Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 958-961, February 1979 Medical Sciences Interactions among Hageman factor, plasma prekallikrein, high molecular weight kininogen, and plasma thromboplastin antecedent (Factor XII/Fletcher factor/Fitzgerald factor/Factor XI) OSCAR D. RATNOFF AND HIDEHIKO SAITO Department of Medicine, The School of Medicine, Case Western Reserve University, Cleveland, Ohio; and University Hospitals of Cleveland, Cleveland, Ohio 44106 Contributed by Oscar D. Ratnoff, December 1, 1978 ABSTRACT To investigate the earliest steps of the intrinsic M sodium citrate, and bovine PTA-deficient plasma was from clotting pathway, Hageman factor (Factor XII) was exposed to blood containing 1/9th vol of 0.13 M sodium citrate buffer (pH Sephadex gels to which ellagic acid had been adsorbed; Hage- 5.0). A standard pool of normal adult plasmas (14) was said to man factor was then separated from the gels and studied in the 1 and PTA. fluid phase. Sephadex-ellagic acid-exposed Hageman factor, contain unit/ml each of HF, PK, HMWK, whether purified or in plasma, activated plasma thromboplastin Plasmas simultaneously deficient in PTA and other factors antecedent, but only when high molecular weight kininogen were prepared by incubating Fletcher or Fitzgerald trait was present. In the absence of plasma prekallikrein, maximal plasmas for 1 hr at 370C with specific immunoglobulins. Plas- activation of plasma thromboplastin antecedent was slightly mas simultaneously deficient in PK and other factors were delayed in plasma, a delay not observed with similarly treated prepared from Fletcher trait plasma. Absorption with specific purified Hageman factor. Thus, high molecular weight kini- immunoglobulins removed more than 99% of PTA and PK, and nogen was needed for expression of Hageman factor's clot- more than 98% of HMWK and plasminogen; immunoabsorp- promoting properties and plasma prekallikrein played a minor role in the interaction of ellagic acid-treated Hageman factor tion did not bring about activation of HF. The mixtures were and plasma thromboplastin antecedent. adsorbed with 1/50th vol of Cy-alumina gel (Calbiochem) for 10 min at room temperature to remove vitamin K-dependent Plasma exposed to negatively charged agents clots through clotting factors, centrifuged at 2000 X g for 10 min to sediment reactions of the intrinsic pathway of thrombin formation. The the Cy-alumina gel and insoluble antigen-antibody precipitates, first step in this pathway is activation of Hageman factor (HF, and stored at -70'C until used. Factor XII). Indirect evidence implies that negatively charged Plasmas simultaneously deficient in HF and other clotting substances bring about a conformational change in HF needed factors were prepared by immunoabsorption of Fletcher or for its clot-promoting activity (1-3). Recent studies suggest that Fitzgerald trait plasmas on columns of insoluble immuno- activation of human (4) and bovine (5) HF also involves its globulin against HF covalently bonded to cyanogen bromide- proteolytic scission within an internal disulfide loop. Once ac- activated agarose (Pharmacia) (15) and elution with barbital/ tivated, HF activates plasma thromboplastin antecedent (PTA, saline buffer (0.025 M sodium barbital in 0.125 M sodium Factor XI). The roles of plasma prekallikrein (PK, Fletcher chloride, pH 7.5). (Unless otherwise noted, the term "buffer" factor) and high molecular weight kininogen (HMWK, Fitz- refers to this buffer.) The contents of tubes containing protein gerald, Williams, Flaujeac, or Reid factor) in the intrinsic were pooled, 1/50th vol of 0.5 M sodium citrate buffer (pH 5.0) pathway are not fully elucidated. This pathway is impaired in was added, and the solution was concentrated to its original plasmas deficient in either PK (6) or HMWK (7). In purified volume by ultrafiltration through an Amicon PM-10 membrane systems, however, while activation of PTA requires the presence (Amicon Corporation, Lexington, MA) at 4VC. Simultaneous of HMWK (8-11), PK is not a necessary reactant (12, 13). One deficiency of HF, PK, and plasminogen was produced by in- explanation for the difference between plasma and purified cubation of immunoglobulin against plasminogen with Fletcher HF thus obscuring trait plasma for 1 hr at 370C before filtration through insoluble systems is that is altered during purification, anti-HF. Alternatively, plasma was depleted of more than 98% a need for PK. of plasminogen by filtration through lysine-agarose (16). The We have reexamined the early steps in the intrinsic pathway treated plasmas, containing no detectable HF, were stored at to determine whether activation of HF in plasma requires PK -70°C in silicone-coated polyethylene containers until used. or HMWK. Plasma containing HF, or purified HF, acquired A crude immunoglobulin fraction was separated from normal clot-promoting properties upon exposure to ellagic acid ad- rabbit serum or from monospecific rabbit antisera to human sorbed to Sephadex gels. Expression of these properties required HF (17), PTA (18), kallikrein (19), HMWK (13), and plas- the presence of HMWK but not PK. minogen (Behring, Sommerville, NJ) (20). The volume of an- tiserum needed was determined by incubating dilutions of MATERIALS AND METHODS immunoglobulin in buffer with equal volumes of pooled plasma Normal human plasma and plasma from individuals with for 1 hr at 37°C and measuring the residual titer of the specific Hageman trait, Fitzgerald trait (HMWK deficiency), and PTA factor. deficiency were separated from venous blood containing 1/50th Ellagic acid, synthesized by James D. Crum (21), was dis- vol of 0.5 M sodium citrate buffer (pH 5.0) (14). Fletcher trait solved at 0.1 mM in 0.05 M glycine buffer (pH 10.0), adjusted (PK deficiency) and Christmas disease (Factor IX deficiency) to pH 7.5 with 1 M sodium acetate buffer (pH 4.8), and filtered plasmas were prepared from blood containing 1/9th vol of 0.13 through Whatman no. 1 paper, leaving a faint yellow-green residue. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: HF, Hageman factor; PTA, plasma thromboplastin vertisement" in accordance with 18 U. S. C. §1734 solely to indicate antecedent; PK, plasma prekallikrein; HMWK, high molecular weight this fact. kininogen; CT, clotting time; PTT, partial thromboplastin time. 958 Downloaded by guest on September 28, 2021 Medical Sciences: Ratnoff and Saito Proc. Natl. Acad. Sci. USA 76 (1979) 959 Sephadex-ellagic acid was prepared by swelling Sephadex Table 1. Effect of Sephadex-ellagic acid upon the coagulant G-10, G-15, G-25, G-100, G-150 or G-200 (Pharmacia) in'A3 vol properties of HF in plasma of buffer for 1 hr. The supernatant was decanted and the pro- Addition to Clotting activity cess was repeated twice, with 0.02% sodium azide added to the PTA-deficient Centrolex Kaolin/Centrolex third wash. The swollen Sephadex was stored at 40C until used. plasma Sec Units/ml Sec Units/ml Ten milliliters settled volume of Sephadex was mixed with 30 ml of 0.1 mM ellagic acid, stirred for several min, and allowed Sephadex-ellagic acid 80.6 0.48 77.9 0.57 to settle, and the supernatant was decanted. Approximately 25% Sephadex-buffer >300 <0.01 63.5 > 1.0 of the ellagic acid was adsorbed to the Sephadex, as estimated Two milliliters of Sephadex G-10-ellagic acid or Sephadex-buffer, by the clot-promoting properties of the supernatant after cen- washed 15 times, was mixed with 2.0 ml of human PTA-deficient trifugation at 2000 X g for 10 min, relative to fresh solutions plasma, then diluted to 4 ml with-buffer and centrifuged, and a sample of ellagic acid, using the partial thromboplastin time (PTT) of of the supernatant was tested on HF-deficient substrate plasma normal plasma as an indicator. The Sephadex-ellagic acid was without further dilution for activated HF (Centrolex added) or total supernatant HF (kaolin/Centrolex added), -incubating the final mixture 8 min washed 6-15 times with 100 ml of buffer, with the before recalcification. Clotting activity was compared to that ofpooled discarded each time after the gel had settled; the last buffer normal plasma that was diluted serially with buffer and assayed for contained 0.02% sodium azide. Sephadex-ellagic acid retained total HF without exposure to Sephadex-ellagic acid or Sephadex- its clot-promoting properties for at least a month at 4VC. The buffer. Clotting activity is expressed both as CT (in sec) and in units 15th wash contained less than 0.1 sM ellagic acid, measured of HF/ml in comparison to the standard. The PTA-deficient plasma by the PTT after centrifugation. Sephadex-buffer was prepared had a titer of 0.78 unit of HF/ml. in the same way except for substitution of buffer for ellagic acid. observed when the treated PTA-deficient plasma was incubated Purified HF (22) varied in specific activity from 63 to 83 with HF-deficient plasma for 8 min before recalcification units/mg of protein. Partially purified PK contained 1.25 (Table 1). The same result was obtained with bovine PTA- coagulant units of PK per ml (3.2 units/mg of protein) and no deficient plasma and also when the supernatant, diluted plasma detectable contamination with other clotting factors; the bulk was recentrifuged at 30,000 X g for 15 min to ensure that no of extraneous protein was IgG (13). Sephadex-ellagic acid was transferred to the substrate plasma. Bovine serum albumin (crystallized, Pentex, Miles) was Sephadex G-25-, G-100-, G-150-, and G-200-ellagic acid were dissolved at a concentration of 1% in buffer. successively less effective, while Sephadex-buffer, without el- Clotting factors in plasma were titrated by a modified ka- lagic acid, was not clot-promoting.