In a previous study of plasma specimens from reactors to clinical dextran, it was concluded that a factor activated by acetone converted the high-molecular-weight kininogen (HMrK) into a non-functional state. We recently identified the HMrK-destroying factor as a plasma kallikrein modification with high plasminogen activator (PGA) activity. The aim of the present work was to investigate whether the level of plasma kallikrein, assayed as PGA on fibrin plates, was higher than normal in plasma from patients reacting to dextran or radiographic contrast media. Plasma specimens from 10 reactors and 16 controls were examined. No difference in PGA level could be detected between reactors and controls in citrated plasma stabilized with benzamidine 9 mM, whereas a significant loss of PGA activity took place in citrated reactor plasma during the acetone activation procedure. The loss of PGA activity could not be due to known inhibitors of plasma kallikrein. It was only partial, and did not influence the amidase effect against the tripeptide substrate H-D-Pro-Phe-Arg-pNA. The results might indicate the presence in plasma of different amounts of an unknown constituent important for the obtainable level of plasma kallikrein with high PGA activity. The significant loss of PGA activity in reactor plasma might indicate an abnormally high level of the unknown plasma factor.
Abstract High molecular weight kininogen (S1) is useful as a sensitive substrate in assays of the kininogenase activity of plasma kallikrein, but considerable difficulties are encountered in the isolation of this kininogen due to loss by “spontaneous” activation. In the present work a satisfactory yield (40–60 %) of a crude preparation of S1 was obtained through gel chromatography on Sephadex G–200 of plasminogen–free 60°‐heated human citrated plasma. Low molecular weight kininogen (S2) was also obtained by the same procedure. The two kininogen preparations did not contain significant amounts of inhibitors of human plasma kallikrein, hog pancreas kallikrein or human plasmin, as judged by the stability of the arginyl–esterase activities of these enzymes in contact with the kininogens. Both S1 and S2 were stable for at least one year when stored at ‐20°. A preparation of human plasma kallikrein reacted specifically with S1, while both hog pancreas kallikrein and human plasmin also reacted with S2. The quantitative data are in accordance with the assumption that hog pancreas kallikrein and plasmin release bradykinin from S1 and kallidin from S2. The rate of release of kinin produced by hog pancreas kallikrein in S2 occurred twice as fast as in S1, while the opposite was registered for plasmin.
The kaolin-induced activation of factor XII (XII) to XIIa was studied in plasminogen-free human citrated plasma treated with acetone in the presence of benzamidine 7.5 mM. XIIa was assayed as prekallikrein (PK) activator. The significance of the concentrations of XII, PK and high molecular weight kininogen (HMrK) was examined using mixtures of normal plasma and plasma genetically deficient in these factors. At the high plasma dilution used (1 + 23 v/v in the kaolin incubate) a joint estimation of the factors was obtained. A reduction in amount of XII, PK or HMrK resulted in a correspondingly reduced yield of XIIa. Plasma kallikrein present was assayed as S-2302 amidase. The concentration of PK in XII-deficient plasma was normal, in HMrK-deficient plasma about 30% of normal. The activation of XII was studied in fresh plasma as well as in plasma stored for 3-6 months at -70 degrees, and the activation with acetone was carried out in the presence and in the absence of benzamidine, EDTA or purified HMrK. In previous work benzamidine was found to protect the cofactor function of purified HMrK in the assay system used, and EDTA was found to inhibit purified human plasma kallikrein assayed as plasminogen activator. The present results support the previous observations, and indicate that acetone treatment of fresh human plasma (benzamidine present) results in the activation of plasma kallikrein in a functional state that requires kinin-free, but otherwise native HMrK as a cofactor for the activation of XII.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: By incubation of human citrated plasma with acetone 25% v/v kallikrein inhibitors were destroyed and prekallikrein activated to kallikrein. When the incubation was carried out in the presence of benzamidine 7 mM, the cofactor capacity of high molecular weight kininogen (HM r K) was protected against destruction by a serine protease which was not plasma kallikrein. By analogy with studies in rat plasma this protease might be a plasminogen activator (Berstad & Briseid 1982; Johansen & Briseid 1983). Factor XII in the plasma preparation was activated to unfragmented factor XII a by adsorption to kaolin, and assayed as prekallikrein activator (PKA). The extent of activation of factor XII was only insignificantly influenced by the 1 + 1 (v/v) dilution of the plasma preparation with a suspension of kaolin. When, however, the preparation was diluted > 1 + 5 (v/v) before incubation with the suspension, a stoichiometric HM r K concentration‐effect curve could be established, allowing the assay of cofactoractive HM r K. Assays of HM r K in plasma preparations from healthy men and women demonstrated an average lower level of cofactor‐active HM r K in the preparations from women. It is suggested that benzamidine is not capable of providing a complete protection of HM r K during the procedure in all plasma samples.
A method described by Bieltvedt & Briseid (1966) and Bieltvedt (1967) for the determination of the inhibition by cardiac glycosides of the isolated, histamine-stimulated guinea pig ileum was modified to estimate the inhibition of the acetylcholine-stimulated rat jejunum. The glycoside concentrations causing 50 % inhibition of submaximal, isotonic contractions were determined, and the procedure was based on 20-minute contact periods between the muscle preparations and the cardioactive substances, and then 22-minute equilibration periods with chemical stimulation. The degree of inhibition was found to depend on the potassium content of the Tyrode solutions used. In a low potassium Tyrode solution (1.8 mM) the inhibition of the rat intestine by cardiac glycosides was found to be of the same order of magnitude as the inhibition of the guinea pig intestine. When the potassium chloride concentration was increased to 2.7 mM, the well known species difference was observed, the concentration of cardiac glycoside required for inhibition of the rat intestine being about 30 times higher than that required for inhibition of the guinea pig intestine. The range of potency of the different cardiac glycosides and aglycones which were tested in the low potassium. Tyrode solution, was the same for the two species. Presupposing an inhibition of Na+, K+-activated transport ATPases as the basic mechanism for the inhibition by cardiac glycosides of the isolated intestine preparations, a theory of qualitative differences between the transport enzymes in the two species is advanced. In the rat intestine the presence is suggested of one ATPase which is rather resistant to inhibition by cardiac glycosides, but sensitive to a reduction in potassium concentration, in addition to a glycoside-susceptible ATPase also present in the guinea pig intestine.
Plasminogen activator (PGA), kininogenase (Kase) and benzoyl arginine ethyl ester (BAEe) activities generated in plasminogen-free rat plasma by incubation with acetone (23% v/v) at 22 degrees were purified. The activities passed unadsorbed through columns of DEAE-Sephadex A-50 (pH 7.8) and arginine methylester-Sepharose 4B (pH 8.5). Part of the activities (rat plasma kallikrein) was adsorbed onto a soybean trypsin inhibitor (SBTI)-Sepharose 4B column at pH 8.5. At pH 7.0 a fraction with higher ratios PGA/BAEe esterase and Kase/BAEe esterase was also adsorbed. Both fractions could be eluted with 5 mM sodium hydroxide. The fraction not adsorbed at pH 8.5, but adsorbed at pH 7.0 was designated low molecular weight plasminogen activator (LMr-PGA), a plasminogen activator fraction with higher molecular weight, but without esterase activity being also present (Berstad & Briseid 1982). LMr-PGA was strongly inhibited by tranexamic acid (AMCA) 0.10 mM, whereas the fraction designated rat plasma kallikrein was not. By polyacrylamide gel electrophoresis Mr-values in the range 120,000 to 130,000 were established for native samples of both rat plasma kallikrein and LMr-PGA, whereas Mr-values of 78,000 to 80,000 were established after treatment with SDS.
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