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.

Purification and partial characterization of plasminogen activator from human uterine tissue.

A procedure was developed for the purification of a plasminogen activator from human uterine tissue. It involves six consecutive steps: (1) extraction of the plasminogen activator from delipidated uterine tissue with 0.3 M potassium acetate buffer, pH 4.2; (2) ammonium sulphate precipitation; (3) zinc chelate-agarose chromatography; (4) n-butyl-agarose chromatography; (5) concanavalin A-agarose chromatography; and (6) gel filtration on Sephadex G-150. The specific activity of the final plasminogen activator preparation was increased by a factor 4500 as compared with the crude extract. The purified plasminogen activator showed a strong tendency to adsorb to surfaces. This could be effectively prevented by Tween-80. The molecular weight of the plasminogen activator was 64 000 as estimated by gel filtration in 1.0 M NaCl and 69 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The plasminogen activator consisted of two chains (molecular weights 31 000 and 38 000) connected by disulphide bridges. The smallest chain contained the serine residue of the active site as deduced from the incorporation of the tritium label of [3H]diisopropylphosphofluoridate.

Interfering factors in the assay of plasminogen activators by the fibrin plate method. Occurrence of different inhibitors against tissue plasminogen activator and urokinase.

The assay of plasminogen activator activities on fibrin plates was re-evaluated with special reference to fibrinolysis inhibitors present in samples and in fibrin plates. The nature, action and stability of inhibiting material were studied in tissue with considerable differences in activator and inhibitor contents: human lung, liver and placenta. Extracts were tested for inhibitory capacity against purified human uterine tissue plasminogen activator, urokinase and plasmin of fibrin plates prepared from different grades of fibrinogen and fibrin. The tissue extracts inhibited fibrinolysis on fibrin plates to varying degrees, dependent on the sample medium, the type of fibrin plate and the kind of plasminogen activator. The influence of inhibitors in the sample and in the fibrin plate was partly abolished by the presence of 2 M KSCN in the sample. The procedure for preparing the samples as described by Astrup and Albrechtsen did not completely eliminate the inhibitory action against the added plasminogen activators. Comparison of urokinase inhibition with tissue activator inhibition by the tissue extracts as to the degree of denaturation in the Astrup and Albrechtsen procedure showed that they have much in common. Nevertheless, some differences were found which indicated the possible existence of separate urokinase and tissue activator inhibitors or of different inhibition mechanisms for these plasminogen activators.

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.

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.

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.

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.

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.

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.

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.

Characterization of changes in psychometric colour attributes of comminuted porcine lean meat during processing.

Changes in colour during the processing of a comminuted porcine lean meat system were characterized using the psychometric colour attributes lightness L (∗)) hue (h (∗))and chroma (C (∗)). The effects of processing temperature, nitrite and air pressure during comminution were examined. The pattern of changes as a function of processing time for L (∗) and C (∗), but not for h (∗), showed clear dependence on processing temperature (15, 30, 40, 50 and 60°C). For C (∗) this dependence was much more complex than foor L (∗). Nitrite dramatically affected the pattern of change in h (∗) and C (∗), but much less so changes in L (∗). Varying the air pressure during comminution did not induce shifts in patterns of changes of the three colour attributes. However, it did have a marked effect on the absolute values and the extent of changes in L (∗) and, to a much lesser extent, in h (∗) and C (∗). The findings of this study furnish the basis for a possible usage of the psychometric colour attributes as parameters for a quantitative kinetic analysis and modelling of the effect of processing factors during the production of comminuted meats.

Shelf-life of vacuum-packaged wild boar meat in relation to that of vacuum-packaged pork: Relevance of intrinsic factors.

In order to study the factors influencing the relatively poor keepability of pork compared with beef, a study with wild boar meat was carried out. Microbiological and sensory quality traits of vacuum-stored wild boar (Sus scrofa scrofa) cuts of M. longissimus dorsi (longissimus) at 0°C were determined after 1, 10, 28, 42, 56, 70, 84 and 98 days and of tenderloins after 3, 35, 49 and 63 days. The amount of glycogen, glucose and glucose-6-phosphate in the longissimus cuts was measured during storage, in order to study the processes that determine shelf-life. Tenderloins developed off-odours after 35 days, probably due to Enterobacteriaceae growth. Unacceptable discolouration of longissimus cuts and off-odour development were noticed after 84-98 days. Shelf-life comparison between wild boar meat and pork stored under similar conditions indicates that the relatively poor keepability of pork is due to intrinsic factors. Glucose depletion is probably triggering the onset of spoilage processes.

The effect of scalding on subcutaneous and ham temperatures and ultimate pork quality.

Scalding of pig carcasses (n = 34) at 60°C for a period of at least 5·5 to 7·5 min gave satisfactory dehairing results, with the exception of autumn hair for which a longer period (9 mins) was required. Temperature curves were recorded for a subcutaneous position in the ham (n = 26) between the rind and the underlying fat layer. These showed a curve starting at about 30·8°C and increasing to an asymptotic value of 53°C during scalding. Results of calculations with a finite element model of a flat layer of muscle covered with a layer of 1·0 cm fat broadly showed the same temperature increase at about 0·5 cm below the surface as the actual values measured. Immediately after dehairing, about 1·5 mins after finishing scalding, the subcutaneous temperature had already dropped to 46·1 ± 3·0°C, which was considerably higher than the muscle temperature at the same position at a depth of 5 cm under the skin (40·6°C). The heat removal and temperatures during the cooling period after scalding were also calculated. It can be concluded that the increase in temperature due to scalding has only a minor influence on muscle temperature and that meat quality (pH, FOP) is not affected.

Training for more accurate visual fat estimation in meat.

Much animal fat in the diet is contained in meat. As fat intake is considered too high in western societies, a more fat-conscious attitude may be desirable. One of the parties involved is the butcher, who sells fresh meat directly to the consumer. In a pre-post experimental design, with an interpolated training phase, the possibility to improve the ability of student butchers to visually estimate fat content of meat, was investigated. A limited number of training sessions, in which immediate feed-back was given of the actual fat percentage after each estimation, led to a large improvement in fat estimation accuracy. A delayed post-test indicated that most of the training effect was preserved after six weeks. Similarities between the observed learning process and informational feed-back learning with numerosity stimuli were discussed. On the basis of these results it is recommended that courses for trainee butchers include a short course on fat estimation in their curriculum. If butchers sell what they think they sell, consumers are more likely to get what they think they get. Increased 'fat awareness' may indirectly contribute to healthier eating habits.

Haematological and clinico-chemical profiles of barrows at the farm and at slaughter.

Haematological and clinico-chemical profiles of blood of healthy stress-resistant swine collected at the farm and at slaughter were determined to investigate whether values of blood variables can be used to establish stress. The values of most variables investigated showed highly significant changes. It is concluded that haematological and clinico-chemical values may be useful in studies to detect, quantify and reduce stress-provoking conditions in stress-resistant swine.