Clement E. Burrowes

Isolation of two functionally different kininogens from human plasma—separation from proteinase inhibitors and interaction with plasma kallikrein☆

Abstract A high (HMW) and a low (LMW) molecular weight kininogen were isolated in highly purified form from human plasma, using QAE-Sephadex chromatography, followed by ammonium sulfate precipitation, gel filtration through Sephadex G-200, re-precipitation with ammonium sulfate, CM-Sephadex and SP-Sephadex chromatography. The initial preparative step was done at room temperature and the remaining procedures at 4°. In aqueous media, the apparent molecular size of the HMW-kininogen was about four times the size of the LMW-kininogen (200,000 vs 50,000). During the process of purification, proteinase inhibitors were separated from the two kininogens: α 1 -antitrypsin and α 2 -macroglobulin from the LMW-kininogen preparations: Cl-inactivator and inter-α-trypsin inhibitor from the HMW-kininogen preparations. There was a well defined functional difference between the two kininogens with respect to kinin generation by plasma kallikrein. This enzyme released kinin at a much faster rate from the HMW-kininogen than from the LMW-kininogen. When equipotent preparations of kininogens were incubated for 10 min with kallikrein, 60 times more enzyme was required to release the same amount of kinin from the LMW-kininogen as from the HMW-kininogen.

The inhibition of activated factor XII (hageman factor) by antithrombin III: The effect of other plasma proteinase inhibitors

Abstract Human factor XII was activated by adsorption onto kaolin in the presence of high molecular weight kininogen. The washed kaolin-containing precipitates activate prekallikrein to kallikrein. When antithrombin III was added to the reaction mixture, the conversion of prekallikrein to kallikrein was inhibited, the degree of inhibition depending on the concentration of antithrombin and the time of incubation. Heparin had a slight enhancing effect with low concentrations of antithrombin and short incubation times. However, the inhibition of the generated kallikrein by antithrombin III was markedly enhanced by heparin. Antithrombin III inhibited also the effect of activated factor XII on the partial thromboplastin time, using factor XII-deficient plasma. Of other plasma proteinase inhibitors used (α1-antitrypsin, α2-macroglobulin, C l -inactivator) only C l -inactivator inhibited activated factor XII.

Interaction between factor XII (Hageman factor), high molecular weight kininogen and prekallikrein.

Abstract Adsorption of factor XII and plasma kallikrein onto kaolin induces activation of factor XII. Trace amounts of high molecular weight (HMW)-kininogen markedly enhances the rate and the amount of activation of factor XII. Activation of factor XII was measured by its capacity to convert prekallikrein to kallikrein. This potentiating effect is dependent on the amount of HMW-kininogen. Low molecular weight kininogen has no enhancing effect. When the reactants are adsorbed to kaolin and the latter separated by centrifugation the prekallikrein-converting activity is detectable only in the kaolin-containing precipitates. Less activation of factor XII occurs when the HMW-kininogen is added not initially to the kaolin-factor XII mixture, but to the prekallikrein, indicating that the HMW-kininogen probably acts when factor XII is being activated, rather than at a later stage. Treatment of HMW-kininogen with excess trypsin, plasmin or kallikrein abolishes its potentiating effect. When HMW-kininogen is treated with increasing concentrations of kallikrein there is an inverse relationship between the generated kinin and the ability of the kininogen to enhance the activation of factor XII.

The inhibition of human plasma Kallikrein by antithrombin III

Abstract Prekallikrein, prekallikrein activator, high molecular weight kininogen and antithrombin III were isolated from human plasma. The prekallikrein was converted to kallikrein by prekallikrein activator. The kallikrein had arginine esterase activity and generated kinin from kininogen. Both activities were inhibited by antithrombin III in concentrations of approximately 1 2 of that of plasma. Physiological concentrations of heparin were required for the rapid and complete inhibition of kallikrein. In the absence of heparin only partial and much slower inhibition was achieved. The proteolytic, i.e. kinincleaving activity of kallikrein was more readily inhibited than the esterolytic activity.

Generation of kinin by plasma kallikrein and plasmin and the effect ofα(in1) antitrypsin and antithrombin III on the kininogenases

low molecular weight Kgn I, which has the bradykinin (BK) sequence at its carboxyl terminus, and Kgn II, with the BK moiety inside the molecule (J.V. Pierce and M.E. Webster, in Hypotensive Peptides, E.G. Erd6s, N. Back, F. Sicuteri, eds., Springer, 1966), were equally good substrates for pepsin. All of the kinin was released in three minutes when 10 mg of Kgn B3.2c~ (containing ca. 15/Jg of BK/mg, as determined with trypsin) was incubated with 50/Jg of 2X crystallized pepsin at pH 2.0 and 37 ~ . The released kinin, equivalent to 18 #g of BK on the isolated guinea pig ileum, was stable in the incubation mixture. It was inactivated by carboxypeptidase B and could be converted to an 8-fold more active peptide upon incubation with human plasma aminopeptidase or dipeptidyl aminopeptidase I (DAP-I). The peptic Kinin was separated from the bulk of protein by gel filtration on Sephadex G-25 and was further purified by SP-Sephadex G-25 and CM-cellulose chromatography. The protein fraction did not generate any additional kinin upon incubation with trypsin or human plasma kallikrein. Several criteria were used to identify the purified peptic kinin as MLBK: a) it has the same retention volume as MLBK on an analytical SP-Sephadex C-25 column which is able to resolve BK, LBK, and MLBK; b) it was converted by treating with DAP-I to BK, identified by the 8-fold activity increase and by its now having the same retention volume as BK on the SP-Sephadex C-25 columnn; and c) it had the same amino acid composition as MLBK. The kinin released from other pure Kgns by pepsin was also identified as MLBK by some of the above criteria. Pepsin, like trypsin and plasmin, cleaves several peptide bonds in Kgns other than the bonds involved in the release of MLBK. The kinin-releasing activity of pepsin extends to pH 5.0 and is inhibited by pepstatin at both pH 2.0 and 5.0 Our highly purified preparations retained full Kgn activity at pH 2.0 after several days at room temperature (ca. 25~ MLBK was first isolated from bovine plasma which had been dialyzed against 0.01 M HCI and then incubated at pH 7.5 (D.F. Elliott and G.P. Lewis, Biochem. J. 95, 435, 1965). Since plasma contains pepsinogen, its activation in the acid dialysis step could explain the release of MBLK observed by Elliott and Lewis. GENERATION OF KININ BY PLASMA KALLIKREIN AND PLASMIN AND THE EFFECT OF oq ANTITRYPSIN AND ANTITHROMBIN III ON THE KININOGENASES M. FLAVIO, HABAL, E. CLEMENT, BURROWES and Z HENRY, M O V A T Division of Experimental Pathology, Department of Pathology, University of Toronto,

The Kinin System of Human Plasma VI. The Action of Plasmin

Summary Plasminogen was isolated from acid euglobulin by gel filtration through Sephadex G-200 and anion exchange chromatography on DEAE-Sephadex A-50. Nonspecific esterases were inactivated with DFP and the plasminogen was rechromatographed. Plasmin (streptokinase activated plasminogen) was shown to act on kininogen (heated plasma), from which it released kinin. This was a slow process. Rapid kinin formation was achieved when plasmin liberated prekallikrein activator from partially purified activated Hageman factor (factor XIIa), which in turn acted on prekallikrein. The active kallikrein enhanced vascular permeability when injected intradermally into guinea pigs, hydrolyzed BAEe, and rapidly liberated kinin from heated plasma substrate. It is concluded that plasmin in high concentrations liberates kinin directly from kininogen. However, in low concentrations (which prevail in vivo), when it functions as an activator, it acts not directly on prekallikrein, as postulated repeatedly, but probably by liberating prekallikrein activator from factor XIIa.

Fibrinolytic activity of adult rat liver cells in primary culture and inhibition of glucocorticoids

Summary Adult rat liver cells, maintained in primary culture in a non-proliferating state, have been observed to flatten and spread-out during the first 3 days in culture. A fibrin-agar overlay technique and the digestion of 125 I-fibrin films have been used to show the potent fibrinolytic activity of liver cells in primary culture. Microscopic observation of liver cells under fibrin-agar overlays demonstrated the potent fibrinolytic activity of individual flattened liver cells. Both the fibrinolytic activity and the flattening of the liver cells are inhibited by dexamethasone and to a lesser degree by hydrocortisone. Fibrin digestion per se is unaffected by dexamethasone supplementation.

Plasminogen activator and thromboplastin activity from sheep alveolar macrophages

Alveolar lavage cells from normal sheep were found to be composed of over 95% macrophages. When the cells were cultured, fibrinolytic and thromboplastin-like activities could be detected within 2-4 hours of incubation. As the number of cultured cells was increased the two activities in the conditioned medium increased proportionately. The cells were separated into two distinct subpopulations by means of a sedimentation velocity cell fractionation technique. The macrophage subpopulations were examined for differences in size, morphology, esterase staining and ability to release plasminogen activator and procoagulant activity respectively. These activities were confined to the large cell subpopulation. The fibrinolytic activity was shown to be plasminogen-dependent and could be inhibited by DFP. On the basis of this the fibrinolytic activity has been designated as plasminogen activator. The procoagulant activity was shown to be thromboplastin in nature because it was Factor VII dependent, inactivated by phospholipase C and not inhibited by DFP. The procoagulant activity has been designated as macrophage thromboplastin. The two activities could be distinguished on the basis of DFP inhibition.

Surface activation of factor XII (Hageman factor) — Critical role of high molecular weight kininogen and another potentiator

When factor XII was adsorbed to kaolin it slowly became activated and converted prekallikrein to kallikrein. In the presence of HMW-kininogen the rate of activation of factor XII and consequently that of prekallikrein was markedly enhanced. The enhancing effect of HMW-kininogen was a dose-dependent phenomenon. In order to enhance the activation of factor XII on a surface the HMW-kininogen molecule had to be intact. Cleavage of HMW-kininogen by kallikrein decreased the enhancing effect of HMW-kininogen, there being an inverse relation between the bradykinin-generated and the capacity to enhance factor XII activation. Another ‘potentiator’ of factor XII activation was isolated from proteins adsorbed to aluminum hydroxide. This potentiator further increased the activation of factor XII, also in a dose-dependent fashion. It was postulated that factor XII is slowly converted into its active form by exposure to negatively charged surfaces; that this process is enhanced by kallikrein and further accelerated by HMW-kininogen and the ‘potentiator’; and that these enhancing substances probably act by opening active sites on the factor XII molecule.

High molecular weight kininogen: its inability to correct the clotting of kininogen-deficient plasma after cleavage of bradykinin by plasma kallikrein, plasmin or trypsin.

Abstract Human high molecular weight (HMW)-kininogen was cleaved with trypsin, plasmin or plasma kallikrein to release bradykinin. ‡ When an excess of enzyme was used and all releasable peptide was released, the residual HMW-kininogen could not correct the clotting deficiency of kininogen-deficient plasma. There was an inverse relationship between the amount of bradykinin released and the capacity of the kininogen to correct the abnormal partial thromboplastin time of kininogen-deficient plasma. These findings and earlier ones indicate that the intact HMW-kininogen molecule is required for contact mediated clotting and activation of factor XII.