Biomedical subjects
Publications and source records attributed to G Wijngaards.
Various preparations of anti-urokinase antibodies were found to quench a part of the plasma fibrinolytic activity. This part was also inhibited by a specific, urokinase inhibitor isolated from placenta. These observations indicate that plasma contains a urokinase-related immuno-reactivity. This activity was found to belong to the factor XII-independent proactivator system.
This study concerns a case of congenital homozygous deficiency in alpha 2-antiplasmin associated with a severe hemorrhagic diathesis. Heterozygous family members also show a mild bleeding tendency. The propositus is a 17-yr-old male born of white parents and showing a severe hemorrhagic diathesis characterized by spontaneous bleeding in the joints since his early childhood. He was originally suspected of having factor XIII deficiency but was found to have normal functions of the coagulation system and the platelets. Except for alpha 2-antiplasmin, all protease inhibitors showed normal plasma values. With the immediate plasmin inhibition test (synthetic substrate), only 2% of normal functional inhibition was detected, while no reaction with monospecific antisera for alpha 2-antiplasmin was observed. Inhibition of activator-induced fibrinolysis in vitro was reduced. No enhanced spontaneous in vitro fibrinolysis was detected nor were there signs of increased in vivo fibrinolysis during an asymptomatic period. During recovery from a hemorrhagic episode, signs of previous consumption of antithrombin III, alpha 2-macroglobulin, factor XIII, and inter-alpha-trypsin inhibitor were noted. After the diagnosis was made, treatment with tranexamic acid (4 daily doses of 1 g) was effective for about 2 yr. Among the 37 family members studied, a separate group of 16 individuals (including the father and mother of the propositus) with approximately one-half normal plasma levels of alpha 2-antiplasmin both functionally (59% +/- 6%) and immunologically 48% +/- 8%) was discovered. The defect appeared to be inherited as an autosomal recessive gene; no ancestral consanguinity could be shown. The group of apparent heterozygotes as a whole showed increased levels of alpha 1-antitrypsin (142% +/- 39%; p less than 0.01), indicating systemic consequences of the deficiency and reduced binding (+/- 50%) of alpha 2-antiplasmin to fibrin. Six exhibited a mild hemorrhagic diathesis for which no explanation was provided by routine screening of coagulation and platelet functions; also, within the group of heterozygotes, the occurrence of the bleeding tendency did not correlate with differences in residual alpha 2-antiplasmin levels and functions. It is concluded that not only the absence of alpha 2-antiplasmin but also a reduction in its plasma level to +/- 60% of normal may predispose to a hemorrhagic diathesis.
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Human plasminogen activators were compared immunologically in both a double-diffusion technique and quenching experiments on the fibrinolytic activities of the activators. Antisera against HMW and LMW urokinase and an antiserum against highly purified tissue plasminogen activator from human uterus were used. It was found that uterine tissue plasminogen activator and urokinase are two immunologically distinct plasminogen activators. The occurrence of the two kinds of plasminogen activators in human tissues and body fluids was studied on the basis of the quenching of the activities by antibodies. In tissue extracts, mainly tissue plasminogen activator was found. Seminal plasma exhibited a high plasminogen activator activity, consisting of both urokinase and tissue plasminogen activator-related activators. Urine contained a small amount of tissue plasminogen activator-related activator in addition to urokinase. The low plasminogen activator activities of saliva and tears were completely attributed to activators related to tissue plasminogen activator.
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3 patients with Behçet's disease were studied for their fibrinolytic status during exacerbation of the disease accompanied with thrombotic complications. All 3 patients exhibited low euglobulin fibrinolytic activity. This, however, could be attributed to an artificially increased coprecipitation of inhibitors, i.e., C1-inactivator in the euglobulin fractions. This phenomenon correlated with and was possibly related to inflammatory reactions in the patients. The most severely diseased patient only showed an increased plasmin inhibition in the plasma and a decreased response to venous occlusion. The increased plasmin inhibition was due to a slow type of inhibition, which could not be attributed to known protease inhibitors in blood. Its physiological relevance to fibrinolysis is questionable. There was no evidence for relevant general deviations in the fibrinolytic system in Behçet's disease; only for the severe case may the decreased fibrinolytic potency revealed by venous occlusion have contributed to the extent of the thrombotic manifestations.
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.
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.
Two fluorogenic peptide amides have been synthesized, i.e. BOC-L-valyl-glycyl-L-arginine 2-naphthylamide (I) and L-valyl-glycyl-L-arginine 2-naphthylamide (II). The kinetic parameters of plasmin, urokinase and human uterine tissue plasminogen activator on substrates I and II have been determined. Quite unexpectedly, the tissue activator appeared to require for its activity a blocked amino terminus on the substrate. This was further corroborated with other synthetic substrates. Plasmin and urokinase did not show this requirement.
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The one-stage prothrombin time test for tissue thromboplastin activity was evaluated using the correction method. A strong influence of the intrinsic system was seen depending on the clotting time and the type of tubes. This test was unsuitable for quantitative comparison of thromboplastin activity of different preparations. Subsequently a two-stage assay for human thromboplastin is described using purified human factor VII, artifically prepared factor VII reagent, and a phospholipid suspension. The correction method, applicated to this two-stage assay, resulted in a rectilinear dose-response curve when the clotting times of a thromboplastin dilution series were plotted against the reciprocal of the concentrations. Using this test system it could be shown that thromboplastin from porcine tissue can activate human factor VII quite well, while that from bovine tissue cannot.
The tissue thromboplastin activity of porcine lung was separated from an aqueous tissue extract by differential centrifugation. Repeated suspension and sedimentation yielded a thromboplastin preparation free of soluble protein. The most effective delipidation of the lyophilized preparation with minimum denaturation of the protein moiety of thromboplastin was achieved using n-heptane/n-butanol extraction. A detergent was required to solubilize the thromboplastin protein moiety from the delipidated preparation. Gel filtration in the presence of detergent permitted further delipidation of the protein moiety and yielded a purified protein moiety. Apparent homogeneity was observed for the purified protein with several fractionation techniques. This apparently homogeneous protein dissociates into the constituent proteins in the presence of dodecyl sulphate. The aggregation of proteins is ascribed to their hydrophobic nature. Gel filtration on Bio-Gel A-1.5 m was found to be useful in the separation of the dodecyl sulphate-protein complexes on a preparative scale. Following the removal of the dodecyl sulphate, the renaturation of the thromboplastin protein moiety was unsuccessful. The purified protein moiety with its low lipid content (1%) is suitable for lipid requirement studies.
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