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Biomedical subjects

B R Binder

Publications and source records attributed to B R Binder.

At least 145 records · Page 8Linked to original sources
PubMed1990

Release of [hydroxyproline3]-kinins by tissue kallikreins of pig, rat and man.

To determine the susceptibility of kininogens containing the recently described [Hyp3]-bradykinin moiety to cleavage by tissue kallikreins, we have studied the release of [Hyp3]-kinins from heat inactivated human plasma by purified tissue kallikreins. Kallikreins from man and pig were employed and compared with purified rat urinary kallikrein which is known to have a different cleavage specificity. Kinins released were separated by a modified reversed phase HPLC method and quantitated by bioassay and radioimmunoassay. Human urinary kallikrein and hog tissue kallikreins released 85-90% of the total kinins as Lys-bradykinin and 10-15% as [Hyp3]-Lys-bradykinin. In contrast, rat urinary kallikrein released 77% as bradykinin, 22% as [Hyp3]-bradykinin and negligible amounts of [Hyp3]-Lys-bradykinin from the identical substrate source indicating that rat tissue kallikreins prefer the Lys-Arg-bond within both unhydroxylated and hydroxylated kininogens. Therefore, hydroxylation of human kininogens apparently does not affect their ability to serve as substrates for tissue kallikreins with different cleavage specificities.

Animals↗
PubMed1990

Plasminogen activators, plasminogen activator inhibitor, and fibronectin in human granulosa cells and follicular fluid related to oocyte maturation and intrafollicular gonadotropin levels.

We determined the relative distribution of tissue plasminogen activator (TPA) antigen, urokinase-type PA antigen, PA inhibitor activity, and fibronectin levels in lysates of human granulosa cells (GC) and the respective follicular fluid (FF) in relationship to oocyte-corona-cumulus complex morphology. In addition, FF gonadotropins were measured to investigate a possible relationship of gonadotropins to PA activity. A significant increase of TPA antigen in GC lysates of intermediate and mature oocyte-corona-cumulus complex was found when compared with immature oocyte-corona-cumulus complex. Urokinase-type plasminogen activator levels and PA inhibitor levels did not reveal any significant differences between the different groups. In FF the concentrations of PA and PA inhibitor were significantly lower than in GC lysates and showed no significant difference between the oocyte-corona-cumulus complex groups. The concentration of fibronectin was significantly elevated in GC lysates of mature follicles. The marked increase of TPA in human GC during oocyte maturation showed a positive correlation with the increase of FF follicle-stimulating hormone and beta-human chorionic gonadotropin in the group of mature oocyte-corona-cumulus complex. The data obtained suggest that in man TPA is the predominant PA involved in the process leading to follicular rupture.

Adult↗
PubMed1990

Relationship between plasma levels of components of the fibrinolytic system and acute-phase reactants in patients with uterine malignancies.

Plasma samples from 17 patients with endometrial cancer and from 52 patients with cervical carcinoma were determined with respect to their levels of components of the fibrinolytic system (tissue-type plasminogen activator antigen, urokinase-type plasminogen activator antigen, plasminogen activator inhibitor activity) and related to the observed alterations of three acute-phase reactants (C-reactive protein, coeruloplasmin, alpha-1-antitrypsin). As shown previously, uterine malignancies, especially at later stages, exhibited significant increases in plasma levels of urokinase-type plasminogen activator antigen as compared to an age-matched control group. In contrast, tissue-type plasminogen activator antigen and plasminogen activator inhibitor activity remained unchanged. Determination of the acute-phase reactants revealed significant changes in the case of C-reactive protein and coeruloplasmin in later tumour stages. However, the increase in urokinase-type plasminogen activator antigen did not correlate with the increase of either C-reactive protein or coeruloplasmin plasma level. These data indicate that the increase in plasma urokinase-type plasminogen activator antigen in patients with uterine malignancies does not follow the pattern of common acute-phase reactants, like C-reactive protein or coeruloplasmin.

Acute-Phase Proteins↗
PubMed1990

Influence of urokinase on cell proliferation and invasion.

The urokinase-dependent plasminogen activating system is regulated not only by zymogen to enzyme conversion of pro-urokinase and inhibition of the active enzyme by plasminogen activator inhibitors, but also by regulated expression of urokinase receptors on the cell surface. Receptor-bound pro-urokinase in turn becomes activated and is capable of activating plasminogen probably bound site by site to urokinase to a cell surface receptor. Plasmin by itself or via activation of pro-collagenase to collagenase is capable of degrading the extracellular matrix, in turn mediating processes like invasion, metastasis and tumour growth. In addition, in some cell lines the urokinase-dependent system mediated via receptor-bound active urokinase is also capable of eliciting a mitogenic response of the cells. Therefore, the urokinase-dependent plasminogen activating system might not only be responsible for mediating extravascular proteolysis but might also be an autocrine mitogen for some cell lines.

Animals↗
PubMed1989

Heparin induced increase of t-PA antigen plasma levels in patients with unstable angina: no evidence for clinical benefit of heparinization during the initial phase of treatment.

Patients with unstable coronary artery disease were randomly treated either with a combination therapy consisting of nitrates and calcium-channel blockers without or with addition of clinical grade heparin administered subcutaneously; in order to evaluate the effect of heparin treatment on the fibrinolytic system, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) plasma levels were related to the clinical course of the disease. In heparinized patients thrombin time was prolonged more than 3-fold the normal range indicating effective heparin treatment. Heparinization led to a significant increase in t-PA antigen plasma levels (p less than 0.0001) within approximately four hours while PAI-1 activities remained unaltered. However, the measurable increase of the anticoagulant and pro-fibrinolytic activities of heparin did not result in a short-term benefit for the heparinized patients because the number of further ischemic attacks per patient during the observation period of three days was not different between the two study groups.

Aged↗
PubMed1989

Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo.

Protein C inhibitor (PCI) and plasminogen activator inhibitor 3 (PAI-3; urinary urokinase inhibitor) are immunologically identical. The role of PCI for urokinase (uPA) inhibition in vivo was investigated. We therefore developed an enzyme-linked immunosorbent assay (ELISA) specific for uPA-PCI complexes: Rabbit anti-PCI IgG was immobilized on a microtiter plate and following incubation with uPA-PCI complex-containing samples, bound uPA-PCI complexes were quantified with a horseradish-peroxidase-linked monoclonal antibody (MoAb) to uPA. Using this assay, time, dose, and heparin-dependent complexes were detected when uPA was incubated with normal plasma or purified urinary PCI, whereas no complexes were measurable using PCI-immunodepleted plasma. Plasma samples (containing 20 mmol/L benzamidine to prevent complex formation ex vivo) from patients undergoing systemic urokinase therapy (1 x 10(6) IU/60 min intravenously [IV]) after myocardial infarction were also studied. uPA present in these plasma samples (up to 1,200 ng/mL) had only 43% to 70% of the specific activity of purified 2-chain uPA, suggesting that a major portion of uPA is complexed to inhibitors. In these plasma samples uPA-PCI complexes were present in a concentration corresponding to 21% to 25% of inactive uPA antigen. These data suggest that at high uPA concentrations, such as during uPA therapy, plasma PCI might contribute significantly to uPA inhibition in vivo.

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

Purification and identification of the lipoprotein-binding proteins from human blood platelet membrane.

As reported previously, homologous plasma lipoproteins specifically bind to the plasma membrane of human blood platelets. The two major lipoprotein-binding membrane glycoproteins were purified to apparent homogeneity and identified by their mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both in the nonreduced and reduced state, by specific antibodies against glycoproteins IIb (GPIIb) and IIIa (GPIIIa), respectively, including the alloantibody anti-PlA1 and monoclonal antibodies. Furthermore, lipoprotein binding to intact platelets is also inhibited in a dose-dependent fashion by preincubation of the platelets with antibodies against these glycoproteins. From these experiments it can be concluded that lipoproteins bind to both components of the glycoprotein IIb-IIIa complex in isolated membranes and intact platelets. High density lipoprotein and low density lipoprotein bind to GPIIIa blotted to nitrocellulose in a way that binding of one species interferes with the binding of the other. Addition of fibrinogen significantly inhibits this binding. The specific binding of fibrinogen to GPIIIa is strongly inhibited in the presence of either of the two lipoproteins. LDL and HDL are specifically bound by isolated GPIIb, too. In our blotting experiments fibrinogen shows no binding to this membrane glycoprotein. On the other hand, fibrinogen significantly interferes with the interaction between GPIIb and the lipoproteins.

Antibodies, Monoclonal↗
PubMed1989

Demonstration of plasminogen activator activity in the intima and media of the normal human aorta and other large arteries: immunological identification of the plasminogen activator(s).

The plasminogen activator activity (PAA) in extracts of the intima, media, and adventitia of the normal human aorta and other large arteries (carotid artery, renal artery and iliac artery) was studied with a sensitive, quantitative spectrophotometric assay using plasminogen and the chromogenic plasmin substrate S-2251. All layers of the arteries showed PAA which was highest in the adventitia, lowest in the media, while in the intima (aorta) PAA was intermediate, but much closer to that of the media. Plasminogen activator inhibition (PAI) was at the same level in all layers of the arteries studied. Plasmin inhibition (PI) was higher in adventitia than in intima (aorta), while in media the PI was intermediate. The PAA was due to the tissue-type plasminogen activator (t-PA), but not to the urokinase-type (u-PA), as judged by addition of respective antibodies. The relatively low PAA found in the intima of large arteries is therefore due to a low plasminogen activator and not a high plasminogen activator inhibitor activity or plasmin inhibitor level.

Adolescent↗
PubMed1989

Functional characterization of monoclonal antibodies directed against fibrin binding domains of tissue-type plasminogen activator.

Fibrin interacts with tissue-type plasminogen activator (tPA) via the finger and the kringle 2 domains. Three monoclonal antibodies against tPA, designated MPW3VPA, MPW6VPA, and MPW7VPA, which react with epitopes in the tPA molecule involved in fibrin binding, were characterized. The IgM monoclonal antibody MPW6VPA, directed against an epitope close to the finger and epidermal growth factor domains, stimulated plasminogen activation only in the absence of CNBr-fibrinogen fragments by increasing kcat in a dose-dependent fashion, an effect which was not restricted to the intact molecule. These results suggest that MPW6VPA mimics the initial effect of fibrin bound to the tPA molecule, which results in a change of kcat values. The MPW6VPA effect was reversed by another antibody, MPW3VPA, also directed against epidermal growth factor and finger domains. The latter antibody also inhibited plasminogen activation by tPA in the presence of CNBr-fibrinogen fragments in a dose-dependent, apparently noncompetitive way. No effect of MPW3VPA was seen in the absence of CNBr-fibrinogen fragments. MPW7VPA directed against kringle 2 of tPA inhibited plasminogen activation by tPA only when CNBr-fibrinogen fragments were present. This inhibition was apparently competitive and dose-dependent. These data suggest that MPW3VPA interferes with the first phase of fibrin binding to tPA, whereas MPW7VPA interferes with the second phase of fibrin binding to the tPA molecule via kringle 2, resulting in Km changes.

Antibodies, Monoclonal↗
PubMed1989

Evaluation of fibrinolytic capacity in plasma during thrombolytic therapy with single (scu-PA) or two-chain urokinase type plasminogen activator (tcu-PA) by a combined assay system for urokinase type plasminogen activator antigen and function.

A combined assay for urokinase type plasminogen activator (u-PA) activity and antigen determination in plasma samples is described. This assay is based on binding of u-PA to an antibody immobilized on a microtiter plate followed by determination of the enzymatic activity of the bound u-PA. Thereafter bound u-PA antigen can be quantified by means of a specific peroxidase labelled monoclonal antibody against u-PA. By use of this assay system u-PA activity and antigen can be determined with lower detection limits of 0.08 IU/ml and 1.0 ng/ml, respectively, and intraassay as well as interassay coefficients of variation of 10% and 12% for activity and 5% and 7% for antigen determinations, respectively. Normal plasma levels of u-PA antigen could be determined to be 1.88 ng/ml +/- 0.61. Furthermore, this assay system allows specific quantification of u-PA antigen and activity during thrombolytic therapy.

Antigens↗
PubMed1989

Growth stimulation of human epidermal cells by urokinase is restricted to the intact active enzyme.

In this study we report on the effect of urokinase fragments on the proliferation of cells of the human epidermal cell line, CCL 20.2, which expresses high-affinity receptors for urokinase and the growth of which is stimulated by intact active 54-kDa urokinase. The 33-kDa fragment containing only the active-site domain, does not bind to the receptors and does not stimulate cell proliferation, while the 17-kDa fragment, containing only the kringle and the growth-factor domains binds to the receptor but does not stimulate growth of the human epidermal cell line. Growth promotion of this tumor cell line by urokinase is therefore restricted to the complete intact and active urokinase molecule.

Cell Division↗
PubMed1989

In vitro generation of dysmorphic erythrocytes.

In hematuria, dysmorphic red blood cells found in the urine sediment are known to indicate glomerular origin of bleeding. To better understand the mechanisms which might cause the typical membrane changes in dysmorphic erythrocytes in vivo, we have exposed normal human erythrocytes in vitro to an osmotic and enzymatic environment similar to that of the nephron. In addition, treated or untreated erythrocytes were exposed to a hemolytic environment. The morphology of the cells was evaluated by light and electron microscopy in comparison of that of dysmorphic erythrocytes obtained in vivo. Neither the passage of erythrocytes in sequence through various solutions each simulating the tubule fluid in a different part of the nephron, nor exposure to urinary or renal enzymes or to serum per se causes the typical membrane alterations. Upon exposure of passaged erythrocytes to a hemolytic environment, however, 50 to 90% of the cells become dysmorphic in a time and dose dependent fashion. By light and electron microscopy these dysmorphic erythrocytes obtained in vitro are indistinguishable from those obtained in vivo. In contrast, non-passaged red blood cells are not affected by the same hemolytic environment. We conclude that osmotically challenged erythrocytes exposed to a hemolytic environment develop dysmorphic membrane alterations.

Adult↗
PubMed1989

Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line.

Binding of urokinase-type plasminogen activator (u-PA) to its receptor has been shown not only to focus proteolytic activity to the cell surface but also to exert a mitogenic effect on the human epidermal tumor cell line CCL 20.2. This report shows that u-PA is an autocrine mitogen in the human melanoma cell line GUBSB and that inhibition of receptor-bound u-PA by specific anti-u-PA antibodies causes a significant suppression of cell proliferation in this system. The GUBSB cell line secretes 70-80% of the u-PA in its active form and expresses high-affinity u-PA receptors with a Kd of 5.2 x 10(-10) M and 2.8 x 10(4) binding sites per cell. Approximately 70% of the u-PA receptors on these cells are occupied by endogenously secreted u-PA. Addition of the monoclonal anti-u-PA antibody MPW5UK (10 nM), directed against the active site of u-PA, twice daily to the cell cultures resulted in a significant decrease of [3H]thymidine incorporation by the tumor cells, whereas a 10 times higher concentration of the monoclonal antibody MPW4UK, which does not inhibit plasminogen activator activity of u-PA, was necessary to achieve the same effect. In addition, diisopropyl fluorophosphate-inactivated u-PA, in a concentration 50-fold higher than the concentration necessary to saturate the u-PA receptor (250 pM), decreased [3H]thymidine incorporation similarly to the specific antibody, proving that active u-PA is required for the mitogenic effect. Inhibition of endogenous u-PA production by cycloheximide reduced [3H]thymidine incorporation significantly; after addition of exogenous u-PA, [3H]thymidine incorporation increased again in the cycloheximide-treated cells. Therefore, inhibition of receptor-bound u-PA might represent a tool not only to inactivate cell-bound proteolytic activity, necessary for invasion, but also to exert a specific antiproliferative effect on certain tumor cells.

Antibodies, Monoclonal↗
PubMed1989

Regulation of plasminogen activation in isolated perfused rat kidney.

To better understand the mechanism and regulation of plasminogen activation within the kidney, the release and excretion of plasminogen activator activities was studied in the isolated perfused rat kidney in the absence and presence of plasminogen substrate. In the absence of plasminogen, the kidneys released a constant amount of plasminogen activator activity into both the urine and the perfusate. On continuous infusion of purified human plasminogen into the perfusate, the release of plasminogen activator activity into the urine slightly increased, and plasmin generated could be detected in both urine and perfusate. With the use of specific antibodies against the tissue-type (t-PA) and the urokinase-type plasminogen activator (u-PA), respectively, the activity in the perfusate could be identified as t-PA, whereas the activity in the urine could be ascribed to u-PA. A bolus injection of either antibody into the plasminogen-supplemented perfusion medium completely inhibited plasminogen activator activity and generation of plasmin in the vascular or tubular compartment. Furthermore, intrarenal inhibition of t-PA activity by the specific antibody significantly increased the concentration of plasminogen in the perfusate, indicating decreased consumption. This effect was accompanied by increased excretion of u-PA into the urine, suggesting that the availability of intact plasminogen in the renal circulation directly or indirectly might participate in the regulation of u-PA excretion into the urine.

Animals↗
PubMed1989

A comparison between activated protein C and des-1-41-light chain-activated protein C in reactions with type 1 plasminogen activator inhibitor.

This study investigates the role of the gamma-carboxyglutamic acid (gla) containing domain of activated protein C in interactions with both platelet-derived and purified type 1 plasminogen activator inhibitor (PAI-1). The activity of human platelet PAI-1 was neutralized to the same extent by bovine activated protein C and bovine des-1-41-light chain-activated protein C. Both forms of activated protein C formed SDS-stable, divalent-cation independent complexes with platelet PAI-1, as demonstrated by immunoblotting using antibodies directed to either protein C or PAI-1. Since activated protein C neutralized PAI-1, the potential inhibition of the enzyme by PAI-1 was studied. Purified PAI-1 inhibited the amidolytic activity of bovine-activated protein C and bovine des-1-41-light chain-activated protein C with a k2 of 2.85 X 10(4) M-1 sec-1 for both proteins. These data suggest that the gla domain of activated protein C is not required for neutralization of PAI-1 activity, for complex formation with PAI-1, or for inhibition of the amidolytic activity of activated protein C by PAI-1.

Animals↗
PubMed1988

Circadian fluctuations of plasminogen activator inhibitor and tissue plasminogen activator levels in plasma of patients with unstable coronary artery disease and acute myocardial infarction.

A decrease in the fibrinolytic potential, mainly due to an elevation of plasminogen activator inhibitor (PAI), has been described in patients with stable coronary artery disease and a previous myocardial infarction. We investigated plasma levels of PAI and tissue plasminogen activator (t-PA) and their possible circadian variations in patients with unstable coronary artery disease (CAD). Sixty-three patients were studied for at least 2 consecutive days during their stay at the coronary care unit (CCU). Diurnal plasma fluctuations in PAI and t-PA and onset of further myocardial ischemic episodes were monitored. As controls we used 22 age-matched patients submitted to the clinic because of non cardiac chest pain or valvular disease who revealed no evidence of CAD. PAI levels were significantly elevated in patients with unstable CAD (p less than 0.0001) but were not influenced by the extent of underlying CAD, history of previous myocardial infarction, known risk factors for CAD, or by extent of myocardial damage. The circadian variation of PAI levels with peak values between midnight and 6 A.M. found in controls was still present in patients but at a higher level. Preservation of circadian pattern in PAI plasma levels despite myocardial ischemic attacks indicates that elevation of PAI is rather not caused by a reactive phenomenon. On the other hand, elevated PAI levels and episodes of severe myocardial ischemia exhibiting a median time of onset at 10 A.M. seem to be closely related.

Aged↗