Hum Genet (1992) 89 : 265-269

9 Springer-Verlag1992

Discovery of a genetic polymorphism of human plasma protein C inhibitor (PCI): genetic survey utilizing isoelectric focusing followed by immunoblotting, immunological and biochemical characterization Toshihiro Yasuda ~, Daita Nadano 1, Reiko lida 1, Yukie Tanaka 2, Masao Nakanaga 3, and Koichiro Kishi ~ 1Department of Legal Medicine, 2Center of Medical Technology, and 3Department of Internal Medicine and Medical Genetics, Fukui Medical School, Matsuoka-cho, Fukui, 910-11 Japan Received September 15, 1991

Summary. The objectives of this study were to determine the genetic basis of the electrophoretic differences of human plasma protein C inhibitors (PCI) from 977 individuals. Three discrete antibodies were produced against the PCI purified from human plasma and peptides that corresponded to the N-terminal 15 amino acid residues and the C-terminal 15 residues of human PCI, the chemical structures of which were determined by cDNA sequence analysis. The combined techniques of polyacrylamide gel isoelectric focusing and immunoblotting with these three different antibodies resolved the plasma PCI into several isoprotein bands, with a pH range of 6-7. These PCI isoproteins, however, were not stained by anti-human kallikrein, anti-human protein C or anti-human urokinase antibodies. Therefore, each of the PCI bands, which were detected by immunoblotting with the anti-PCI antibody and the two different anti-peptide antibodies, were derived from free PCI, and not an inactive PCI species. Two common phenotypes, designated PCI 1 and 1-2, were recognized, and family studies showed that they represented homozygosity or heterozygosity for two autosomal codominant alleles, PCI*I and PCI*2. A population study of plasma samples collected from 977 Japanese individuals indicated that the frequencies of the PCI*I and PCI*2 alleles were 0.988 and 0.012, respectively.

Introduction In 1980, Marlar and Griffin (1980) first reported the existence of an inhibitor of activated protein C (APC) in plasma. This has been designated protein C inhibitor (PCI); it has been purified from human plasma and characterized by Suzuki et al. (1983; 1984). Later, the cDNA for PCI was characterized, the amino acid sequence of PCI showing a high degree of homology with members Correspondence to: K. Kishi

of the superfamily of serine protease inhibitors, the "serpins" (Suzuki et al. 1987). Furthermore, it was suggested that the liver was the main site of PCI synthesis (Francis and Thomas 1984; Morito et al. 1985). Immunological and functional assays have shown that PCI is identical to plasminogen activator inhibitor-3 (PAI-3), which is known to be a urinary urokinase inhibitor (Heeb et al. 1987; Stief et al. 1987). PCI is a single chain-glycoprotein, with a molecular weight of 57000; it inhibits APC by forming a 1 : 1 covalent complex with the enzyme in a heparin-dependent manner, followed by cleavage of a small peptide (with 33 amino acids at the carboxyl terminus) from the inhibitor to yield the modified inactive form. This inhibitory mechanism is similar to that of other serpins (Suzuki et al. 1983). Factor Xa, factor XIa, plasma kallikrein, thrombin, tissue plasminogen activator and urokinase are also possible target enzymes for PCI (Meijers et al. 1988; Pratt et al. 1989; Espafia et al. 1989). Such broad protease specificity of PCI led us to suspect that it may play important roles in both procoagulant and antifibrinolytic pathways. Indeed, plasma PCI level is known to be low in patients with depressed liver function or disseminated intravascular coagulation (DIC) (Suzuki et al. 1989). Purified PCI displays microheterogeneity when subjected to isoelectric focusing (IEF) (Suzuki et al. 1983), DEAE-Sephacryl column chromatography (Meijers et al. 1988) and SDS polyacrylamide gel electrophoresis (PAGE) (Laurell et al. 1988; Laurell and Stenflo 1989), although whether there are several intact plasma PCIs has yet to be elucidated. Moreover, the genetic aspects of PCI have attracted little attention so far, because of the lack of a suitable analytical method with a high enough resolution and sensitivity to ascertain whether there is a multiplicity of PCI forms and, if so, the nature of its origin. Isoelectric focusing of thin layer polyacrylamide gel electrophoresis (IEF-PAGE) combined with immunoblotting with a specific antibody has proven to be a valuable technique for investigating possible heterogeneity or new genetic polymorphism inherent in pepsinogen

266 (Kishi a n d Y a s u d a 1987), d e o x y r i b o n u c l e a s e I (Kishi et al. 1989; Y a s u d a et al. 1989b) a n d 4 3 - k D a g l y c o p r o t e i n ( M i z u t a et al. 1989). W e have p r o d u c e d t h r e e d i f f e r e n t a n t i b o d i e s against p u r i f i e d P C I a n d c h e m i c a l l y synthesized p e p t i d e s that c o r r e s p o n d to the N - t e r m i n a l a n d Ct e r m i n a l p a r t s o f the P C I m o l e c u l e . T h e c o m b i n e d techniques of I E F - P A G E a n d i m u n o b l o t t i n g with t h e s e specific a n t i b o d i e s have b e e n u s e d to a n a l y z e the p l a s m a PCI isoproteins. In this p a p e r , we d e s c r i b e a n o v e l a n a l y t i c a l m e t h o d for, the result o f a g e n e t i c survey of, a n d p a r t i a l c h a r a c t e r i z a t i o n o f h u m a n p l a s m a P C I . This is the first r e p o r t of g e n e t i c p o l y m o r p h i s m in h u m a n p l a s m a P C I .

Materials and methods

acrylamide and 0.6% w/v N,N'-methylenebisacrylamide), 2.3ml sucrose-glycerol solution (20% w/v, 10% v/v, respectively), 95 mg urea, I ml distilled water, 1501~1 ampholine 6-8, 80 gl ampholine 5-7, 50 lal ampholine 3.5-10, 5 gl N,N,N',N'tetramethylethylenediamine, and 40btl 1.2% w/v ammonium persulfate were mixed, deaerated and then poured into a gel mold and polymerized. Wicks were made from strips of filter paper and soaked in the electrode solutions: 0.5M HsPO4 at the anode and 0.5M NaOH at the cathode. The samples (5 btl) were applied to the gel with a plastic applicator (Pharmacia LKB, Bromma, Sweden) at a distance of i cm from the anode wick, and the gel was run at 3 W for 4 h, under cooling at 12~ with a Multiphor apparatus (Pharamcia LKB).

Immunoblotting. All operations were carried out at room temperature. Capillary blotting transfer of proteins onto a prewetted Durapore strip (Millipore, Bedford) and visualization of plasma PCI patterns were performed according to the method described in our previous papers (Kishi and Yasuda 1987; Yasuda et al. 1989a; Kishi et al. 1989).

Materials and biological samples Acrylamide, N,N'-methylenebisacrylamide and N,N,N',N'-tetramethylethylenediamine were purchased from Nakarai Tesque (Kyoto, Japan); ampholine 3.5-10, 5-7 and 6-8 were from Pharmacia LKB (Uppsala, Sweden); DEAE-Affi-Gel Blue, ammonium persulfate and peroxidase-labeled goat anti-rabbit immunoglobulin were from Bio-Rad Lab. (Richmond, Calif.); Clostridiumperfringens sialidase (type V) was from Sigma (St.Louis, Mo.); all other chemicals were of reagent grade or the purest grade commerically available. Antibodies against human protein C and cq-antitrypsin were obtained from Dakopatts (Glastrup, Denmark); anti-human urokinase and anti-human plasma kallikrein antibodies were from Japan Chem. Res. (Kobe, Japan) and Protogen (Switzerland), respectively. For the genetic survey, blood was obtained from healthy Japanese individuals, who were laboratory workers, students and volunteer families, who lived in the Fukui Prefecture and who gave their informed consent. The plasma was separated and samples were frozen immediately at -80~ until required for analysis.

Preparation of antibody Human PCI for immunization was purified from 600ml human plasma. The details of the purification procedures and the biochemical characterization of the purified PCI will be published elsewhere. The purified PCI (total ca. 150 btg) was mixed with Freund's complete adjuvant and injected into a Japanese white rabbit, subcutaneously. The antibody obtained reacted strongly with human plasma and with purified PCI in a double-diffusion test, and inhibited the inhibitory activity of PCI towards APC. Two peptides, HRHHPREMKKRVEDL (PCI-N) and IVDNNILFLGKVNRP (PCI-C), which corresponded to the N- and C-terminal 15 residues of PCI, respectively (Suzuki et al. 1987), were synthesized with a peptide synthesizer (Applied Biosystems, Japan, model 430A). The crude products were purified by high-performance liquid chromatography. A total of 5 mg of each of the two peptides PCIN and PCI-C, were mixed with Freund's complete adjuvant and injected into a Japanese white rabbit, subcutaneously. The IgG fraction was purified using DEAE-Affi-Gel Blue and used as the relevant specific antibody.

Analysis of plasma PCI isoproteins using IEF followed by immunoblotting IEF-PAGE. IEF-PAGE analysis was carried out as described in previous papers (Yasuda et al. 1988; Kishi et al. 1990a). The polyacrylamide gels (0.5 • 90 • 120mm) used for IEF-PAGE were prepared as follows: 1.4ml of the monomer solution (19.4% w/v

Results

Isoprotein patterns of plasma PC1 detected by immunoblotting with specific antibodies following IEF-PA GE T h e c o m b i n a t i o n of I E F - P A G E and i m m u n o b l o t t i n g with the a n t i - P C I a n t i b o d y was e x p e c t e d to p r o d u c e high b a n d r e s o l u t i o n a n d specific d e t e c t i o n of P C I i s o p r o t e i n s in p l a s m a , a n d was c o n s i d e r e d to b e the m o s t effective technique for the i n v e s t i g a t i o n of p o s s i b l e m o l e c u l a r h e t e r o geneities a n d g e n e t i c p o l y m o r p h i s m s o f h u m a n p l a s m a P C I . E a c h of the p l a s m a P C I p a t t e r n s w e r e r e s o l v e d into s e v e r a l m a j o r , a n d a few m i n o r , b a n d s on the r e g i o n of t h e gel that c o r r e s p o n d e d to p H 6 - 7 , as shown in Fig. 1. T w o d i f f e r e n t P C I p a t t e r n s f r o m the s a m p l e s f r o m diff e r e n t i n d i v i d u a l s w e r e d e m o n s t r a t e d consistently a n d r e p r o d u c i b l y . A s this i m m u n o l o g i c a l visualization techn i q u e was v e r y sensitive, less t h a n 21~1 p l a s m a was req u i r e d for analysis o f the P C I p a t t e r n s . If the c o n c e n t r a tion of p l a s m a P C I is a s s u m e d to b e 5 lal/ml (Suzuki et al. 1983; E s p a f i a a n d Griffin 1989), t h e n the limit of d e t e c tion of P C I with o u r m e t h o d is a b o u t 10 ng. T h e P C I p u r i f i e d f r o m o u t d a t e d p l a s m a has b e e n rep o r t e d to f o r m a c o m p l e x with p l a s m a k a l l i k r e i n ( L a u r e l l a n d Stenflo 1989). T h e r e f o r e , it is p o s s i b l e that t h e kallikrein-PCI complex, other protease-PCI complexes,

Fig. 1.A-C. IEF-PAGE patterns of plasma PCI in plasma samples from different individuals detected by immunoblotting with the anti-PCI antibody (A), the anti-PCI-N antibody (B) and the antiPCI-C antibody (C) with a mixture of ampholine 3.5-10, 5-7 and 6-8 containing 95 mg urea. Anode is at the top. These isoprotein patterns are designated phenotype PCI 1 (lane 1) and 1-2 (lane 2)

267 and free PCI may be detected by the anti-PCI antibody. This was shown not to be the case by the following resuits: no band that corresponded to PCI isoproteins detected by the anti-PCI antibody was stained by the antikallikrein antibody. Treatment of the sample with 1 M ammonia, which induces dissociation of the kallikreinPCI complex, induced no change in the PCI patterns. None of the other antibodies investigated, anti-al-antitrypsin, anti-protein C and anti-urokinase, demonstrated a band pattern that corresponded to those stained by the anti-PCI antibody.

Detection of PCI isoproteins on IEF-PAGE by immunoblotting with two different peptide-antibodies One of the murine monoclonal antibodies raised against intact PCI is known to bind specifically to the N-terminal 15 residues of PCI (Kuhn et al. 1990). Anti-PCI-C, the antibody specific to the peptide that corresponds to the C-terminal 15 residues, is expected to be a useful tool for the discrimination between active and modified inactive forms of PCI. The affinities of both the anti-PCI-C and anti-PCI-N antibodies were found to be as high for the blotted PCI protein as that of the anti-PCI antibody. Therefore, it was still possible that the inactive forms of PCI, which are formed by cleavage between Arg364 and Ser365 at the reactive site, and between Arg357 and Leu358, and Arg362 and Leu363 at two additional sites by several proteases in plasma (Suzuki et al. 1984, 1987; Laurell and Stenflo 1989), may have contributed to the PCI patterns to some extent. The PCI patterns detected by I E F - P A G E and immunoblotting with the two peptide-antibodies, anti-PCI-N and anti-PCI-C, were almost identical to those detected by the anti-PCI antibody (Fig. 1). As the maximum dilutions of the peptide-antibodies required to produce a suitable electrophoretogram were both 1:100, i.e., nearly equal to that of the anti-PCI antibody required, these peptide-antibodies may be useful tools for the analysis of plasma PCI.

Phenotypic variations of plasma PCI The technique of I E F - P A G E followed by immunoblotting with each of the three antibodies demonstrated, reproducibly, two different PCI patterns among 977 plasma samples from different individuals. These two phenotypes have been tentatively designated PCI 1 and PCI 12, and were clearly distinguishable from each other (Fig. 1). The PCI 1 phenotype resolved into seven major bands, with a few minor bands, which were distributed on the gel at a p H range of 6-7. The PCI 1-2 phenotype consisted of fourteen major bands, one set of seven major bands of PCI 1 and the other set of seven major bands of PCI 2: these seven bands all migrated more cathodally than did those of phenotype 1. Unfortunately, the PCI 2 phenotype was not found in this survey. The PCI 1-2 phenotype appeared to be a simple mixture of the PCI 1 and PCI 2 bands: there was no evidence for the existence of "hybrid" isoproteins. About 100 plasma samples, which included both phenotypes, were stored at - 8 0 ~ for a period of 5 years and retained sufficient antigenic ac-

Fig. 2. IEF-PAGE patterns of plasma PCI before (lane1) and after (lanes 2 and 3) sialidase treatment, and detected by immunoblotting with anti-PCI antibody. The plasma sample was treated with an equal volume of 5 units/ml sialidase as described in a previous paper (Yasuda et al. 1989b). The PCI patterns detected by the anti-PCI antibody were identical to those detected by the anti-PCIC and anti-PCI-N antibodies. Anode is at the top. Lanes 1, 2 phenotype 1; lane 3 phenotype 1-2

tivities that PCI-phenotyping could be carried out reliably. Sialidase treatment of plasma prior to I E F - P A G E simplified the PCI patterns by diminishing some of the anodal bands (Fig. 2); this enabled the PCI-phenotypes to be classified more easily. Moreover, each of the three different antibodies (anti-PCI, anti-PCI-N and anti-PCIC) produced the same PCI patterns when used in the immunoblotting detection test.

Family studies Thirty six different families, with a total of 53 children, were studied to ascertain the genetic transmission of plasma PCI phenotypes; the data are summarized in Table 1 and examples of the more informative pedigrees are presented in Fig. 3. Unfortunately, we were unable to find any 1-2 x 1-2 and 2 x 2 matings. Analysis of the family data presented in Table 1 showed that the inheritance of the phenotypes was consistent with the segregation of two codominant alleles at a single PCI gene locus; we designated these allelels PCI*I and PCI*2. Thus, individuals with the PCI phenotypes 1, 1-2, and 2 are presumed to have the genotypes PCI*I/PCI*I, PCI*I/PCI*2, and PCI*2/PCI*2, respectively. Sex linkage of the PCI locus can be formally excluded, based on the data from

Table 1. Transmission of the plasma PCI phenotypes in 36 families. To confirm the genetic transmission of patterns 1, 1-2 and 2, studies were performed in 36 different families including 53 children. The results are in agreement with an autosomal codominant transmission of the two alleles No. of families (n = 36)

Mating FxM

No. of children (n = 53)

Phenotypes

27 2 2 4 1

1x 1 1xl-2 1-2x 1 1 x ND ~ 1-2 x ND a

37 4 4 4 4

37 1 1 4 1

1

1-2 3 3 3

a Not determined because one parent was unavailable for study

Family.1Fami2ly ~ 268

r-L' 1-2l ~]1-2 ~

1~ 1~2 1.02 1-2

Fig. 3. Pedigrees of two families in which PCI*I and PCI*2 alleles show segregation. ?, Dead several families in which the father to son transmission of the PCI*2 allele occurred. Distribution of PCI phenotypes

In order to determine the frequency of each of the PCI patterns, the plasma of 977 unrelated Japanese individuals, with no known diseases, was analyzed. The group consisted of 641 males and 336 females, who ranged in age from 1 day to 80 years. The distribution of the phenotypes in this population is shown in Table 2; 954 had phenotype 1 and 23 had phenotype 1-2. There was no association of the PCI patterns with sex, as the frequency in each category did not differ significantly between males and females (Table 2). The frequencies of each of the alleles, as shown by a population study based on the HardyWeinberg law, were 0.988 for PCI*I and 0.012 for PCI*2. Discussion There are some doubts as to whether there are different types of plasma PCI, although a little information about this has been obtained from purified PCIs extracted from a mixture of pooled plasma samples collected from several volunteers (Suzuki et al 1983; Meijers et al. 1988; Laurell and Stenflo 1989). It was unknown whether the reported heterogeneity was the result of intra-individual or inter-individual variations, or a combination of the two. The combination of I E F - P A G E and immunoblotting with specific antibodies has been demonstrated to be the most suitable technique for the investigation of the heterogeneity and genetic polymorphism of body fluid components with low concentrations (Kishi et al. 1990b), such as PCI. The plasma PCI isoprotein patterns

Table 2. Frequency of plasma PCI phenotypes 1, 1-2 and 2 in a Japanese population of 977 subjects (641 males and 336 females), Numbers in parentheses indicate percentage in each category. The slight differences in frequency between males and females are not significant. The plasma PCI allele frequencies from the data are PCI*I: 0.988 and PCI*2: 0.012. The phenotype distribution was in close agreement with that predicated by the Hardy-Weinberg equation (Z2 = 0.1413, dfl, 0.75 > P>0.5)

PCI phenotypes

Total group

1

954 (97.65)

1-2 2

were resolved into several sharp bands by immunoblotting with anti-PC! antibody, as shown in Fig. 1. Next, two different anti-peptide antibodies (anti-PCI-N and anti-PCI-C) that were produced against the synthetic peptides corresponding to the N- or C-terminal part of the PC! molecule were used instead of the anti-PCI antibody. These observed PCI patterns were almost identical to those detected by the anti-PCI antibody. It has been reported that fresh plasma yields a biologically active inhibitor, whereas outdated plasma yields cleaved and inactive inhibitor (Laurell et al. 1988; Laurell and Stenflo 1989). If the inactive forms, which are free of the C-terminal part of PCI because of specific proteolytic cleavage with proteases (Suzuki et al. 1984, 1987; Laurell and Stenflo 1989), were responsible, even partially, for the PCI patterns, then the corresponding bands would be detected with the anti-PCI or anti-PCI-N antibodies, but not with the anti-PCI-C antibody, which is specific to the C-terminal 15 amino acid residues of plasma PCI. The plasma PCI patterns detected by the anti-PCI-C antibody were, unexpectedly, almost identical to those detected by the other two antibodies and, therefore, this possibility could be ruled out. Next, we examined whether any complexes between PCI and plasma proteases could, in part, contribute to the formation of a new band among the PC! bands detected by anti-PCI. The plasma separated on I E F - P A G E showed a very different pattern from PCI, when stained with anti-kallikrein, anti-protein C or anti-urokinase antibodies. In the light of this result, we excluded such a possibility and concluded that each of the bands revealed by anti-PCI were derived from free plasma PCI species, which showed intra-individual variations, and furthermore, that there were, among different individuals, three different PCI patterns that were attributable to the inter-individual variations of free PCI. Hence, the multiplicity of plasma PCI species is based on at least two kinds of variation, intra- and inter-individual. The simplification and reduction of the PCI bands produced by desialylation (Fig. 2) was considered to be a result of differences in the sialylated carbohydrate moieties of the PCI molecule; this also demonstrates PCI heterogeneity. At least two different PCI patterns among the 977 plasma samples could be distinguished with I E F - P A G E and immunostaining with anti-PCI. These two phenotypes were designated PCI 1 and PCI 1-2, as shown in Fig. 1. Our family studies showed that the genetic polymorphism of plasma PCI may be explained by the exis-

Sex Male

Expected no.

X:

Female

626 (97.66)

328 (97.62)

953.69

0.0001

23

(2.35)

15

(2.34)

8

(2.38)

23.17

0.0012

0

(0.00)

0

(0.00)

0

(0.00)

0.14

0.1400

977.00

0.1413

977 (100.0)

641 (100.0)

336 (100.0)

269 tence of two c o m m o n alleles, PCI*I and PCI*2, at a single gene locus. T h e p h e n o t y p e s P C I 1 and P C I 2 (not detected) result f r o m the h o m o z y g o u s g e n o t y p e s PCI*I/ PCI*I and PCI*2/PCI*2, respectively, whereas the P C I 1 - 2 p h e n o t y p e is seen in individuals h e t e r o z y g o u s for the two alleles. This hypothesis is s u p p o r t e d by the close a g r e e m e n t b e t w e e n the p h e n o t y p e distribution o b s e r v e d and that predicted by the H a r d y - W e i n b e r g equation. T h e d e m o n s t r a t i o n of male to male transmission of the PCI*2 allele (Table 1) and the identity of the male and female h e t e r o z y g o u s p h e n o t y p e (Table 2) effectively rule out X-linkage of the P C I locus. T h e frequencies of PCI*I and PCI*2 alleles, were 0.988 and 0.012, respectively. W e conclude, therefore, that P C I 1, P C I 1 - 2 and P C I 2 are inherited as simple autosomal c o d o m i n a n t traits. L o w levels of P C I in h u m a n plasma have b e e n reported in patients with several insufficiencies of the liver (Francis and T h o m a s 1984) and circulatory system (Marlar et al. 1985; Suzuki et al. 1989; H e e b et al. 1989). P C I would be expected, therefore, to play i m p o r t a n t roles in helping to control the D I C process and provide useful diagnostic m a r k e r s of D I C . H o w e v e r , the existence of genetic p o l y m o r p h i s m o f plasma P C I has not b e e n considered hitherto. Therefore, in order to ascertain whether the P C I system is a useful diagnostic m a r k e r , the association b e t w e e n P C I - p h e n o t y p e and possible associated diseases must be investigated. T h e plasma P C I - p h e n o t y p e is expected, in the near future, to b e c o m e a valuable m a r k e r for diagnostic and genetic programs.

Acknowledgements. The authors wish to thank Dr. K. Mizuta, Department of Biochemistry and Biophysics, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima, Japan, for her excellent advice and encouragement. We wish to express our appreciation to Dr.T.Taniguchi, Department of Biochemistry, Fukui Medical School, for the preparation of the figures, and to Miss Y. Ikehara, Miss E. Tenjo and Mrs. F. Nakamura for their excellent technical and secretarial assistance. This work was supported in part by grants from the Research Foundation for Traffic.Preventive Medicine, the Uehara Memorial Foundation and the Research Foundation for Cancer and Cardiovascular Diseases, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

References Espafia F, Griffin JH (1989) Determination of functional and antigenic protein C inhibitor and its complexes with activated protein C in plasma by ELISA's. Thromb Res 55 : 671-682 Espafia F, Berrettini M, Griffin JH (1989) Purification and characterization of plasma protein C inhibitor. Thromb Res 55 : 369384 Francis RB Jr, Thomas W (1984) Behaviour of protein C inhibitor in intravascular coagulation and liver disease. Thromb Haemost 52: 71-74 Heeb M J, Espafia F, Geiger M, Collen D, Stump DC, Griffin JH (1987) Immunological identity of heparin-dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3. J Biol Chem 262 : 15813-15816 Heeb MJ, Mosher D, Griffin JH (1989) Activation and complexation of protein C and cleavage and decrease of protein S in plasma of patients with intravascular coagulation. Blood 73: 455-461

Kishi K, Yasuda T (1987) Newly characterized genetic polymorphism of uropepsinogen group A (PGA) using both isoeleetric focusing and immunoblotting. Hum Genet 75 : 209-212 Kishi K, Yasuda T, Awazu S, Mizuta K (1989) Genetic polymorphism of human urine deoxyribonuclease I. Hum Genet 81: 295 -297 Kishi K, Yasuda T, Ikehara Y, Sawazaki K, Sato W, Iida R (1990a) Human serum deoxyribonuclease I (DNase I) polymorphism: pattern similarities among isozymes from serum, urine, kidney, liver, and pancreas. Am J Hum Genet 47: 121126 Kishi K, Yasuda T, Mizuta K (1990b) New genetic markers detected in human urine: DNase I, RNase 1, and 43-kDa glyeoprotein. Prog Clin Biol Res 344: 891-913 Kuhn LA, Griffin JH, Fisher CL, Greengard JS, Bouma BN, Espafia F, Tainer JA (1990) Elucidating the structural chemistry of glycosaminoglycan recognition by protein C inhibitor. Proc Natl Acad Sci USA 87: 8506-8510 Laurell M, Stenflo J (1989) Protein C inhibitor from human plasma: characterization of native and cleaved inhibitor and demonstration of inhibitor complexes with plasma kallikrein. Thromb Haemost 62: 885-891 Laurell M, Carlson TH, Stenflo J (1988) Monoclonal antibodies against the heparin-dependent protein C inhibitor suitable for inhibitor purification and assay of inhibitor complexes. Thromb Haemost 60 : 334-339 Marlar RA, Griffin JH (1980) Deficiency of protein C inhibitor in combined factor V/VIII deficiency disease. J Clin Invest 66: 1186-1189 Marlar RA, Endres-Brooks J, Miller C (1985) Serial studies of protein C and its plasma inhibitor in patients with disseminated intravascular coagulation. Blood 66: 59-63 Meijers JCM, Kanters DHAJ, Vlooswijk RAA, Erp HE van, Hessing M, Bouma BN (1988) Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor. Biochemistry 27 : 4231-4237 Mizuta K, Yasuda T, Kishi K (1989) Biochemical and genetic studies on GP43, a 43-kD glycoprotein detected immunologically in human urine and serum. Biochem Genet 27: 731-743 Morito F, Saito H, Suzuki K, Hashimoto S (1985) Synthesis and secretion of protein C inhibitor by the human hepatoma-derived cell line, Hep G2. Biochim Biophys Acta 844 : 209-215 Pratt CW, Macik BG, Church FC (1989) Protein C inhibitor: purification and proteinase reactivity. Thromb Res 53 : 595-602 Stief TW, Radtke K-P, Heimburger N (1987) Inhibition of urokinase by protein C-inhibitor (PCI): evidence for identity of PCI and plasminogen activator inhibitor 3. Biol Chem Hoppe Seyler 368 : 1427-1433 Suzuki K, Nishioka J, Hashimoto S (1983) Protein C inhibitor: purification from human plasma and characterization. J Biol Chem 258:163-168 Suzuki K, Nishioka J, Kusumoto H, Hashimoto S (1984) Mechanism of inhibition of activated protein C by protein C inhibitor. J Biochem 95 : 187-195 Suzuki K, Deyashiki Y, Nishioka J, Kurachi K, Akira M, Yamamoto S, Hashimoto S (1987) Characterization of cDNA for human protein C inhibitor: a new member of the plasma serine protease inhibitor superfamily. J Biol Chem 262:611-616 Suzuki K, Deyashiki Y, Nishioka J, Toma K (1989) Protein C inhibitor: structure and function. Thromb Haemost 61 : 337-342 Yasuda T, Sato W, Mizuta K, Kishi K (1988) Genetic polymorphism of human serum ribonuclease 1 (RNase 1) Am J Hum Genet 42: 608-614 Yasuda T, Ikehara Y, Takagi S, Mizuta K, Kishi K (1989a) A hereditary double double-banded variation in the vitamin Dbinding protein (GC) system analyzed by immunoblotting: duplication of the 1F and 1A2 genes? Hum Genet 82 : 89-91 Yasuda T, Mizuta K, Ikehara Y, Kishi K (1989b) Genetic analysis of human deoxyribonucleae I by immunoblotting and the zymogram method following isoelectric focusing. Anal Biochem 183 : 84-88