Biomedical subjects

O F Wagner

Publications and source records attributed to O F Wagner.

43 records · Page 3Linked to original sources

PubMed1990

Plasma concentrations of endothelin in man: arterio-venous differences and release during venous stasis.

Endothelin-1 is a recently described endothelium-derived vasoconstricting peptide. Plasma concentrations of immunoreactive (IR-) endothelin were investigated in six healthy young men applying a radioimmunoassay after extraction of endothelin from plasma. In venous plasma a mean concentration of 1.3 +/- 0.4 pmol l-1 was found, whereas the mean concentration in arterial plasma was 0.9 +/- 0.4 pmol l-1 (P less than 0.005). During venous stasis for 10 min the mean plasma concentration of IR-endothelin increased about twofold, from basal 1.1 +/- 0.3 pmol l-1 to 2.1 +/- 0.3 pmol l-1 (P less than 0.01). This manoeuvre may prove helpful to investigate the control of endothelin in vivo under a variety of pathological conditions.

Adult↗

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

Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA.

Plasminogen activation is catalyzed both by tissue-type-(t-PA) and by urokinase-type plasminogen activator (u-PA). This reaction is controlled by plasminogen activator inhibitor type 1 (PAI-1) that is either present in plasma or bound to fibrin, present in a thrombus. We studied the mechanism of in vitro inhibition of both t-PA and u-PA activity by PAI-1 bound to fibrin. It is shown that activation of latent PAI-1 unmasks a specific fibrin-binding site that is distinct from its reactive site. This reactive site of activated PAI-1 bound to fibrin is fully exposed to form complexes with t-PA and u-PA, that are unable to activate plasminogen. Upon complex formation with either one of the plasminogen activators, PAI-1 apparently undergoes a conformational change and loses its affinity for fibrin. Consequently, complexes of u-PA and PAI-1 dissociate from the fibrin matrix and are encountered in the fluid phase. In contrast, t-PA/PAI-1 complexes remain bound to fibrin. By employing recombinant t-PA deletion-mutant proteins, that precisely lack domains involved in fibrin binding, we demonstrate that binding of t-PA/PAI-1 complexes is mediated by both the "finger" (F) and the "kringle-2" (K2) domain of t-PA. A model is proposed that explains inhibition of the fibrinolytic process, at the level of plasminogen activation by t-PA, directed by PAI-1 bound to fibrin. An implication of the proposed model is that t-PA/PAI-1 complexes and free t-PA compete for the same binding sites on fibrin.

Animals↗

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↗

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

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↗

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↗