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1.
Cell Chem Biol ; 28(12): 1728-1739.e5, 2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34352225

RESUMO

Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.


Assuntos
Antifibrinolíticos/farmacologia , Antitrombinas/farmacologia , Inativadores de Plasminogênio/farmacologia , Inibidores de Serina Proteinase/farmacologia , Trombose Venosa/tratamento farmacológico , Animais , Antifibrinolíticos/química , Antitrombinas/química , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Hidrolases/metabolismo , Inativadores de Plasminogênio/química , Inibidores de Serina Proteinase/química , Trombose Venosa/metabolismo
2.
Chem Commun (Camb) ; 51(75): 14263-6, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26264862

RESUMO

The concept of enzyme immobilization via an inhibitor-derived peptide was developed. This method of immobilization was shown to be advantageous over physical adsorption and covalent bonding in retaining the enzymatic activity. Moreover, the surface-immobilized enzyme exhibited resistance against its inhibitor due to the occupation of an inhibitor binding site on the enzyme.


Assuntos
Enzimas Imobilizadas/química , Inativadores de Plasminogênio/química , Ativador de Plasminogênio Tecidual/química , Sítios de Ligação , Enzimas Imobilizadas/metabolismo , Ligantes , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Propriedades de Superfície , Ativador de Plasminogênio Tecidual/metabolismo
3.
Bioorg Khim ; 41(5): 565-78, 2015.
Artigo em Russo | MEDLINE | ID: mdl-26762094

RESUMO

Comparative in vitro study of the kinetics of various reactions involved in the process of thrombolysis initiated by streptokinase (SK) and staphylokinase (STA) was carried out. It was shown that at the interaction of an equimolar ratio of plasminogen (Pg) with SK or STA the rate of formation and the specific esterase activity of the complex plasmin (Pm) · SK are higher than those of the complex Pm · STA. The catalytic efficiency (kcat/Km) of hydrolysis of the chromogenic plasmin substrates by Pm · SK complex was 2 times higher than by Pm · STA complex. In the absence of fibrin catalytic efficiency (kPg/K(Pg)) of activation of Glu-plasminogen and Lys-plasminogen glycoform II by Pm · SK complex was higher than by Pm · STA complex, but the pres- ence of fibrin increased kPg/K(Pg)) activation of both plasminogens by Pm · STA complex significantly stronger than by Pm · SK complex due to the decrease in K(Pg)). In contrast to STA (15.5 kDa), SK molecule (47 kDa) creates significant steric hindrances for the interaction of plasmin in Pm · SK complex with protein inhibi- tors. In addition, SK caused greater fibrinogen degradation than STA. It is shown that Pm · SK and Pm · STA complexes lyse fibrin clots in buffer with similar rates, while the rate of lysis of plasma clots, immersed in plas- ma, by Pm · STA complex are significantly higher than those by Pm · SK complex. It was revealed that the species specificity of STA and S K is determined mainly by the rate of formation and the efficiency of Pm · SK and Pm · STA complexes in the activation of autologous plasminogen. The lysis efficiency of plasma clots of mammals fell in the series: human > dog > rabbit for SK and the dog > human > rabbit for STA. The results show that in the purified system SK is a more effective activator of plasminogen than STA. In the system con- taining fibrin and α2-AP, the activator and fibrinolytic activities of STA are higher than those of SK, due to the increased stability in plasma and fibrin specificity of STA, the fast reaction of the complex Pm · STA with α2AP and the ability of the STA to recyclization in the presence of α2AP.


Assuntos
Fibrina/química , Fibrinólise , Metaloendopeptidases/química , Ativadores de Plasminogênio/química , Inativadores de Plasminogênio/química , Plasminogênio/química , Estreptoquinase/química , Animais , Cães , Humanos , Cinética , Metaloendopeptidases/genética , Ligação Proteica , Coelhos , Proteínas Recombinantes/química , Especificidade da Espécie , Especificidade por Substrato
4.
Cell Mol Life Sci ; 68(5): 785-801, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21136135

RESUMO

The plasmin-antiplasmin system plays a key role in blood coagulation and fibrinolysis. Plasmin and α(2)-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into soluble fragments. However, besides plasmin(ogen) and α(2)-antiplasmin the system contains a series of specific activators and inhibitors. The main physiological activators of plasminogen are tissue-type plasminogen activator, which is mainly involved in the dissolution of the fibrin polymers by plasmin, and urokinase-type plasminogen activator, which is primarily responsible for the generation of plasmin activity in the intercellular space. Both activators are multidomain serine proteases. Besides the main physiological inhibitor α(2)-antiplasmin, the plasmin-antiplasmin system is also regulated by the general protease inhibitor α(2)-macroglobulin, a member of the protease inhibitor I39 family. The activity of the plasminogen activators is primarily regulated by the plasminogen activator inhibitors 1 and 2, members of the serine protease inhibitor superfamily.


Assuntos
Antifibrinolíticos/metabolismo , Plasminogênio/fisiologia , Antifibrinolíticos/química , Sítios de Ligação , Coagulação Sanguínea/fisiologia , Fibrinólise/fisiologia , Humanos , Modelos Biológicos , Modelos Moleculares , Plasminogênio/química , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/fisiologia , Inativadores de Plasminogênio/química , Inativadores de Plasminogênio/fisiologia , Estrutura Terciária de Proteína , Serina Proteases/química , Serina Proteases/fisiologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/fisiologia , alfa-Macroglobulinas/química , alfa-Macroglobulinas/fisiologia
5.
Bioorg Med Chem Lett ; 20(3): 966-70, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20056540

RESUMO

Inactivators of plasminogen activator inhibitor-1 (PAI-1) have been identified as possible treatments for a range of conditions, including atherosclerosis, venous thrombosis, and obesity. We describe the synthesis and inhibitory activity of a novel series of compounds based on bis-arylsulfonamide and aryl sulfonimide motifs that show potent and specific activity towards PAI-1. Inhibitors containing short linking units between the sulfonyl moieties and a 3,4-dihydroxy aryl substitution pattern showed the most potent inhibitory activity, and retained high specificity for PAI-1 over the structurally-related serpin anti-thrombin III (ATIII).


Assuntos
Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inativadores de Plasminogênio/química , Inativadores de Plasminogênio/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo , Humanos
6.
Bioorg Med Chem Lett ; 15(1): 93-8, 2005 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-15582418

RESUMO

A series of non-amide-linked 6-substituted-2-naphthamidine urokinase plasminogen activator (uPA) inhibitors are described. These compounds possess excellent binding activities and selectivities with significantly improved pharmacokinetic profiles versus previously described amide-linked inhibitors.


Assuntos
Naftalenos/farmacocinética , Inativadores de Plasminogênio/farmacocinética , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Modelos Moleculares , Naftalenos/química , Inativadores de Plasminogênio/química , Especificidade por Substrato
7.
Bioorg Med Chem ; 12(10): 2797-805, 2004 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15110861

RESUMO

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) was performed on a series of indole/benzoimidazole-5-carboxamidines as urokinase plasminogen activator (uPA) inhibitors. The ligand molecular superimposition on template structure was performed by atom/shape-based RMS fit, multifit, and RMSD fit methods. The removal of two outliers from the initial training set of 30 molecules improved the predictivity of the models. The statistically significant model was established from 28 molecules, which were validated by evaluation of test set of nine compounds. The atom-based RMS alignment yielded best predictive CoMFA model (r2(cv) = 0.611, r2(cnv) = 0.778, F value = 43.825, r2(bs) = 0.842, r2(pred) = 0.616 with two components) while the CoMSIA model yielded (r2(cv) = 0.499, r2(cnv) = 0.976, F value=96.36, r2(bs) = 0.993, r2(pred) = 0.694 with eight components). The contour maps obtained from 3D-QSAR studies were appraised for the activity trends of the molecules analyzed. The results indicate that the steric, electrostatic, and hydrogen bond donor/acceptor substituents play significant role in uPA activity and selectivity of these compounds. The data generated from the present study can be used as putative pharmacophore in the design of novel, potent, and selective urokinase plasminogen activator inhibitors as cancer therapeutics.


Assuntos
Antineoplásicos/química , Desenho de Fármacos , Inativadores de Plasminogênio/química , Relação Quantitativa Estrutura-Atividade , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Amidinas/química , Antineoplásicos/farmacologia , Benzimidazóis/química , Indóis/química , Conformação Molecular , Inativadores de Plasminogênio/farmacologia
8.
Rinsho Byori ; 42(4): 346-51, 1994 Apr.
Artigo em Japonês | MEDLINE | ID: mdl-8176842

RESUMO

Tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) are the most important factors involved in the regulation of blood fibrinolysis. t-PA converts zymogen plasminogen to plasmin on the surface of endothelial cells to maintain blood fluidity and on the surface of the thrombus to efficiently lyse the thrombus. In circulating plasma, PAI-1 inactivates t-PA by forming an equimolar complex with t-PA to suppress the hyperfibrinolysis of the blood. Both proteins are synthesized by the vascular endothelial cells and secreted from the cells in resting state and in response to several stimuli such as thrombin, endotoxin, cytokines and growth factors. In various diseases such as DIC, thromboembolism and bacterial infection, the plasma concentrations of those two factors vary as a consequence of several stimuli, representing the altered fibrinolytic balance caused by the disease. Measurements of these factors and evaluation of the fibrinolytic balance will be important for determination of the most appropriate method of treatment. Moreover, the high plasma concentration of PAI-1 will be a prognostic marker of the disease and may be a risk factor of thrombotic events. In clinical treatment, t-PA is now widely used as a valuable fibrinolytic agent especially in myocardial infarction.


Assuntos
Endotélio Vascular/fisiologia , Inativadores de Plasminogênio/fisiologia , Ativador de Plasminogênio Tecidual/fisiologia , Endotélio Vascular/citologia , Humanos , Inativadores de Plasminogênio/química , Ativador de Plasminogênio Tecidual/química
9.
Biochimie ; 75(10): 911-5, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-7906150

RESUMO

Amyloid beta protein (beta/A4) is deposited in senile plaques of patients with Alzheimer's disease. This protein is derived from a larger membrane-associated protein, termed amyloid precursor protein (APP). The constitutive processing of APP occurs at the central portion of beta/A4, resulting in the release of large N-terminal peptides. We have purified these peptides from the culture medium of cDNA-transfected COS-1 cells. Some of the isoforms contain the Kunitz-type protease inhibitor (KPI) domain and strongly inhibit trypsin, chymotrypsin and plasmin, but do not inhibit kallikrein, prolyl endopeptidase or granzyme A. The peptides also do not inhibit cysteine proteases such as cathepsin B or calpain. Soluble APPs lacking the KPI domain fail to inhibit any of these proteases. The results indicate that the KPI domain in soluble APPs has protease inhibitory activity against certain serine proteases.


Assuntos
Precursor de Proteína beta-Amiloide/química , Inativadores de Plasminogênio/farmacologia , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/isolamento & purificação , Precursor de Proteína beta-Amiloide/farmacologia , DNA Complementar , Eletroforese , Humanos , Dados de Sequência Molecular , Inativadores de Plasminogênio/química , Inibidores de Serina Proteinase/isolamento & purificação , Solubilidade , Transfecção
10.
J Invest Dermatol ; 99(2): 193-200, 1992 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1629632

RESUMO

Transforming growth factor-beta (TGF beta) is the most potent known inhibitor of keratinocyte growth. Pericellular proteolytic activity is usually high in proliferating and malignant cells and decreased in resting or growth-arrested cells. We have therefore analyzed the effects of TGF beta 1 on the production of plasminogen activator activity by normal human keratinocytes and a mouse keratinocyte cell line under serum-free conditions. The plasminogen activator activity of the culture medium was analyzed using caseinolysis-in-agar and zymography assays, immunoblotting, and Northern hybridization analysis for the plasminogen activators (PA) and PA inhibitor-1 (PAI-1). Alterations of radiolabeled polypeptides were observed in fluorograms of gels. It was found that like in human epidermoid carcinoma cells picomolar concentrations of TGF beta 1 (0.2-20 ng/ml) enhanced total plasminogen activator activity in both keratinocyte cell systems. Zymographic and immunoblotting analyses of the medium indicated that the activator was of the urokinase type (u-PA). Immunoprecipitation and Concanavalin A affinity chromatography of the culture medium indicated that the cells also started to produce PAI-1. Analysis of the pericellular matrix preparations of the keratinocytes showed that PAI-1 is deposited to the pericellular space. Evidently due to elevated u-PA activity PAI-1 was removed from the extracellular matrix more rapidly in TGF beta 1-treated cells than from control cultures. Northern hybridization analysis of human keratinocytes showed that TGF beta 1 rapidly elevated both u-PA and PAI-1 mRNA levels. Comparison of the temporal induction profiles indicated that the mRNA for u-PA increased more slowly but was more persistent than that of PAI-1. Actinomycin D inhibited the induction of both u-PA and PAI-1 mRNA, suggesting that the induction was due to increased transcription. The results suggest that enhanced plasminogen activator activity can be associated with growth inhibition also in nonmalignant cells like cultured human or murine keratinocytes.


Assuntos
Queratinócitos/metabolismo , Ativadores de Plasminogênio/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Células Cultivadas , Dactinomicina/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Masculino , Camundongos , Ativadores de Plasminogênio/genética , Inativadores de Plasminogênio/química , RNA Mensageiro/análise , Ativador de Plasminogênio Tipo Uroquinase/genética
11.
Thromb Haemost ; 68(1): 60-3, 1992 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-1514173

RESUMO

The effects of human recombinant plasminogen activator inhibitor (rPAI-1) on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) were studied in a rabbit model of jugular vein thrombosis. Two functionally distinct rPAI-1 preparations were used in these experiments, including latent rPAI-1 (approximately 2 units of t-PA neutralizing activity per micrograms protein) and reactivated rPAI-1 (approximately 150 units/micrograms). Simultaneous intravenous infusion over 4 h of 1.7 mg/kg of reactivated rPAI-1 (inhibitory capacity approximately 0.5 mg/kg rt-PA) with 0.5 mg/kg of rt-PA completely prevented lysis of a jugular venous thrombus, whereas an equivalent amount of latent PAI-1 did not significantly influence clot lysis. These findings demonstrate that reactivated human rPAI-1 efficiently neutralizes thrombolysis with rt-PA in vivo. Since previous studies have suggested that elevated endogenous levels of PAI-1 do not attenuate the thrombolytic potency of rt-PA in the endotoxin-treated model, we compared the stability of complexes formed by 125I-rt-PA with reactivated human rPAI-1 and with rabbit PAI-1 in vitro. Our findings indicate that both forms of PAI-1 form SDS-stable complexes following incubation with 125I-rt-PA. Thus, it seems likely that elevated levels of active PAI-1 can negate the thrombolytic effects of rt-PA in vivo and argues against the possibility that t-PA can dissociate from PAI-1 and have its activity restored in the presence of a thrombus.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Inativadores de Plasminogênio/farmacologia , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Animais , Radioisótopos do Iodo , Veias Jugulares , Inativadores de Plasminogênio/química , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tecidual/química
12.
J Biol Chem ; 267(17): 11693-6, 1992 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-1601844

RESUMO

Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, is a serine proteinase inhibitor (serpin) that forms a 1:1 stoichiometric complex with its target proteinase leading to the formation of a stable inactive complex. The active, inhibitory form of PAI-1 spontaneously converts to a latent form that can be reactivated by protein denaturants. In the present study we have isolated another molecular form of intact PAI-1 that, in contrast with active PAI-1, does not form stable complexes with t-PA but is cleaved at the P1-P1' bond (Arg346-Met347). Other serine proteinases, e.g. urokinase-type plasminogen activator and thrombin, also cleaved this "substrate" form of PAI-1. Fluorescence spectroscopy revealed conformational differences between the latent, active, and substrate forms of PAI-1. This observation confirms our hypothesis that the three functionally different forms of PAI-1 are the consequence of conformational transitions. Thus PAI-1 may occur in three interconvertible conformations: latent, inhibitor, and substrate PAI-1. The identification of two distinct conformations of PAI-1 which interact with their target protease either as an inhibitor or as a substrate is a previously unrecognized phenomenon among the serpins. Conversion of substrate PAI-1 to its inactive degradation product may constitute a pathway for the physiological regulation of PAI-1 activity.


Assuntos
Inativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Hidrólise , Inativadores de Plasminogênio/química , Inativadores de Plasminogênio/isolamento & purificação , Conformação Proteica , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Especificidade por Substrato
13.
Hum Genet ; 89(3): 265-9, 1992 May.
Artigo em Inglês | MEDLINE | ID: mdl-1318261

RESUMO

The objectives of this study were to determine the genetic basis of the electrophoretic differences of human plasma protein C inhibitors (PCI) from 977 individuals. Three discrete antibodies were produced against the PCI purified from human plasma and peptides that corresponded to the N-terminal 15 amino acid residues and the C-terminal 15 residues of human PCI, the chemical structures of which were determined by cDNA sequence analysis. The combined techniques of polyacrylamide gel isoelectric focusing and immunoblotting with these three different antibodies resolved the plasma PCI into several isoprotein bands, with a pH range of 6-7. These PCI isoproteins, however, were not stained by anti-human kallikrein, anti-human protein C or anti-human urokinase antibodies. Therefore, each of the PCI bands, which were detected by immunoblotting with the anti-PCI antibody and the two different anti-peptide antibodies, were derived from free PCI, and not an inactive PCI species. Two common phenotypes, designated PCI 1 and 1-2, were recognized, and family studies showed that they represented homozygosity or heterozygosity for two autosomal codominant alleles, PCI*1 and PCI*2. A population study of plasma samples collected from 977 Japanese individuals indicated that the frequencies of the PCI*1 and PCI*2 alleles were 0.988 and 0.012, respectively.


Assuntos
Inativadores de Plasminogênio , Polimorfismo Genético/genética , Proteína C/antagonistas & inibidores , Alelos , Sequência de Aminoácidos , Povo Asiático/genética , Feminino , Frequência do Gene , Humanos , Immunoblotting , Focalização Isoelétrica , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Inativadores de Plasminogênio/química , Inativadores de Plasminogênio/imunologia , Inibidor da Proteína C
15.
Nature ; 355(6357): 270-3, 1992 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-1731226

RESUMO

Human plasminogen activator inhibitor-1 (PAI-1) is the fast-acting inhibitor of tissue plasminogen activator and urokinase and is a member of the serpin family of protease inhibitors. Serpins normally form complexes with their target proteases that dissociate very slowly as cleaved species and then fold into a highly stable inactive state in which the residues that flank the scissile bond (P1 and P1';) are separated by about 70 A. PAI-1 also spontaneously folds into a stable inactive state without cleavage; this state is termed 'latent' because inhibitory activity can be restored through denaturation and renaturation. Here we report the structure of intact latent PAI-1 determined by single-crystal X-ray diffraction to 2.6 A resolution. The three-dimensional structure reveals that residues on the N-terminal side of the primary recognition site are inserted as a central strand of the largest beta sheet, in positions similar to the corresponding residues in the cleaved form of the serpin alpha 1-proteinase inhibitor (alpha 1-PI). Residues C-terminal to the recognition site occupy positions on the surface of the molecule distinct from those of the corresponding residues in cleaved serpins or in the intact inactive serpin homologue, ovalbumin, and its cleavage product, plakalbumin. The structure of latent PAI-1 is similar to one formed after cleavage in other serpins, and the stability of both latent PAI-1 and cleaved serpins may be derived from the same structural features.


Assuntos
Inativadores de Plasminogênio/química , Sítios de Ligação , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Difração de Raios X
16.
FEBS Lett ; 295(1-3): 102-6, 1991 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-1765139

RESUMO

Type-1 inhibitor of plasminogen activators (PAI-1) occurs in purified preparations in a latent form that can be activated with denaturants; in vivo, latency is prevented by binding to vitronectin. We have compared latent, denaturant-activated and reactive centre-cleaved human PAI-1 with respect to thermal stability and affinity to monoclonal antibodies. By both criteria, latent and cleaved PAI-1 are very similar or indistinguishable, and clearly different from active PAI-1. Our findings suggest that the conformations of latent and reactive centre-cleaved PAI-1 are similar and resemble the so-called relaxed (R) serpin conformation, while that of active PAI-1 is different and resembles the stressed (S) serpin conformation.


Assuntos
Inativadores de Plasminogênio/química , Anticorpos Monoclonais , Sítios de Ligação , Sítios de Ligação de Anticorpos , Estabilidade de Medicamentos , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G , Modelos Moleculares , Inativadores de Plasminogênio/sangue , Inativadores de Plasminogênio/isolamento & purificação , Conformação Proteica , Termodinâmica
17.
J Invest Dermatol ; 97(5): 803-11, 1991 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1919045

RESUMO

The expression of urokinase-type plasminogen activator (u-PA) and its type-1 inhibitor (PAI-1) was examined in vivo in mouse wounds by in situ hybridization and immunohistochemistry. u-PA mRNA was present in both basal and suprabasal keratinocytes in the regenerative epithelial outgrowths at the edge of the wounds. In the same area, PAI-1 mRNA was only present in the basal keratinocytes. u-PA protein was detected in keratinocytes in several layers of the epithelial outgrowth, whereas PAI-1 protein was confined to the basal keratinocytes and to the area of the basal membrane. The two proteins and their mRNA were not detected in normal epidermis or in normal-looking epidermis adjacent to the wounds. Fibroblast-like cells and fairly large stellate cells (possibly macrophages) in the granulation tissue underneath the wound contained both the two proteins and their mRNA. The large stellate cells, showing a strong hybridization signal for PAI-1 mRNA, were especially abundant at the border between the necrotic wound and the newly formed granulation tissue. The specificity of these results was supported by the use of two different non-overlapping antisense probes, sense mRNA probes, antibody preparations preabsorbed with purified proteins, and Northern analysis of tissue extracts. The localized and regulated expression of u-PA and PAI-1 seen in this study may reflect that plasminogen activation plays a role in the migration of keratinocytes and connective tissue cells during reepithelialization and tissue remodeling in wound healing.


Assuntos
Inativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Cicatrização/fisiologia , Animais , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Hibridização de Ácido Nucleico , Inativadores de Plasminogênio/química , RNA Mensageiro/análise
18.
Blood ; 78(5): 1254-61, 1991 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-1715220

RESUMO

Plasminogen activator inhibitor 1 (PAI-1) is the fast-acting inhibitor of both tissue-type and urokinase-type plasminogen activators (t-PA, u-PA) and is an essential regulatory protein of the fibrinolytic system. In the presence of either the protein vitronectin or the glycosaminoglycan heparin, PAI-1 is also an efficient inhibitor of thrombin. To assess whether these cofactors turn PAI-1 into a general protease inhibitor or whether their influence is restricted to thrombin, the second-order association rate constants between PAI-1 and the human plasma proteases t-PA, u-PA, plasmin, thrombin, Factor Xa (FXa), and Factor XIIa (FXIIa) in the absence and in the presence of either vitronectin or heparin are determined. In addition, the role of the PAI-1 reactive site P3 to P3' residues for the specificity of inhibition was studied by using PAI-1 reactive site mutants. Our results show that: (1) Heparin exclusively increases the rate of inhibition of thrombin by PAI-1, whereas in the presence of heparin the rate of inhibition of the other proteases is not altered; (2) Vitronectin is an obligatory cofactor for the inhibition of thrombin by PAI-1. In addition, vitronectin moderately increases the rate of inhibition by PAI-1 of u-PA and of plasmin, but does not alter the rate of inhibition of t-PA, FXa, or FXIIa; (3) Apart from the important role of the P1 residue, no consensus can be presented on the nature of other residues within the P3 to P3' region with regard to target protease specificity.


Assuntos
Antitrombina III/antagonistas & inibidores , Fator XII/antagonistas & inibidores , Inibidores do Fator Xa , Fibrinolisina/antagonistas & inibidores , Glicoproteínas/farmacologia , Heparina/farmacologia , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio/farmacologia , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Antitrombina III/química , Sequência de Bases , Fator XII/química , Fator Xa/química , Fibrinolisina/química , Dados de Sequência Molecular , Ativadores de Plasminogênio/química , Inativadores de Plasminogênio/química , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tipo Uroquinase/química , Vitronectina
19.
J Biol Chem ; 266(21): 13852-8, 1991 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-1856219

RESUMO

Plasminogen activator inhibitor 1 (PAI-1) is sensitive to oxidative inactivation, and it has been suggested that specific oxidation of a methionine residue, Met347, situated in the P1' position of the reactive center may be the cause of the inactivation. To test this hypothesis we have purified and biochemically characterized mutant proteins of PAI-1 in which Met347 and either of two other methionines, Met266 or Met354, has been replaced with oxidation-resistant valine residues. The mutant proteins were found to be equally sensitive to oxidation as wild-type PAI-1, suggesting that a specific oxidation of the P1' Met347 is not responsible for the inactivation. When PAI-1 was oxidized, circular dichroism analysis revealed a rapid conformational change that correlated to the loss of inhibitory activity. The oxidation sensitivity of PAI-1 was enhanced dramatically in the presence of 0.001% sodium dodecyl sulfate, and the circular dichroism spectrum was significantly different from that of untreated PAI-1, suggesting that the increased sensitivity to oxidation may be caused by a conformational change in the inhibitor molecule. Taken together, our data suggest that the oxidative inactivation of PAI-1 is not caused by the specific oxidation of the P1' methionine but results from a conformational change in the protein structure.


Assuntos
Inativadores de Plasminogênio/química , Sequência de Bases , Dicroísmo Circular , Análise Mutacional de DNA , Humanos , Peróxido de Hidrogênio/química , Técnicas In Vitro , Metionina/química , Dados de Sequência Molecular , Oligonucleotídeos/química , Oxirredução , Conformação Proteica , Proteínas Recombinantes , Dodecilsulfato de Sódio/química , Relação Estrutura-Atividade , Succinimidas/química
20.
Biol Reprod ; 45(1): 49-56, 1991 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1878436

RESUMO

Two major plasminogen activator inhibitors (PAI-1 and PAI-2) increase in the peripheral circulation during pregnancy in humans. PAI-1 is of vascular endothelial origin whereas PAI-2 is produced primarily by human placental tissues. This study was undertaken to determine a) if PAI-1 and PAI-2 are also present in the baboon and b) their association with pregnancy. Citrated plasma was obtained from pregnant baboons sequentially at 15 +/- 3-day intervals between Days 30 and 140 of pregnancy. PAI activity increased significantly (p less than 0.05) at Day 120 (15.3 IU/ml) and 140 (21.8 IU/ml) of gestation and returned to baseline (2.6 IU/ml) 48 h post cesarean section. Placental tissues obtained at cesarean section during the third trimester were either placed in explant culture, fixed for immunocytochemistry, or frozen for RNA extraction. Western blot analysis of tissue culture media (TCM) indicated that the polyclonal antibody to PAI-1 reacted with a major band (Mr 47 000) in TCM from placental tissues while the PAI-2 antibody reacted primarily with a doublet (Mr 67 000 and 69 000) in these same media. PAI-1 was immunocytochemically localized primarily in the chorioamniotic tissue (CAM-D) and PAI-2 was found predominantly in placental villi. Slot blot hybridization with cDNAs to PAI-1 and PAI-2 indicated that the mRNA for PAI-2 was found primarily in placental villi, whereas the mRNA for PAI-1 was present in all three tissue compartments.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Papio/metabolismo , Placenta/química , Inativadores de Plasminogênio/imunologia , Animais , Anticorpos/imunologia , Feminino , Immunoblotting , Imuno-Histoquímica , Placenta/metabolismo , Inativadores de Plasminogênio/química , Inativadores de Plasminogênio/metabolismo , Gravidez , RNA Mensageiro/metabolismo
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