Proc. Natl. Acad. Sci. USA Vol. 86, pp. 8319-8322, November 1989 Biochemistry Coagulation factor XII (Hageman factor) Washington D.C.: Inactive factor XIIa results from Cys-571 -* Ser substitution ToSHIYUKI MIYATA*, SHUN-ICHIRo KAWABATA*, SADAAKI IWANAGA*t, ISAO TAKAHASHIt, BARBARA ALVING§, AND HIDEHIKO SAITOt *Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, Japan; tFirst Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Aichi 446, Japan; and §Coagulation Laboratory, Department of Hematology, Walter Reed Army Institute of Research, Washington, DC 20327-5100 Communicated by Oscar D. Ratnoff, August 3, 1989

ABSTRACT Structural studies on a congenital abnormal abnormal factor XII proteins from the plasma ofan individual coagulation factor XII (Hageman factor), factor XII Washing- with CRM' Hageman trait (factor XII Washington D.C.) has ton D.C., have been performed to identify the defect respon- been isolated and partially characterized (14). This abnormal sible for its lack of procoagulant activity. Amino acid sequence factor XII has the following properties: (i) it has the same analysis of a tryptic peptide isolated from the abnormal factor molecular weight as normal factor XII, (ii) it shows no XII indicated that Cys-571 (equivalent to Cys-220 in the clot-promoting activity but does show the same specific chymotrypsin numbering system) had been replaced by serine. antigenicity as purified normal factor XII in immunoassay, No other substitutions in the active-site triad-namely, His- (iii) limited proteolysis ofthe abnormal factor XII exposed to 393, Asp-442, and Ser-544-were found. We propose that the a mixture of plasma and glass yields a two-chain a-XIIa with Cys-571 -* Ser replacement found in this factor XII variant normal-sized heavy and light chains, and (iv) this abnormal destroys the formation ofthe disulfide linkage between Cys-540 factor XII is fragmented by trypsin in the same way as normal and Cys-571, giving rise to an altered conformation of the factor XII, but no prekallikrein-activating activity and no active-site serine residue or the secondary substrate-binding incorporation of [3H]diisopropyl phosphorofluoridate into a site and, thus, leads to the loss of enzyme activity. catalytic domain are observed. These results suggest that the abnormality in this enzyme resides at or near the active-site Coagulation factor XII (Hageman factor) is a plasma protein triad of the molecule. that was originally found as a functionally deficient agent in The present study was undertaken to further elucidate the plasmas from individuals with Hageman trait (1). Under structural abnormality of factor XII Washington D.C. that certain conditions, this protein appears to initiate several host would account for the functional impairment ofthe molecule. defense plasma reactions, including blood coagulation, fi- The results indicate that the impairment of the serine prote- brinolysis, and kinin generation (2-4). Human factor XII is a ase activity is due to replacement of Cys-571 with serine in zymogen of a serine protease with an approximate Mr of the catalytic domain. 80,000 and consists of a single-chain glycoprotein composed of 596 amino acid residues (5-9). When normal blood comes MATERIALS AND METHODS into contact with negatively charged surfaces, such as glass, kaolin, dextran sulfate, and sulfatide, factor XII is bound to Preparation ofAbnormal Factor XII and Its p-XII. Plasmas the surface and converted to an active enzyme (factor XIIa) from an individual with CRM' Hageman trait (14) were by a complex reaction involving plasma prekallikrein and obtained, after informed consent, by plasmapheresis, using high-molecular-weight kininogen (2-4, 10-12). Factor XII acid/citrate/dextrose anticoagulant. Factor XII was purified undergoes, on the surface, specific limited proteolysis by according to a published method (16) from plasma samples of kallikrein and transforms into two active enzyme forms, this individual and from normal individuals that served as a a-factor XIIa (a-XIIa) and 8-factor XIIa (,B-XIIa). a-XIIa is control. Normal factor XII was a gift from Kazuo Fujikawa composed oftwo polypeptide chains with Mr values of52,000 (University of Washington, Seattle). The normal factor XII and 28,000, whereas ,B-XIIa consists of two chains with Mr and abnormal factor XII were treated by trypsin, and the values of 2,000 and 28,000 (5). The heavy chain of a-XIIa is resulting 83-XII was isolated by a DEAE-Sephacel column (5). composed of 353 amino acid residues and contains four The 8-XII samples thus prepared were reduced and S- different domains (6, 7). The light chain of a-XIIa is a pyridylethylated (17). catalytic domain with 243 amino acid residues that contains Tryptic Peptide Mapping by HPLC. The S-pyridylethylated the active-site triad of serine protease (5, 7). abnormal 83-XII (320 gg) and normal /3-XII (210 Ag) were The plasma of the majority of subjects with Hageman trait suspended separately in 20 ,ul of 50 mM Tris-HCl, pH 8.0/8 also lacks immunologically identifiable factor XII. Very rare M urea. After dilution with 60 ,ul of 50 mM Tris-HCI, pH 8.0, individuals with this disorder, however, whose plasma con- 20 ,l of trypsin (0.35 mg/ml) was added, and the digestion tains nearly normal amounts of nonfunctional but immuno- was performed at 37°C for 14 hr. Each digest was chromato- logically identifiable factor XII, have been reported (13-15). graphed on a reversed-phase C4 column (0.46 x 15 cm, Because these plasmas contain cross-reacting material Cosmosil 5C4-300; Nakarai Chemical, Kyoto) on a Beckman (CRM) recognized by the specific antiserum, these cases model 344 HPLC system. HPLC separation of the digest was have been called CRM+ (CRM-positive) variant. Studies of performed with a linear gradient of 0-42% (vol/vol) aceto- the nonfunctional factor XII in CRM+ Hageman trait may nitrile in 0.1% trifluoroacetic acid for 84 min and 42-60% provide a unique opportunity to examine the structure- acetonitrile in 0.1% trifluoroacetic acid for 9 min at a flow rate function relationship of this plasma protein. One of the of 0.5 ml/min. The effluent was monitored by measuring

The publication costs of this article were defrayed in part by page charge Abbreviations: >PhNCS, phenylthiohydantoin; a-XIIa, ca-factor payment. This article must therefore be hereby marked "advertisement" XI1a; 8-XIIa, 8-factor XIIa; CRM, cross-reacting material. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

8319 Downloaded by guest on September 23, 2021 8320 Biochemistry: Miyata et al. Proc. Natl. Acad. Sci. USA 86 (1989) absorbance at 214 nm. The fractions containing several peptides were rechromatographed on a reversed-phase C18 column (0.21 x 15 cm, Chemcosorb 7-ODS-H; Chemco Scientific, Osaka) with the acetonitrile/trifluoroacetic acid system. Amino Acid Analysis and Sequence Determination. Samples were hydrolyzed at 110'C for 20 hr in 5.7 M HCl containing 1% phenol in tubes sealed under reduced pressure. After evaporation, the hydrolysates were coupled with phenyl isothiocyanate, and the phenylthiocarbamoyl amino acids were analyzed on the Pico-Tag system (Waters/Millipore), I ~~~~~~~~~z according to the method of Bidlingmeyer et al. (18). Se- 0.1 VX , quence analysis of the peptide was performed by an Applied Biosystems model 470 A gas-phase sequencer (19) equipped with an Applied Biosystems model 120 A on-line phenylthio- C4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. hydantoin (>PhNCS) analyzer. 1. Abnormal T RESULTS ds e ea xT5sedHo0.4 T9 30c Tryptic Peptide Mapping of Abnormal and Normal ,-XII. ITS T9 The S-pyridylethylated 83-XII derived from abnormal and T15 normal factor XII were each digested with trypsin, and the resulting peptides were separated on a reversed-phase C4 d column (Fig. 1). A peak eluted at 21.3 min appeared to be S -40 4-vinylpyridine used for the S-alkylation ofthe ,3-XII because 0.4' UJ tk 02 M T0.2- a-orT2C-3 this peak did not contain any amino acids in the HCI TIOC I- hydrolysate. Except for the peaks eluted at 4.5 min (Figs. 1 ~~~~~~20 A and B), 54.0 min and 67.8 min (Fig. 1A), and 45.9 min (Fig. TI z 1B), all peptides from both samples were obtained as a single 0.1. peptide. The impure peptides were purified further by re- chromatography as described, and the purified peptides were subjected to further analyses. The two chromatograms in Fig. numere sqetalfrm the 12 temiu of 40ctlyi 0 a I0 1 differ in some ways. Three peptides eluted at the retention 0 30 60 time of 35.4 min (T1OC), 50.7 min (T12'), and 60.9 min (T9N) Time, min appeared only in the digest of abnormal 83-XII (Fig. 1B), and also the heights of three peptide peaks eluted at the retention FIG. 1. HPLC of peptides derived from normal (Upper) and time of 40.5 min (T2C-3), 62.1 min (T2), and 66.0 min (T9) abnormal (Lower)m-factorX1i after trypsin (T) digestion. Tryptic were greater than those of the digest of the normal control. digests were separately subjected to HPLC on a 0.4 x 30 cm Most differences between the two chromatograms in Fig. 1, Cosmosil 5C18-P column; for further details, see text. Numbers on the peptide peaks are abbreviations for the tryptic peptides derived except for peptide T12', could be explained mainly by uneven from A-pyridylethylated normal and abnormalP 1X; peptides are digestion of S-pyridylethylated f-XII by trypsin. In the numbered sequentially from the NH2 terminus of the catalytic abnormal molecule, complete cleavage of the Arg- domain of factor XII (5, 7). Positions of these peptides in the 2,II 474-Pro-475 bond was seen, which yielded the peptides T8 molecule are shown in Table 1. The peptides, named T13-14, T2-3, and T9, whereas the extent of this cleavage was fairly and T8-9, show a peptide consisting of, for example, T13 and T14, reduced in the normal molecule. In addition, peptide bonds which was not cleaved by trypsin under the conditions used. T8N, of Arg-398-Pro-399, His-393-Cys-394, Tyr-462-Val-463, T9N, TioC, and T2C-3 are named to correspond to their NH2(N)- or Phe-507-Leu-508, and Met-527-Leu-528 were partially COOH(C)-terminal source peptide. cleaved by trypsin in the abnormal molecule, generating the peptides T2, T2C-3, T8N, T9N, and T10C. One clear differ- containing one cysteine and two serines, as expected from ence between A and B of Fig. 1 was the appearance ofpeptide the sequence data (6, 7). However, the amino acid compo- T12' in the chromatogram from the abnormal molecule. sition of peptide T12' derived from the abnormalg3-X11 Amino Acid Composition of Tryptic Peptides Derived from showed one more seine and one less -pyridylethylcysteine Abnormal f-XII. Table 1 shows the amino acid compositions than that of peptide T12. To determine the basis for this of all tryptic peptides derived from abnormal B-XII. Peptides difference, sequence analysis of the aberrant peptide T12' were numbered sequentially from the NH2 terminus of the was performed. catalytic domain of factor XII. The data indicated a high Amino Acid Sequence of Aberrant Peptide T12'. The se- purity of all peptides and allowed unambiguous identification quence of peptide T12' was determined with duplicate sam- of their positions in the known amino acid sequence for the ples by automated Edman degradation. As shown in Table 2, catalytic domain of factor XII. Approximately 99% of the peptide T12' differed from the normal T12 peptide by a single entire amino acid residues of the abnormal j3-XII molecule amino acid at position 571-a cysteine in the normal peptide was assigned by these analyses. Among these peptides, the and a serine in the abnormal peptide. This result indicates peptides T2-3 and T13-14 contained 2 mol of basic residues that a Cys --+ Ser substitution at position 571 has shifted the (arginine or lysine) per mol of peptide, and this result is due retention time from 54 min for the normal peptide T12 to 52.8 to incomplete hydrolyses of the Arg-398-Pro-399 and Lys- min for the aberrant peptide T12'. The Cys --+ Ser substitution 576-Pro-577 peptide bonds. Two free arginines, Arg-413 (T4) at position 571, on the other hand, has to provide a half- and Arg-558 (T11), generated in the tryptic digest were not Cys-540, because both cysteine residues correspond to the identified; they might be coeluted in the breakthrough frac- "&serine disulfide loop" found in chymotrypsin and other tion with salts, even in the rechromatography on a reversed- serine proteases. To confirm the free sulfhydryl group at phase C18 column (data not shown). The peptide T12 derived position 540, abnormalB1-XII was S-pyridylethylated without from normal j8-XII consisted of 16 amino acid residues any reduction. However, the sample after acid hydrolysis did Downloaded by guest on September 23, 2021 Biochemistry: Miyata et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8321 Table 1. Amino acid compositions (residues per molecule) of tryptic peptides obtained from the abnormal 3-XII Amino acid T1 T2 T2-3 T2C-3 T5 T6 T7 T8 T9N T9 T10 T10C T12' T13-14 T15 Asp 0.5 2.3 2.2 1.4 0.7 1.5 0.8 4.0 2.9 1.2 2.2 Glu 1.3 4.1 3.8 2.2 1.6 2.9 8.6 10.0 4.8 4.3 1.3 0.3 1.0 Ser 1.9 1.8 0.4 0.9 0.9 1.8 2.8 2.1 2.7 3.2 1.2 2.7 1.0 Gly 1.8 2.9 3.1 1.2 2.2 3.4 2.9 6.6 4.5 3.8 1.0 His 2.6 2.3 1.1 1.8 1.0 1.1 0.7 1.1 Arg 1.0 0.3 1.9 2.0 1.4 1.1 1.1 1.3 0.5 1.2 1.4 1.0 1.3 1.0 Thr 1.0 2.0 1.0 1.0 2.0 2.0 1.2 1.0 0.9 1.0 1.0 Ala 1.0 7.4 7.1 1.4 1.1 1.9 4.1 5.0 4.9 4.9 3.8 2.4 Pro 2.0 3.1 2.0 1.0 1.1 3.1 2.2 2.0 2.5 1.0 1.0 Tyr 1.8 2.3 1.0 0.9 1.0 1.3 1.1 2.7 Val 2.0 1.3 3.3 2.0 0.7 1.1 2.2 2.5 2.3 2.1 1.0 2.1 1.1 Met 0.7 0.6 0.3 0.5 0.6 1.4 0.3 PECys 3.0 3.2 1.2 1.6 1.2 1.2 2.0 4.2 3.1 Q0 Ile 2.3 2.1 0.5 1.4 0.3 1.9 1.1 Leu 1.9 3.4 5.1 3.3 0.8 4.1 4.6 2.1 4.5 5.4 2.1 2.2 1.2 Phe 1.6 1.3 1.0 3.6 3.9 1.3 1.2 Trp ND ND ND ND ND ND Lys 0.7 GaINH2 + Total 8 36 50 19 13 3 18 27 33 38 45 30 16 17 5 354 363 363 394 414 427 430 448 475 475 513 528 559 575 592 Position* 362 398 412 412 426 429 447 474 507 512 557 557 574 591 596 Amount analyzed, pmol 866 75 55 350 294 693 189 73 116 84 58 193 625 236 625 PECys, S-Pyridylethylcysteine; ND, not determined; GalNH2, galactosamine. *Positions of each tryptic peptide from the abnormal -XII correspond to those taken from the amino acid sequence (6, 7). Peptide T12' was identified as an aberrant peptide derived from the abnormal ,3-XII. not yield S-pyridylethylcysteine, indicating that Cys-540 residue (His-57) and consisting of50 amino acid residues, was must be a mixed disulfide form (data not shown). digested further with a-chymotrypsin, and the resulting pep- Among the tryptic peptides isolated from normal and tides were separated by HPLC. The histidine residue (His-57) abnormal 13-XII, three peptides-T7, T2-3, and T10- in one of these peptides was identified as >PhNCS-His. contained the functionally important residues Asp-102, His- Peptide T10, containing the active-site serine residue (Ser- 57, Ser-195, Asp-189, and Asp-194 of a-chymotrypsin. Of 195), the side-chain-binding site Asp-189 (in trypsin-like these residues, the aspartate residue (Asp-102) in peptide T7 protease), and the NH2-terminal ion-pair-interacting residue was identified by the direct Edman degradation method. Asp-194 and consisting of 45 amino acid residues, was Peptide T2-3, the peptide containing the active-site histidine digested with Staphylococcal V8 protease, and the resulting peptides were separated by HPLC. The serine residue (Ser- Table 2. Amino acid sequence of aberrant peptide T12' derived 195) and two aspartate residues (Asp-189 and Asp-194) were from pyridylethylated abnormal t3-XII found in one of these peptides and identified by the direct Peptide T12' Edman degradation method (data not shown). Thus, -47% of the entire amino acid sequences of the abnormal 83-XII 375 pmol of 750 pmol of molecule was confirmed by the Edman method. peptide peptide >PhNCS >PhNCS DISCUSSION amino amino Normal Cycle Position acid pmol acid pmol peptide Collectively, the data described thus far make it very unlikely that the molecular abnormality of factor XII Washington 1 559 Leu 163.2 Leu 267.7 Leu D.C. is due to replacement of any amino acid residues 2 560 Thr 58.2 Thr 178.4 Thr associated directly with the active-site catalytic triad or the 3 561 Leu 208.9 Leu 315.4 Leu substrate-binding pocket. Therefore, the replacement ofcys- 4 562 Gln 44.1 Gln 175.4 Gln teine by serine at position 571 from the NH2 terminus of 5 563 Gly 60.9 Gly 202.8 Gly abnormal factor XII appears responsible for the loss of 6 564 Ile 120.8 Ile 162.6 Ile catalytic activity. Because the genetic codon for Cys-571 has 7 565 Ile 175.5 Ile 195.7 Ile been determined as TGT (7-9, 20), we concluded that the 8 566 Ser 20.7 Ser 71.1 Ser patient's genomic DNA encoding for factor XII has a AGT or 9 567 Trp 53.9 Trp 32.8 Trp TCT nucleotide mutation. 10 568 Gly 39.8 Gly 83.7 Gly Cys-571 (equivalent to Cys-220 in chymotrypsin) is a highly 11 569 Ser 14.6 Ser 36.2 Ser conserved amino acid residue among serine proteases, in- 12 570 Gly 29.7 Gly 66.5 Gly cluding pancreatic enzymes, blood coagulation factors, fi- 13 571 Ser 13.9 Ser 27.0 Cy, brinolytic enzymes, and complement factors. A reasonable 14 572 Gly 18.6 Gly 53.4 Gly assumption from the disulfide location in other serine prote- 15 573 Asp 9.2 Asp 27.7 Asp ases is that a disulfide linkage between Cys-540 and Cys-571 16 574 Arg ND Arg ND Arg is formed in factor XII (5, 7). Based on the crystallographic ND, not quantitatively determined. data for a bovine trypsin-pancreatic trypsin inhibitor com- Downloaded by guest on September 23, 2021 8322 Biochemistry: Miyata et al. Proc. Natl. Acad. Sci. USA 86 (1989) Norma Factor XI Factor)l Wwhhgton D.C. supported by a Grant-in-Aid for Scientific Research from the Min- istry of Education, Science and Culture of Japan, Uehara Memorial 4F7 Foundation, and the Aichi Blood Disease Research Foundation. 1. Ratnoff, 0. D. & Colopy, J. E. (1955) J. Clin. Invest. 34, 47718~ 602-613. CEDQAAERRLTL 2. Ratnoff, 0. D. & Saito, H. (1979) Curr. Top. Hematol. 2, 1-57. 3. Cochrane, C. G. & Griffin, J. H. (1982) Adv. Immunol. 33, G 1 241-306. 4. Colman, R. W. (1984) J. Clin. Invest. 73, 1249-1253. 5. Fujikawa, K. & McMullen, B. A. (1983) J. Biol. Chem. 258, A. QiteD5 40 2 10924-10933. -DA GD- 6. McMullen, B. A. & Fujikawa, K. (1985) J. Biol. Chem. 260, 5328-5341. FIG. 2. Comparison of the serine disulfide loop of normal factor 7. Cool, D. E., Edgell, C.-J. S., Louie, G. V., Zoller, M. J., XII with that of factor Xll Washington D.C. Cys-571 is replaced by Brayer, G. D. & MacGillivray, R. T. A. (1985) J. Biol. Chem. serine in factor XII Washington D.C. The secondary substrate- 260, 13666-13676. binding site Ser-Trp-Gly (SWG in one-letter code) is boxed. Cys-540 8. Que, B. G. & Davie, E. W. (1986) Biochemistry 25, 1525-1528. may have been masked with a mixed disulfide form, such as cysteine 9. Tripodi, M., Citarella, F., Guida, S., Galeffi, P., Fantoni, A. & or glutathione. Cortese, R. (1986) Nucleic Acids Res. 14, 3146. 10. Kirby, E. P. & McDevitt, P. J. (1983) Blood 61, 652-659. 11. Fujikawa, K., Heimark, R. L., Kurachi, K. & Davie, E. W. plex (21), this disulfide bond is exposed on the surface, the (1980) Biochemistry 19, 1322-1330. so-called serine disulfide loop, and lies close to the specificity 12. Tans, G. & Griffin, J. H. (1982) Blood 59, 69-75. hole. Therefore, its integrity is probably important for en- 13. Saito, H., Scott, J. G., Movat, H. Z. & Scialla, S. J. (1979) J. zyme activity. A comparison of the serine disulfide loop of Lab. Clin. Med. 94, 256-265. normal factor XII with factor XII Washington D.C. is shown 14. Saito, H. & Scialla, S. J. (1981) J. Clin. Invest. 68, 1028-1035. in Fig. 2. This serine disulfide loop contains the active-site 15. Berrettini, M., Lammle, B., Ciavarella, G. & Ciavarella, N. serine residue (Ser-195) and residues Ser-214-Trp-215- (1985) Thromb. Haemostasis 54, 120 (abstr.). The 16. Takahashi, 1. & Saito, H. (1988) J. Biochem. (Tokyo) 103, Gly-216. peptide backbone of these residues is thought 641-643. to interact with the side chains of the substrate to properly 17. Hermodson, M. A., Ericsson, L. H., Neurath, H. & Walsh, orient the bond that is to be cleaved (22, 23). Therefore, we K. A. (1973) Biochemistry 12, 3146-3153. propose that the Cys -* Ser substitution found in this factor 18. Bidlingmeyer, B. A., Cohen, S. A. & Tarvin, T. S. (1984) J. XII variant destroys the formation of disulfide linkage be- Chromatogr. 336, 93-104. tween Cys-540 and Cys-571 and may form a mixed disulfide 19. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. & Dreyer, bond between Cys-540 and a cysteine residue or a glu- W. J. (1981) J. Biol. Chem. 256, 7990-7997. tathione, conformation of the res- 20. Cool, D. E. & MacGillivray, R. T. A. (1987) J. Biol. Chem. altering active-site serine 262, 13662-13673. idue or the secondary substrate-binding site, which leads to 21. Huber, R., Kukla, D., Bode, W., Schwager, P., Bartels, K., loss of the enzyme activity. Deisenhofer, J. & Steigemann, W. (1974) J. Mol. Biol. 89, 73-101. We are grateful to Dr. Kazuo Fujikawa, University ofWashington, 22. Steitz, T. A., Henderson, R. & Blow, D. M. (1969) J. Mol. Seattle, for providing us with normal factor XII. We thank Chizuko Biol. 46, 337-348. Sueyoshi and Satsuki Kajiyama for performing amino acid analyses 23. Segal, D. M., Powers, J. C., Cohen, G. H., Davies, D. R. & and Nobuko Ueno for typing this manuscript. This work was Wilcox, P. E. (1971) Biochemistry 10, 3728-3738. Downloaded by guest on September 23, 2021