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Biomedical subjects

B R Binder

Publications and source records attributed to B R Binder.

At least 163 records · Page 9Linked to original sources
PubMed1988

Clot lysis mediated by cultured human microvascular endothelial cells.

The lysis of fibrin clots on the surface of cultured human omental tissue microvascular endothelial cells (HOTMEC) and cultured human umbilical vein endothelial cells (HUVEC) was studied. Fibrin clots were made by mixing fibrinogen, plasminogen and thrombin on the surface of both cell types. Clot lysis was seen only on the surface of HOTMEC, which were found to synthesize about 100-fold more tissue plasminogen activator (tPA) antigen than HUVEC. Clot lysis of HOTMEC could be blocked by anti-tPA IgG but was not affected by the incorporation of exogenous plasminogen activator (PAI) into the clot in concentrations (75 arbitrary units) exceeding the tPA activity (21 +/- 2.5 IU) of the cells. Thus, it is likely that tPA secreted by HOTMEC is protected from inhibition by PAI in the presence of fibrin and endothelial cells. The stimulation of EC to release an excess of tPA over PAI, in contrast to the secretion of an excess of PAI over tPA found in unstimulated cells in the absence of fibrin, is obviously no prerequisite for the initiation of fibrinolysis on the surface of HOTMEC. As thrombin was used for clot formation, its influence on tPA and PAI synthesis of both cell types was investigated. In contrast to HOTMEC, which were not affected by alpha-thrombin, HUVEC revealed a dose-dependent increase in tPA and PAI synthesis upon incubation with the enzyme. This increase in tPA production by HUVEC was not sufficient to lyse the clots within 48 hours. Furthermore, HUVEC behaved differently towards thrombin as these cells in contrast to HOTMEC revealed the typical shape change reaction upon incubation with the enzyme.

Capillaries↗
PubMed1988

Correlation of two different assays for urinary kallikrein in normotensive and hypertensive subjects.

Possible differences in structure-function relationship of urinary kallikrein between normotensive and hypertensive individuals were analysed using two different assay systems which detect two distinct entities of the enzyme. A monospecific goat anti-human urinary kallikrein antibody was characterized by inhibition studies with the purified active enzyme and by trypsin activation of endogenous urinary prokallikrein. Analysis of the data revealed that the antibody is directed against active kallikrein by recognizing an epitope which is different from the catalytic site of the enzyme but which is being exposed together with the active site during trypsin activation of the proenzyme. A direct radioimmunoassay for urinary kallikrein was developed and correlated with the kinin generating activity of the enzyme by assessing endogenous active and trypsin activated kallikrein in the urine of normotensive and hypertensive subjects. Significant positive correlations were found between the two assays for both active and total kallikrein in normotensive and hypertensive subjects and the slopes of the respective regression lines were identical. These data do not provide evidence for a defective enzyme, a defective activation of the proenzyme or for the presence of an inhibitor of urinary kallikrein in essential hypertension.

Adult↗
PubMed1988

[Persistent changes in tissue-type plasminogen activator and plasminogen activator inhibitor fibrinolytic parameters in patients following juvenile ischemic cerebral infarct].

In diseases associated with thrombotic or thromboembolic complications, a reduction in the fibrinolytic potential may contribute to the risk to develop thrombosis. To investigate whether juvenile cerebral infarction is associated with a permanent defect of the fibrinolytic system we measured the main components of the fibrinolytic system, tissue plasminogen activator (t-PA) and its fast acting inhibitor (PAI) in plasma samples of 21 patients (aged 21-44 years) 3-24 months after the acute event. The data obtained were compared to those from thirteen healthy young volunteers (22-46 years). A direct effect of known risk factors on the fibrinolytic system could be excluded because patients avoided their risk factors immediately after the ischemic cerebral attack. Hypertension and the combination of oral contraceptives and smoking had been the most striking original risk factors. Levels of t-PA antigen and t-PA activity before and after venous occlusion, or PAI activity were not different between patients and controls suggesting that at least a permanent decrease in the activity of the fibrinolytic system does not exist in these patients. However, our findings do not exclude that a temporary defect in fibrinolysis might have contributed to the acute onset of the thrombotic cerebral event possibly induced by the risk factors originally present.

Adult↗
PubMed1988

Plasminogen activation by tissue plasminogen activator in the presence of stimulating CNBr fragment FCB-2 of fibrinogen is a two-phase reaction. Kinetic analysis of the initial phase of slow plasmin formation.

Plasminogen activation by tissue-type plasminogen activator (t-PA) is stimulated by fibrin. In a purified system maximal fibrin-enhanced plasmin formation occurs with a delay after an initial phase of slow plasmin formation (lag phase). In the present study purified stimulating CNBr-fragment FCB-2 of fibrinogen was used, and kinetics of plasminogen activation by t-PA were analyzed with respect to the lag phase. At constant FCB-2 concentration the duration of the lag phase decreased with increasing concentrations of t-PA and plasminogen. During this period the rate of plasmin formation/min increased linearly with time with a slope dependent on the initial concentrations of FCB-2, plasminogen, and t-PA. Plasmin pretreatment of FCB-2 resulted in a dose- and time-dependent shortening of the lag phase, and at plasmin concentrations greater than or equal to 1 nM and preincubation times greater than or equal to 3 min maximal plasmin formation occurred without a lag phase. Kinetics during the phase of maximal and constant plasmin formation were not influenced by plasmin pretreatment of FCB-2. We therefore conclude that maximal t-PA-dependent plasmin formation in a system stimulated by purified FCB-2 requires plasmin modification of FCB-2.

Cyanogen Bromide↗
PubMed1988

Identification of [hydroxyproline3]-lysyl-bradykinin released from human kininogens by human urinary kallikrein.

The types of kinins released from purified native, single chain human high and low molecular mass kininogens (HMMKs and LMMKs, respectively) by purified human urinary kallikrein were separated by reverse-phase HPLC and quantitated by the rat uterus bioassay. [Hyp3]-lysyl-bradykinin, a recently discovered kinin, represented up to 58% of the biological activity released from 4 individual HMMK preparations purified from 4 different healthy volunteers. In contrast, the majority of the biological activity released from LMMKs purified from pooled plasma was identified as Lys-bradykinin and [Hyp3]-lysyl-bradykinin represented only 6.4 +/- 3.8%. These findings indicate posttranslation hydroxylation of human kininogens and suggest a preference of HMMKs for this modification.

Amino Acid Sequence↗
PubMed1988

Urokinase-type plasminogen activator as a marker for the formation of distant metastases in prostatic carcinomas.

Plasma levels of urokinase-type plasminogen activator have been investigated in 80 patients with prostatic carcinoma by means of a radioimmunoassay. A total of 30 patients with disseminated prostatic carcinoma had significantly elevated levels of urokinase-type plasminogen activator, whereas the plasma levels in patients without metastases did not differ from a healthy age matched control group. Sensitivity of elevated urokinase-type plasminogen activator levels in patients with prostatic carcinoma for the presence of metastases was 80 per cent. Therefore, urokinase-type plasminogen activator appears to be a reliable marker for the formation of metastases in prostatic carcinoma.

Acid Phosphatase↗
PubMed1988

Mitogenic effect of urokinase on malignant and unaffected adjacent human renal cells.

Primary cultures of renal cell carcinomas and of the corresponding normal adjacent kidney tissue from 6 patients were analyzed for the effects of exogenously added urokinase-type plasminogen activator on cell proliferation as compared to the effects of tissue type plasminogen activator, plasmin and dihydrocortisone. Cell proliferation was studied over a period of up to 5 days by measuring 3H-thymidine incorporation as well as cell viability and cell count; conditioned media of the cultures were also analyzed for their plasminogen activator and plasminogen activator inhibitor content. Addition of urokinase stimulated cell proliferation in a time and dose dependent fashion; after 3 days 3H-thymidine incorporation was significantly increased in malignant renal cells (188.3 +/- 28.7%), while it reached in normal renal cells approximately 130% of the 3H-thymidine incorporation of untreated cultures. Tissue-type plasminogen activator had no effect and plasmin decreased cell proliferation slightly while dihydrocortisone inhibited cell proliferation significantly (34.1 +/- 4.9%) in malignant cells. It is concluded that urokinase-type plasminogen activator itself exhibits a mitogenic effect also on primary cultures of renal cell carcinomas.

Adult↗
PubMed1988

Elevated tissue type plasminogen activator in human granulosa cells correlates with fertilizing capacity.

An increased production of plasminogen activators, able to convert plasminogen into plasmin, has been found in experiments in vivo on rat ovarian granulosa cells at the time of ovulation, indicating an involvement in follicular rupture. The granulosa cells of 49 follicles from 20 patients undergoing in-vitro fertilization were obtained by laparoscopy and tested for the content of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and inhibitor of plasminogen activator (PAI). In the respective follicular fluids the concentrations of oestradiol (E2), progesterone (P) and testosterone (T) were determined and the levels of these enzymes and of the follicular steroid content were related to the fertilizing behaviour of the respective oocytes. Follicles containing oocytes which could be fertilized, revealed significantly higher follicular fluid E2 and P levels and significantly lower T levels than follicles with unfertilized oocytes. The respective granulosa cells of fertilized oocytes exhibited higher levels of t-PA compared to their unfertilized counterparts, whereas no significant difference occurred in the levels of u-PA and PAI. These data suggest that successful fertilization of human oocytes is associated with a high content of t-PA in granulosa cells and high E2 and P levels in the follicular fluid.

Adult↗
PubMed1988

Competition of activated protein C and urokinase for a heparin-dependent inhibitor.

Human urine contains a hitherto unrecognized heparin-dependent inhibitor of activated protein C (APC) (Mr approximately 50,000) that coelutes from heparin-Sepharose together with the only observed peak of urokinase inhibitory activity at a position (0.35 M NaCl) similar to that of plasma protein C (PC) inhibitor. Based on functional assays and immunoblot studies, urokinase and APC compete for this crude inhibitor in the absence or presence of heparin. These results suggest that the same heparin-dependent urinary inhibitor that is immunologically different from several known protease inhibitors is responsible for the observed inhibition of APC and urokinase. In the absence of heparin this inhibitor inhibits APC and urokinase with similar rates, and heparin enhances its inhibitory activity toward both enzymes with more pronounced stimulation of its PC inhibitory activity than its urokinase inhibitory activity. Half-maximal stimulation of inhibition of APC occurs at about 2 mU/ml and maximal stimulation (approximately 10-fold increase of the pseudo-first-order rate constant) at greater than or equal to 50 mU/ml of heparin. This is the first demonstration of competition between APC and urokinase for a heparin-dependent inhibitor. These results may therefore represent a new link between the two major antithrombotic pathways, the PC pathway and the fibrinolytic system.

Chromatography, Affinity↗
PubMed1988

Plasminogen activators and plasminogen activator inhibitor in malignant and non-malignant ascitic fluid.

Ascitic fluid from tumour patients (hepatoma, gastric cancer, gallbladder cancer, colorectal cancer, ovarian cancer) and from non-malignant diseases (liver cirrhosis, congestive heart failure) were compared with respect to their content of determinants of the fibrinolytic system, tissue-type plasminogen activator antigen (t-PAag) and activity (t-PAact), urokinase-type plasminogen activator antigen (u-PA) and plasminogen activator inhibitor activity (PAI). Furthermore, SDS-polyacrylamide slab-gel electrophoresis (SDS-PAGE) was performed to evaluate molecular weight distribution of the detectable fibrinolytic parameters. In malignant ascites, PAI activity was three to four times higher, and increased complex formation of PAI with t-PA could be demonstrated, compared with non-malignant ascitic fluid. Tissue-type plasminogen activator antigen and activity showed a similar concentration in ascites of both study groups. Urokinase-type plasminogen activator antigen was detectable neither in ascites of malignant nor in ascites of non-malignant origin. It is concluded that t-PA is the physiological plasminogen activator in ascites and that increased PAI levels followed by increased complex formation between t-PA and PAI might reflect a reaction of the peritoneum.

Adult↗
PubMed1988

Comparison of fibrinolytic activities of human and bovine endothelial cells.

Human aortic (HAE), human umbilical vein (HUVE), and bovine aortic (BAE) endothelial cells were compared in their synthesis and release of fibrinolytic components during culturing. After isolation, the cultures were grown to confluency and then studied under identical conditions for release of tissue plasminogen activator (t-PA) antigen and plasminogen activator inhibitor (PAI) into serum-free medium. HAE cells released 10 times more t-PA antigen than HUVE cells, and the respective cell lysates also contained comparably higher values. Free PAI capacity was found in the conditioned media of both HAE and HUVE cells. BAE cell t-PA release was much lower than that of the HAE cells, and free inhibitor capacity was not found in the conditioned medium. BAE cells contained significant amounts of PA activity in cell membrane-bound form. This PA activity on the cell surface was not stimulated by addition of CNBr fibrinogen fragments but could be partially inhibited by activated bovine PAI and antibodies against human t-PA and urokinase PA, respectively.

Animals↗
PubMed1988

Effect of anti plasminogen monoclonal antibodies on whole blood clot lysis.

Several monoclonal antibodies (MABs) against specific parts of the plasminogen molecule were developed. One of them, MPW1PG, recognizes only the native form Glu-plasminogen, while the other one, MPW2PG, reacts equally well with Glu-plasminogen and its proteolytically degraded derivative Lys-plasminogen as confirmed by immunoblotting experiments. Both MABs do not alter the cleavage of the low molecular weight substrate S-2251 by plasmin in a purified system but rather increase the rate of plasmin formation by urokinase and t-PA in a system without fibrin. In the presence of fibrin MPW1PG has no effect on plasmin formation by t-PA, while MPW2PG exhibits mixed type inhibition. To investigate whether this effect can also be seen in a whole blood clot lysis system, the Chandler loop was used (PVC tubes, 4 mm inner diameter, 28 mm length), 125I-fibrinogen was added to citrated whole blood (0.36% sodium citrate) to about 5,000 cpm/10 microliter sample. 1.5 ml of the mixture were pipetted into each tube, the tubes were closed, put on a rotary plate, tilted to an angle of 23 degrees, and rotated at 16 rpm. 10 microliter samples were taken for radioactive counting before clotting by addition of 10 microliter 3.02M CaCl2 (100%-value), 45 min after clotting (0%-value), and 30, 60, 120, 180, 360 minutes after addition of activators. Lysis was started one hour after recalcification. Different amounts of MABs were added either 30 min before recalcification (MAB endogen) or together with the activator (MAB exogen) Percent lysis was calculated from the radioactivity released into the fluid phase. Double determinations were done in all experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1988

Plasma urokinase-type plasminogen activator correlates to bone scintigraphy in prostatic carcinoma.

Urokinase-type plasminogen activator (u-PA) levels in the plasma of 35 patients suffering from disseminated prostatic carcinoma have been determined. According to the grade of dissemination, found by bone scintigraphy, the patients were divided into three groups. Patients with a higher degree of dissemination revealed significantly higher levels of plasma u-PA compared to patients with a lower degree of dissemination. Therefore plasma u-PA seems to be a possible marker for progression in patients suffering from disseminated prostatic carcinoma.

Aged↗
PubMed1988

Plasminogen activation in diabetes mellitus. Kinetics of plasmin formation with tissue plasminogen activator and plasminogen from individual diabetic donors and with in vitro glucosylated plasminogen.

Functional abnormalities of tissue plasminogen activator (t-PA) (high Km in the presence of fibrin) and plasminogen (Pg) (substrate inhibition in the fibrin-stimulated system) from uncontrolled type I diabetics are reversible upon normalization of metabolic parameters. Therefore the effect of in vitro glucosylation of Pg was studied and similar but less pronounced substrate inhibition as with diabetic Pg was observed. However, the activation of the Pg fraction most likely containing bound cis-OH groups (e.g. glucose) was normal. These data suggest that either glucosylation in vitro is less stable than in vivo or that in diabetics protein modifications other than glucosylation occur. In this respect we can also show that acetone, a representative of ketone bodies and also elevated in poorly controlled diabetics, does not affect Pg activation in vitro.

Diabetes Mellitus, Type 1↗
PubMed1988

Increased plasma levels of urokinase-type plasminogen activator with endometrial and cervical cancer.

Plasma concentrations of urokinase-type plasminogen activator (competitive radioimmunoassay), tissue-type plasminogen activator (sandwich enzyme-linked immunosorbent assay), and plasminogen activator inhibitor (functional assay) were measured in 17 women with endometrial cancer and 52 women with cervical carcinoma. Significantly increased plasma urokinase-type plasminogen activator antigen levels were found (11.3 +/- 4.7 ng/mL) in cervical cancer patients when compared with an age-matched control group (7.4 +/- 0.6 ng/mL). Women with endometrial cancer (9.9 +/- 2.0 ng/mL) showed a similar pattern of plasma urokinase-type plasminogen activator antigen levels. Patients with advanced cervical cancer (International Federation of Gynecology and Obstetrics stages III and IV) revealed higher plasma urokinase-type plasminogen activator antigen levels than those with stages I and II disease. Compared with an age-matched control group, neither carcinoma group showed elevated plasma concentrations of tissue-type plasminogen activator and plasminogen activator inhibitor.

Adenocarcinoma↗
PubMed1988

Reduction in pO2 decreases the fibrinolytic potential of cultured bovine endothelial cells derived from pulmonary arteries and lung microvasculature.

The effect of anoxia on the fibrinolytic potential of cultured endothelial cells derived from bovine pulmonary artery and bovine lung microvasculature was studied. Both cell types reacted with an increase in plasminogen activator inhibitor (PAI) activity and a decrease in the plasminogen activator (PA) activity in the media after incubation under anoxic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fibrin autography and reverse fibrin autography indicated that the change in fibrinolytic potential was due to an impaired release of PA and not an increase in the production of PAI. Although anoxia did not affect the viability of the cells as judged by 51Cr release, their metabolism was influenced, which is reflected by increases in the levels of lactate in cell lysates and media. Furthermore, the effect of short-term anoxia on PA and PAI could not be reversed by reoxygenation for 24 hours. The results are discussed in terms of helping to explain the tendency of reocclusion after successful thrombolytic therapy, the development of pulmonary hypertension, and the thrombotic tendency of areas with an impaired circulatory supply.

Animals↗
PubMed1987

Effect of urokinase on the proliferation of primary cultures of human prostatic cells.

The effects of exogenously added urokinase type plasminogen activator, tissue type plasminogen activator, plasmin and thrombin on the proliferation of primary cultures of cells derived from prostatic hyperplasia or prostatic carcinomas were investigated by measuring the incorporation of 3H-thymidine into the cultures. Addition of urokinase type plasminogen activator (1.35 x 10(-9) M) or thrombin (10(-7) M) to the culture medium caused a two-fold increase of 3H-thymidine incorporation, regardless of the origin of the prostatic cells. Tissue type plasminogen activator did not alter the rate of 3H-thymidine incorporation, whereas plasmin caused a 25% decrease of 3H-thymidine incorporation in all cultures.

Cell Division↗