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Function and Immunochemistry of Prekallikrein-High Molecular Weight Kininogen Complex in Plasma

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Function and Immunochemistry of Prekallikrein-High Molecular Weight Kininogen Complex in Plasma Function and Immunochemistry of Prekallikrein-High Molecular Weight Kininogen Complex in Plasma Cheryl F. Scott, Robert W. Colman J Clin Invest. 1980;65(2):413-421. https://doi.org/10.1172/JCI109684. Research Article Plasma from individuals with high molecular weight (HMW) kininogen deficiency has been reported to be deficient in prekallikrein as measured by radial immunodiffusion, prekallikrein coagulant activity, and/or kaolin-activated arginine esterase activity. The discovery that prekallikrein and HMW kininogen circulate as a complex in plasma led us to reevaluate the antigenic and functional properties of prekallikrein in HMW kininogen-deficient plasma as well as in normal plasma. The low prekallikrein antigen level in an individual with HMW kininogen deficiency was corrected to the normal range (80- 95%) by the addition of 0.2 U/ml of purified HMW kininogen. A similar increase in apparent prekallikrein antigen was observed when purified prekallikrein and HMW kininogen were combined. The correction of the apparent prekallikrein defect in this HMW kininogen-deficient plasma coincided with the formation of a prekallikrein-HMW kininogen complex as demonstrated by immunoelectrophoresis. Similar findings were demonstrated with purified prekallikrein and HMW kininogen by immunoelectrophoresis as well as crossed immunoelectrophoresis. The coagulant activity in HMW kininogen-deficient plasma was increased in a dose-dependent manner by the addition of HMW kininogen, reaching 85% of normal level at a concentration of 0.2 U/ml. Kaolin-activated arginine esterase activity (kallikrein) in HMW kininogen-deficient plasma was fully corrected when HMW kininogen was added to the deficient plasma after depletion of kallikrein inhibitors. The functional and antigenic concentration of prekallikrein in plasma from four […] Find the latest version: https://jci.me/109684/pdf Function and Immunochemistry of Prekallikrein-High Molecular Weight Kininogen Complex in Plasma CHERYL F. SCOTT and ROBERT W. COLMAN, Coagulation Unit, Hematology-Oncology Section, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 A B S T R A C T Plasma from individuals with high mo- Addition of HMW kininogen increased the apparent lecular weight (HMW) kininogen deficiency has been prekallikrein activity in native normal plasma (as meas- reported to be deficient in prekallikrein as measured ured by esterase activity) but not in normal plasma by radial immunodiffusion, prekallikrein coagulant ac- in which inhibitors were inactivated. The apparent pre- tivity, and/or kaolin-activated arginine esterase activity. kallikrein antigen concentration (as measured by radial The discovery that prekallikrein and HMW kininogen immunodiffusion or electroimmunodiffusion) increased circulate as a complex in plasma led us to reevaluate upon addition of HMW kininogen. Immunoelectropho- the antigenic and functional properties of prekallikrein resis as well as gel filtration of normal plasma revealed in HMW kininogen-deficient plasma as well as in nor- the presence of free prekallikrein (17-38% of the total) mal plasma. in addition to the HMW kininogen-prekallikrein com- The low prekallikrein antigen level in an individual plex previously reported. with HMW kininogen deficiency was corrected to the This study emphasizes the influence of HMW kinino- normal range (80-95%) by the addition of 0.2 U/ml of gen on both functional and immunologic determina- purified HMW kininogen. A similar increase in ap- tions of prekallikrein. parent prekallikrein antigen was observed when puri- fied prekallikrein and HMW kininogen were combined. INTRODUCTION The correction of the apparent prekallikrein defect in this HMW kininogen-deficient plasma coincided with Plasma from individuals with severe deficiency of high the formation of a prekallikrein-HMW kininogen com- molecular weight kininogen (HMW kininogen)l have plex as demonstrated by immunoelectrophoresis. Simi- abnormalities in all Factor XII-dependent pathways lar findings were demonstrated with purified prekalli- including coagulation, kinin formation, fibrinolysis, krein and HMW kininogen by immunoelectrophoresis and permeability enhancement (1-5). Associated pre- as well as crossed immunoelectrophoresis. kallikrein deficiency, although insufficient to account The coagulant activity in HMW kininogen-deficient for these abnormalities, has been described in all but plasma was increased in a dose-dependent manner by one of these individuals (3). Close functional relation- the addition of HMW kininogen, reaching 85% of nor- ships, however, do exist between these two proteins. mal level at a concentration of 0.2 U/ml. Kaolin-activated The activated form of prekallikrein, kallikrein, is the arginine esterase activity (kallikrein) in HMW kinino- enzyme which releases bradykinin from HMW kinino- gen-deficient plasma was fully corrected when HMW gen (6), whereas HMW kininogen in turn potentiates kininogen was added to the deficient plasma after de- the activation of prekallikrein on a surface by Factor pletion of kallikrein inhibitors. The functional and anti- XII (7, 8) or in the fluid phase by Factor XII fragments genic concentration ofprekallikrein in plasma from four (XIIf) (9). The former activity may involve the increased other HMW kininogen-deficient individuals was simi- binding of prekallikrein to the surface when complexed larly corrected to normal after adding HMW kininogen. with HMW kininogen. The intimate physical and func- tional interrelations between these two proteins sug- This work was presented in part at the International Sympo- gested that the assay methods used for prekallikrein sium on Kinins, Tokyo, Japan, 8 November 1978 and published as in abstract form: 1979. Clin. Res. 27: 460A. IAbbreviations used in this paper: B-2, sodium barbital Dr. Scott's and Dr. Colman's present address is Specialized buffer, pH 8.6; Cl-inhibitor, CIS-inactivator; EID, electro- Center for Thrombosis Research, Temple University Health immunodiffusion; HMW kininogen, high molecular weight Sciences Center, Philadelphia, Pa. 19140. kininogen; XIIf, Factor XII fragments; IEP, immunoelec- Received for publication 23 January 1979 and in revised trophoresis; RID, radial immunodiffusion; TAMe, p-tosyl-L- form 20 September 1979. arginine methyl ester. J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/80/02/0413/09 $1.00 413 Volume 65 February 1980 413-421 may be affected by the presence or absence of HMW between these two fractions regarding functional or immuno- kininogen and give rise to an apparent deficiency of logic activity. Antiserum to kallikrein completely neutralized the amidolytic activity of the activated enzyme. Kallikrein prekallikrein in HMW kininogen-deficient plasma. was prepared by incubating 50 ,il of the prekallikrein described This study provides evidence that both immuno- above (1 U/ml) with 10 ,ul XIIf (50 ,ug/ml) at 37°C for 8 min. chemical and functional assays are thus influenced and HMW kininogen was purified by a modification of the that the addition of purified HMW kininogen to plasma method of Habal and Movat (12) by employing chromatog- not only raphy on QAE-Sephadex, ammonium sulfate precipitation, congenitally deficient in that protein corrects chromatography on CM-Sephadex and chromatography on SP- the defects in the Factor XII-dependent pathways, but Sephadex. An additional step was added to the published also results in normalization of the apparent prekalli- procedure. A 2.5 x 60-cm column containing SP-Sephadex krein deficiency. Furthermore, the nature of prekalli- was equilibrated with 0.03 M sodium acetate buffer, pH 6.0, krein in normal plasma has also been reinvestigated, containing 0.05 M NaCl. After application ofthe HMW kinino- gen from the CM-Sephadex steps and washing with the start- both functionally and immunochemically. Plasma pre- ing buffer, a linear gradient from 0.2 to 0.4 M NaCl in the kallikrein was found to exist in two forms, the major same buffer was used to elute the HMW kininogen. The puri- portion complexed to HMW kininogen as well as a fied HMW kininogen emerged near the end of the gradient minor component in an uncomplexed state. and had a specific activity of 15 U/mg coagulant activity. The protein was a single component of 120,000 daltons on sodium dodecyl sulfate-disc gel electrophoresis. No contamination METHODS was detected by functional assays for Factor XI, Factor XII, Materials. Antisera to immunoglobulin (Ig)G, CIS-inacti- or prekallikrein coagulant activity, or by immunological as- vator (Cl-inhibitor), a1-antitrypsin, a2-macroglobulin, anti- says for a2-macroglobulin, Cl-inhibitor, antithrombin III, a,- thrombin III, and radial immunodiffusion (RID) plates for IgG antitrypsin, or kallikrein. (L-C Partigen [Behring Diagnostics, American Hoechst Corp., The Factor XII fragments were prepared as described pre- Sommerville, N. J.]). H-D-Pro-Phe-Arg-p-nitroanilide-HCl viously (13). No contamination with Factor XII or high molec- (S-2302, Ortho Diagnostics Inc., Raritan, N. J.) was used as a ular weight activator (13) was detected in purified XIIf. There synthetic substrate of kallikrein. Tris and p-tosyl-L-arginine was no detectable contamination with Factor XI, plasminogen, methyl ester (TAMe [Sigma Chemical Co., St. Louis, Mo.]); plasmin, HMW kininogen, prekallikrein, or kallikrein. For kaolin (Fisher Scientific Co., Pittsburgh, Pa.); HSA agarose use in functional studies, XIIf was stabilized with bovine (Litex-Denmark); sodium
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