Ulla Hamberg

PLASMA PROTEASE AND KININ RELEASE WITH SPECIAL REFERENCE TO PLASMIN

Earlier studies have indicated that bradykinin can be released in human plasma by a spontaneously activated protease after acid-heat treatment to destroy kininase and protease inhibitors.1-3 During those studies the similarity between the acid-heat stability of plasminogen and bradykininogen, as well as the generation of plasmin simultaneously with the release of bradykinin from human denatured plasma, was noted.2 The peptide released during certain experimental conditions was shown to be identical with bradykinin by N-terminal end-group determination, the amino acid proportions, and the biological activity, when compared with synthetic bradykinin. Similar results were obtained after activation in human plasma with streptokinase3 and urokinase.4 Preliminary results indicated that a mixture of plasma kinins might have been formed. The presence of PTHarginine5.6 in an end-group assay showed that bradykinin was present. The fact that activation of the endogenous protease or the fibrinolytic system released bradykinin suggests a related mechanism of pathological plasma-kinin release observed in various states such as liver cirrhosis, carcinoid syndrome, anaphylactic shock, and inflarnmati~n,~-l~ which are also noted for an increased fibrinolytic activity.11J2 While it is possible to show kinin release with plasmin or the activation of fibrinolysis in vitro, the demonstration appears to be more difficult to achieve in vivo under physiological conditions. Back,lsJ4 however, was able to demonstrate a transient hypotensive reaction, presumably due to a release of vasoactive peptides by intravenous (i.v.) injection of human plasmin in the dog, which could be abolished by the administration of the pancreatic trypsin inhibitor (TrasyloF) .l4 It appears possible that the physiological and pathological bradykinin release proceeds through different pathways, and that release of plasma kinin by plasmin may occur mainly in pathological conditions. It is already well known that the kininogen is subject to the action of various proteases such as trypsin, snake venom proteases, plasma protease, bacterial proteaseP et cetera. It should further be noted that in many cases investigating the actions of these so-called kininogenins16 or kininogenases, analyses of the peptides split off have not been included. The present studies deal with the possible participation of the fibrinolysin system in the release of plasma kinins such as those found in the pathophysiological system that exists in the postmortem human plasma in cases of sudden death.17 In this type of plasma, the proteins have been subjected to at least two of the endogenous proteolytic activation processes, and were incoagulable because of the lack of fibrinogen or a defective coagulation mechanism.18 The results reported here concern the bradykininogen level in the postmortem human plasma, which was found to be decreased but not exhausted, despite strong proteolysis of the blood-coagulation process followed by complete lysis of the clots. At the same time it was noted that peptide split products deriving from the plasma protease reaction may play an important role in potentiating the action of bradykinin under pathophysiological conditions.

Isolation and immunologic properties of a heterogenous antigen with the characteristics of the heavy chain of human plasma kininogen

Kininogen antigen was purified from human plasma fraction IV by ion exchange chromatography, gel filtration and affinity chromatography with antibody specific immunoadsorbents. The immunologically pure glycoprotein had a mol. wt of approximately 60,000 and only one polypeptide chain by SDS-PAGE. An extensive charge heterogeneity by isoelectric focusing and gel filtration on polyacrylamide agarose could only in part depend on a comparatively high sialic acid content, but may be caused by differences in the carbohydrate structures sustained by lectin-binding heterogeneity on Con A-Sepharose. This antigen shares a dominating determinant with native plasma kininogens shown by complete patterns of identity in immunochemical analyses and with the monospecific antisera developed in rabbits against the heterogeneous components. The similar size, amino acid composition, low histidine content, lack of N-terminal amino acid and antigenic homogeneity fit all the so far known characteristics of the human kininogen heavy chain. Notably the antigenic determinant is resistant to degradation by activated kallikrein. This antigen with unimpaired immunologic activity may be a useful tool for preparation of antiserum for immunochemical determination of human plasma kininogen.

Conformation and sequence dependent antigenic determinants in human low molecular weight kininogen

Conformation and sequence-dependent antigenic determinants were investigated using a kinin-free low molecular weight kininogen isolated from Cohns plasma fraction IV. This antigen contains the determinants of the apparently intact heavy chain common to the high molecular weight and low molecular weight kininogens. Straightforward reduction and carboxymethylation destroyed the immunoreactivity of this molecule. Antiserum prepared against the reduced protein recognized both reduced and unreduced antigen showing the presence of both types of antigenic determinant. The corresponding antibodies were separated using immunoadsorbent columns. As shown by the higher avidity of the antibodies, the conformation-dependent determinants dominate the antigenic structure.

Identification in human plasma of low Mr protein fragments with antigenic determinants of kininogen

Identification in human plasma of low Mr protein fragments with antigenic determinants of kininogen

On the Mechanism of Bradykinin Potentiation

Bradykinin potentiating effects have been demonstrated with various compounds, some of them due to a sensitization during bioassay with proteolytic enzymes (1), specific fibrinopeptides (2,3) or peptides isolated from snake venom (4). In earlier reported work we found that the bradykinin-induced contraction of isolated guinea pig ileum was influenced by tryptic peptides split from plasma proteins or obtained by a spontaneous activation of an endogenous plasma protease (5). Since kininase inhibitors have been shown to produce similar effects (6,7) we have explored the possibility that a competitive enzyme inhibition may regulate the biological activity of bradykinin. Several kininases may have to be accounted for (6). The importance of the carboxyl end arginine for the activity of bradykinin (8) appears first to suggest an enzyme mechanism correlated to a carboxypeptidase-like action (9). As shown below a competitive inhibition of car boxypeptidase B action is obtained with the tryptic peptides isolated from human plasma, which may be released simultaneously with bradykinin.

Kininogen by the SRI Method in Human Serum During an Acute Phase Inflammatory Reaction

The concentration of several serum proteins changes during the acute phase in inflammatory states.1 Kininogen belongs to this group of acute phase reactants as shown in our earlier studies and also with plasma from patients with rheumatoid arthritis.2 In early studies plasma kininogen was measured exclusively by estimation of the bradykinin equivalent by bio- assay on the isolated rat uterus or guinea pig ileum. 3 More recently a bradykinin enzyme immunoassay4, radioimmunoassay5 and rocket Immunoelectrophoresis6 were reported for the determination of HMr kininogen in plasma. A method designated to determination of the total plasma and serum kininogen by single radial immunodiffusion (SRI) was recently presented by us in detail.7 The SRI method was compared with and correlated to kininogen determined by the bradykinin equivalent and gives the corresponding normal values in human plasma and serum. This presentation deals with its application in a study of kininogen investigated in a serum material collected from patients during periods of treatment with immunotherapy in renal cell carcinoma.8–12 An infiammatory reaction provoked during the treatment was shown by the increase of a1-acid glycoprotein and some other acute phase reactants.

Determination of human plasma kininogen by a single radial immunodiffusion method and the bradykinin equivalent.

An SRI method is presented for the quantitation of native human plasma kininogen using a monospecific high avidity antiserum prepared against the conformational determinants of kininogen heavy chain (HC antigen). The SRI value of total human plasma kininogen averaged 0.260 +/- 0.052 g/l (+/- 2 SD) using a standard curve of normal human plasma in each assay (measuring range 0.016-0.260 g/l). The SRI data, confirmed by RIA, were compared with determination of the bradykinin equivalent by bioassay 4.30 +/- 0.052 mg/l (+/- 2 SD), generally accepted as a measure of total plasma kininogen. The SRI method does not discriminate between LMr and HMr kininogen but detects an increase of total kininogen independently of bradykinin release as a consequence of activated proteolysis. It is suggested that the SRI method can be applied to study levels of kininogen in clinical research.

Kininogen molecular heterogeneity by affinity chromatography on concanavalin A and Ricinus communis lectins

Kininogen molecular heterogeneity by affinity chromatography on concanavalin A and Ricinus communis lectins

Kininogen as a pregnancy-associated plasma protein.

Pregnancy induces marked changes in plasma protein concentrations. Some proteins are secreted by the placenta into the maternal circulation and are thus called pregnancy-specific plasma proteins. Pregnancy-specific beta-l-glycoprotein (SP1) was the first protein of this group.1 Other plasma proteins also undergo changes during normal pregnancy and in response to contraceptive doses of estrogens.2,3 In early studies several authors reported determination of kininogen during pregnancy, delivery and puerperium4–6 indicating increased kininogen during pregnancy, decreasing values during labor and increasing kininogen content in puerperium. In all these studies kininogen was determined by the estimation of the bradykinin equivalent by bioassay on the rat uterus or guinea pig ileum. Suzuki7 found in a preliminary report a 100% increase of plasma kininogen compared with non- pregnancy levels showing that bradykinin was not released from kininogen during the pregnancy periods. In particular interest has more recently been focused on the HMr (high molecular weight) kininogen due to its function as a cofactor in the activation of F XII and the onset of coagulation. Maki et al.8 reported increase of HMr kininogen during pregnancy and decrease with the start of labor which suggested that the vasoactive bradykinin was relesed during labor through the activation of plasma kallikrein and brought back to normal level after delivery. Similar findings were recently reported by Suzuki et al.9 applying the bradykinin equivalent to determine kininogen while Maki et al.8 were first to utilize immunological determination with HMr specific antiserum in pregnancy.

The Fibrinolytic Activation Mechanism in Human Plasma

Some recent advances are presented with special reference to molecular aspects. Emphasis is given to the formation of certain inhibitor complexes involving the human α2-macroglobulin. As shown with streptokinase-activated human plasma (Hamberg 1966a,b; Hamberg & Savolainen 1967, 1968) an association with proteins occurs, which lacks the proteolytic action on fibrin while retaining esterase and plasminogen activator activity. The complex formation is further stressed by a correlation to molecular size found with various inhibitors demonstrated below, and the concentration-dependent inhibition with ε-aminocaproic acid. The significance of these effects produced by steric hindrance for the interpretations of fibrinolytic enzyme activity and the proactivator reaction are discussed.