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Biomedical subjects

G Wijngaards

Publications and source records attributed to G Wijngaards.

At least 19 recordsLinked to original sources
PubMed2001

Purification and substrate specificity of transglutaminases from blood and Streptoverticillium mobaraense.

A procedure for a fast and simple purification of bovine plasma transglutaminase was developed, which resulted in a homogeneous enzyme preparation. Two different procedures were developed for the purification of pig erythrocyte transglutaminase, both of which resulted in partial purification. Both enzymes were used in cross-linking reactions of alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, casein, hemoglobin, glycinin, and myosin. The substrate specificity was compared to that of bacterial transglutaminase isolated from Streptoverticillium mobaraense. The bacterial transglutaminase caused cross-linking of a wider range of proteins and, thus, exhibited a lower substrate specificity than the blood transglutaminases. In addition, differences exist in the necessity of the addition of reducing agents. These differences allow specific applications of blood and bacterial transglutaminases at protein cross-linking in single or complex protein systems.

Animals↗
PubMed2001

Effects of pH and heat treatments on the structure and solubility of potato proteins in different preparations.

The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.

Calorimetry, Differential Scanning↗
PubMed1987

alpha 2-Antiplasmin Enschede: dysfunctional alpha 2-antiplasmin molecule associated with an autosomal recessive hemorrhagic disorder.

alpha 2-Antiplasmin (alpha 2-AP) is a major fibrinolysis inhibitor, whose complete, congenital absence has been found to be associated with a distinct hemorrhagic diathesis. We studied a 15-yr-old male with a hemorrhagic diathesis after trauma from early childhood on. This bleeding tendency was associated with a minimal alpha 2-AP level recorded functionally in the immediate plasmin inhibition test: less than or equal to 4% of normal. However, a normal plasma concentration of alpha 2-AP antigen (83%) was found. His sister (5 yr old) showed similar results (2 and 92%). In their family, eight heterozygotes could be identified by half-normal activity results and normal antigen concentrations. The inheritance pattern is autosomal recessive. On analysis, the alpha 2-AP of the propositus was homogeneous in all respects tested, suggesting a homozygous defect. We designated the abnormal alpha 2-AP as alpha 2-AP Enschede. alpha 2-AP Enschede showed the following characteristics: (a) complete immunological identity with normal alpha 2-AP; (b) normal molecular weight (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); (c) normal alpha-electrophoretic mobility; (d) presence in plasma of both molecular forms excluding an excessive conversion to the less reactive non-plasminogen-binding form; (e) quantitatively normal binding to lys-plasminogen and to immobilized plasminogen kringle 1-3; and (f) normal Factor XIII-mediated binding to fibrin. Functional abnormalities were found in: (i) no inhibition of amidolytic activities of plasmin and trypsin, even on prolonged incubation; (ii) no formation of plasmin-antiplasmin complexes in plasma with plasmin added in excess; and (iii) no inhibition of fibrinolysis by fibrin-bound alpha 2-AP. In the heterozygotes, the presence of abnormal alpha 2-AP did not interfere with several functions of the residual normal alpha 2-AP. One-dimensional peptide mapping showed an abnormal pattern of papain digestion. We conclude that in this family, abnormal antiplasmin molecules, defective in plasmin inhibition but with normal plasminogen-binding properties, have been inherited. The residual plasminogen-binding properties do not protect against a hemorrhagic diathesis.

Adolescent↗
PubMed1986

Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture.

Culture fluid of a monkey kidney cell culture was harvested every two days, for a two week period, in order to obtain urokinase in the zymogen form. Pro-urokinase was isolated by immunoadsorption chromatography and gel filtered on Sephadex G-150, which resulted in three peaks with pro-urokinase activity. SDS-polyacrylamide gel electrophoresis showed that the first peak contained 55 kd pro-urokinase, aggregated with high molecular weight contaminants, whereas the second and third peaks consisted of almost pure 55 kd and 30 kd pro-urokinase, respectively. The latter form represented a relatively unknown and inactive precursor of low molecular weight urokinase, which was, like 55 kd pro-urokinase, activatable with plasmin. In comparison with tissue-type plasminogen activator, 55 kd and 30 kd pro-urokinase only bound weakly to purified fibrin clots and fibrin-sepharose columns. The extent of binding of the two pro-urokinases and their plasmin-activated forms to fibrin-sepharose decreased in the following order: 55 kd pro-urokinase 30 kd pro-urokinase 55 kd urokinase 30 kd urokinase. These results indicate that the two precursors exhibited stronger binding to fibrin-sepharose than the corresponding active enzymes, and the two 55 kd forms exhibited stronger binding than the corresponding 30 kd forms. This indicates the importance of both the zymogen nature and an intact NH2-terminal part of the molecules for binding to fibrin.

Animals↗
PubMed1985

Appropriate milieu for the assay of alpha-2-antiplasmin activity with chromogenic substrates.

Inhibition reference curves for alpha 2-antiplasmin showed a deviation from linearity at low inhibitor concentrations using the chromogenic substrate S-2251. Extrapolation of these curves to zero inhibition gave higher amidolytic activities than actually recorded with the free enzyme plasmin. It was further found that comparison of purified alpha 2-antiplasmin and that in plasma was prevented by the deviation. The difference between calculated and measured plasmin activity could not be attributed to instability of plasmin. It was established that the observations (1) were specific for high concentrations of S-2251, (2) were not the same with another plasmin substrate, chromozym PL, and (3) were related to the addition of plasma proteins. Apparently, the problem was related to the solubility state of S-2251. A solution to this problem is the addition of nonionic detergents, notably Tween 80 (0.01%) or Triton X-100 (0.03%), which prevent all deviations.

Chromogenic Compounds↗
PubMed1984

Fibrinolytic activators and inhibitors in terminal renal insufficiency and in anephric patients.

Fibrinolytic factors were measured before and after DDAVP-infusion in 18 patients on chronic, regular haemodialysis, 11 of whom underwent bilateral nephrectomy, and in 7 patients in whom non-functioning kidneys were still present. Baseline fibrinolytic activity was normal or high in all but two cases. Before haemodialysis, the response to DDAVP-infusion was greatly reduced in the majority of patients as compared with healthy controls, irrespective of the baseline level. This was in accordance with mean t-PA-antigen levels which increased only slightly after DDAVP. When DDAVP was given after haemodialysis, previous non-responders showed a normal increase in fibrinolytic activity. The level of free t-PA-inhibitors was normal in most cases as were levels of alpha 2-antiplasmin and alpha 2-macroglobulin.

Antigens↗
PubMed1984

Stanozolol-induced changes in fibrinolysis and coagulation in healthy adults.

The effect of orally-administered stanozolol, 5 mg b.d. on fibrinolysis, coagulation and on various haematological and biochemical parameters have been studied in 16 healthy adults, 8 males and 8 females. Statistically significant enhancement of extrinsic (tissue-type) plasminogen activator activity was detected in all subjects studied. This was associated with significant increases in plasma plasminogen and a concomitant reduction in histidine-rich glycoprotein. There were no changes in plasma urokinase activity. Changes in the coagulation system included significant reduction in plasma fibrinogen and elevation of protein C and antithrombin III. Changes in plasma lipids included significant reduction of HDL cholesterol associated with an increase in LDL triglycerides. No change occurred in total cholesterol. There were no major differences between the sexes, nor were there serious side effects. The effects of stanozolol on extrinsic (tissue-type) plasminogen activator activity, "free" plasminogen, protein C and antithrombin III, argue strongly in favour of its therapeutic potential.

Adult↗
PubMed1984

Comparison of enzymes of tears, lacrimal gland fluid and lacrimal gland tissue in the rat.

A variety of enzymes were identified in rat tears and lacrimal gland fluid. The use of a tapered glass cannula in the opening of the excretory duct was found to be an useful method to collect samples of rat lacrimal gland fluid, i.e., the fluid directly originated from the main excretory duct of the lacrimal gland, uncontaminated by secretions from the Harderian gland and from desquamating conjunctival and corneal epithelial cells. Based upon comparison of the enzyme pattern in tears, lacrimal gland fluid and the lacrimal gland tissue, we concluded that the lacrimal gland is the primary tissue source for peroxidase (POD), amylase (AMY) and total protein in rat tears, while plasminogen activator and lactatedehydrogenase (LDH) may be present in tears primarily as secretion products from other ocular tissue sources. beta-hexosaminidase (beta-hex) is released from the lacrimal gland and from other ocular tissue sources as well.

Amylases↗
PubMed1984

Intrinsic plasma fibrinolysis: involvement of urokinase-related activity in the factor XII-independent plasminogen proactivator pathway.

A portion of human blood fibrinolytic activity can be quenched by antibodies to urokinase. We attempted to identify this portion and its position in the three pathways (extrinsic, intrinsic factor XII dependent, and intrinsic factor XII independent) of plasminogen activator activity that have been defined in the DEFs of plasma. Activity quenching in various plasmas (including plasma adsorbed by agarose-bound AUK) demonstrated the involvement of a discrete activator activity of 40 to 50 BAU/ml with little variation among individuals (43 +/- 6 (SD) BAU/ml, n = 13). The quenching did not involve, quantitatively or qualitatively, the extrinsic (tissue-type) plasminogen activator activity varying between 1 and 146 BAU/ml in baseline plasma and in plasma obtained after stimulation by venous occlusion, exercise, or DDAVP administration. In confirmation, extrinsic activator activity was recovered in plasma adsorbed by agarose-bound AUK. The quenching also did not involve the factor XII-dependent activities; it was quantitatively normal in plasma with Hageman (n = 3) and Fletcher (n = 2) traits, and AUK did not interfere with the generation of factor XII-dependent fibrinolytic activity by addition of purified activated factor XII in plasma with Hageman trait. There was no effect of AUK on kallikrein generation, kallikrein activities, or the APPT, nor were these aspects altered in depleted plasma. In conclusion, the quenching involved the factor XII-independent system of intrinsic fibrinolysis. Addition of purified urinary urokinase did not result in restoration of missing activity in plasma adsorbed by AUK-agarose. The quenching, therefore, probably involved an apparent urokinase-related activator component in plasma.

Dextran Sulfate↗
PubMed1984

The influence of L-asparaginase therapy on the fibrinolytic system.

Fibrinolytic factors were assessed during L-asparaginase administration, to study whether their changes may predispose to a haemorrhagic or thrombotic diathesis. The total level of alpha 2-antiplasmin declined, as well as the ratio of the plasminogen-binding form of alpha 2-antiplasmin to the non-plasminogen-binding form. After cessation of L-asparaginase administration, the ratio increased to 1.6 times that of the pretreatment value. These data indicate that the plasminogen-binding form of alpha 2-antiplasmin is the form primarily synthesized in vivo. L-Asparaginase therapy reduced plasma levels of plasminogen and histidine-rich glycoprotein ( HRG ) and influenced the equilibrium between HRG , plasminogen and HRG -plasminogen complex, with a more pronounced decrease of plasminogen (62% +/- 8) and HRG (76% +/- 11) in comparison to the free-plasminogen levels (51% +/- 6). alpha 2-Macroglobulin was only slightly influenced by L-asparaginase and may consequently play a more pronounced role in inhibition. This is suggested by moderate declines in functional tests of plasmin, urokinase and tissue activator inhibition by patients plasma, and by the ratio of inhibition of these enzymes over alpha 2-antiplasmin. Thus the bleeding tendency described during L-asparaginase therapy can be ascribed not only to a temporary deficiency of coagulation factors but also to temporary alpha 2-antiplasmin deficiency.

Adolescent↗
PubMed1983

Inhibition of tissue-type plasminogen activator by conditioned medium from cultured human and porcine vascular endothelial cells.

Conditioned serum-free medium both from confluent porcine aortic endothelial cells and from human venous and arterial umbilical cord endothelial cells will inhibit plasminogen activation by tissue-type plasminogen activator. Inhibitory activity in the conditioned medium increases with time, and depends upon protein synthesis. Also, the conditioned medium directly inhibits the amidolytic activity of tissue-type plasminogen activator. Inhibitory activity can be removed from conditioned medium by absorption with immobilized tissue-type plasminogen activator.

Animals↗
PubMed1983

Fibrinolytic activity of the gastric mucosa in Ménétrièr's disease.

Biopsies of the gastric mucosa were collected from two patients with Ménétrièr's disease before, during, and after treatment with cimetidine and studied for fibrinolytic activity. The fibrinolytic activity in the homogenates or in the potassium thiocyanate soluble fractions increased markedly during the treatment. Immunological characterization of the activity indicated that its major component was an endothelium-derived type of plasminogen activator, and that the increase in activity was caused solely by an increase in the concentration of this component. This suggests an increase in the vascularity of the mucosal layers during treatment thus reflecting a repair process.

Cimetidine↗
PubMed1983

Immunological characterization and possible origin of plasminogen activator in human tear fluid.

Human tear fluid has plasminogen activator activity. The type of plasminogen activator activity in unstimulated and stimulated tears was determined, using antibodies that specifically neutralize tissue plasminogen activator or urokinase. All plasminogen activator activity was tissue plasminogen activator-related in both types of tears. Correlations between activities of beta-hexosaminidase, lysozyme and lactate dehydrogenase with tissue plasminogen activator activity indicate that the contribution to plasminogen activator activity of conjunctival and corneal epithelium is more important in unstimulated tears than stimulated tears. In stimulated tears the tissue plasminogen activator activity originates mainly from the lacrimal gland. It is suggested that a constant concentration of plasminogen activator is released from the lacrimal gland and that this concentration is independent of the secretion rate of tear fluid and that the release from the conjunctiva is due to desquamation of cells.

Adult↗
PubMed1983

A new life-long hemorrhagic disorder due to excess plasminogen activator.

A life-long bleeding disorder is described, characterized by hemorrhage occurring after surgery, injury, or dental extraction, and finally by spontaneous intracerebral bleeding. No abnormality of platelet function or plasma coagulation was demonstrable, but grossly enhanced overall fibrinolytic activity was present. The patient had, additionally, a hyperlipidemia with gross arterial atheroma and a family history of myocardial infarction but not of any hemorrhagic disorder. Laboratory studies led to the conclusion that the enhanced fibrinolysis was due to consistently greatly raised levels of a plasma plasminogen activator physically and immunologically related to that in human tissues and blood vessel endothelium. No deficiency of any known inhibitor of fibrinolysis was detected. Free plasmin was not detectable in functional assays but continuous intravascular plasmin generation clearly occurred as evidenced by presence of plasmin-alpha 2-antiplasmin complexes and of fibrin/fibrinogen-related antigens. Excessive production of plasminogen activator appeared to have occurred throughout life and to be independent of the hyperlipidemia. The pathologically increased fibrinolytic activity may have accounted for the complete absence of detectable thrombotic vascular occlusion at autopsy despite extensive arterial disease with severe narrowing of coronary and cerebral arteries.

Blood Coagulation Tests↗
PubMed1982

A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma.

An indirect spectrophotometric assay for extrinsic plasminogen activator has been devised, which is based on the parabolic assay of Drapier et al. (5). The system contains activator, plasminogen, the synthetic plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251, Kabi) and a mixture of soluble fibrinogen fragments prepared by treatment of fibrinogen with cyanogen bromide. The addition of these fibrinogen fragments considerably enhances the sensitivity and specificity of the method owing to specific stimulation of the plasminogen activation by extrinsic plasminogen activator. The assay conditions were optimized and the application for extrinsic plasminogen activator measurements in plasma euglobulin fractions is demonstrated.

Fibrin↗
PubMed1982

Activation of plasminogen by tissue activator is increased specifically in the presence of certain soluble fibrin(ogen) fragments.

Tissue activator-mediated plasminogen activation is potentiated both by fibrin and by some soluble fibrin(ogen) fragments. The potentiating effect of the different fragments decreases in the order fibrin monomer greater than D-dimer greater than Y greater than D EGTA greater than Dcate greater than X. Fibrinogen and the fragments Ecate, E EGTA and E fibrin have almost no effect. The existence of a fibrin polymer is apparently not a prerequisite for this potentiating effect. The plasminogen activation by various urokinase preparations is not potentiated by fibrin and fibrin(ogen) fragments.

Fibrin↗