to 0.2 units/ml. In the great majority of patients, only a modest elevation of the PTT occurred two and four hours after heparin therapy. However, in 10% to 15% the PTT was prolonged two times or more and in a similar number, PTT after surgery was shorter than baseline values despite heparin. No correlation between PTT prolongation and weight, ponderal index, age, or sex was found. Significant bleeding occurrred in three patients, two from the group of hyperresponders to heparin. Recent aspirin ingestion was implicated in one patient and our evidence indicates that low-dose heparin potentiates aspirin-induced prolongation of bleeding time in certain individuals. Local hematoma formation and discomfort from the injections was not a problem.
Asunto(s)
Hemostasis Quirúrgica , Heparina/administración & dosificación , Complicaciones Posoperatorias/sangre , Adulto , Anciano , Aspirina/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Humanos , Inyecciones Subcutáneas , Masculino , Persona de Mediana Edad , Tromboplastina/análisisRESUMEN
The contact-dependent intrinsic pathway of fibrinolysis involving factor XII, prekallikrein (PK) and pro-urokinase (pro-UK) remains poorly understood. Casein autography of washed, intact platelets revealed both PK and pro-UK. Accordingly, platelets may mediate physiological thrombolysis by this pathway since factor XIIa activates PK and kallikrein activates pro-UK. Acid washing dissociated PK but not pro-UK from platelets. Exogenous pro-UK was specifically incorporated by platelets from the ambient fluid and similarly could not be dissociated from intact platelets. Therefore, platelets may also mediate an effect from therapeutically administered pro-UK by prolonging its half-life.
Asunto(s)
Plaquetas/enzimología , Fibrinólisis , Precalicreína/metabolismo , Terapia Trombolítica , Activador de Plasminógeno de Tipo Uroquinasa/sangre , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Factor XIIa/farmacología , Humanos , Concentración de Iones de Hidrógeno , Calicreínas/farmacología , Precalicreína/análisis , Proteínas Recombinantes/sangre , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
Factor XII has long been implicated in the intrinsic pathway of fibrinolysis, but the mechanism by which it triggers plasminogen activation and targets fibrinolysis has not been established. In the present study, the assembly and function of activated Factor XII (F.XIIa), prourokinase (pro-u-PA), high molecular weight kininogen (H-kininogen), and prekallikrein on human umbilical vein endothelial cells (HUVEC) was investigated. 125I-prekallikrein was shown to bind to HUVEC via receptor-bound H-kininogen in the presence of 50 microM ZnCl2. After the addition of F.XIIa, 78% of the 125I-prekallikrein initially bound to HUVEC was converted to 125I-kallikrein. However, only 6% of the HUVEC-bound 125I-pro-u-PA was thereby activated. This discrepancy was shown to be related to rapid dissociation (> 50% within 15 min) of prekallikrein/kallikrein, but not pro-u-PA, from HUVEC. Increasing the level of cell-bound kallikrein increased the portion of cell-bound pro-u-PA activated, indicating that their co-localization was important for this pathway. Finally, F.XIIa was shown to trigger plasminogen activation on HUVEC via this pathway. This assembly of reactants on the endothelium suggests a mechanism whereby local fibrinolysis may be triggered by blood coagulation.
Asunto(s)
Endotelio Vascular/metabolismo , Precursores Enzimáticos/metabolismo , Fibrinólisis/fisiología , Coagulación Sanguínea/fisiología , Células Cultivadas , Endotelio Vascular/citología , Activación Enzimática , Factor XII/metabolismo , Humanos , Quininógenos/metabolismo , Plasminógeno/metabolismo , Precalicreína/metabolismo , Proteínas Recombinantes/metabolismo , Venas Umbilicales , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Fibrin formation from fibrin monomer (FM) complexes was studied in experimental animals utilizing a previously described technique for quantitating fibrin deposition. A uniform thrombin infusion was used to produce FM, fibrinolysis being inhibited by EACA. In vivo complex formation between FM and 125I-fibrinogen was demonstrated chromatographically. A direct correlation was found between blood fibrinogen concentration and fibrin deposition in organs. By contrast, an inverse correlation between fibrinogen concentration and both enzymatic or non-enzymatic fibrin formation was found in vitro. The mechanism by which fibrinogen potentiates FM precipitation in vivo could not be explained by coprecipitation of fibrinogen in the complex which could not be demonstrated. The inhibitory effect of HN2 on fibrin deposition despite the associated hyperfibrinogemia induced by this drug is believed to underscore the importance of leukocytes in certain types of fibrin deposition. A correlation between the leukocyte count and fibrin formation from FM was also found. It was concluded that the risk of intravascular fibrin deposition is increased by a raised fibrinogen level especially when accompanied by leukocytosis.
Asunto(s)
Trastornos de la Coagulación Sanguínea/sangre , Fibrina/metabolismo , Fibrinógeno/análisis , Recuento de Leucocitos , Animales , Vasos Sanguíneos/metabolismo , Endotoxinas/farmacología , Mecloretamina/farmacología , Conejos , Trombina/farmacologíaRESUMEN
Intravascular coagulation was induced by two appropriately spaced doses of endotoxin and by infusion of thromboplastin. The resulting fibrin deposition was measured by a previously described quantitative technique. Evidence of thrombin elaboration was obtained indirectly by measurement of fibrin monomer (FM) and by the detection and isolation of a thrombin-induced anticlotting activity. Venous segments were isolated at intervals and examined for thrombus formation following 40 minutes of stasis. Endotoxin triggered thrombin elaboration was not detectable in the circulation for at least one hour and was not accompanied by any thrombosis in isolated venous segments. No thrombin elaboration was found in leukopenic rabbits given endotoxin. In the thromboplastin infused animals, the quantity of fibrin deposited in the organs was comparable to that found after endotoxin. However, thrombin was found in the blood immediately and was associated with thrombosis in the isolatet venous segments. Less thrombin-induced anticoagulant activity was found after thromboplastin than after endotoxin. The findings suggest that endotoxin-induced intravascular coagulation is probably not caused by a mechanism of systemic hypercoagulability due to the release of thromboplastic material into the blood stream. A focal process of thrombin elaboration involving leukocytes is postulated. The study is believed relevant to patients with disseminated intravascular coagulation in whom venous thromboembolism is rarely found despite evidence of extensive microvascular fibrin deposition.
Asunto(s)
Trastornos de la Coagulación Sanguínea/sangre , Endotoxinas , Fibrina/metabolismo , Leucocitos/fisiología , Trombina/metabolismo , Animales , Trastornos de la Coagulación Sanguínea/inducido químicamente , Vasos Sanguíneos/metabolismo , Coagulación Intravascular Diseminada/sangre , Fibrinógeno/metabolismo , Leucopenia/sangre , Mecloretamina , Conejos , TromboplastinaRESUMEN
A re-examination of the affinity of pro-urokinase (pro-UK), HMW and LMW-urokinase (UK) to fibrin/Celite was undertaken in order to explain how the chance purification of pro-UK from freshly voided urine by fibrin/Celite affinity chromatography may be reconciled with the subsequent observations that pro-UK failed to bind significantly to fibrin clots in plasma. A significant pH dependence of pro-UK binding to fibrin/Celite was found. Substantial binding of pro-UK (native or recombinant from E. coli), but not of the two-chains forms, was seen at about pH 6.5, which is in the normal pH range of pooled, freshly voided urine. By contrast, at pH 7.4 fibrin binding of pro-UK was much reduced, though it was still significantly greater than that of HMW or LMW-UK. This finding helps to explain the fibrin-binding of pro-UK in freshly voided urine but not in blood. In order to determine if this pH dependence was the sole explanation for why pro-UK could not be isolated by this method from stored urine, the stability of pro-UK in urine was evaluated by incubating 125I-labeled pro-UK in urine. Incubation for up to 4 days (37 degrees C) was not accompanied by any degradation of the single-chain pro-UK as evidenced by autoradiography under reducing conditions. It was concluded that the alkaline shift in pH which occurs in urine left standing, rather than the degradation of pro-UK, explained why freshly voided urine was found to be essential. Clot lysis studies at pH 6.5 and 7.4 showed no promotion of fibrinolysis at the pH which favored fibrin/Celite binding. Therefore, while the present study defines the conditions under which pro-UK may be purified from urine by fibrin/Celite chromatography, it provides no evidence that this binding phenomenon plays any role in fibrinolysis.
Asunto(s)
Tierra de Diatomeas/metabolismo , Fibrina/metabolismo , Activadores Plasminogénicos/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Estabilidad de Enzimas , Humanos , Concentración de Iones de Hidrógeno , Activadores Plasminogénicos/orina , Unión Proteica , Activador de Plasminógeno de Tipo Uroquinasa/orinaRESUMEN
We previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-)(302)pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (approximately 2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK. These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.
Asunto(s)
Precursores Enzimáticos/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Catálisis , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Escherichia coli/genética , Fibrinolisina/farmacología , Fibrinólisis , Glicosilación , Humanos , Técnicas In Vitro , Mutagénesis Sitio-Dirigida , Plasminógeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Clots formed from platelet rich plasma were found to be lysed more readily by low concentrations of pro-urokinase (pro-UK) than clots formed from platelet poor plasma. This was not a non-specific effect since the reverse occurred with tissue plasminogen activator. A mechanical explanation due to platelet-mediated clot retraction was excluded by experiments in which retraction was inhibited with cytochalasin B. Therefore, a platelet-mediated enzymatic mechanism was postulated to explain the promotion of fibrinolysis. Casein autography of isolated platelets revealed a approximately 90 kDa band of activity which comigrated with plasma prekallikrein (PK)/kallikrein, a known activator of pro-UK. Furthermore, treatment of platelets with plasma PK activator (PPA), consisting essentially of factor XIIa, induced activation of pro-UK and of chromogenic substrate for kallikrein (S-2302). This activity corresponded to approximately 40-200 pM kallikrein per 10(8) washed and gel filtered platelets per ml. The activation of pro-UK by PPA-pretreated platelets was dose-dependent and inhibited by soybean trypsin inhibitor but not by bdellin, a specific inhibitor of plasmin, nor by the corn inhibitor of factor XIIa. Kinetic analysis of pro-UK activation by kallikrein showed promotion of the reaction by platelets. The KM of the reaction was reduced by platelets by approximately 7-fold, while the kcat was essentially unchanged. In conclusion, PK was shown to be tightly associated with platelets where it can be activated by factor XIIa during clotting. The activation of pro-UK by platelet-bound kallikrein provides an explanation for the observed platelet mediated promotion of pro-UK-induced clot lysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Plaquetas/fisiología , Factor XIIa/fisiología , Fibrinólisis/efectos de los fármacos , Precalicreína/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Adulto , Plaquetas/química , Compuestos Cromogénicos , Retracción del Coagulo , Activación Enzimática/efectos de los fármacos , Fibrinólisis/fisiología , Humanos , Plasminógeno/farmacología , Precalicreína/análisis , Proteínas Recombinantes/farmacología , Estimulación Química , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
Safety and efficacy of the thrombolytic agent pro-urokinase (pro-UK) in the treatment of deep vein thrombosis of the lower limbs (DVT) have been investigated in an open, uncontrolled, pilot study. Fifteen patients were infused with 800.000 IU (5 mg)/h of pro-UK over 24 h (120 mg), together with unfractionated heparin adjusted to maintain the activated partial thromboplastin time between 1.5 and 2.5 times the basal value. Efficacy was assessed comparing venographic changes in the 11 evaluable limbs before and after pro-UK infusion. The Marder score decreased from a median pre-thrombolysis value of 28 (range 4-40) to 16 (3-38) (p < 0.05). One major hemorrhagic event (retroperitoneal bleeding 4 days after the end of the pro-UK infusion) occurred. Fibrinogen, alpha 2-antiplasmin and plasminogen significantly decreased from baseline values after 12 and 24 h, fibrin(ogen) degradation products significantly increased. Changes in hemostasis parameters were unrelated to thrombolytic efficacy. The results of this pilot study indicate that pro-UK is thrombolytic in DVT and that it can be administered simultaneously with conventional heparin treatment.