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J Mimuro

Publications and source records attributed to J Mimuro.

At least 55 records · Page 3Linked to original sources
PubMed1989

Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells.

The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.

Animals↗
PubMed1989

Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin.

Type 1 plasminogen activator inhibitor (PAI-1) binds to the extracellular matrix of cultured bovine aortic endothelial cells. Bovine plasma and bovine lung extract contain protein(s) that bind to PAI-1 and prevent this interaction. One of these proteins was purified approximately 425-fold from ammonium sulfate-fractionated plasma using standard chromatographic procedures together with affinity chromatography on PAI-1-Sepharose. The final product consisted of a major polypeptide of Mr 65,000 and two minor polypeptides of Mr 80,000 and 57,000. NH2-terminal amino acid sequence analysis of the Mr 65,000 polypeptide revealed that it was homologous with vitronectin, and antiserum against this purified binding protein recognized vitronectin and vice versa. Immunological analysis using these antisera demonstrated that the three peptides were immunologically related, and that vitronectin was present in the extracellular matrix of bovine endothelial cells and also in bovine lung.

Amino Acid Sequence↗
PubMed1989

Retinal pigment epithelial cells produce a latent fibrinolytic inhibitor that is antigenically and biochemically related to type 1 plasminogen activator inhibitor produced by vascular endothelial cells.

Conditioned media from retinal pigment epithelial (RPE) cells in culture contain active and latent plasminogen activator inhibitors (PAIs). Latent activity is unmasked by denaturants and accounts for the vast majority of total inhibitor activity. Activation by denaturants is an unusual characteristic previously described for PAI-1, the inhibitor produced by vascular endothelial cells. This property is not shared by PAI-2 or protease nexin. Reverse fibrin autography demonstrates that the PAI activity in RPE-conditioned media (RPE-CM) comigrates with purified endothelial cell-derived PAI-1 and has an apparent Mr of 50,000. Immunoblotting with a monospecific antiserum directed against endothelial cell-derived PAI-1 demonstrates a cross-reacting protein in RPE-CM at 50 kDa, and this same antiserum is able to immunoprecipitate a 50 kDa protein from [35S]methionine-labeled RPE-CM. These data suggest that RPE cells produce a PAI that is biochemically and immunologically related to PAI-1.

Cells, Cultured↗
PubMed1989

Clot lysis induced by a monoclonal antibody against alpha 2-plasmin inhibitor.

A monoclonal antibody (MoAb) to alpha 2-plasmin inhibitor designated JTPI-1 inhibited antiplasmin activity by interfering with formation of alpha 2-plasmin inhibitor (alpha 2-PI)-plasmin complex. With this MoAb, we observed plasma clot lysis in vitro and evaluated the potential of JTPI-1 to serve as a new therapeutic agent for thrombolysis. After adding 125I-labeled fibrinogen to plasma, clots were made by adding thrombin and calcium and were then resuspended in normal plasma containing various concentrations of JTPI-1. The presence of JTPI-1 enhanced release of the soluble 125I-labeled fibrin degradation fragment from the clots in a dose-dependent manner. With tissue plasminogen activator (t-PA)-depleted plasma, we showed that induction of clot lysis by JTPI-1 was dependent on fibrin-bound endogenous t-PA. Regulation of fibrinolysis initiated on the fibrin surface by fibrin-bound t-PA and plasminogen is mediated by alpha 2-PI cross-linked to fibrin by activated factor XIII. JTPI-1 bound to this cross-linked alpha 2-PI neutralized its activity and induced partial digestion of fibrin by plasmin. This resulted in additional binding of Glu-plasminogen to fibrin during the incubation. When 1.2 mumol/L JTPI-1 and 5 U/mL exogenous t-PA were present in the suspending plasma, the rate of clot lysis was essentially the same as that induced by 60 U/mL exogenous t-PA alone. These results suggest that JTPI-1 may be useful in reducing the amount of t-PA administered for thrombolytic therapy.

Antibodies, Monoclonal↗
PubMed1987

Monoclonal antibodies to discrete regions in alpha 2-plasmin inhibitor.

Three monoclonal antibodies to alpha 2-plasmin inhibitor (alpha 2PI) were characterized. The first, JTPI-1, was directed against the reative site of alpha 2PI and inhibited antiplasmin activity by interfering with the formation of alpha 2PI-plasmin complexes. The avidity of JTPI-1 to the preformed alpha 2PI-plasmin complex was markedly lower than that to free alpha 2PI, which made this antibody useful for measuring the free alpha 2PI in plasma. The second, JTPI-2, recognized an epitope in the C-terminal fragment of alpha 2PI (11,000 daltons [11 K]) that was cleaved from alpha 2PI by plasmin upon complex formation but remained noncovalently attached to the complex. However, binding of JTPI-2 to alpha 2PI was not inhibited by the C-terminal 26-residue peptide containing the plasminogen-binding site and had no effect on the function of alpha 2PI. These data suggested that JTPI-2 recognized an epitope between the C-terminal 26-residue peptide and the reactive site. The third, JTPI-3, bound the alpha 2PI-plasmin complex (150 K) as well as alpha 2PI. Binding was inhibited by the N-terminal 12-residue peptide of alpha 2PI, but factor XIII-catalyzed cross-linking of alpha 2PI to fibrin was not inhibited by JTPI-3. These results suggested that the antibody recognized an epitope near the N terminus. These three monoclonal antibodies were useful for analyzing the mechanism of interaction between alpha 2PI and plasmin.

Antibodies, Monoclonal↗
PubMed1987

Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor.

The extracellular matrix (ECM) of cultured bovine aortic endothelial cells (BAEs) was analyzed by immunoblotting and reverse fibrin autography and shown to contain type 1 plasminogen activator inhibitor (PAI-1). Most PAI-1 in the ECM formed complexes with exogenously added tissue-type plasminogen activator (tPA), demonstrating that this PAI-1 was functionally active. The resulting tPA/PAI-1 complexes were recovered in the reaction solution, indicating that the PAI-1 in such complexes no longer bound to ECM. The PAI-1 could not be removed by incubating ECM in high salt (2 mol/L NaCl), sugars (1 mol/L galactose, 1 mol/L mannose), glycosaminoglycans (10 mmol/L heparin, 10 mmol/L dermatan sulfate), or epsilon-aminocaproic acid (0.1 mol/L). However, PAI-1 could be extracted from ECM by treatment with either arginine (0.5 mol/L) or potassium thiocyanate (2 mol/L), or by incubation under acidic conditions (pH 2.5). ECM depleted of PAI-1 by acid extraction was able to bind both the active and latent forms of PAI-1. In this instance, most of the bound PAI-1 did not form complexes with tPA, indicating that the latent form was not activated as a consequence of binding to ECM. Although the PAI-1 activity in conditioned medium decayed with a half-life (t 1/2) of less than 3 hours, the t 1/2 of ECM-associated PAI-1 was greater than 24 hours. These data suggest that PAI-1 is produced by cultured BAEs in an active form and is then either released into the medium where it is rapidly inactivated or into the subendothelium where it binds to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.

Animals↗
PubMed1987

Level of protein C determined by combined assays during disseminated intravascular coagulation and oral anticoagulation.

We have developed a variation of the solid-phase enzyme-linked immunosorbent assay (ELISA) to enable measurement of the activity and antigen levels of protein C (PC) in human plasma. With this assay it is possible to do both tests with the same sample and same microtiter plate coated with anti-PC monoclonal antibody (MCA)JTC-4, which inhibited neither activation of PC nor activity of activated PC (APC). Even in patients undergoing heparin treatment for severe disseminated intravascular coagulation, there were no detectable differences between amidolytic activity and antigen levels of PC in patients' plasma. In addition, there was a strong correlation between the immunologic levels of PC in patients' plasma determined both by polyclonal ELISA using peroxidase-labeled immunopurified antiprotein C-IgG and those found with MCA ELISA using peroxidase-labeled MCAJTC-5, which does not bind to APC. In contrast, when oral anticoagulation therapy was started, immunologic levels of plasma PC estimated by peroxidase-labeled MCAJTC-1, a MCA that recognizes a gamma-carboxyglutamic acid domain-related conformational change of PC induced by metal ions, decreased more rapidly than did either the PC level determined by polyclonal ELISA or the percent prothrombin time. This suggested that comparison of MCAJTC-1-recognized PC levels and prothrombin time may be necessary at the beginning of oral anticoagulation therapy to treat patients safely.

Administration, Oral↗
PubMed1986

Release of alpha 2-plasmin inhibitor from plasma fibrin clots by activated coagulation factor XIII. Its effect on fibrinolysis.

When blood coagulation takes place in the presence of calcium ions, alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked to fibrin by activated coagulation Factor XIII (XIIIa) and thereby contributes to the resistance of fibrin to fibrinolysis. It was previously shown that the cross-linking reaction is a reversible one, since the alpha 2PI-fibrinogen cross-linked complex could be dissociated. In the present study we have shown that the alpha 2PI-fibrin cross-linking reaction is also a reversible reaction and alpha 2PI which had been cross-linked to fibrin can be released from fibrin by disrupting the equilibrium, resulting in a decrease of its resistance to fibrinolysis. When the fibrin clot formed from normal plasma in the presence of calcium ions was suspended in alpha 2PI-deficient plasma of buffered saline, alpha 2PI was gradually released from fibrin on incubation. When alpha 2PI was present in the suspending milieu, the release was decreased inversely to the concentrations of alpha 2PI in the suspending milieu. The release was accelerated by supplementing XIIIa or the presence of a high concentration of the NH2-terminal 12-residue peptide of alpha 2PI (N-peptide) which is cross-linked to fibrin in exchange for the release of alpha 2PI. When the release of alpha 2PI from fibrin was accelerated by XIIIa or N-peptide, the fibrin became less resistant to the fibrinolytic process, resulting in an acceleration of fibrinolysis which was proportional to the degree of the release of alpha 2PI. These results suggest the possibility that alpha 2PI could be released from fibrin in vivo by disrupting the equilibrium of the alpha 2PI-fibrin cross-linking reaction, and that the release would result in accelerated thrombolysis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Purpura fulminans in a Chinese boy with congenital protein C deficiency.

Severe and recurrent purpura fulminans developed in a Chinese boy at one day of age. Results of coagulation studies performed on the patient during attacks were compatible with the diagnosis of disseminated intravascular coagulation. Subsequent investigations have revealed that the patient is homozygous and that his parents are heterozygous for protein C deficiency. Cryoprecipitate and fresh frozen plasma induced a remission, and administration of warfarin has been successful in preventing recurrence of attacks for as long as 8 months without infusion of any plasma components. None of the family members who are heterozygous for protein C deficiency have had thrombotic episodes.

Combined Modality Therapy↗
PubMed1984

Histidine-rich glycoprotein and alpha 2-plasmin inhibitor in inhibition of plasminogen binding to fibrin.

The plasma proteins alpha 2-plasmin inhibitor and histidine-rich glycoprotein were compared directly with respect to their effectiveness in inhibiting the binding of plasminogen to fibrin under the same experimental conditions. At their physiological concentrations, the presence of alpha 2-plasmin inhibitor more markedly decreased the binding of plasminogen to fibrin than histidine-rich glycoprotein. Significance of these findings in inhibition of fibrinolysis is discussed.

Depression, Chemical↗
PubMed1984

Differential binding of plasminogen to crosslinked and noncrosslinked fibrins: its significance in hemostatic defect in factor XIII deficiency.

In spontaneous fibrinolysis of an alpha 2-plasmin inhibitor-deficient plasma clot or tissue-type plasminogen activator-induced fibrinolysis in a purified system without alpha 2-plasmin inhibitor, the lysis was faster when factor XIII-mediated crosslinking of fibrin to fibrin did not occur. During the initial period, the binding of plasminogen to fibrin steadily increased with incubation time. The initial level and subsequent increase of the binding, which may be critical for the subsequent development of fibrinolysis, were more remarkable when fibrin was not crosslinked. The amount of glu- or lys-plasminogen bound to noncrosslinked fibrin was around 4 or 1.5 times larger than the amount of the respective plasminogen bound to crosslinked fibrin. Plasmin was also found to be bound to noncrosslinked fibrin twice as much as the amount bound to crosslinked fibrin. Structural changes induced by crosslinking of fibrin alpha-chain may reduce either the affinity or the number of available complementary sites to lysine binding sites of plasmin(ogen), thereby decreasing the binding of plasmin(ogen) to fibrin. These results suggest that an increased affinity of noncrosslinked fibrin for plasmin(ogen) is contributory to the accelerated fibrinolysis observed in factor XIII deficiency, in addition to an absence of crosslinking of alpha 2-plasmin inhibitor to fibrin.

Enzyme Activation↗