Proc. Nati. Acad. Sci. USA Vol. 73, No. 11, pp. 4179-4183, November 1976 Immunology Identification of prekallikrein and high-molecular-weight kininogen as a complex in human plasma (Hageman factor/bradykinin generation/contact activation) ROBERT J. MANDLE*, ROBERT W. COLMANt, AND ALLEN P. KAPLAN** * Allergic Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014; and t The Coagulation Unit of the Hematology-Oncology Section, Department of Medicine, University of Pennsylvania, Philadelphia, Pa. 19104 Communicated by K. Frank Austen, August 19, 1976 ABSTRACT Prekallikrein and high-molecular-weight ki- (Fletcher trait) was a gift from Dr. C. Abildgaard (University ninogen were found associated in normal human plasma at a of California, Davis, Calif.). Kininogen-deficient plasma was molecular weight of 285,000, as assessed by gel filtration on obtained from Ms. Williams and was collected as described Sephadex G-200. The molecular weight of prekallikrein in plasma that is deficient in high-molecular-weight kininogen was (3). 115,000. This prekallikrein could be isolated at a molecular Preparation of Plasma Proteins. Fresh plasma used for the weight of 285,000 after plasma deficient in high-molecular- isolation of prekallikrein and HMW kininogen was collected weight kininogen was combined with plasma that is congeni- in 0.38% sodium citrate. Hexadimethrine bromide (3.6 mg) in tally deficient in prekallikrein. Addition of purified 125I-labeled 0.1 ml of 0.15 M saline was added for each 10 ml of blood prekallikrein and high-molecular-weight kininogen to the re- drawn. The tubes were then centrifuged at 900 X g for 20 min spective deficient plasma yielded a shift in the molecular weight of prekallikrein, and complex formation could be demonstrated at 40 and the plasma was separated with plastic pipettes. Plastic by incubating prekallikrein with high-molecular weight kini- columns and test tubes were used throughout the chromato- nogen. This study demonstrates that prekallikrein and high- graphic procedures to minimize contact activation of Hageman molecular-weight kininogen are physically associated in plasma factor and nonspecific adsorption to glass surfaces. Samples as a noncovalently linked complex and may therefore be ad- were concentrated by ultrafiltration (Amicon Corp., Lexington, sorbed together during surface activation of Hageman factor. Mass.) through a UM-10 membrane. The complex is disrupted when these proteins are isolated by Hageman Factor Fragments. Prealbumin fragments of ion exchange chromatography. Hageman factor were purified by chromatography of plasma Activation of Hageman factor upon a negatively charged sur- on QAE-Sephadex twice, Sephadex G-100, and SP-Sephadex, face initiates the intrinsic coagulation pathway, the fibrinolytic and elution from alkaline disc gels after electrophoresis as re- pathway, and the generation of the vasoactive peptide bra- ported (6). dykinin. Recent investigations from several laboratories have Prekallikrein. Two liters of fresh plasma were dialyzed shown that the proteins of the kinin-forming pathway, namely, against 0.003 M phosphate buffer (pH 8.3) and passed over a prekallikrein (1, 2) and high-molecular-weight (HMW) kini- 20 X 100 cm column of QAE-Sephadex equilibrated with the nogen (3-5), are both required for optimal activation and same buffer. The effluent, containing a mixture of proteins of function of Hageman factor. Since prekallikrein and HMW gamma globulin mobility, was then fractionated by sequential kininogen are intimately associated functionally, it appeared chromatography on SP-Sephadex and Sephadex G-150, as de- possible that they might be physically associated in plasma. In scribed (7), followed by passage over an immunoadsorbent this paper we demonstrate that prekallikrein and HMW kini- prepared with antisera to human IgG and ,B2 glycoprotein I. nogen circulate in plasma as a noncovalently linked complex. For preparation of the immunoadsorbent, 100 ml of sheep Formation of this complex was observed: (a) when prekalli- antiserum to human IgG and 132 glycoprotein I was made 45% krein-deficient plasma was combined with plasma deficient in ammonium sulfate and stirred at 24° for 1 hr. The mixture in HMW kininogen, (b) after prekallikrein-deficient plasma was centrifuged at 1200 X g for 90 min at 40, and the precipi- and plasma deficient in HMW kininogen were reconstituted tate was washed twice in 45% ammonium sulfate. The washed with prekallikrein and HMW kininogen, respectively, and (c) precipitate was dissolved in distilled water, dialyzed against when purified prekallikrein was incubated with HMW kini- three changes of 10 liters of 0.003 M phosphate buffer-0. 15 M nogen. NaCl (pH 7.5) for 24 hr at 40, and coupled to 250 ml of Seph- arose 4B by the cyanogen bromide method (8). The Sepharose MATERIALS AND METHODS was poured into a 5 X 20 cm plastic column, washed with one column volume of 1.0% ethanolamine, and then equilibrated Apoferritin and catalase (Calbiochem, San Diego, Calif.), blue in 0.003 M phosphate buffer-0.35 M NaCl (pH 8.0). Samples dextran, ovalbumin, ribonuclease A, and chymotrypsinogen were dialyzed against this buffer prior to application to the (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.), chloramine immunoadsorbant. T (Matheson, Coleman and Bell Co., Norwood, Ohio), sodi- HMW Kininogen. HMW kininogen was isolated by chro- um[125I] iodide (New England Nuclear Co., Boston, Mass.), and matography of normal human plasma on QAE-Sephadex A-50, human transferrin (Sigma Chemical Co., St. Louis, Mo.) were ammonium sulfate precipitation, fractionation on SP-Sephadex, obtained as indicated. Plasma deficient in Hageman factor and and Sephadex G-200 gel filtration according to the method of containing 0.38% sodium citrate was obtained from Sera Tec Habal et al. (9). The preparation was quantitated functionally Biologicals, New Brunswick, N.J. Prekallikrein-deficient plasma by its ability to selectively correct the coagulation defect in Williams trait plasma as described (3). It was free of any de- Abbreviation: HMW kininogen, high-molecular-weight kininogen. tectable Hageman factor or prekallikrein. f To whom reprint requests should be addressed. The protein content of samples was approximated by ab- 4179 Downloaded by guest on September 27, 2021 4180 Immunology: Mandle et al. Proc. Natl. Acad. Sci. USA 73 (1976) sorbance at 280 nm with Al%m assumed to equal 10, or was determined by the Lowry method (10); the color reaction was 0.7 read at an optical density of 700 nm in a Beckman spectro- 0.6 photometer. Gel filtration on Sephadex G-150 (7), alkaline disc 0.5 E gel electrophoresis (11), and sodium dodecyl sulfate gel elec- 0.4 trophoresis (12) were performed as described. Prekallikrein was w radioiodinated by the Chloramine T method (13) using sodium 0.3 0.2 F:z [125I]iodide. The iodinated protein was immediately fraction- E ated on a 2 X 100 cm column of Sephadex G-50 equilibrated 0.1 8 in 0.003 M phosphate buffer-0.15 M NaCl (pH 8.0). The initial 0co peak of radioactivity was completely separated from the peak 'IC 0.7 0.6 of free iodine and contained over 99% trichloroacetic acid- I- precipitable counts. The 125I-labeled prekallikrein was mixed 0.5 0. with a 100-fold excess of nonradiolabeled prekallikrein in order 0.4 'S to assess binding to HMW kininogen. Radioactivity was de- 0.3 termined in a sodium iodide well scintillation counter (Nuclear 0.2 Chicago-Searle, Des Plaines, Ill., model 1185) with automatic 0.1 subtraction of background and an efficiency of 82%. Coagulation Assays. The partial thromboplastin time was 50 60 70 80 90 100 110 120 measured by the method of Proctor and Rapaport (14). Hage- TUBE NO. man factor, prekallikrein, and HMW kininogen were deter- FIG. 1. Sephadex G-200 gel filtration of normal human plasma mined by a modification of the procedure for determining (upper panel) and plasma deficient in Hageman factor (lower panel). partial thromboplastin time, with congenitally deficient plasma The column fractions were assayed for prekallikrein (A), HMW ki- (3). ninogen (o), and unactivated Hageman factor (0): Protein content Assays of Kinin-Forming Proteins. The proteolytic activity was estimated by the Lowry method and the color reaction was read of kallikrein was routinely measured by its ability to release at 700 nm (0). bradykinin from heat-inactivated plasma (11). Twenty-five the upper panel of Fig. 1, the prekallikrein and HMW kinino- microliters of kallikrein source were incubated with 0.2 ml of gen eluted in the same fractions, between the first and second substrate for 2 min of 370 and the bradykinin generated was protein peaks at an approximate molecular weight of 285,000, quantitated by bioassay as described (11). while unactivated Hageman factor was eluted between the Prekallikrein was determined by incubation of 25 ,l of second and third protein peaks at a molecular weight of proenzyme source with 25 ,ul of Hageman factor fragments (25 110,000-120,000. When plasma deficient in Hageman factor jig/ml) for 5 min at 370 and the kallikrein generated was de- was fractionated in an identical fashion (lower panel, Fig. 1), termined. prekallikrein and HMW kininogen were again found together Unactivated Hageman factor was assayed for its kinin-gen- at a molecular weight of 285,000. The elution position of pre- erating capacity by adding 10 ,l of sample to 200 ,g of plasma kallikrein and Hageman factor in the normal plasma chro- deficient in Hageman factor containing 0.9 mg/ml of EDTA. matogram was then confirmed, as assessed by their kinin- Fifty microliters of a suspension of kaolin in 0.15 M NaCl (10 generating ability. The location of each protein as assessed by mg/ml) were added, the mixture was incubated for 2 min at kinin generation was identical to its elution profile as assessed 370C and applied to the bioassay.