Biomedical subjects

T C Wun

Publications and source records attributed to T C Wun.

62 records · Page 4Linked to original sources

PubMed1987

Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis.

A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above.

Acids↗

PubMed1987

Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level.

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA-inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT-1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI-2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.

Antigens↗

PubMed1985

Human leucocyte urokinase inhibitor--purification, characterization and comparative studies against different plasminogen activators.

A plasminogen activator inhibitor (PA-I) which inhibits primarily plasminogen activator of the urokinase type (u-PA) was isolated from the cytosol of human peripheral leukocytes. The inhibitor was isolated using ion exchange chromatography, gel filtration and FPLC. This inhibitor has an apparent molecular weight of 45 kDa, determined by SDS-PAGE, and a pI of 5.5-5.7. The inhibitor is a fast reacting inhibitor, is thermally unstable and is inactivated outside the pH range 7-9. Treatment of cytosol to pH 9 for 30 min at 37 degrees C resulted in a large increase in inhibitory activity. Antibodies against human placental UK-I completely quenched the inhibitory activity of human leucocyte UK-I.

Blood Proteins↗

PubMed1985

Spontaneous fibrinolysis in whole human plasma. Identification of tissue activator-related protein as the major plasminogen activator causing spontaneous activity in vitro.

Spontaneous fibrinolysis of plasma clots was studied by following the lysis of the clots formed in 125I-fibrinogen-supplemented citrated plasma. Lysis of the clots invariably follows sigmoidal kinetics with S50 (the time required for 50% clot lysis) ranging from 3.5 to 4.7 days in 8 samples of pooled blood bank plasma and in the majority of apparently healthy donor plasmas. The spontaneous lysis of factor XII-deficient and prekallikrein-deficient plasmas was found to be similar to that of normal plasma. Addition of ellagic acid or antibodies against kallikrein or urokinase to normal pooled plasma did not alter significantly its rate of spontaneous lysis. On the other hand the addition of antibody against tissue activator (t-PA) inhibited over 80% of the spontaneous fibrinolysis in a 7-day incubation period at 37 degrees C, and the clot visually persisted for more than a month. Therefore, the factor XII-dependent components and prourokinase/urokinase system do not contribute significantly in whole plasma fibrinolysis in vitro, while the t-PA-related protein appears to be the major plasminogen activator responsible for initiating spontaneous fibrinolysis in whole plasma. Exogenous addition of increasing amounts of purified HeLa cell t-PA to normal pooled plasma in the ng/ml range cause progressively faster clot lysis. By extrapolation, normal pooled plasma is found to contain endogenous tissue activator in an amount functionally equivalent to 2 ng/ml of purified 60-kDa t-PA. The molecular nature of the t-PA-related proteins in plasma was studied by zymographic and immunological methods. The major t-PA-related protein in plasma was found to have a molecular mass of 100 kDa as determined by zymography. By incubating purified HeLa 60-kDa t-PA with a t-PA-depleted plasma, the 100-kDa component can be generated in plasma, suggesting that the latter is formed as a result of the binding of 60-kDa t-PA to a binding protein in plasma.

Antibodies↗

PubMed1982

A proenzyme form of human urokinase.

A culture of the human epidermoid carcinoma HEp 3 produces a plasminogen activator of Mr = 53,000 which we have purified to apparent homogeneity from serum-free conditioned medium by the combination of immunoaffinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The highly purified protein has the following properties: 1) It is indistinguishable from urinary urokinase in electrophoretic mobility, in immunodiffusion, and in autoradiographically visualized tryptic peptide maps obtained from the 125I-labeled proteins. 2) The HEp 3 protein differs from urinary urokinase in the following respects: (a) although the apparent molecular weights of the two are identical (Mr = 53,000), the urinary enzyme consists of two polypeptide chains, whereas the HEp 3 protein is a single chain form. (b) Urinary urokinase can be labeled easily by incubation with radioactive diisopropylfluorophosphate but the HEp 3 protein cannot. (c) When assayed by the hydrolysis of a synthetic chromogenic peptide substrate, the HEp 3 enzyme has less than 1% of the catalytic activity of urinary urokinase. 3) On controlled exposure to plasmin, the HEp 3 protein is converted to an active enzyme that is identical with urinary urokinase in molecular weight, polypeptide chain composition, diisopropylfluorophosphate labeling, and specific catalytic activity. We conclude that the HEp 3 protein is a proenzyme that can be converted to active two-chain urokinase by plasmin, probably by a single proteolytic nick in the polypeptide chain.

Carcinoma, Squamous Cell↗

PubMed1982

Isolation and characterization of urokinase from human plasma.

The presence of activators of the fibrinolytic system in blood plasma has been assumed for a long time but never convincingly documented by the isolation of characterized and physiologically plausible enzymes. The low catalytic efficiency of previously identified plasma plasminogen activators, which has made their physiological significance uncertain, prompted us to search for other plasma enzymes, resembling especially the potent urinary activator, urokinase. We report here the detection of a urokinase-like activity in human plasma, and the isolation of the enzyme from whole plasma protein fractions. The purified enzyme is indistinguishable from the 53,000-dalton components of human urinary urokinase in the following respects: apparent Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, double immunodiffusion, amino acid analysis, two-dimensional tryptic peptide maps, catalytic efficiency with synthetic peptide substrates, and inhibitor spectrum. The results suggest (a) that the enzyme is present in plasma in a latent form whose nature remains to be defined, and (b) that the circulating concentration is at least 5 to 10 microgram/liter, and sufficient to generate substantial levels of plasmin, particularly if activation were somehow confined by localization at specific sites.

Amino Acids↗

PubMed1977

Binding properties of neutral diamide ligands for alkaline-earth cations.

The complexation of a series of aromatic and alicyclic N,N,N',N'-tetra-n-propyl amides of 1,2-ethylenedioxydiacetic acids with group IIA metal-ion bromides in anhydrous methanol was investigated by ultraviolet absorption spectroscopy. These synthetic ligands were previously found to show selectivity toward divalent over monovalent cations with respect to extraction of ions into bulk organic phase (Borowitz, I.J., Lin, W-O., Wun, T-C., Bittman, R., Weiss, L., Diakiw, V., and Borowitz, G.B. (1977), Tetrahedron, in press). At low concentrations, ligands bearing benzene and naphthalene rings form 1:1 ligand to divalent cation complexes with each of the alkaline-earth metals, but ligands in the cyclohexyl series are stoichiometrically bound to cations in more than one type of complex. Binding isotherms obtained by Scatchard analysis and by the method of continuous variation revealed ligand to divalent ion mole ratios of 2:1, 3:2, and 4:3 for binding of N,N,N',N'-tetra-n-propyl-cis-1,2-cyclohexanedioxydiacetamide with Ca2+, Sr2+, and Ba2+, respectively. In contrast, Scatchard analysis of ultraviolet spectral changes showed that a 1:1 complex is formed between this ligand and Na+ with an apparent association constant of 56 +/- 2M-1; the constant for binding with K+ was smaller (11 M-1). The order of apparent association equilibrium constants for complexation of group IIA cations with this series of neutral ligands was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+; for example, for N,N,N',N'-tetra-n-propyl-1,2-phenylenedioxydiacetamide the apparent binding constants at 25 degrees C were 7.33 +/- 0.25 X 10(4) M-1 for Ca2+, 1.23 +/- 0.03 X 10(4) for Sr2+, 4.42 +/- 0.09 X 10(3) for Ba2+, and 4.04 +/- 0.24 X 10(2) for Mg2+. The divalent cation binding properties of these synthetic diamide ligands are discussed in relation to those of other synthetic ligands and of two naturally occurring ligands.

Azo Compounds↗

PubMed1977

Ionophorous properties of neutral diamide ligands toward calcium.

The ability of a series of aromatic and alicyclic analogues of 1,2-ethylenedioxydiacetic acids bearing N,N,N',N'-tetra-n-propyl amide or N-methyl-N-carbethoxypentyl amide linkages to enhance the rate of 45Ca2+ efflux from vesicles was studied. The ligands were less potent in enhancing membrane permeability to Ca2+ than A23187 and X537A. Lipid-soluble anions markedly increased the rate and extent of Ca2+ transport mediated by these neutral ligands. The abilities of these synthetic diamide ligands and naturally occurring ionophores to transport Ca2+ across bilayer membranes were sensitive to the lipid composition of the vesicle. The mechanism of Ca2+ transport mediated by this series of synthetic ligands is discussed.

Azo Compounds↗