RESUMO
Thrombolysis with tissue plasminogen activator (tPA) has been a disappointment and has now been replaced by an endovascular procedure whenever possible.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.
Assuntos
Fibrina/metabolismo , Fibrinólise/efeitos dos fármacos , Ativadores de Plasminogênio/farmacologia , Sinergismo Farmacológico , Produtos de Degradação da Fibrina e do Fibrinogênio , Humanos , Modelos Biológicos , Ativador de Plasminogênio Tecidual/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
MLL-AF4 fusion is the most common consequence of chromosomal translocations in infant leukaemia and is associated with a poor prognosis. MLL-AF4 is thought to be required in haematopoietic stem cells to elicit leukaemia and may be involved in tumour phenotype specification as it is only found in B-cell tumours in humans. We have employed the invertor conditional technology to create a model of MLL-AF4, in which a floxed AF4 cDNA was knocked into Mll in the opposite orientation for transcription. Cell-specific Cre expression was used to generate Mll-AF4 expression. The mice develop exclusively B-cell lineage neoplasias, whether the Cre gene was controlled by B- or T-cell promoters, but of a more mature phenotype than normally observed in childhood leukaemia. These findings show that the MLL-AF4 fusion protein does not have a mandatory role in multi-potent haematopoietic stem cells to cause cancer and indicates that MLL-AF4 has an instructive function in the phenotype of the tumour.
Assuntos
Linfócitos B/patologia , Linhagem da Célula , Transformação Celular Neoplásica , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Animais , Linfócitos B/metabolismo , Feminino , Genes Letais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Integrases/metabolismo , Linfoma de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Fenótipo , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
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.
Assuntos
Ativadores de Plasminogênio/farmacologia , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Carboxipeptidase B , Carboxipeptidases/farmacologia , Sinergismo Farmacológico , Fibrinolisina/farmacologia , Fibrinólise , Humanos , Lisina/sangue , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
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.
Assuntos
Fibrinolisina/farmacologia , Fibrinólise , Lisina , Ativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Resistência a Medicamentos , Fibrinólise/efeitos dos fármacos , Humanos , Metionina , Dados de Sequência Molecular , Mutação , Ativadores de Plasminogênio/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/genética , ValinaRESUMO
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.
Assuntos
Precursores Enzimáticos/metabolismo , Fibrina/metabolismo , Fibrinólise , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Cães , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/isolamento & purificação , Fibrinogênio/análise , Humanos , Cinética , Peso Molecular , Oxirredução , Coelhos , Especificidade da Espécie , Ativador de Plasminogênio Tipo Uroquinase/isolamento & purificaçãoRESUMO
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.
Assuntos
Proteínas Inativadoras do Complemento 1/farmacologia , Fibrina/metabolismo , Plasminogênio/efeitos dos fármacos , Serpinas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteína Inibidora do Complemento C1 , Eletroforese em Gel de Poliacrilamida , Fibrinólise , Humanos , Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
The MTG8 (ETO) locus is involved in a reciprocal exchange with runx1 in the t(8;21) of acute myeloid leukemia. It is a member of a small gene family encoding transcriptional regulators that interact with corepressors and histone deacetylase. However, the physiologic cellular processes controlled by MTG8 are not known. In order to gain an insight into the latter, we have generated mutant mice with an insertional inactivation at the locus, which disrupts transcription of exon 2. The postnatal viability of homozygous mutants was greatly reduced. In approximately 25% the midgut was missing, whereas practically all pups surviving past the first 2 days showed severe growth impairment, which was likely due to a gross disruption of the gut architecture. The latter phenotype could be traced back to late embryonic development. No difference in gut cell differentiation or proliferation was found compared to wild-type littermates. Levels of factors known to be involved in gut morphogenesis were also unchanged. MTG8 is expressed in the outermost layers of the developing gut from at least E9.5. Thus, MTG8 plays a novel, essential role in the gastrointestinal system.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Fatores de Transcrição/fisiologia , Animais , Fenômenos Fisiológicos do Sistema Digestório , Regulação da Expressão Gênica/fisiologia , Marcação de Genes , Camundongos , Especificidade de Órgãos , Proteínas Proto-Oncogênicas/fisiologia , Proteína 1 Parceira de Translocação de RUNX1RESUMO
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.
Assuntos
Hemorragia/induzido quimicamente , Hemostasia/efeitos dos fármacos , Mutação , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/administração & dosagem , Ativador de Plasminogênio Tipo Uroquinase/administração & dosagem , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Modelos Animais de Doenças , Cães , Estabilidade Enzimática/genética , Artéria Femoral , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Terapia Trombolítica/efeitos adversos , Trombose/tratamento farmacológicoRESUMO
The affinity maturation of antibodies is driven by somatic hypermutation which is localized to specific segments of the coding genes. The information available on this process derives from studies in vivo. With the intention of developing new approaches, we have constructed a fusion gene between a kappa chain and a selectable neomycin resistance gene, neor. The neor gene, which includes the SV40 small t intron and polyadenylation site, but not the upstream elements nor its first 12 amino acids, is an in-frame substitution of the FR2-CDR3 fragment of a rearranged V kappa OX1-J kappa 5 gene. Expression of neor activity is therefore dependent on the upstream immunoglobulin sequence. A stop codon was placed in the CDR1 region so that only mutants survive treatment with geneticin sulphate (G418). The effectiveness of the system was tested by transfecting the NS0 myeloma cell line and isolating spontaneous mutants. Neomycin-resistant clones arose at an estimated rate of 1 x 10(-8)/cell division, and over 90% were authentic structural mutants. Unlike the somatic hypermutations, the majority arose by in-frame deletions including the stop codon, although up to 30% involved a point mutation. The reporter gene was then modified by substituting all the sequences downstream of the J kappa 5 with others known to be required for full hypermutation in vivo. Different cell lines were transfected and G418-resistant clones analyzed. No significant increase in the rate of reversion or in the generation of point mutations versus deletions was detected, even using conditioned culture medium. In the presence of azacytidine however, a mutant involving multiple events (single base addition and deletion plus two point mutations) was detected. The reporter gene system therefore seems suitable to test culture conditions and modifications of the host cells aimed at the derivation of an in vitro assay of somatic hypermutation.
Assuntos
Genes de Imunoglobulinas , Genes Reporter , Mutação , Sequência de Aminoácidos , Animais , Linfócitos B , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Análise Mutacional de DNA , DNA Recombinante/genética , Resistência Microbiana a Medicamentos/genética , Rearranjo Gênico de Cadeia Leve de Linfócito B , Camundongos , Dados de Sequência Molecular , Neomicina/farmacologia , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
To determine environmental requirements for erythroid burst formation in primary culture, we added human bone marrow cells to serum-depleted methylcellulose, agar, or fibrin clot cultures. Optimal BFU-E proliferation was present in cultures prepared with Iscove's modified Dulbecco medium, 248 micrograms/ml transferrin, 1.63 micrograms/ml ferric chloride, 117 micrograms/ml bovine serum albumin, and each of seven preparations of erythropoietin. Burst number was comparable to that in serum containing culture. Reducing sodium dodecyl sulfate electrophoresis of commercial albumin preparations showed them to contain abundant lipoproteins. Results of experiments with human plasma albumin found to be greater than 98% pure by one- and two-dimensional gel electrophoresis and delipidated albumin indicate that an albumin source is needed for burst formation to occur. Together with albumin, exogenous phosphatidylcholine but not phosphatidylserine augmented burst number. Bursts routinely appeared in serum-depleted culture without added burst-promoting activity (BPA). However, leukocyte-conditioned medium (LCM) and its high-speed supernatant and pellet fractions enhanced burst formation. Antimembrane IgG capable of neutralizing BPA reduced burst number to a level below that achieved in LCM-depleted culture, suggesting that endogenous BPA was inactivated. We conclude that human marrow BFU-E proliferation requires iron-saturated transferrin, albumin, and erythropoietin. Exogenous BPA and phospholipids enhance but are not essential for burst formation to proceed in primary culture.
Assuntos
Células da Medula Óssea , Células-Tronco Hematopoéticas/citologia , Sangue , Divisão Celular , Células Cultivadas , Meios de Cultura , Eritropoese , Eritropoetina/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Cinética , Leucócitos/citologia , Fosfolipídeos/isolamento & purificação , Albumina Sérica/isolamento & purificação , Albumina Sérica/fisiologiaRESUMO
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.
Assuntos
Plaquetas/enzimologia , Fibrinólise , Pré-Calicreína/metabolismo , Terapia Trombolítica , Ativador de Plasminogênio Tipo Uroquinase/sangue , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Fator XIIa/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Calicreínas/farmacologia , Pré-Calicreína/análise , Proteínas Recombinantes/sangue , Ativador de Plasminogênio Tecidual/farmacologiaRESUMO
A simple roller bottle was constructed to house three dialysis tubes, each with a capacity of 75 ml. Cells were grown inside the dialysis tubing, which was immersed in ordinary DMEM medium without serum supplement. Cultures of hybridomas at medium or low density (2 X 10(5) cells/ml) could be expanded directly in the dialysis tubes to attain a high cell density of the order of 10(7)/ml. Continuous gentle stirring of the cells was possible, since the design causes a bubble to oscillate along the length of each tube. The six cell lines tested all gave antibody concentrations of between 1.1 and 2.3 mg/ml at 20 days. Such an in vitro apparatus obviates the need to employ ascites production, because it is as simple or simpler than the injection of mice, and the in vitro product is very rich in antibody, whilst containing low amounts of contaminating proteins.
Assuntos
Anticorpos Monoclonais/biossíntese , Células Cultivadas , Hibridomas , Ascite/imunologia , Divisão Celular , Meios de Cultura Livres de Soro , Equipamentos e Provisões , Imunoglobulina G/análiseRESUMO
A procedure is described which permits high-yield direct cloning of newly established hybridomas on STO fibroblast feeders in soft agarose. Several thousand independent clones are typically obtained from each fusion (1 X 10(8) spleen cells). These are screened using a colony replica assay in which secreted antibodies diffuse through an agar overlay and bind to antigen immobilised on nitrocellulose. Bound antibodies are then detected with enzyme-labelled second antibody. The procedure is fast and efficient and permits the isolation and selection of antigen-specific clones in less than 2 weeks from fusion. It has been successfully employed for the derivation and selection of high-affinity anti-hapten antibodies. Other potential applications of the assay are in the detection of non-immobilised antigens by an indirect method using anti-globulin on nitrocellulose, in the generation of bispecific antibodies and the selection and characterisation of antibody specificities generated by the expression of antibody fragments in bacteria or yeasts.
Assuntos
Anticorpos Monoclonais/biossíntese , Hibridomas/imunologia , Animais , Células Clonais , Ensaio de Imunoadsorção Enzimática , CamundongosRESUMO
An anti-peroxidase-anti-biotin hybrid hybridoma rat cell line, capable of producing a bispecific monoclonal antibody, has been derived to explore its use in conjunction with a luminol immunodetection system. Luminescence was detected using x-ray film. The method was sufficiently sensitive and effective, but was less sensitive than autoradiographic methods using high-specific-activity 32P-labeled probes. Exposure times, on the other hand, were of the order of seconds rather than days. The direct binding of both peroxidase and biotin by the bispecific monoclonal antibody is simpler but less sensitive than the more conventional indirect method using a commercial peroxidase coupled with anti-rat antibody as a developing antibody.
Assuntos
Técnicas Imunológicas , Medições Luminescentes , Animais , Anticorpos Monoclonais , Autorradiografia , Biotina/imunologia , Histocitoquímica , Luminol , Naftóis , Hibridização de Ácido Nucleico , RatosRESUMO
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.
Assuntos
Endotélio Vascular/metabolismo , Precursores Enzimáticos/metabolismo , Fibrinólise/fisiologia , Coagulação Sanguínea/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Ativação Enzimática , Fator XII/metabolismo , Humanos , Cininogênios/metabolismo , Plasminogênio/metabolismo , Pré-Calicreína/metabolismo , Proteínas Recombinantes/metabolismo , Veias Umbilicais , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
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.
Assuntos
Terra de Diatomáceas/metabolismo , Fibrina/metabolismo , Ativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Estabilidade Enzimática , Humanos , Concentração de Íons de Hidrogênio , Ativadores de Plasminogênio/urina , Ligação Proteica , Ativador de Plasminogênio Tipo Uroquinase/urinaRESUMO
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.
Assuntos
Precursores Enzimáticos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Catálise , Ativação Enzimática/efeitos dos fármacos , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Escherichia coli/genética , Fibrinolisina/farmacologia , Fibrinólise , Glicosilação , Humanos , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Plasminogênio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
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)
Assuntos
Plaquetas/fisiologia , Fator XIIa/fisiologia , Fibrinólise/efeitos dos fármacos , Pré-Calicreína/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Adulto , Plaquetas/química , Compostos Cromogênicos , Retração do Coágulo , Ativação Enzimática/efeitos dos fármacos , Fibrinólise/fisiologia , Humanos , Plasminogênio/farmacologia , Pré-Calicreína/análise , Proteínas Recombinantes/farmacologia , Estimulação Química , Ativador de Plasminogênio Tecidual/farmacologiaRESUMO
Endothelial cells (ECs) in culture synthesize and secrete urokinase-type plasminogen activator (u-PA), but the normal vascular endothelium is believed to synthesize only tissue plasminogen activator (t-PA), which is thought to be responsible for intravascular fibrinolysis. More recently, animal studies have shown that the biological role of u-PA in fibrinolysis has been underestimated, prompting a re-examination of its synthesis by the endothelium. In this study, we investigated whether u-PA was synthesized by non-atherosclerotic endothelial cells in vivo by testing ECs dislodged by venipuncture from 12 normal volunteers and 17 patients admitted for plasmapheresis. The ECs were isolated with an anti-endothelial monoclonal antibody coupled to immunomagnetic beads and characterized by morphology and by labelling for vWF, CD31, and UEA-1 binding. U-PA antigen was found in 50% of the ECs from the normal subjects and in 60% of those from patients. U-PA enzymatic activity on zymograms was detected in 50% of the normal samples and 60% of the patient samples, with the latter being more frequently and more strongly positive. U-PA mRNA was found in all the normal and patient samples tested. The results indicate that u-PA is synthesized by the venous endothelium in vivo but that its expression is highly variable.
Assuntos
Endotélio Vascular/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Adulto , Biomarcadores , Células Cultivadas , Endotélio Vascular/citologia , Indução Enzimática , Feminino , Fibrinólise , Humanos , Separação Imunomagnética , Hibridização In Situ , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , RNA Mensageiro/análise , Ativador de Plasminogênio Tipo Uroquinase/genética , VeiasRESUMO
Adhesion of platelets to endothelium has been shown to induce important changes in endothelial properties. In this study, we examined the effect of platelet-endothelial cell interactions on the expression of urokinase-type plasminogen activator (u-PA) by human microvascular endothelial cells. After incubation of endothelial cells with platelets, a dose-dependent increase in the expression of u-PA Ag was observed and reached a plateau for a ratio of 300 platelets per endothelial cells. The u-PA Ag upregulation resulted from an increase in u-PA mRNA that originated from a synthesis by endothelial cells since no u-PA mRNA was detected in platelets. The platelet-induced u-PA synthesis was inhibited when the endothelial cells were pre-treated with phospholipase C to remove the u-PA receptor, or when the platelets were incubated with an antibody that blocks the binding of u-PA to u-PAR. Taken together, these data indicate that u-PA present on the platelet surface interacts with u-PAR on the endothelial cells and induces the u-PA synthesis. This mechanism may represent a physiological control of platelet-mediated intravascular fibrin deposition.