Tohoku J. exp. Med., 1981, 133, 67-80

Transcription

1 Tohoku J. exp. Med., 1981, 133, Abnormalities in the Contact Activation through Factor XII in Fujiwara Trait: A Deficiency in Both High and Low Molecular Weight Kininogens with Low Level of Prekallikrein SACHIKO OH-ISHI,* AKINORI UENO, YASUHIRO UCHIDA, MAKOTO KATORI, HISAT0M0 HAYASHI, I HIROMICHI KOYA,1 KOICHI KITAJIMA~' and IKURO KIMURA~ Department of Pharmacology, Kitasato University School of Medicine, Sagamihara, Kanagawa 228 and tthe Second Department of Internal Medicine, Okayama University Medical School, Okayama 700 OH-ISHI, S., IJENO, A., UCHIDA, Y., KATORI, M., HAYASHI, H., KOYA, H., KITAJIMA, K, and KIMURA, I. Abnormalities in the Contact Activation through Factor XII in Fujiwara Trait: A Deficiency in Both High and Low Molecular Weight Kininogens with Low Level of Prekallikrein. Tohoku J. exp. Med., 1981, 133 (1), Fujiwara trait, the first case of kininogen deficiency in Japan previously reported which did not show any clinical symptom except the prolonged activated partial thromboplastin time was further examined. The activated partial thromboplastin time of the patient was corrected by addition of normal, Factor XII deficient or Fletcher plasma, but not corrected by Fitzgerald or Williams plasma. It was also corrected by addition of highly purified bovine or human high molecular weight (HMW) kininogen, but not by low molecular weight (LMW) kininogen. When total kininogen was measured as the amount of bradykinin released by trypsin, only a trace amount was detected in Fujiwara as well as Williams plasma. No immunoreactive protein against anti-human-hmw-kininogen nor anti-human-lmw-kininogen was found in Fujiwara plasma. Acetonekaolin-activated plasma kallikrein was not generated by Fujiwara plasma. Substitution with normal plasma in various ratios showed the generation of various plasma kallikrein activities. Calculations with these activities of mixed plasma gave the prekallikrein content of Fujiwara trait plasma about 30% of the normal level. These results suggest that Fujiwara trait is very similar to Williams trait in that both plasmas were deficient in HMW and LMW kininogens with reduced content of prekallikrein. Fujiwara trait; kininogen deficiency; high-molecular-weight kininogen; prekallikrein Recent findings in deficiencies of the plasma components of contact activation have been clarifying the mechanism of the contact activation, especially, the initiation of Factor XII activation. However, with the plasma of these Received for publication, February 22, Present address: Department of Pharmacology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1, Shirokane, Minatoku, Tokyo. 67

2 68 S. Oh-ishi et al. deficiencies, such as prekallikrein and kininogen deficiencies, the clotting time activated in vitro was markedly prolonged, but no clinical bleeding tendency was reported. Such defects reported were pre kallikrein deficiency (Fletcher factor deficiency) (Wuepper 1973), and the deficiency in high molecular weight (HMW) kininogen, like Fitzgerald trait (Saito et al. 1975; Donaldson et al. 1976) as well as deficiency in total kininogen, Flaujeac trait (Lacombe et al. 1975; Wuepper et al. 1975) and Williams trait (Colman et al. 1975). A female Japanese patient, 59 years old, as well as her twin sister, was found in Okayama University to show prolonged activated partial thromboplastin time (APTT) without any bleeding sign (Hayashi et al. 1978a). Coagulation factors I, II, V, VII and VIII were examined to be normal and Factor XII was functionally normal, when assayed with the deficient plasma. The prolonged APTT was corrected by Factor XII deficient or Fletcher trait plasma. Furthermore, bovine HMW kininogen corrected it, but bovine low molecular weight (LMW) kininogen did not. The deficiency was designated as Fujiwara trait and some of the above results were reported (Hayashi et al. 1978b, c; Oh-ishi et al. 1978a, b). In this paper, we describe examination of the plasma kallikrein-kinin system and its components in Fujiwara trait plasma by using new assay methods and compared with similar deficiencies already reported, such as Fitzgerald (Saito et al. 1975) and Williams trait (Colman et al. 1975). The contents of kininogens and prekallikrein were assayed by previously reported methods (Uchida and Katori 1978; Oh-ishi and Katori 1979). The same assays were also applied to Fitzgerald, Williams, Fletcher and Factor XII deficient plasmas. MATERIALS AND METHODS Fujiwara and normal human plasmas were collected from blood drawn into plastic tubes containing 1/10 volume of 3.8% sodium citrate and then centrifuged at 3000 rpm for 15 min. Most experiments on Fujiwara trait were done with the plasma of propositus, but some were also done with the plasma from the twin sister (Fujiwara 2). Kaolin (k-5, Fisher Sci. Co., Fair Lawn, N.J.), Thrombofax reagent (Ortho Diagnostics Inc., Raritan, N.J.), Factor XII deficient plasma and bovine Factor XI deficient plasma (bade Division American Hospital Supply Co., Miami, Fla), human Factor XI deficient plasma (GK , George-King, Bio-Medical, Inc., KS) and Carbobenzoxy-Lphenylalanyl-L-arginine 4-methylcoumarinyl-7-amide (Z-Phe-Arg-MCA, Peptide Institute Inc., Osaka) were purchased. Highly purified bovine high molecular weight (HMW) and low molecular weight (LMW) kininogens were gifts from Drs. S. Iwanaga and H. Kato, Kyushu University (Suzuki et al. 1972; Komiya et al. 1974). Fitzgerald plasma was a gift from Drs. H. Saito and O.D. Ratnoff, Case Western Reserve University (Saito et al. 1975), and Williams and Fletcher plasmas gifts from Dr. R.W. Colman, the University of Pennsylvania and Dr. A.P. Kaplan, State University of New York at Stony Brook (Colman et al. 1975). Highly purified human HMW and LMW kininogens and their antiserum from goat were gifts from Drs. J.V. Pierce and J.J. Pisano, National Institutes of Health (Liu et al. 1977). Assay procedure for APTT. Fifty t1 of each plasma sample and 50 yl of Thrombofax reagent were mixed, and then 50,u1 of kaolin suspension (5 mg/ml in 0.15 M NaCI) was added. The mixture was preincubated at 37 C for exactly 2 min and then 50 yl of warmed M CaCl2 solution was added, and clotting time was measured. The APTT of the mixture of

3 Contact Activation in Fujiwara Trait d of Fujiwara and 25 d of the other deficient plasmas were assayed in the same way (Table 1). For the experiment in Table 2, 25 µl of kininogen solution in saline and 25 µl of patient or normal plasma were mixed, and assayed in the same way as above. For the APTT in Fig. 1, 25 µl of normal, Fujiwara or Fitzgerald plasma was mixed with 25 µl of serial dilution of heated plasma (60 C, 1 hr) and then assayed as above. Assay for Factor Xl. One stage assay based on the kaolin-cephalin-clotting time (Austen and Rhymes 1975) was used. Bovine Factor XI deficient plasma (bade, 25 µl) was mixed with 25µl of varying dilutions of normal human plasma (a mixture of 5 healthy plasmas). Factor XI content was calculated as 100% in the mixture of 1:5 dilution of normal plasma in 0.15 M NaCI. Standard curve of Factor XI was obtained by the serial dilution of normal plasma. Assay procedure was in the same way as APTT. A separate experiment was done by addition of 25 µl heated normal plasma in the mixture. When human Factor XI deficient plasma (GK ) was used instead of bovine Factor XI deficient plasma, assay system contained 25 d of XI deficient plasma, 50 µl of normal plasma dilution (1:10, 1:40, 1:160, 1:640 in 0.01 M Tris-0.15 M NaCI buffer ph 7.4, containing 1 mg/ml bovine serum albumin), (Bouma and Griffin 1977), and 50µl kaolincephalin suspension (10 mg in Thombofax reagent), and was preincubated for 8 min at 37 C, and then 50 µl of 0.05 M CaCl2 was added and clotting time recorded. Assay method for total kininogen. The method used was previously reported (Uchida and Katori 1978). Briefly, acid treated plasma (ph 2.0) was incubated with trypsin and the released bradykinin was assayed on rat uterus. Assay method for prekallikrein. The method used was previously reported (Oh-ishi and Katori 1979). Briefly, the plasma kallikrein activity generated by the activation with acetone and kaolin was measured by peptidyl-fluorogenic substrate, Z-Phe-Arg-MCA. Fifty µl plasma sample was activated in 1 ml acetone-iris buffer, ph 8.0, with 1 mg kaolin. After certain intervals, 20 µl aliquot of the solution was incubated with Z-Phe-Arg MCA (5 X 10-5 M) for 10 min at 37 C, and the released aminomethylcoumarin was measured by spectrofluorometer. One unit of the activity was defined as the enzyme activity that released 10-' M aminomethylcoumarin in 10 min under the described condition (Oh-ishi and Katori 1979). Immunodiffusion. Double diffusion by Ouchterlony method was performed overnight at room temperature using 100 agarose in 0.02 M Tris-HCl buffer, ph 8.0, containing 0.15 M NaCI (Ouchterlony and Nilsson 1978). Agarose plate used was 3 mm thick and diameter of each well was 3 mm and distance of each well 2 mm. Sample solution of 5 µl was put into each well. Kinin formation of.fujiwara plasma. One hundred µl of plasma, 1 mg of kaolin, 0.25 mg of 1,10-phenanthroline, and 0.25 mg of EDTA-2Na were mixed in 900 µl of 0.1 M Tris-0.15 M NaCI buffer, ph 8.0, and incubated at 37 C for 1 hr. The reaction was terminated by boiling the tubes for 20 min and the released bradykinin was assayed on rat uterus. In the second series of experiment, 100 µl of heated plasma (60 C for 30 min) was added to the mixture mentioned above. In the third series of experiment, 50 µl of plasma was heated at 60 C for 30 min and incubated with partially purified plasma kallikrein, 0.05 TAME unit (Oh-ishi and Katori 1979). The released bradykinin was assayed in the same way. RESULTS APTT of Fujiwara plasma and its correction by addition of various plasmas As shown in Table 1, APTT of Fujiwara trait plasma was markedly prolonged. This coagulation abnormality was fully corrected by addition of one part of normal, Fletcher or Factor XII deficient plasma to one part of Fujiwara plasma. However, it was not corrected by addition of Fitzgerald or Williams plasma,

4 70 S. Oh-ishi et al. TABLE 1. Prolonged APTT of Fujiwara trait plasma and by addition of normal or other deficient plasma its correction indicating the same factor(s) is missing in Fujiwara trait plasma as Fitzgerald and Williams. Fig. 1 shows the correction of the prolonged APTT of Fujiwara trait or Fitzgerald trait plasma by addition of heated (60 C for 1 hr) human plasma. Normal plasma, Fujiwara or Fitzgerald plasma alone showed 106, 1147 or 436 sec, respectively. In this assay, one part of various dilutions of heated normal plasma in saline (shown as % in abscissa) was mixed with one part of Fujiwara or Fitzgerald plasma. The full corrections of both plasmas were obtained when 20% or more heated plasma was added. Fig. 1. Correction of prolonged ATPP of Fujiwara and Fitzgerald trait by addition of heated plasma. Twenty-five al of test plasma was mixed with 25 yl of heated normal plasma (60 C, 1 hr) in various dilutions, and then 50 yl of Thrombofax reagent, 50 µl of kaolin suspension (5 mg/ml) were added, and preincubated for exactly 2 min, and then, 50,al of M CaC12 was added, and clotting time was measured. On abscissa, 100 heated plasma means that 25 d of undiluted heated plasma was added to 25,x1 test plasma. Fujiwara (.), Fitzgerald (o) and normal (.) plasmas mixed with 25,ul of 0.15 M NaCI showed 1147, 436 and 106 sec, respectively.

5 Correction of APTT by addition of kininogen Contact Activation in Fujiwara Trait 71 The prolonged APTT of Fujiwara or Fitzgerald trait plasma was corrected by addition of highly purified human or bovine HMW kininogen in proportion to the amount added, as shown in Table 2, whereas highly purified human or bovine LMW kininogen failed to correct it. Human HMW kininogen corrected the prolonged APTT to the normal value, but with bovine HMW kininogen the full correction was not found even at 25 µg/tube. TABLE 2. Effect of HMW and LMW kininogen on prolonged APTT o f Fujiwara and Fitzgerald trait plasmas Assay of Factor XI in Fujiwara trait plasma Result of one stage assay of Factor XI, using bovine Factor XI-deficient plasma, is illustrated at the top of Fig. 2. By this procedure, a standard curve of Factor XI was obtained with various dilutions of normal plasma mixed with Factor XI-deficient plasma. Then, the values of the lines obtained with dilutions of sample plasmas mixed with Factor XI-deficient plasma, were read from the standard curve. The content of Factor XI in Fujiwara or Fitzgerald plasma was obtained as about 25-30% of normal value. The results were the same when the preincubation time was increased to 8 min. The bottom graph in Fig. 2

6 72 S. Oh-ishi et al. illustrates the results of the Factor XI assay in the presence of heated plasma. Twenty-five µl of heated plasma was added to whole system. A standard curve for Factor XI was not significantly changed from the above graph, but the lines of Fitzgerald and Fujiwara plasmas shifted to the left, indicating the Factor XI levels of both plasmas being 60-70% of normal value. Further, when Factor XI of Fujiwara or Fitzgerald plasma was assayed by using human Factor XI deficient plasma, the standard curve of normal plasma gave a linear line between 170 and 220 sec (1/10 to 1/640 dilutions), Fujiwara or Fitzgerald plasmas showed normal value, 60 to 140%. Fig. 2. Assay of Factor XI in Fujiwara and Fitzgerald triat plasmas. One stage assay based on kaolin-cephalin clotting time using bovine Factor XIdeficient plasma was performed. Fifty µl of Factor XI-deficient plasma was mixed with 50 µl various dilutions of normal plasma, and assayed in the same way as APTT. Standard curves for Factor XI were obtained ( -.). Fifty µl of undiluted plasma and 1:2 and 1:4 dilutions of Fujiwara (.-.) or Fitzgerald (o---o) plasma were mixed with 50 µl of Factor XI-deficient plasma and APTT was assayed. In the bottom figure, 25 µl of heated human normal plasma (60 C, for 1 hr) was added to the test system, and assayed as above. Kinin formation of Fujiwara trait plasma By kaolin activation, Fujiwara trait plasmas of twin Patients 1 and 2 (Hayashi et al. 1978a) generated kinin less than 0.03 µg/ml plasma, whereas normal citrated plasma generated 0.67 µg (Table 3). When heated plasma (60 C for 30 min) was added to each plasma and then activated by kaolin, Fujiwara plasma generated kinin of 0.75 and 1.00 µg, which corresponded approximately amount of kinin released from kininogen of the heated plasma added. to the Normal plasma generated 2 µg under the same procedure, which is the sum of kinin from HMW kininogen of the heated plasma plus normal plasma. Fifty µl of Fujiwara or normal plasma was heated at 60 C for 30 min, and then

7 Contact Activation in Fujiwara Trait 73 TABLE 3. Kinin formation of Fujiwara plasma incubated with partially purified human plasma kallikrein, 0.05 TAME unit (Ohishi and Katori 1979). Again, Fujiwara plasma yielded very little kinin, whereas normal plasma generated 1.13,ag/ml. Total kininogen in.fujiwara trait plasma When total kininogen was measured and expressed as µg bradykinin equivalent ml plasma, as shown in Table 4, the levels of kininogen in Fletcher trait and Factor XZZ-deficient plasma were almost in normal range but Fujiwara and Williams plasmas were 0.05 and 0.02, tg, respectively. Fitzgerald plasma yielded 1.84 µg, about a half of the normal level as reported (Saito et al TABLE 4. Total kininogen of Fujiwara trait and other related plasmas When Fujiwara plasma was activated with acetone kallikrein activity was found. However, if this deficient normal or heated plasma, it generated plasma kallikrein plasma kallikrein activity generated after acetone-kaolin and kaolin, no plasma plasma was mixed with activity. Fig. 3 shows activation of Fujiwara

8 74 S. Oh-ishi et al. plasma mixed with various ratios of normal plasma. Normal plasma (100%), at the top, generated the full activity 30 min after kaolin addition. Fujiwara plasma alone with kaolin (0% of normal plasma) showed no activity even after 180 min of incubation. When Fujiwara trait plasma was substituted by various ratios of normal plasma, the plasma kallikrein activity generated was increased with the content of normal plasma, although the level would be the sum of both Fig. 3. Time course of the activation of prekallikrein in Fujiwara trait plasma by acetone and kaolin. Ordinate shows the arbitrary unit of amidase activity of generated plasma kallikrein. One unit indicates the released fluorescence of 10-7 M aminomethylcoumarin in 10 min incubation at 37 C. Abscissa indicates the time after addition of kaolin (min). Figures at the right hand side indicate the content of normal plasma in the mixture. plasmas. As the rate of activation was low in the lower percent of normal plasma, such as 1 or 0.2%, the activity was still increasing even at 180 min. The mixtures of more than 5% of normal plasma showed the plateau 120 min after activation. The mean activities at 120 min from three experiments were plotted against the percentage of normal plasma (Fig. 4). The linear relationship was obtained and the extrapolation to the ordinate (0% of normal plasma) indicates that Fujiwara trait plasma may contain prekallikrein of 29% of normal level. In the same way, Fitzgerald or Williams trait plasma contains prekallikrein of 23 or 2'5%, respectively (Fig. 5). Plasma kallikrein activity in Fujiwara plasma was again examined in the same way, when Factor XII-deficient, Fletcher, Williams or heated plasma was added (Fig. 6). Neither the mixture of Fujiwara and Williams plasmas nor Fujiwara plasma alone showed activity. Fujiwara plasma mixed with Factor XII-deficient plasma showed more than half of the normal level, but the mixture with heated or Fletcher plasma showed only 13-16% of the normal level. These data suggest that

9 Contact Activation in Fujiwara Trait 75 Fig 4. Prekallikrein content of Fujiwara trait plasma in percent of normal value after substitution with normal plasma. Abscissa indicates the ratio of normal plasma in Fujiwara plasma. Ordinate shows the percent activity of plasma kallikrein 120 min after kaolin addition, when the activities reached the plateau. The experimental values showed a linear line and the extrapolation at ordinate, 0% normal plasma, expresses the prekallikrein content of Fujiwara trait plasma. Fig. 5. Prekallikrein contents of Fitzgerald and Williams plasmas in percent of normal value after substitution with normal plasma. Ordinate and abscissa are the same as in Fig. 4. Extrapolations at ordinate express prekallikrein contents of Williams (.---.) and Fitzgerald (o-o), 25.3 and 22.8%, respectively. prekallikrein content of Fujiwara trait plasma is about 26-32%. Fig. 7 shows the effect of bovine HMW and LMW kininogen on plasma kallikrein activation of Fujiwara trait plasma. An addition of LMW kininogen showed no

10 76 S. Oh-ishi et al. TABLE 5. Total kininogen and preka as percent of normal level deficient plasmas llikrein contents expressed in Fujiwara and related Fig. 6. Plasma kallikrein activities of Fujiwara trait plasma mixed with various plasmas. Ordinate and abscissa are the same as in Fig. 3. A-., mixture of Fujiwara trait and Factor XII-deficient plasmas (1:1); mixture of Fujiwara trait and heated normal human plasmas (1:1); -, mixture of Fujiwara trait and Fletcher plasmas (1:1); -, mixture of Fujiwara trait and Williams trait plasmas (1:1), o-o, Fujiwara trait plasma; a-o, normal human plasma. effect, about but the 16-22% addition of HMW kininogen caused activation of the normal level. of kallikrein up to Immunodiffusion of deficient plasmas Fig. 8 shows immunodiffusion study by Ouchterlony method using agarose gel. Against anti-human-hmw-kininogen antiserum (center well in A), Fujiwara (3), Williams (1), Fitzgerald (5) plasmas, and human LMW kininogen (6) showed no precipitin line, but normal plasma (2) and human HMW kininogen (4) showed precipitin lines. Against anti-human LMW-kininogen antiserum (center well in B), Fujiwara (3) and Williams (1) plasmas showed no precipitin line, but normal (2),

11 Contact Activation in Fujiwara Trait 77 Fig. 7. Effect of HMW and LMW kininogens on plasma kallikrein activation. Fifty µg bovine HMW or LMW kininogen was added to each 50 µl Fujiwara, Williams or Fitzgerald plasma. -, Fujiwara+HMW; A-, Williams+HMW; -i, Fitzgerald+HMW; o-o, Fujiwara+LMW; a-a, Williams+LMW; o-o, Fitzgerald -{---LMW; x-x normal. Fig. 8. Immunodiffusion of various plasmas against anti-human HMW (A) or anti-human LMW (B) kininogen antisera. Center well contained 5 µl of anti-hmw kininogen antiserum from sheep (A) or 5µl of anti-lmw kininogen antiserum from sheep (B). Each well contained 5 µl of the following plasma; 1, Williams; 2, normal; 3, Fujiwara; 4, human HMW kininogen (B4, RC d F1, 0.75 mg/ml); 5, Fitzgerald; 6, human LMW kininogen, (B2, RC , 0.37 mg/ml). 1% Agarose gel of 3 mm thickness in 0.02 M Tris-HC1 buffer, ph 7.5, containing 0.15 M NaCI was used. Well diameter was 3 mm and a distance of each well 2 mm. Diffusion time was overnight at room temperature. Fitzgerald precipitin (5) plasma and human lines. HMW (4) and LMW (6) kininogens showed DISCUSSION The abnormally prolonged APTT of Fujiwara with plasma deficient in Factor XII or prekallikrein, trait was corrected by mixing but not with plasma deficient

12 78 S. Oh-ishi et al. in kininogens, such as Fitzgerald or Williams. The results suggested that this trait would be a deficiency of kininogens, especially HMW kininogen. The fact that the addition of highly purified bovine or human HMW kininogen corrected the prolonged APTT but LMW kininogen did not, also confirmed the conclusion. The biochemical assay of kininogen showed that the twin patients lacked both forms of kininogens, and further, immunodiffusion experiments demonstrated that the Fujiwara trait as well as Williams trait plasmas lacked immunoreactive protein of both forms of kininogens, HMW and LMW. By Factor XI assay, using bovine Factor XI-deficient plasma, the content of Factor XI in Fujiwara and Fitzgerald trait plasma appeared to be about 25-30%, but when assayed in the presence of heated normal human plasma, which was used as a source of kininogens, the value turned out to be about 60-70%. Further, by using human Factor XI-deficient plasma, Fujiwara and Fitzgerald plasma showed normal values. These facts might suggest that one stage kaolin-cephalin assay may require more HMW kininogen level than that bovine plasma contains, or may require another factor, which is missing in bovine plasma. Saito et al. (1976) reported that bovine plasma contained a very low level of functionally active HMW kininogen, and the other report described normal level of Factor XI in Fitzgerald trait plasma, when human Factor XI-deficient plasma was used (Saito et al. 1975). Bovine HMW kininogen corrected the APTT of Fujiwara and Fitzgerald trait plasmas but the potency was rather low compared with that of human HMW kininogen. Bovine HMW kininogen, even at 25 µg/tube, did not produce the normal values of the APTT. These results are the same as those reported by Waldmann et al. (1976). However, human HMW kininogen corrected it completely to the normal value as shown in Table 3. The reason was not known, but the species difference might be important since addition of human heated plasma corrected it to the normal value. The full correction of the APTT was achieved by addition of around 20% of heated plasma as shown in Fig. 1. In this experiment, the heated plasma was diluted by saline at different ratios and added to the same volume (25,ul) of Fujiwara trait plasma, so that the content of HIM kininogen in the final incubation mixture would be half. This value corresponds well to the data from Wuepper et al. (1975), in which addition of 12.5% of normal plasma to Flaujeac plasma showed the full correction. Kinin formation by contact activation of Fujiwara trait plasma was also impaired, and neither kinin nor plasma kallikrein activity was detected following kaolin activation. However, Fujiwara plasma substituted with some amount of normal plasma, heated plasma, Fletcher plasma or Factor XII-deficient plasma showed some additional activity of plasma kallikrein over the amount expected from the substituting plasma. These results suggest that Fujiwara plasma itself contained prekallikrein and was activated by the assistance of the factor in added plasma. The content of prekallikrein in these HIM kininogen-deficient plasmas could

13 Contact Activation in Fujiwara Trait 79 hardly be measured by amidase assay using the peptide-mca substrate after activation by kaolin, when assayed individually. However, in our assay procedure (Ohishi and Katori 1979), when the HMW kininogen-deficient plasma was mixed with normal plasma at certain ratios, and incubated as long as the activity reaches the plateau, which was considered as the full activation, the prekallikrein amount in the plasma could be measured as shown in Figs. 3 and 4. In the previous report on the assay method of prekallikrein (Oh-ishi and Katori 1979), we reported the activation of prekallikrein required 10% of Factor XII and 10% of HMW kininogen. Kaolin-acetone activation of the deficient plasma mixed with normal plasma reached the plateau within 180 min of incubation (Fig. 3), when the full activation of plasma kallikrein activity developed. In this way we estimated Fujiwara trait plasma contains about 30%, and Fitzgerald and Williams, 23-25% of normal level of prekallikrein. Reduced levels of prekallikrein in HMW kininogen deficiency were also reported by other authors (Colman et al. 1975; Saito et al. 1975; Wuepper et al. 1975; Donaldson et al. 1976). Table 5 summarizes the contents of kininogen and prekallikrein in various deficient plasmas. Fujiwara trait plasma is closely similar to Williams plasma, since both plasmas lacked HMW kininogen as well as LMW kininogen with reduced amount of prekallikrein, whereas Fitzgerald trait plasma was slightly different, since it contained LMW kininogen. Acknowledgments We are grateful to Drs. S. Iwanaga and H. Kato, Kyushu University, for highly purified bovine HMW and LMW kininogens, and Drs. J.V. Pierce and J.J. Pisano, NIH, for purified HMW and LMW kininogens as well as their antisera. We are also grateful to Drs. H. Saito and O.D. Ratnoff, Case Western Reserve University, for Fitzgerald and Fletcher trait plasmas, to Dr. R.W. Colman, the University of Pennsylvania, and Dr. A.P. Kaplan, State University of New York at Stony Brook, for Williams and Fletcher plasmas. This work was supported by Scientific Research Grants from the Ministry of Education, Science and Culture, Japan (310507). References 1) Austen, D.E.G. & Rhymes, IL. (1975) A Laboratory Manual of Blood Coagulation. Blackwell Scientific Pub., Oxford, pp ) Bouma, B.N. & Griffin, J.H. (1977) Human blood coagulation factor XI. Purification, properties, and mechanism of activation by activated factor XII. J. biol. Chem., 252, ) Colman, R.W., Bagdasarian, A., Talamo, R.C., Scott, C.F., Seavey, M., Guimaraes, J.A., Pierce, J.V. & Kaplan, A.P. (1975) Williams trait: Human kininogen deficiency with diminished levels of plasminogen proactivator and prekallikrein associated with abnormalities of the Hageman factor-dependent pathways. J. din. Invest., 56, ) Donaldson, V.H., Glueck, H.I., Miller, MA., Movat, H.Z. & Habal, F. (1976) Kininogen deficiency in Fitzgerald trait: Role of high molecular weight kininogen in clotting and fibrinolysis. J. Lab. din. Med., 87, ) Hayashi, H., Koya, H., Kitajima, K. & Kimura, I. (1978a) Coagulation factor deficiency

14 80 S. Oh-ishi et al. apparently related to the Fitzgerald trait : The first case in Japan. Acta med. okayama, 32, ) Hayashi, H., Koya, H., Kitajima, K., Kimura, I., Oh-ishi, S., Ueno, A., Uchida, Y. & Katori, M. (1978b) Fujiwara trait: The first case of a possible kininogen deficiency in Japan, Part I. Proceedings of the 40th General Meeting of the Japan Haematological Society. Acta haematol. jap., 41, 243. (Japanese) 7) Hayashi, H., Koya, H., Kitajima, K., Kimura, I., Katori, M. & Oh-ishi, S. (1978c) Fujiwara trait : Kininogen deficiency in identical Japanese female twins. Proceedings of 17th Congress of the International Society of Hematology (July 26-29, Paris), p ) Komiya, M., Kato, H. & Suzuki, T. (1974) Bovine plasma kininogen I. Further purification of high molecular weight kininogen and its physicochemical properties. J. Biochem., 76, ) Lacombe, M-J., Varet, B. & Levey, J-P. (1975) A hitherto undescribed plasma factor acting at the contact phase of blood coagulation (Flaujeac Factor) : Case report and coagulation studies. Blood, 46, ) Liu, C.Y., Scott, C.F., Bagdasrian, A., Pierce, J.V., Kaplan, A.P. & Colman, R.W. (1977) Potentiation of the function of Hageman factor fragments by high molecular weight kininogen. J. din. Ivest., 60, ) Oh-ishi, S., Ueno, A., Uchida, Y., Katori, M., Hayashi, H., Koya, H., Kitajima, K., & Kimura, I. (1978a) Fujiwara trait : The first case of a possible kininogen deficiency in Japan, Pat II. Proceedings of the 40th General Meeting of the Japan Hematological Society, Acta haematol. jap., 41, 243. (Japanese) 12) Oh-ishi, S., Ueno, A., Uchida, Y., Katori, M., Hayashi, H., Koya, H., Kitajima, K. & Kimura, I. (1978b) Fujiwara trait: The first case of kininogen deficiency in Japan. Proceedings of 7th International Symposium on Kinin (November 6-9, Tokyo). Advanc, exp. Med. Biol., 120B, ) Oh-ishi, S. & Katori, M. (1979) Fluorometric assay for plasma prekallikrein using paptidylmethylcoumarinylamide as a substrate. Thrombosis Res., 14, ) Ouchterlony, 0. & Nillson, L.A. (1978) Immunodiffusion and immunoelectrophoresis. In: Handbook of Experimental Immunology, Vol. 1, 3rd Ed., edited by D.M. Weir, Blackwell Scientific Pub., Oxford, ) Saito, H., Ratnoff, O.D., Waldmann, R. & Abraham, J.P. (1975) Fitzgerald trait: Deficiency of a hitherto unrecognized agent, :Fitzgerald factor, participating in surfacemediated reactions of clotting, fibrinolysis, generation of kinins, and the property of diluted plasma enhancing vascular permeability (PF/DIL). J. din. Invest., 55, ) Saito, H., Goldsmith, G. & Waldmann, R. (1976) Fitzgerald factor (high molecular weight kininogen) clotting activity in human plasma in health and disease in various animal plasmas. Blood, 48, ) Suzuki, T., Kato, H. & Komiya, M. (1972) Biochemical properties of kininogens in plasma. In: Pharmacology and the Future of Man. Proceedings of the 5th International Congress of Pharmacology. vol. 5, S. Karger, pp ) Uchida, Y. & Katori, M. (1978) An improved method for determination of the total kininogen in rabbit and human plasma. Biochem. Pharmacol., 27, ) Waldmann, R., Scicli, A.G., McGregor, R.K.., Carretero, O.A., Abraham, J.P., Kato, H., Han, Y.N. & Iwanaga, S. (1976) Effect of bovine high molecular weight kininogen and its fragments on Fitzgerald trait plasma. Thrombosis Res., 8, ) Wuepper, K.D. (1973) Prekallikrein deficiency in man. J. exp. Med., 138, ) Wuepper, K.D., Miller, R.D. & Lacombe, M-J. (1975) Flaujeac trait: Deficiency of human plasma kininogen. J. din. Invest., 56,