Biomedical subjects

B R Binder

Publications and source records attributed to B R Binder.

At least 181 records · Page 10Linked to original sources

PubMed1987

Effect of the cyanogen-bromide-2 fragment of fibrinogen on plasminogen activation by single-chain urokinase-type plasminogen activator.

Activation of Glu-plasminogen by single-chain urokinase-type plasminogen activator (sc-uPA), isolated from human urine, was studied in a purified system in the absence and presence of the cyanogen bromide fibrinogen fragment, FCB 2, and compared to plasminogen activation by two-chain high-Mr urokinase. Plasminogen activation by sc-uPA was significantly increased by the FCB-2 fibrinogen fragment, an effect brought about by decrease of apparent Km and increase of apparent kcat. During the course of plasminogen activation by scu-PA, two-chain urokinase was formed from 125I-sc-uPA to a significant degree only when a concentration of 30 nM plasmin was reached in the incubation mixture; this was only the case in the system stimulated by FCB-2 fibrinogen fragment and only after 30 min. Formation of two-chain urokinase was not, however, related to the increase in the rate of plasmin formation induced by the FCB-2 fibrinogen fragment.

Cyanogen Bromide↗

PubMed1987

Tissue-type plasminogen activator and urokinase: differences in the reaction pattern with the active-site titrant 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride.

The applicability of 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride (MUGB) as active-site titrant for tissue-type plasminogen activator (t-PA) was studied in comparison to urokinase. Although t-PA was capable of cleaving MUGB, active-site titration of t-PA (one-chain form as well as two-chain form) with MUGB was not possible, whereas MUGB titration of urokinase could be performed. We therefore studied the kinetics of the interaction of these two plasminogen activators with MUGB. The equilibrium dissociation constant, KS, for the interaction between MUGB and urokinase was 2.9 X 10(-6) M, and for the interaction with t-PA 3.13 X 10(-5) M. However, one main requirement for active-site titration, namely a stable acyl enzyme intermediate (ES'), was only fulfilled for MUGB urokinase but not for MUGB t-PA. Whereas for the reaction of MUGB and urokinase the first-order acylation rate constant k2 was found to be about 10(6)-times higher than the first-order deacylation rate constant k3 (k2 = 3.76 X 10(-1) s-1, k3 = 3.7 X 10(-7) s-1), the k2/k3 ratio for the reaction of MUGB and t-PA (one- and two-chain form) was 0.77 to 3.85. Therefore, urokinase and t-PA differ in their reaction with this fluorogenic substrate and MUGB cannot be used for active-site titration of tPA.

Binding Sites↗

PubMed1987

Monoclonal antibodies against plasminogen and alpha-2-antiplasmin: binding to native and modified antigens.

Two monoclonal antibodies against plasminogen and two against alpha-2-antiplasmin were characterized regarding their binding properties towards their respective native and modified antigens and towards the plasmin-alpha-2-antiplasmin complex. Using an ELISA system, the two monoclonal antibodies against plasminogen (MPW1PG, MPW2PG), reacted equally well with purified Glu-plasminogen; binding of one (MPW1PG) to the purified plasmin-alpha-2-antiplasmin complex was, however, more than three orders of magnitude weaker than that of the other. Similarly, both antibodies against alpha-2-antiplasmin (MPW2AP, MPW3AP) reacted equally well with native alpha-2-antiplasmin but binding of MPW3AP to the plasmin-alpha-2-antiplasmin complexes was more than one order of magnitude weaker than that of the other. These results were confirmed by immunoblotting by which it was made evident that the antiplasminogen antibody MPW1PG reacted only with the free or complexed Glu-form of plasminogen or plasmin, while MPW2PG reacted equally well with the Glu- and Lys-form. The alpha-2-antiplasmin antibody MPW2AP reacted with native and complexed alpha-2-antiplasmin while MPW3AP reacted only with the native antigen and with a 8,000 dalton cleavage product of the complex.

Antibodies, Monoclonal↗

PubMed1987

Enhanced fibrinolysis caused by tissue plasminogen activator release in hemodialysis.

The effect of hemodialysis on components of the fibrinolytic system was measured in 22 patients. Plasma levels of t-PA antigen, t-PA activity, u-PA antigen and plasminogen activator inhibitor were determined by means of immunological and functional assays. During hemodialysis, 11 patients exhibited an increase in t-PA antigen within the first hour to about three times the starting values (P less than 0.05), followed by a decrease to about double of the initial values until the end of the treatment. Eleven patients showed a continuous increase up to 200% of the starting values until the end of hemodialysis. u-PA levels did not change significantly during the time of investigation (P greater than 0.5).

Adolescent↗

PubMed1987

Proliferation of a human epidermal tumor cell line stimulated by urokinase.

Several tumor cells secrete significantly increased amounts of the plasminogen activator urokinase, a trypsinlike serine protease, whose biological function in tumor biology is unclear. In this study we report that cells of the human epidermal tumor cell line CCL 20.2 express about 80,000 high-affinity urokinase receptors per cell that bind active as well as diisopropylfluorophosphate-treated high-molecular-weight (HMW) urokinase. Low-molecular-weight (LMW) urokinase is not bound to the receptor. Occupation of these receptors by active HMW urokinase stimulates cell proliferation independently in the presence of plasminogen in the culture medium. LMW urokinase has again no effect on cell proliferation. Calculated on a molar basis, this effect is about 28% of that of epidermal growth factor. Active HMW urokinase might therefore provide an autocrine receptor-mediated growth-promoting mechanism for tumor cells similar to those described for other growth factors.

Cell Division↗

PubMed1987

Pattern of fibrinolytic parameters in patients with gastrointestinal carcinomas.

Changes in the plasma levels of components of the fibrinolytic system have been investigated in 80 patients suffering from gastrointestinal carcinomas. Urokinase antigen (RIA), tissue-type plasminogen activator antigen (ELISA) and plasminogen activator inhibitor (functional assay) were determined. Patients with pancreatic and colorectal carcinoma and metastases as well as those without metastases revealed significantly increased plasma urokinase levels. Those with gall bladder or gastric carcinoma did not show significantly elevated urokinase antigen levels compared to age-matched controls. Determination of tissue-type plasminogen activator antigen in all four carcinoma groups did not reveal significant differences when compared to an age-matched healthy control group. The concentrations of plasminogen activator inhibitor were significantly increased in all carcinoma groups; there being no differences between the patient groups with or without metastases. No correlations between the different parameters of the fibrinolytic system could be obtained.

Adult↗

PubMed1987

Evaluation of fibrinolytic capacity by a combined assay system for tissue-type plasminogen activator antigen and function using monoclonal anti-tissue-type plasminogen activator antibodies.

An assay system has been developed that allows consecutive quantification of tissue-type plasminogen activator (t-PA) activity and t-PA antigen in the same plasma sample. In the first step t-PA is bound to an immobilized IgM monoclonal anti-t-PA antibody and functional activity of bound t-PA is quantified by its plasminogen-activating activity. In the second step the amount of bound t-PA antigen is determined by using a different peroxidase-labeled monoclonal anti-t-PA antibody. In this combined assay system t-PA functional activity was found to depend not only on the amount of t-PA antigen but also on the amount of plasminogen activator inhibitor (PAI), whereas in the t-PA antigen assay PAI did not affect the results. In plasma samples obtained from normal controls t-PA activity was detected only in post-venous occlusion plasma (3.7 +/- 2.5 IU/ml), whereas 2.7 +/- 0.5 ng/ml t-PA antigen was found before and 12.6 +/- 4.4 ng/ml after venous occlusion. Using this combined assay system to study plasma samples from patients who did not respond to venous occlusion with shortening of the euglobulin clot lysis time (ECLT), it was possible not only to confirm that in none of these patients could t-PA activity be detected in the postocclusion plasma samples but also to subdivide that group of patients into a group of about 39% not reacting with normal t-PA antigen release to venous occlusion and into a second group of about 61% that reacted with normal t-PA antigen release.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1986

Method for the determination of fast acting plasminogen activator inhibitor capacity (PAI-cap) in plasma, platelets and endothelial cells.

A titration assay for the determination of the fast acting plasminogen activator inhibitor capacity (PAI-cap) was developed. Most of the hitherto published assays for fast acting PAI are reported to have the disadvantage of non-parallel titration curves of tissue plasminogen activator (t-PA) in buffer and plasma. In the assay reported here, this problem has been overcome by adequate predilution of the samples, thus achieving parallel titration curves regardless of the individual PAI-cap of a sample. Provided that values are calculated from parallel titration curves, reproducible PAI-cap values at different dilutions of a sample are obtained. This assay can be applied for the determination of PAI-cap in plasma, serum and other biological fluids as platelet releasates and endothelial cell conditioned medium. PAI-cap of plasma of 10 healthy male volunteers ranged from 15.3 to 32.3 arbitrary inhibitor units AU/ml (24.5 +/- 5.2, mean +/- SD). The alternative use of three different anticoagulants (citrate, EDTA, heparin) had no influence on PAI-cap determinations. Serum generated from blood contained a mean of PAI-cap of 129% in comparison to the plasma of the same donor indicating the release of PAI from cells during clotting. Plasma PAI-cap changed during the day with a constant decrease from the highest levels in the morning (100%) to low levels in the evening (62.9%). Platelets aggregated by thrombin released plasminogen activator inhibitor amounting to a mean PAI-cap of 4.33 +/- 2.98 AU per 2.5 X 10(8) cells.

Anticoagulants↗

PubMed1986

Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor from the conditioned media of a human melanoma cell line.

24 established melanoma cell cultures were screened for their secretion of plasminogen activators and plasminogen activator inhibitors into the culture medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by conventional and reverse fibrin autography. Among the cell lines investigated, 22 cell lines predominantly secreting tissue type plasminogen activator (t-PA) and four cell lines additionally secreting urokinase were found. The conditioned media of two cell lines (KRFM and MJZJ) were found to contain plasminogen activator inhibitor (PAI) activity at a Mr position of approximately 50,000. The PAI of one of the two melanoma cell (MJZJ)-conditioned media found to contain PAI activity was purified to apparent homogeneity employing concanavalin A-Sepharose chromatography, gel filtration on Sephadex G-150, chromatography on Affi-Gel blue, and affinity chromatography on a Sepharose 4B immobilized monoclonal anti-t-PA IgG column. The purified melanoma PAI was found to be a single chain protein, acid stable, immunologically related to the endothelial derived PAI. In contrast to endothelial PAI, melanoma PAI presented itself in the conditioned media of the melanoma cells and in the purified preparation to an appreciable extent in its active form.

Cell Line↗

PubMed1986

Monoclonal antibodies against human high molecular weight urinary urokinase: application for affinity purification of urinary prourokinase.

Monoclonal antibodies against urinary urokinase were obtained by immunizing mice with purified human high molecular weight urokinase. Five antibodies were selected and denominated MPW1UK, MPW2UK, MPW3UK, MPW4UK, and MPW5UK, respectively. All selected antibodies reacted with high and low molecular weight urokinase. Cleavage of the low molecular weight paranitroanilide substrate pyro-Glu-Gly-Arg-pNA by urokinase was not inhibited by the antibodies and only one antibody (MPW5UK) inhibited plasminogen activation by urokinase. The ability of MPW5UK to bind to coated urokinase was 100-fold higher than that of the other antibodies. MPW5UK was used to prepare an immunosorbent for the purification of urokinase antigen from freshly voided crude urine. One-chain prourokinase was separated from two-chain urokinase by chromatography of the urokinase antigen containing mixture on agmatine Sepharose. As judged by SDS gel electrophoresis one-chain prourokinase as well as two-chain urokinase were purified to apparent homogeneity by this two-step procedure; the yields were 18% and 47% for single-chain prourokinase and two-chain urokinase, respectively, as calculated from total urokinase antigen contained in the starting material.

Animals↗

PubMed1986

Sandwich ELISA for t-PA antigen employing a monoclonal antibody.

A sandwich enzyme-linked immunosorbent assay (ELISA) for determination of tissue-type plasminogen activator (t-PA) was developed. 96-well flat-bottom polystyrene plates were coated with polyclonal (goat) anti t-PA IgG (10 micrograms/ml). After addition of samples monoclonal (murine) anti t-PA IgG (1.5 micrograms/ml) was added. Finally, peroxidase labelled anti-mouse IgG (goat) was used to quantify the bound second antibody. The assay can be used for determination of t-PA antigen in purified systems, in cell culture supernatants, and in human plasma, provided that EDTA (0.005 M) is present in the sample. In 78 healthy volunteers, t-PA antigen levels at rest were 0.4 - 15.2 ng/ml (4.3 +/- 2.7, means +/- S.D.); A significant positive correlation between t-PA antigen and age could be demonstrated.

Adult↗

PubMed1986

Hemodynamics of the isolated perfused rat kidney in the absence and presence of kallikrein substrate.

Rat kidneys were isolated and perfused for 120 minutes at 80 mm Hg with Krebs-Henseleit buffer containing 7 gm% bovine albumin fraction V. Analysis of the bovine albumin revealed the presence of a contaminating kallikrein substrate which exhibited a MW of 55,000 upon gel-filtration on Sephadex G-200, similar to the reported MW of bovine low molecular weight kininogen. The mean values for renal perfusate flow rate (RPF) and glomerular filtration rate (GFR) in kidneys perfused with this bovine albumin depleted of the kallikrein substrate were significantly lower when compared to standard perfusions. In two other groups of isolated kidneys perfused with kallikrein substrate depleted bovine albumin, purified bovine low molecular weight kininogen (bLMWK, 8.5 micrograms protein in 50 microliters per minute) or synthetic bradykinin (Bk, 1 ng in 10 microliters per minute) were infused after the first 30 minute clearance period into the renal artery. The induced hemodynamic changes were similar in that RPF and GFR significantly increased. These studies show that depletion of bovine albumin from kallikrein substrate results in a concomitant decrease of RPF and GFR, while overall renal function is unaffected. Infusion of both bLMWK or BK to kallikrein substrate depleted bovine albumin has similar effects resulting in a simultaneous increase in RPF and GFR. This suggests that endogenous kallikrein from the perfused kidney releases kinin from infused bLMWK and indicates that kinin primarily affects the afferent arterioles.

Animals↗

PubMed1986

Urinary kallikrein excretion in healthy young infants.

Nineteen healthy hospitalized children aged between 3 weeks and 10 years and 20 others aged between one month and 16 years have been investigated for their excretion rate of active and total urinary kallikrein. Twelve hour urine samples were obtained between 7 p.m. and 7 a.m. and blood was drawn at the end of the urine collection period. Urinary kallikrein activity was measured by a synthetic substrate and a direct RIA and urinary sodium excretion, urine volume and plasma renin activity (PRA) were determined. Urinary kallikrein activity was found to be between 0 and 6 micrograms/24h and constituted approximately 10-20% of total kallikrein. When urinary kallikrein excretion rate was correlated with the sodium excretion rate the relationship was found to be positive and significant as was the correlation found between urine volume and urinary kallikrein excretion rate. No correlation could be found between PRA and urinary kallikrein excretion. Although a tendency of higher total kallikrein excretion was seen in older children, the amount excreted from all children per kilogram body weight was constant at 0.8 microgram/kg.

Child↗

PubMed1986

Urinary kallikrein excretion during inhibition of endogenous angiotensin II in the pig.

This study was performed to assess the possible contribution of endogenous angiotensin II (AII) to the regulation of urinary kallikrein excretion. The AII antagonist saralasin or the saline vehicle was infused into the aorta above the renal arteries of pigs under halothane-O2/N2O anaesthesia. Systemic and renal functional parameters were followed for 140 min and during stimulation of the reninangiotensin system by haemorrhage. Urinary kallikrein excretion, determined as kininogenase activity, was increased immediately upon both initiation and termination of the 2 h saralasin infusion into pigs not subjected to haemorrhage. Renal cortical blood flow (RCBF) was maintained, in both saline and saralasin-treated animals at blood pressures as low as 70 mm Hg, while glomerular filtration rate was dissociated during saralasin infusion. As long as RCBF was maintained, urinary kallikrein excretion rate was elevated during the progressive hypotension in both saline and saralasin-treated animals. These findings confirm a close relationship between the maintenance of RCBF and increased activity of the kallikrein-kinin system whether or not AII is antagonized, and indicate that during haemorrhage the kallikrein-kinin system is stimulated by a mechanism not involving AII.

Angiotensin II↗

PubMed1986

Nonenzymatic glucosylation as a contributing factor to defective fibrinolysis in diabetes mellitus.

Kinetics of activation of plasminogen purified from the plasma of 3 uncontrolled diabetic patients by tissue type plasminogen activator revealed substrate inhibition in the fibrin-stimulated system. After improvement of metabolic parameters activation of plasminogen was found to be normal in 1 of these patients and partially improved in another patient. Studies performed to investigate a possible nonenzymatic glucosylation of plasminogen showed that purified control plasminogen incorporates 14C-glucose in a dose- and time-dependent manner and that in vitro glucosylation of control plasminogen results in functional abnormalities of plasminogen, which resemble those observed with plasminogen from diabetic patients as far as substrate inhibition in the fibrin-stimulated system is concerned.

Carbon Radioisotopes↗

PubMed1986

[The vascular wall and fibrinolysis--physiologic aspects].

It is the aim of the present review to analyze from a theoretical point of view the functioning of the fibrinolytic system which would be optimally beneficial for the organism. Such a theoretically designed system is further compared with the fibrinolytic system in man. In fact, for an optimal function of the fibrinolytic system fibrin-directed, coagulation-independent activation is required. Furthermore, cross-linked fibrin, necessary for repair mechanisms, should be less attacked by the fibrinolytic system and, in contrast to the injured vessel wall, the intact endothelial cell should provide mechanisms favoring fibrinolysis. These requirements are fulfilled in vivo by the special molecular design of the fibrinolytic system.

Animals↗

PubMed1985

Chromogenic substrate autography: a method for detection, characterization, and quantitative measurement of serine proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing in polyacrylamide gels.

A chromogenic substrate autography is described which allows characterization and quantification of serine proteases in crude systems after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. Separation of samples containing proteases by either method is followed by an overlay of the gels on an indicator film prepared by incorporation of a suitable paranitroanilide substrate into agarose. Positions of the proteases are revealed by the formation of yellow-colored zones which can be quantified by densitometry at 405 nm. The technique proved suitable for determination of molecular weights, isoelectric points, and quantitative measurements of amidolytic activities of urokinase, tissue plasminogen activator, tissue and plasma kallikrein, and thrombin in biological fluids and purified preparations.

Animals↗

PubMed1985

Urokinase antigen in plasma of patients with liver cirrhosis and hepatoma.

Plasma urokinase antigen levels were studied in 78 patients suffering from liver diseases. Blood was drawn before any specific medication was initiated. Impairment of liver function was comparable in all patients. In both groups of cirrhotic liver disease (alcoholic and non-alcoholic), normal levels of plasma urokinase antigen were found as compared to age-matched control groups. In both groups of patients with hepatomas (with or without a history of liver cirrhosis), however, significantly increased plasma urokinase antigen levels could be determined. These data indicate that an increase in plasma urokinase antigen might rather relate to malignant growth in liver disease than to impaired liver function.

Adult↗