F12-1

1983 62: 448-456 DL Tankersley, BM Alving and JS Finlayson XII Activation of factor XII by dextran sulfate: the basis for an assay of factor http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: Copyright 2011 by The American Society of Hematology; all rights reserved. 20036. the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by For personal use only. by guest on November 16, 2011. bloodjournal.hematologylibrary.orgFrom 448 Blood, Vol. 62. No. 2 (August), 1983: pp. 448-456 Activation of Factor XII by Dextran Sulfate: The Basis for an Assay of Factor XII By Donald L. Tankersley, Barbara M. Alving, and J. S. Finlayson A system was developed for studying the activation of factor XII (Hageman factor) in the presence .gf dextran sulfate (DS). Salient features of the system included low ionic strength (0.08). low concentration of factor XII (ap- proximately 1 I 1 0.000 that in normal plasma), and an excess of exogenous prekallikrein (PK). In this system, factor XII was rapidly converted to the 80,000 molecular weight (mol wt) form of factor Xlla (a-factor-Xlla). Once formed. the factor Xlla converted PK to kallikrein at a rate that was proportional to the amount of factor XII originally present in the incubation mixture. This system was used to construct a simple sensitive assay for factor XII in plasma R ECIPROCAL ACTIVATION of factor XII and PK occurs when plasma contacts a negatively charged surface, such as glass or kaolin.’ High molecu- lan weight kininogen, a contact-activation cofactor that circulates as a complex with PK2, accelerates the activation process by binding PK to the surface, where it is cleaved by surface-bound factor XIIa.3 High molecular weight kininogen may also enhance contact- activation by protecting kallikrein from protein- ase inhibitors.4 As an initial step, functional assays for factor XII require the conversion of the zymogen to its active form, whereupon its ability to clot factor-X Il-deficient plasma on to activate PK can be determined. Insoluble agents, such as kaolin or celite, are frequently employed to bring about factor XII activation. When these are used in factor XII assays that involve PK activation, it may be desirable to remove them by centnifugation to avoid interference (light-scattering) in spectrophotometnic analyses for the resulting kalli- krein.5’6 However, substantial amounts of a-factor- XIIa can remain bound to the insoluble agent and thus be removed during centrifugation. The reported use of the soluble polycation, DS, as the activating agent in an assay for PK7 prompted our investigations into its use in an assay for factor XII. Because we intended to employ this assay for monitor- ing the purification of factor XII from plasma, we From the Divison of Blood and Blood Products, National Center for Drugs and Biologics. Bethesda. MD, and the Department of Hematology. Walter Reed Army Institute of Research. Washing- ton. D.C. Submitted November 29. 1982; accepted March 1 7. 1983. Address reprint requests to Dr. Donald L. Tankersley. Division of Blood and Blood Products, National Centerfor Drugs and Biolog- ics. 8800 Rockville Pike. Bethesda, MD 20205. © I 983 by Grune & Stratton, Inc. 0006-4971/83/6202-0034$01 .00/0 and other biologic samples. The kallikrein produced was measured spectrophotometrically with the chromogenic substrate (H-D-Pro-Phe-Arg-p-nitroanilide (5-2302). This assay was shown to be independent of the high molecular weight kininogen and the PK content of the sample being analyzed. The measurements obtained were consistent with fundamental enzymologic principles and, if desired, could be processed with a simple calculator program to achieve linear standard curves. When applied to the quan- titation of factor XII in plasma. the assay yielded values in close agreement with those determined by coagulant assay or by radial immunodiffusion. considered it essential for the assay to be independent of other contact factors that might be present in (or absent from) the samples analyzed. The method devel- oped fulfilled this requirement and, moreover, proved to be suitable for the measurement of factor XII in unfractionated plasma,8 in plasma containing Poly- brene, or in serum obtained by clotting plasma with thrombin or by recalcification in glass. Chemicals MATERIALS AND METHODS DEAE-Sephadex A-SO, CM-Sephadex C-SO, and Sephadex G- 1 50 were obtained from Pharmacia Fine Chemicals, Inc., Piscata- way, NJ. The chromogenic substrate for kallikrein, H-D-Pro-Phe- Arg-p-nitroanilide (S-2302), was purchased from Kabi Diagnostica, Stockholm, Sweden. Dextran sulfate sodium salt (DS), mol wt 500,000, was obtained from Sigma Chemical Co., St. Louis, MO. Human thrombin, lot H-I, was obtained from Dr. D. L. Aronson. Human albumin, 5%, was obtained from Cutter Laboratories, Inc., Berkeley, CA; it contained no detectable PK-activator activity and 0.2 when incubated with DS plus NHP, and <0.01 when incubated with DS alone. These fractions were pooled and concentrated to a volume of I 3.5 ml to afford a PK concentration sufficient for the PK-DS assay. PK-DS Assayfor Factor XII The PK-DS substrate solution used for the assay of factor XII was prepared in the following manner. To 18 ml of 0.05 M Tris-HC1, 0.05 M NaCI, pH 8.0 buffer was added I 00 jzl (I 58 �zg) of PK, 2 ml of 5% human albumin, and I 10 �zl of DS ( I mg/ml in H2O). Aliquots (45 z1) of this solution were then carefully pipetted into the bottom ofconical polypropylene tubes of 1.5 ml capacity (Eppendorf). The solution was kept on ice during the pipetting procedure (-2 hr) and a positive displacement pipettor (Scientific Manufacturing Industries, Emeryville, CA) was used. The tubes were promptly frozen at - 70#{176}C.Alternatively, the crude PK preparation (13.5 ml, see above) was cooled on ice and treated with I .6 ml of 5% albumin, 0.8 ml of 0.05 M Tris-HCI, I .0 M NaCI, pH 8.0, and 88 z1 of DS (1 mg/mI). This solution was then aliquotted as described above. Plasma samples were diluted I : I ,000 with 0.05 M Tris-HCI, 0.05 M NaCI, I mg albumin/mI, pH 8.0, in polypropylene tubes. Other materials to be assayed (column fractions, purified factor XII, etc.) were similarly diluted such that the approximate factor XII content was 0.Ol%-O.2% of that of NHP (i.e., 2-50 ng/ml). Standards were prepared by diluting NHP I :500 in the same buffer; this solution represented a factor XII content of 200% NHP in this assay, and it was further diluted to provide 3-8 different levels of factor XII (see Fig. 4). Diluted samples were stable for at least 4 hr at room temperature, and could be frozen and thawed several times without affecting the measured factor XII content. A total of 20 samples (including standards) could be conveniently assayed in one run, with additions being performed at 15-sec intervals. The tubes of PK-DS substrate, prepared as described above, were warmed to 37#{176}Con a hot-block for 15-20 mm, then S �zl of diluted sample was admixed. After 10 mm, 500 j�l of 0.2 mM 5-2302, 0.05 M Tris-HCI, 0.05 M NaCI, pH 8.0, prewarmed to 37#{176}C,was added, and the mixture was further incubated for exactly S mm; then 100 MI of 20% acetic acid was added to stop the reaction. The absorbance at 405 nm was determined against a blank consisting of 500 MI of 5-2302 solution and I SO �l of 20% acetic acid; results were reported as �A�5/S mm. The �A�5/S mm values observed for the standards were used to construct a standard curve, as described in Results. A programmable calculator (Texas Instruments TI-59) was used to generate the least-squares standard curve and to interpolate the factor XII levels in samples from the measured �A.,,5/5 mm. The program listing, documentation, and user instructions will be supplied by the authors upon request. Other Methods Protein solutions were concentrated by positive-pressure ultrafil- tration in Amicon stirred cells equipped with PM-IO membranes. Protein concentrations were determined by Lowry assay with bovine albumin as standard, or by spectrophotometry (A2,,�), employing previously determined absorption coefficients. Radial immunodiffu- sion of factor XII was performed by the procedure of Mancini et al.;’2 goat antiserum to human factor XII was provided by Dr. John Griffin, Scripps Clinic, La Jolla, CA. It was adsorbed with 1/10 volume of factor-XI1-deficient plasma and used at a final dilution of 1:90 in 1% agarose. Plasma samples were assayed undiluted, and standards consisted of purified factor XII diluted in factor-XII- deficient plasma to provide concentrations of 0, 8, 16, 32, and 64 sg/ml. Factor XII coagulant activity was determined by the acti- vated partial thromboplastin time with factor-XII-deficient plasma as substrate.’3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- For personal use only. by guest on November 16, 2011. bloodjournal.hematologylibrary.orgFrom >- I- I- C-) C’) C�,J C,, 100 80 60 40 20 0 �_ 0 2 4 6 8 10 DEXTRAN SULFATE CONCENTRATION (pg/mI) Fig. 2. Inhibition of kallikrein by DS. PK (1 58 �zg/ml) and �-factor-Xlla (1 pg/mI) were incubated in 0.05 M Tris-HCI, 0.05 M NaCl. pH 8.0. for 2 hr at 30’C to form kallikrein. Portions (5 .tI) of this solution were added to 70 �sl of the same buffer containing DS (0-10 �g/ml). and the activities of these mixtures were deter- mined after 2 mm (�) and 40 mm (#{149}). : � � � � � � � I.., A B CD E� F GH” 1J 450 TANKERSLEY. ALVING, AND FINLAYSON PAGE) was carried out on 6% gels, as described by Weber and Osborn.’4 Figure 1 shows SDS-PAGE gels of the purified proteins used in this work. Unreduced factor XII and a-factor-XIIa both demonstrated a major band corresponding to a mol wt of 80,000. Reduction of a-factor-XIla resulted in the appearance of bands corresponding to mol wts of 53,000 and 28,000, whereas no change was noted upon reduction of factor XII. fl-factor-XIIa appeared as a single band (mol wt 28,000), whether reduced or unreduced. Unre- duced samples of PK and of kallikrein both demonstrated two closely spaced bands of mol wts 82,000 and 84,000, with the latter predomi- nating (-70%). Upon reduction, PK was unchanged, whereas kalli- krein revealed major bands at mol wts of 50,000, 34,000, and 32,000 and two minor bands at 29,000 and 21,000. (The latter bands arise from further proteolysis of the 50,000 mol wt heavy chain.) These SDS-PAGE patterns for PK and kallikrein are consistent with the results reported by others.’5 RESULTS Inhibition ofKallikrein by DS The development of a factor XII assay that depends on the DS-induced activation of factor XII and subse- quent factor-XIIa-catalyzed activation of PK requires the accurate quantitation of the kallikrein produced. As shown in Fig. 2, DS significantly inhibited the hydrolysis of S-2302 by kalliknein. The inhibition appeared to be similar to that observed when kallikrein is exposed to surfaces,’6 in that the process was inre- vensible and had both an immediate and a time- dependent phase. Furthermore, the loss of kallikrein activity produced by DS was considerably less when . . �. Fig. 1. SDS-polyacryl- amide gel electrophoresis (6% gels) of purified proteins used in this study. Gels A and B contained 3 �g of factor XII; gels C and D. 4 pg of a-factor- Xlla; gels E and F. 3 �sg of $-factor-Xlla; gels G and H. 3 1�9 of PK; and gels I and J. 3 �ig of kallikrein. The samples ap- plied to gels B. D. F. H, and J were reduced with dithiothrei- tol (10 mM) before application. Anode is at bottom. For personal use only. by guest on November 16, 2011. bloodjournal.hematologylibrary.orgFrom 0.4 C E U, I- I- C., C,) C,�1 0.2 0.l 0 0 2 4 6 INCUBATIONTIME (nan) Fig. 3. Activation of PK in the presence of DS. Aliquots (45 p1) of a solution containing PK (7.8 pg/mI). DS (5.5 pg/mI). and albumin (5 mg/mI) in 0.05 M Tris-HCI. 0.05 M NaCI. pH 8.0. were treated with 5 pl of NHP (1 :1 .000 dilution; �), factor XII (20 ng/ml; #{149}).a-factor-Xlla (20 ng/ml; A). or $-factor-Xlla (20 ng/ml; v). and incubated at 37’C for the times indicated. The amount of kallikrein produced (i�A40,/5 mm) was determined by hydrolysis of 0.2 mM S-2302. ASSAY OF FACTOR XII WITH DEXTRAN SULFATE 451 crude kallikrein samples (containing primarily IgG) were used, or when whole human plasma was activated with DS.7 Albumin, Tween 20, and gelatin were found to be effective in preventing both the surface-mediated loss of kallikrein and the inhibition by DS. In addition to preventing the inhibition of kalliknein by DS, however, gelatin interfered with the DS-induced activation of factor XII, whereas albumin and Tween 20 did not. We chose an albumin level of 5 mg/mI for use in further studies. At this level, DS (5 j�g/ml) pro- duced <10% inhibition of kallikrein (3 �tg/ml) in 10 mm. Activation of PK The rate of PK activation by NHP, factor XII, and activated forms offactor XII was examined both in the presence of DS and in its absence. In the presence of DS (5 zg/ml) and albumin, rates of PK activation by NHP ( I : I ,000), factor XII (20 ng/ml), and a-factor- XIIa (20 ng/ml) were approximately the same, and were considerably greater than that produced by an equal (mass) concentration of fl-factor-XIIa (Fig. 3). The brief lag phase (-0.5 mm) observed in the PK activation by NHP or by factor XII was absent when a- on f3-factor-XIIa was the activating species. When DS was omitted from the PK-albumin mix- tune, no activation occurred with NHP on with factor XII. The omission of DS resulted in a twofold increase in the rate of activation by f.�-factor-XIIa; that is, DS inhibited this activation by 50%. In contrast, DS greatly accelerated the activation of PK by a-factor- XIIa (-35-fold); hence, in the absence of DS, the activation rate was only 3% of that observed in its presence. The NHP used in these experiments contained factor XII, as determined immunochemically, at a concentration of 25 sg/ml (see below). Thus, the observation (Fig. 3) that a 1:1,000 dilution of NHP activates PK-DS (after the lag period) slightly faster than does 20 ng a-factor-XIIa/ml suggests that all, or nearly all, of the factor XII in the diluted plasma is converted to a-factor-XIIa during the first 0.5-1 mm of incubation. Once formed, a-factor-XIIa catalyzes the conversi