Anne Marie Vennerød

Factor XII-induced fibrinolysis: studies on the separation of prekallikrein, plasminogen proactivator, and factor XI in human plasma.

Abstract A protein fraction containing plasma prekallikrein, factor XII-dependent plasminogen proactivator, and precursor factor XI was prepared by ion exchange chromatography of human plasma. The fraction was concentrated and subjected to further separation. No separation of plasma prekallikrein and plasminogen proactivator was obtained, either by chromatography on CM-Sephadex C-50, hydroxylapatite, or Sephadex G-100 Superfine, or by isoelectric focusing. Plasma prekallikrein separated clearly from precursor factor XI by chromatography on CM-Sephadex C-50, hydroxylapatite, and Sephadex G-100 Superfine. Isoelectric focusing revealed microheterogeneity of prekallikrein, and each peak had plasminogen preactivator activity. Studies with purified C1-inactivator indicated that the affinity of the inhibitor against plasma kallikrein and plasminogen activator is the same. A highly purified preparation of plasma prekallikrein (34.5 BAEe U/mg) also had plasminogen proactivator activity, and the plasminogen-activating capacity of the preparation calculated per BAEe proesterase U (0.16 Ploug unit/BAEe U) was unchanged during the purification procedure. It is concluded that plasma prekallikrein may be a mediator of the factor XII-dependent fibrinolytic pathway of human plasma.

Determination of factor XII in human plasma with arginine proesterase (prekallikrein) I. Preparation and properties of the substrate

Abstract An arginine proesterase was purified from human plasma by ion exchange chromatography and was concentrated on hydroxylapatite. Upon activation the preparation yielded 0.96 BAEe esterase units per mg protein. The preparation contained traces of factor XI, but was devoid of factor XII. One major arginine proesterase peak was observed after analytical gel filtration on Sephadex G-100 Superfine and disc electrophoresis at pH 4.3. The proesterase was activated by incubation with kaolin-treated plasma and was identified as plasma prekallikrein. Upon incubation with kaolin-treated plasma the activation proceeded at a constant rate to about 30% consumption of the proesterase. It is concluded that the preparation may serve as a substrate for determination of factor XII in plasma.

Isolation and characterization of a prealbumin activator of prekallikrein from acetone-activated human plasma

Abstract A prealbumin activator of prekallikrein was isolated from acetone-activated human plasma by preparative disc gel electrophoresis in 15% polyacrylamide. The purification factor was 70, and the recovery was 20–30%. The purified preparation was functionally free of factors II, IX, and X, and it did not clot fibrinogen. It had a weak BAEe esterase activity, but was inactive towards kininogen, fibrin, and plasminogen. C1-inactivator, α 2 -macroglobulin, and α 1 -mantitrypsin could not be demonstrated by immunochemical techniques. The preparation was homogeneous on disc gel electrophoresis. The prekallikrein activator in the preparation had an estimated molecular weight of 32,000, and the isoelectric point was 4.2. The preparation also activated plasminogen proactivator and the proenzyme form of factor XI. It is concluded that the preparation contained factor XII f , and that preparative polyacrylamide disc gel electrophoresis of acetone-activated plasma may represent a simple method for the purification of factor XII f .

Purification of human factor IX.

Abstract Factor IX was isolated from human plasma by a six-step procedure employing adsorption to DE-52 cellulose in batches, aluminium hydroxide adsorption, affinity chromatography on heparin-coupled Sepharose, preparative polyacrylamide disc gel electrophoresis, immunosorbent absorption, and DE-52 cellulose chromatography. Determination of factor IX activity and antigen revealed a specific activity of about 290 U per absorbancy unit at 280 nm. Analytical polyacrylamide disc gel electrophoresis showed one major and three to four minor bands with lower electrophoretic mobility. Disc gel electrophoresis in the presence of 10 M urea gave a double major band and two additional minor bands with higher electrophoretic mobility. One major band was observed in SDS polyacrylamide gel electrophoresis, and no change in mobility took place after reduction.

Determination of factor XII in human plasma with arginine proesterase (prekallikrein) II. Studies on the method

Abstract When kaolin-activated human plasma was incubated with partially purified plasma prekallikrein, a straight-linear relationship was established between the amount of activator and the initial rate of activation of the prekallikrein substrate. An almost identical regression line was obtained after incubation of the substrate with kaolin-activated dilutions of normal plasma in factor XII deficient plasma. A very close correlation existed between the plasma prekallikrein-activating capacity and the clot-promoting effect in factor XII deficient plasma, and it is concluded that determination of the prekallikrein-activating capacity may be well suited for determination of the concentration of factor XII in plasma.

Cold-promoted activation of factor VII in human plasma: studies on the associated acyl-argin?ne esterase activity

Abstract Samples of citrated plasma from untreated normals, women using an oestrogen-containing oral contraceptive, and pregnant women near term were studied regarding activation of factor VII upon cooling at C°C for 18 hours. The samples with markedly shortened thrombotest clotting time also had significantly increased BAEe esterase activity and reduced prekallikrein activity. Concomitantly with the increase in BAEe esterase activity the cooled plasma increased in kininogenase activity and was depleted of kininogen. The esterase and kininogenase activity of cold-activated plasma had the same elution volume upon gel filtration as purified α 2 -macroglobulin-kallikrein complex. Kinetic data also suggested that α 2 -macreglobulin-kallikrein complex is formed during cold activation of plasma. Increased factor XII activity, determined as the prekallikrein-activating capacity, was present in the plasma samples from women using an cestrogen-containing oral contraceptive and from pregnant women near term. Both kallikrein and factor XII may be involved in the cold-promoted activation of factor VII.

Studies of human factor IX by a high-titred sheep antiserum against factor IX

Abstract In order to produce a high-titred antiserum against factor IX a sheep was immunized with highly purified human factor IX. One ml of the antisera obtained from three different bleedings neutralized 90% of the activity of factor IX in 130–200 ml standard plasma. The antisera gave one main precipitin band and occasionally an additional weak precipitin band against normal plasma in double immunodiffusion. The purified factor IX preparation had a specific activity of 290 U per absorbancy unit at 280 nm. However, it was not homogenous in various polyacrylamide gel electrophoresis techniques. In analytical polyacrylamide gel electrophoresis one main band and several additional weaker bands with lower electrophoretic mobility were seen. When this gel was submitted to electrophoresis into an agarose gel containing the antiserum against factor IX, one precipitin arc was seen corresponding to each of the bands. Reactions of identity was seen between the precipitin arcs, thus demonstrating the presence of factor IX antigen in all the bands. The minor bands with lower electrophoretic mobility disappeared on polyacrylamide disc gel electrophoresis in the presence of 10M urea and on SDS polyacrylamide electrophoresis. They may represent aggregates of factor IX, or complexes between factor IX and other proteins.

ON THE STABILITY OF ADRENALINE IN INJECTIONS: A COMPARISON OF CHEMICAL AND BIOASSAY METHODS.

The stability of adrenaline in some injections has been investigated by chemical assays based on determinations of adrenochrome and of adrenolutine, while the rat blood pressure method and the rat uterus inhibition method served as biological control procedures. The colorimetric method, in which adrenaline is oxidised to adrenochrome by means of potassium ferricyanide in acid solution, proved unable to detect any deterioration of adrenaline in procaine and adrenaline injections. The corresponding fluorimetric method, in which the adrenochrome formed is rearranged to adrenolutine by addition of strong alkali, gave results which agreed well with the biological results. In injections, adjusted to pH 3 with hydrochloric acid, adrenaline was stable for at least 20 months at 4° in solutions heated at 120° for 20 min., at 100° for 20 min. or unheated. Replacement of the hydrochloric acid by sodium metabisulphite (pH 3·6) prevented the discoloration. In injections of procaine and adrenaline, adjusted to pH 3 with hydrochloric acid, the adrenaline content decreased over the 20 month period, and was most pronounced in the solutions that had been heated before storage. Sodium metabisulphite significantly increased the stability of adrenaline in these injections and also prevented any colour formation.

Possibility of Immunodiagnosis in Ovarian Cancer

An antigenic material possibly associated with ovarian cancer has been demonstrated by the Ouchterlony technique in the sera from 42(66.7%) of 63 patients with primary ovarian malignancy of various histological types. It was more frequently found in the sera of preoperative patients in the later stages of the disease and those in relapse. Positive reactions could also be elicited from control sera of a very few persons without ovarian cancer. in none of the sera examined could the corresponding antibodies by demonstrated. The antigenic material was found to be an alpha2- or beta-glycoprotein on preliminary identification. There is an antigenic relationship between this material and fetal or normal adult ovaries. The possibility of immunodiagnosis of ovarian cancer is briefly discussed.