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At least 19 recordsLinked to original sources
PubMed1989

Functional characteristics of receptor-bound urokinase on human monocytes: catalytic efficiency and susceptibility to inactivation by plasminogen activator inhibitors.

We compared urokinase-type plasminogen activator (u-PA) in fluid phase and u-PA bound with its receptor on human blood monocytes with respect to proteolytic activity and susceptibility to inactivation by the plasminogen activator inhibitors PAI-1 and PAI-2. Receptor-bound u-PA is catalytically twice as efficient as fluid-phase u-PA. Fluid-phase u-PA is susceptible to rapid inhibition by PAI-1 and PAI-2 at an estimated PAI:u-PA molar ratio of 2:1. In contrast, u-PA bound to endogenously occupied receptors is inhibited by PAI-2 only at PAI:u-PA molar ratios of 20:1, but is not inhibited by PAI-1, u-PA/PAI-1 and u-PA/PAI-2 complexes bind to the receptor with a tenfold lower affinity than u-PA itself. Thus, competition of u-PA/PAI complexes with fluid-phase u-PA for binding to the receptor is unlikely to affect the overall plasminogen activator activity of the monocyte. These findings demonstrate that the activity of receptor-bound u-PA can be modulated by PAI-2, but not by PAI-1, to adjust the cell's proteolytic activity to different local situations.

Catalysis↗
PubMed1991

The oxidative inactivation of plasminogen activator inhibitor type 1 results from a conformational change in the molecule and does not require the involvement of the P1' methionine.

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.

Base Sequence↗
PubMed1986

Inactivation of plasminogen activator inhibitor by oxidants.

The rapidly acting plasminogen activator inhibitor (PAI) purified from cultured bovine aortic endothelial cells (BAEs) was inactivated during iodination with chloramine T and other oxidizing iodination systems. Inactivation was observed in the absence of iodine, suggesting that the loss of activity resulted from the oxidizing conditions employed. In an attempt to further study the nature of this inactivation, the PAI was treated with chloramine T under conditions that specifically oxidize methionine and cysteine residues. Both PAI inhibitory activity and the ability of the PAI to form complexes with tissue-type PA were decreased in a dose-dependent manner by such treatment. The PAI was more sensitive to oxidative inactivation than urokinase, elastase, and alpha 1-protease inhibitor. Incubation of the chloramine T inactivated PAI with methionine sulfoxide peptide reductase in the presence of dithiothreitol (DTT) restored more than 90% of the PAI activity. The reductase is a DTT-dependent enzyme that specifically converts methionine sulfoxide to methionine. Little activity was restored by either the reductase or DTT alone. These results indicate that the oxidation of at least one critical methionine residue is responsible for the loss of PAI activity upon iodination. In this respect, the BAE PAI resembles alpha 1-protease inhibitor, a well-characterized elastase inhibitor that also is inactivated by oxidants. Both inhibitors are members of the serine protease inhibitor superfamily (Serpins), and both have a methionine residue in their reactive center.

Animals↗
PubMed1988

Acid treatment of plasma for the inactivation of plasminogen activator inhibitor-1 (PAI-1).

The present study was initiated to assess the effectiveness of various acid treatments of blood or plasma in the inactivation of PAI-1. It was shown that a frequently used treatment of blood or plasma with 1 M acetate buffer, pH 3.9, only partially inactivated PAI-1. The inactivation of PAI-1 in plasma was found to depend upon pH and temperature, showing an optimal inactivation at a pH less than or equal to 3, at 37 degrees C.

Acetates↗
PubMed1987

Poly-D-lysine dependent inactivation of tissue plasminogen activator by a class PAI-2 inhibitor (minactivin).

Two-chain tissue plasminogen activator (t-PA) was found to be inactive in a coupled colorimetric assay for plasminogen activators, but a high level of activity was obtained in the presence of poly-D-lysine. This stimulated activity was strongly inhibited by minactivin, a urokinase inhibitor, but unstimulated enzyme could be shown to be unaffected by minactivin. In the presence of poly-D-lysine minactivin was a very successful competitive inhibitor of t-PA with respect to the substrate, plasminogen. The Ki for minactivin determined by the Henderson method was 2.5 X 10(-12) M, compared to the Km for plasminogen determined as 0.6 X 10(-6) M. The value of Ki for minactivin with u-PA, determined under the same conditions, was 1.6 X 10(-11) M.

Drug Interactions↗
PubMed1979

Inactivation of the plasminogen activator from HeLa cells by peptides of arginine chloromethyl ketone.

The binding specificities of human urinary urokinase (EC 3.4.99.26) and HeLa cell plasminogen activator were studied using peptidyl chloromethyl ketone inhibitors. A 125I-labeled fibrin assay has been developed to yield kinetic information. Reagents of the sequence X-Gly-ArgCH2Cl were the most effective. The susceptibility of the HeLa cell plasminogen activator differed from that of urokinase in several respects indicating the utility of this type of inhibitor in distinguishing between proteases of this specificity.

Amino Acid Chloromethyl Ketones↗
PubMed1986

Proteolytically induced variations in the enzymatic properties of tissue plasminogen activator. Activations, inactivations and reactivations.

Tissue plasminogen activator was treated with Sepharose-bound trypsin or chymotrypsin. Trypsin rapidly converted the one-chain activator to the two-chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active-site labeling revealed that after the conversion to the two-chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded. Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50,000/52,000 and 17,000/20,000 respectively. N-terminal sequence analysis identified cleavage sites at peptide bonds 420-421 and 423-424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations. However, treatment of two-chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin-digested one-chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420-421 and 423-424, convert the active one-chain activator into an 'inactive' zymogen, which is again 'activated' by plasmin cleavage.

Amino Acid Sequence↗
PubMed1992

Thrombolytic actions of reptilase.

In thrombolytic model in vitro, reptilase (Rep, defibrase) did not show appreciable thrombolytic actions on red and white thrombi. After daily iv infusion of Rep 0.25 IU for 10 d, the time of 50% lysis of euglobulin (ELT1/2) was shortened from 9.3 +/- 0.8 to 6.7 +/- 1.0 h (P < 0.01), alteplase activity was increased from 1.9 +/- 0.7 to 3.7 +/- 0.9 IU.ml-1, and plasminogen inactivator (PI) activity reduced from 4.3 +/- 0.6 to 1.8 +/- 0.9 AU.ml-1 (all P < 0.01). The findings indicate that the thrombolytic action of Rep shown in vivo may not be from the direct action on thrombi but from the influence on alteplase and PI activity.

Adult↗
PubMed1987

Conversion of the active to latent plasminogen activator inhibitor from human endothelial cells.

The plasminogen activator inhibitor from human endothelial cells (PAI-1) exists in two forms in the culture medium: an active form that binds to and inactivates plasminogen activators and a latent form that in its native state has no anti-activator activity. Inhibitor activity associated with the latent form can be generated by treatment with protein denaturants and makes up more than 98% of the total inhibitor activity in conditioned medium. Plasminogen activator inhibitor activity is also found in cell cytosol. This inhibitor activity is stable to SDS-treatment but is not enhanced by it. We investigated the relationship between this active cell-associated inhibitor and the latent PAI-1 found in the conditioned medium. Both intracellular and extracellular inhibitors were immunoprecipitated by a monoclonal antibody produced against the latent inhibitor from HT1080 fibrosarcoma cells and electrophoresis on SDS gels of various acrylamide concentrations demonstrated that both forms had the same Mr. Incubation of cytosol inhibitor at 37 degrees C resulted in a decline in inhibitor activity with a half-life of approximately 4 hours, a rate of decline similar to that of the active PAI-1 in conditioned medium, with less than 10% of the original activity present after eight hours. This decline is accelerated at higher temperatures and is not affected by the presence of a variety of protease inhibitors. Approximately 90% of the activity can be regenerated after SDS treatment suggesting that the cell associated inhibitor, during incubation at 37 degrees C, converts to a form similar to that found in conditioned medium. Despite these similarities, the apparent Stoke's radii of the active intracellular inhibitor and the latent inhibitor in conditioned medium were significantly different with values of 2.77 nm and 2.40 nm for active and latent PAI-1, respectively. Incubation of the active form at 37 degrees C resulted in the shift of the Stoke's radius to that similar to the latent PAI-1 (2.45 nm). Thus, the active and latent PAI-1, while being immunologically similar and of the same apparent Mr, can be differentiated by their behavior on gel permeation columns. This suggests that the intracellular inhibitor is a precursor to the latent form.

Cells, Cultured↗
PubMed1987

The resistance of fibrin-stimulated tissue plasminogen activator to inactivation by a class PAI-2 inhibitor (minactivin).

Solid phase fibrin was an efficient stimulator of the tissue-type plasminogen activator (t-PA), and the plasmin produced could be detected by colorimetric assay of the soluble phase above the fibrin. However the fibrin-stimulated activity of t-PA was not inhibited by minactivin. This result was in contrast to that obtained with poly-D-lysine (PL) stimulated t-PA, where minactivin was a potent inhibitor. However, if PL was added to fibrin-bound t-PA, the enzyme once again became susceptible to minactivin inhibition. This occurred without release of t-PA from the fibrin matrix. Minactivin alone did not bind to fibrin or to the t-PA fibrin complex. It was therefore concluded that minactivin normally has no significant role in the regulation of t-PA mediated fibrinolysis, but this effect can be induced by PL.

Drug Interactions↗
PubMed1986

Involvement of both heparanase and plasminogen activator in lymphoma cell-mediated degradation of heparan sulfate in the subendothelial extracellular matrix.

The effect of plasminogen on the ability of highly metastatic ESb mouse lymphoma cells to degrade heparan sulfate (HS) in the subendothelial extracellular matrix (ECM) was studied. A metabolically sulfate-labeled ECM was incubated with the lymphoma cells, and labeled degradation products were analyzed by gel filtration on Sepharose 6B. Heparanase-mediated release of low-Mr (0.5 less than Kav less than 0.85) HS cleavage products was stimulated fourfold in the presence of plasminogen. Incubation of plasminogen alone with the ECM resulted in its conversion into plasmin, which released high-Mr (Kav less than 0.33) labeled proteoglycans from the ECM. Heating the ECM (80 degrees C, 1 hr) abolished its ability to convert plasminogen into plasmin, yet plasminogen stimulated, through its activation by the ESb plasminogen activator, heparanase-mediated release of low-Mr HS fragments. Heparin inhibited both the basal and plasminogen-stimulated degradation of HS side chains but not the total amount of labeled material released from the ECM. In contrast, aprotinin inhibited the plasminogen-stimulated release of high- as well as low-Mr material. In the absence of plasminogen, degradation of heated ECM by ESb cells was completely inhibited by aprotinin, but there was only a partial inhibition of the degradation of native ECM and no effect on the degradation of soluble HS proteoglycan. These results demonstrate that proteolytic activity and heparanase participate synergistically in the sequential degradation of ECM HS and that the ESb proteolytic activity is crucial for this degradation when the ECM-associated protease is inactivated. Plasminogen may serve as a source for the proteolytic activity that produces a more accessible substrate to the heparanase.

Animals↗
PubMed1985

Preparation and characterization of human bone marrow-derived macrophages.

Bone marrow-derived macrophages were prepared from human bone marrow mononuclear cells following cultivation in GCT-conditioned medium (GCT-CM) and purification by adherence to fibronectin-coated flasks. The growth of bone marrow mononuclear cells in GCT-CM was dependent on the shape of the culture vessels, being increased in round-bottomed versus flat-bottomed wells. Proliferation was confined to nonadherent cells; like blood monocytes, bone marrow-derived macrophages did not incorporate [3H]thymidine in response to GCT-CM or human serum. Purified macrophages from this source expressed nonspecific esterase and OKM1, OKla, FMC 17, 32, and 34 and 25F9 antigens but lacked Mo2. They expressed high levels of an inactivator of plasminogen activator, minactivin, and gave a substantial metabolic burst in response to phorbol myristate acetate or opsonized (but not unopsonized) zymosan. Bone marrow-derived macrophages acted as accessory cells in the response of T lymphocytes to phytohemagglutinin. The results suggest that liquid bone marrow cultures are useful in the study of the differentiation of human mononuclear phagocytes.

Antibodies, Monoclonal↗
PubMedDate not supplied

A new canine model of proximal internal carotid embolism.

A new model of internal carotid artery embolism was developed using autologous clot. The clot was prepared by incubating blood at room temperature for 2 hours to inactivate plasminogen activators and then refrigerating it at 4 degrees C for 22 hours. The purpose of the experiment was to devise a model of the intravascular lesion and not of stroke itself. The dog was chosen as the experimental animal since it has a maxillocarotid artery which permits collateral flow beyond proximal internal carotid artery occlusions. A volume of clot measuring 0.25 to 0.30 cc was sufficient to occlude the artery for 48 hours in 80% of the animals without causing major strokes. We have used this model to study clot radiolabeling and suggest it may also have application for evaluating thrombolytic drugs.

Animals↗
PubMed1990

Effect of peptides on the inactivation of tissue plasminogen activator by plasminogen activator inhibitor-1 and on the binding of tissue plasminogen activator to endothelial cells.

The effectiveness of tissue plasminogen activator (tPA) in thrombolytic therapy is dependent upon the rate at which therapeutically administered tPA reaches the clot site and the proportion of that tPA which is enzymatically active. Interactions between tPA and its main plasma inhibitor (PAI-1) and between tPA and the endothelial cells lining blood vessels are two factors which may limit efficacy. In an attempt to identify the regions of the tPA molecule involved in these interactions, we have examined a series of synthetic peptides with amino acid sequences corresponding to different regions of the tPA molecule for their ability to protect tPA from inactivation by PAI-1 and for their ability to reduce the binding of tPA to endothelial cells. Three peptides were identified which were especially effective at maintaining tPA activity in the presence of PAI-1 and three others were found which had a lesser effect. These same peptides were also found to inhibit the binding of tPA to endothelial cells. This suggests that the same regions of the tPA molecule are involved in both processes. None of the peptides inhibited the binding of tPA to fibrin. These peptides may serve as models for the development of agents for enhancing the activity of both endogenous tPA and of tPA administered in thrombolytic therapy.

Amino Acid Sequence↗
PubMed1990

Prourokinase activation on the surface of human rhabdomyosarcoma cells: localization and inactivation of newly formed urokinase-type plasminogen activator by recombinant class 2 plasminogen activator inhibitor.

Recombinant class 2 plasminogen activator inhibitor (PAI-2) was used in an approach to probe the formation and location of enzymatically active urokinase-type plasminogen activator (u-PA) sites on the surface of cultured human rhabdomyosarcoma cells (RD cells). Activation of pro-u-PA on the cell surface and consequent binding of PAI-2 was dependent on the addition of native plasminogen to serum cultures of the cells. Inhibition of the enzyme activity of surface-bound u-PA by the added PAI-2 resulted in a 79% reduction in the capacity of the RD cells to generate cell surface-associated plasmin activity from bound plasminogen. Under these conditions, the PAI-2 probe was localized at focal adhesions of RD cells, where it colocalized with both extracellular u-PA and intracellular vinculin antigens in double immunofluorescence labeling. Specificity of the probe's interaction with cell surface-bound u-PA was confirmed by blocking with a monoclonal antibody to human u-PA, which could also inhibit the formation of bound plasmin activity. These results showed the assembly of the plasmin-generating system at focal adhesions and the accessibility of bound u-PA on which it depends to added PAI-2. Therefore, PAI-2 has the potential both to localize at sites of tumor expression of functionally active u-PA and simultaneously to inhibit cell surface plasminogen activation.

Cell Line↗
PubMed1991

A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator.

c[Arg-aB-(CH2+SCH3 phi)-Gly4] was designed and studied as a mechanism-based inactivator (suicide substrate) for plasminogen activators (u-PA and t-PA) and plasmin. This compound inhibited u-PA and fulfills criteria expected for the involvement of an enzyme-activated inhibitor: first-order and irreversible process, saturation kinetics, protection by substrate. The limiting first-order rate constant kinact and the apparent enzyme-inhibitor dissociation constant KI were 0.021 s-1 and 9 microM, respectively at pH 7.5 and 25 degrees C. The activation of plasminogen by u-PA is compromised after this enzyme has been treated by the reagent. Plasmin and t-PA were inactivated 40- and 2330-fold less efficiently than u-PA, respectively.

Amino Acid Sequence↗
PubMed1988

Susceptibility of plasminogen activators to suicide inactivation.

Halomethylated derivatives of dihydrocoumarins are efficient enzyme-activated inhibitors ("suicide" substrates) of plasminogen activators. Kinetic analysis indicate that the one-chain and two-chain forms of the human plasminogen activator are inhibited by 3,4-dihydro-3-benzyl-6-chloromethylcoumarin through a mechanism-based inactivation characterized by the following kinetic parameters (4 degrees C, pH 6.8) : k2 equal to 0.02 s-1 and 0.03 s-1 (for one- and two-chain tissue plasminogen activators, respectively) and Ki equal to 0.16 mM for both forms. Human urokinase and human tissue-type plasminogen activator can be discriminated on the basis of their inhibition by this suicide substrate. The design of a new series of suicide substrates of serine proteases (functionalized cyclopeptides possessing a potential alkylating function closely related to that found in halomethylated derivatives of dihydrocoumarins) is described.

Chemical Phenomena↗
PubMed1991

Species specificity of the fibrinolytic effects of activated protein C.

Activated protein C (APC) has been shown to stimulate fibrinolysis in both in vitro and in vivo experimental systems. In order to test the importance of protein S in the fibrinolytic activity of APC we have compared the activity of APC, prepared from rabbit, bovine and human plasma, in the stimulation of whole blood clot lysis, the inactivation of plasminogen activator inhibitors and anticoagulant activity. When whole blood clot lysis was performed using tissue plasminogen activator in either human or rabbit blood, APC was found to enhance clot lysis in a species specific manner that paralleled the pattern observed for its anticoagulant activity. Bovine APC, was the poorest stimulator of fibrinolysis in human plasma. However, if bovine protein S was also added to human plasma, bovine APC was as effective in promoting fibrinolysis as human APC. In contrast, no species specific effects on the inactivation of plasminogen activator inhibitor activity was observed. Though substantial effects of APC on plasminogen activator inhibitor levels were made by rabbit, human and bovine activated protein C in human plasma, there was no effect of activated protein C on the rate of clot lysis of human plasma. These results suggest that protein S is important for the expression of the fibrinolytic activity of activated protein C and that the effect of protein S may be useful for the differentiation of fibrinolytic effects of activated protein C that may be related to the inactivation of plasminogen activator inhibitors and those that are not.

Animals↗