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Immunochemical Studies of Plasma Kallikrein

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Immunochemical Studies of Plasma Kallikrein Immunochemical Studies of Plasma Kallikrein Andranik Bagdasarian, … , Pat Wong, Robert W. Colman J Clin Invest. 1974;54(6):1444-1454. https://doi.org/10.1172/JCI107892. Research Article A monospecific antibody against human plasma kallikrein has been prepared in rabbits with kallikrein further purified to remove gamma globulins. The antisera produced contained antikallikrein and also anti-IgG, in spite of only 8% contamination of kallikrein preparation with IgG. The latter antibody was removed by adsorption of antisera with either Fletcher factor-deficient plasma or with purified IgG. Both kallikrein and prekallikrein (in plasma) cross-react with the antibody with no apparent difference between the precipitation arcs developed during immunoelectrophoresis and no significant difference in reactivity when quantified by radial immunodiffusion. Kallikrein antibody partially inhibits the esterolytic and fully inhibits the proteolytic activity of kallikrein. In addition, the antibody inhibits the activation of prekallikrein, as measured by esterase or kinin release. The magnitude of the inhibition is related to the molecular weight of the activator used. Thus, for the four activators tested, the greatest inhibition is observed with kaolin and factor XIIA, while large activator and the low molecular weight prekallikrein activators are less inhibited. With the kallikrein antibody, the incubation of kallikrein with either plasma or partially purified C1 esterase inactivator results in a new precipitin arc, as detected by immunoelectrophoresis. This finding provides physical evidence for the interaction of the enzyme and inhibitor. No new arc could be demonstrated between kallikrein and α2-macroglobulin, or α1-antitrypsin, although the concentration of free […] Find the latest version: https://jci.me/107892/pdf Immunochemical Studies of Plasma Kallikrein ANDRANiK BAGDASARIAN, BISWAJrr LAHmi, RiCHARu C. TALAMO, PAT WONG, and ROBERT W. COLMAN From the Coagulation Unit, Hematology-Oncology Section, Department of Medicine, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104 and the Department of Pediatrics and Medicine, Harvard Medical School, Boston, Massachusetts 02115 A B S T R A C T A monospecific antibody against human viduals contained 103±13 /g/ml prekallikrein antigen. plasma kallikrein has been prepared in rabbits with kal- Although in mild liver disease, functional and immuno- likrein further purified to remove gamma globulins. The logic kallikrein are proportionally depressed, the levels antisera produced contained antikallikrein and also of prekallikrein antigen in plasma samples from patients anti-IgG, in spite of only 8% contamination of kallikrein with severe liver disease remains 40% of normal, while preparation with IgG. The latter antibody was removed the functional kallikrein activity was about 8%. These by adsorption of antisera with either Fletcher factor- observations suggest that the livers of these patients deficient plasma or with purified IgG. Both kallikrein have synthesized a proenzyme that cannot be converted and prekallikrein (in plasma) cross-react with the anti- to active kallikrein. body with no apparent difference between the precipita- INTRODUCTION tion arcs developed during immunoelectrophoresis and no significant difference in reactivity when quantified Plasma kallikrein is a proteolytic enzyme that releases by radial immunodiffusion. the nonapeptide bradykinin from the plasma a2-globulin Kallikrein antibody partially inhibits the esterolytic kininogen. Bradykinin, when present systemically in and fully inhibits the proteolytic activity of kallikrein. nanogram amouts, profoundly lowers the blood pressure In addition, the antibody inhibits the activation of pre- and, when locally injected, mimics many of the features kallikrein, as measured by esterase or kinin release. of the inflammatory response (1). Kallikrein exists in The magnitude of the inhibition is related to the molecu- a precursor form, prekallikrein, which is converted to lar weight of the activator used. Thus, for the four the active protease by factor XIIA (Hageman factor) activators tested, the greatest inhibition is observed with and its proteolytically produced derivatives (2-5). As- kaolin and factor XIIA, while large activator and the says for kallikrein and prekallikrein have been based low molecular weight prekallikrein activators are less on the action of the enzyme on synthetic substrates (6) inhibited. or its ability to release bradykinin (7). It seemed de- With the kallikrein antibody, the incubation of kalli- sirable also to measure prekallikrein immunochemically krein with either plasma or partially purified Cf esterase to observe fluctuations of the protein as well as the inactivator results in a new precipitin arc, as detected by enzymatic acitvity in disease and to ascertain whether immunoelectrophoresis. This finding provides physical nonfunctional forms of kallikrein might characterize evidence for the interaction of the enzyme and inhibi- congential or acquired disorders. tor. No new arc could be demonstrated between kal- This study describes the further purification of kal- likrein and a2-macroglobulin, or ai-antitrypsin, although likrein (8) and the use of such a preparation and plasma the concentration of free kallikrein antigen decreases congenitally lacking prekallikrein (9) to obtain a mono- after interaction with the former inhibitor. specific precipitating antibody to prekallikrein and kal- By radial immunodiffusion, plasma from healthy indi- likrein. The interaction of the antibody with prekalli- krein is assessed by its ability to inhibit the conversion Dr. Bagdasarian is the recipient of special Postdoctoral of prekallikrein to kallikrein and the effect on kallikrein Fellowship HL52189 from the National Institutes of Health. is measured by inhibition of the proteolytic and estero- Drs. Talamo and Colman are the recipients of Career De- lytic activity of kallikrein. The antibody is also used as velopment Awards AM31937 and HL48075, respectively. Received for publication 10 April 1974 and in revised a tool to demonstrate complex formation between kal- form 5 August 1974. likrein and some of its natural inhibitors. Finally, de- 1444 The Journal of Clinical Investigation Volume 54 December 1974-1444-1454 velopment of an immunochemical assay permitted as- sessment of changes in kallikrein antigen level in liver disease. METHODS 10.9 2.0 Reagents. L-tosyl arginine methyl ester (TAMe) 1 was 0.0 2 4 1.8 purchased from Sigma Chemical Co., Inc., St. Louis, Mo., EE0.8 1.6 14E. and dissolved in 0.1 M sodium phosphate containing 0.15 0.7-0 ~~~~~~~~~~~~~~~~E M NaCi, pH 7.65. Agarose was purchased from Sigma 0.6 1.21 .2 0.5 Chemical Co. Special agar-noble was obtained from Difco - G 1.00 Laboratories, Detroit, Mich. Complete Freunds' adjuvant 0.4 0.6 was obtained from Difco. Polystyrene dishes for immuno- 0.3 o 0.6 diffusion were obtained from Hyland Div., Travenol Labor- 0.2 0.4 w atories, Inc., Costa Mesa, Calif. Sephadex G-200 and CM50 0.1- 0.2 .4 Sephadex beads were obtained from Pharmacia Fine Chemi- cals, Inc., Piscataway, N. J. DE52 cellulose was obtained 20 40 60 8 100 120 140 from Whatman. Hexadimethrene bromide (Polybrene) was FRACTION NUMBER obtained from Abbott Laboratories, North Chicago, Ill. FIGURE 1 Gel filtration of partially purified kallikrein. Antisera. Antibodies prepared in rabbits against human Starting material was 32.4 mg of the second carboxymethyl plasma proteins, anti-a-macroglobulin (a2-M), anti-Cl-in- Sephadex chromatography of kallikrein (8). The specific activator, and anti-al-antitrypsin (a,-AT) were purchased activity of the kallikrein was 9.3 umol TAMe hydrolyzed/ from Behringwerke AG, Marburg-Lahn, West Germany. ml/min. A 2.e X 97-cm column was packed with Sephadex Plasma samples. To obtain human, monkey, rabbit, rat, G-200 and equilibrated with 0.016 M sodium phosphate, goat, and dog plasma, nine vol of blood from healthy pH 8.0, in 0.13 M NaCl. Fractions of 1.3 ml were collected. donors was drawn directly into plastic tubes containing The flow rate was 15 ml/h 4rCat in a downward direction. 1 vol of 3.8% citrate and was immediately centrifuged at Circles denote the absorption at 280 nm while the triangles 2,500 g at 4VC to remove the cells. Plasma was then stored represent the esterase activity. Pools A, B, and C include at - 70'C. For certain experiments, hexadimethrene bro- the fractions denoted on the graph. mide was added to the citrate to produce a final concentra- tion of 50 ,g/ml plasma. Factor XII-deficient plasma was obtained from a donor with congenital deficiency of factor to Davis (16), as described earlier (4). Duplicate gels were XII. Fletcher factor-deficient plasma, which lacks func- run. One gel was stained and the other was sliced in 2-mm tional and immunochemical kallikrein (9), from a single segments and eluted overnight at 4oC with 5 mM potassium donor was a gift from Dr. C Abilgaard, University of phosphate in 0.15 M NaCl containing l0' M KCN, pH 8.0. California at Davis. Plasma samples were obtained from Preparation of activators of prekallikrein. These en- patients included in a previous study (10) measuring func- zymes were obtained in a partially purified state as a by- tional kallikrein and bradykinin. The criteria for diagnosis product of kallikrein preparation and further purified as have been described (10). Patients with prothrombin times described earlier (4). Large activator was a single band more than 3 s from control were
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