the journal of vol. 258, no. 1, issue of january …the journal of biological chemistry prlnted in...

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Prlnted in U. S. A. Vol. 258, No. 1, Issue of January 10, pp. 163-168, 1983

Protein C Inhibitor PURIFICATION FROM HUMAN PLASMA AND CHARACTERIZATION*

(Received for publication, May 24, 1982)

Koji SuzukiS, Junji Nishioka, and Senichiro Hashimoto From the Department of Clinical Pathology, Mie University Hospital, Tsu-city, Mie 514, Japan

Protein C inhibitor was isolated from human plasma using conventional chromatographic technique con- sisting of barium citrate adsorption, polyethylene gly- col fractionation, DEAE-Sepharose CL-GB treatment, ammonium sulfate fractionation, dextran sulfate-agarose chromatography, gel filtration on ACA-44, and DEAE-Sephacel chromatography. The purified protein C inhibitor is a single polypeptide chain with an appar- ent M, = 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor is heteroge- neous in PI: six PIS exist between pH 7.4 and 8.6. The inhibitor was shown to be different from the already known plasma protease inhibitors by chemical and immunological analyses. It migrates to the late al-globulin region on agarose gel electrophoresis. The inhibitor reduced the amidolytic activity of activated protein C noncompetitively by forming a 1:l molar complex with the enzyme, determined by the use of a fluorogenic substrate toward activated protein C (Boc-Leu-Ser- Thr-Arg-4-methylcoumaryl-7-amide). The inhibition constant (Ki) of the inhibitor against activated protein C was 5.8 X M. The inhibitor also blocked the prolongation of activated partial thromboplastin time by activated protein C. The immunoglobulin which was produced by the inhibitor completely removed the inhibitory activity present in normal human plasma against activated protein C. This suggests that the inhibitor which we have isolated is the only inhibitor in plasma against activated protein C.

Protein C is a vitamin K-dependent serine protease zymo- gen isolated from bovine and human plasma (1-4). Activated protein C, formed as a result of cleavage of protein C by a thrombin or Factor X activator from Russell’s viper venom, has strong anticoagulant properties due to the selective pro- teolytic inactivation of both coagulant cofactors Factor V (Va), functioning as a receptor protein for Factor Xa and prothrombin, and Factor VI11 (VIIIa) (5-8). In addition to its anticoagulant properties, activated protein C can stimulate fibrinolysis in vitro and in uiuo (9, 10). Recently, the presence of a cofactor for thrombin-catalyzed protein C activation was demonstrated by Esmon and Owen (11, 12). Subsequently, they isolated and characterized the cofactor protein (13).

Unlike other serine proteases in blood coagulation, activated protein C is not inactivated by antithrombin 111, not even in the presence of heparin (14). Recently, Marlar and

* This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$. To whom correspondence should be addressed.

Griffin (15) have reported the presence of an inhibitor for activated protein C which, except in very rare cases of patients with combined Factor V and Factor VI11 deficiency, may be different from other plasma protease inhibitors in human beings. In this report, we shall describe the purification and characterization of the inhibitor of activated protein C, des- ignated PCI,’ from human plasma.

EXPERIMENTAL PROCEDURES”

MaterlalS

DEAE-Sepharose CL-68, DE=-Sephacel, SP-Sephadex C-50, QAE-Sephadex A-50, cyanogen bromlde activated Sepharose 48. protein A-sepharose CL-48, dextran Sulfate IMr=500,000, sulfur content-17 $ 1 and M standards were obtalned from Pharmacla F l n e Chem., Uppsala, Sweden. Ultrog61 AcA-44 and PAGplate were from LKB Pmdwtor AB. Bmma, Sweden. Bentamidine chloride. phenylmethylsulfonyl fluoride ( P M S F ) . p-Chlo=Ome=CUTlben=OiC acld (PCMB). soybean trypsm mhlbltor, bovme 9erm albmrn and Russel’s viper venom were from Szgma Chem.

Buchs. Germany. Polyethyleneglyc01-6.000 (PEG-6,000) and sodium dodecyl su1- St. LOIYS, HO. D~1SOprOpy1 fluorophosphate IDFP) was from Fluka AG Chem..

fate (SDS) were from Nakaral Chem.. Kyoto, Japan. The synthetlc fluorogenlc

PIOteln Research FoundatLon. Osaka, Japan. substrate towards activated protein C , Boc-Le“-Ser-ThT-ATg-MCA (16). was from

Inter-o-trypsin inhlbitor, 02-macroglobulm, antithrombin ;II1and c mactiva- tor were from Behrinqwerke AG, narburg, Gemany. Rabblt antiserum-for 02- plasmin Inhibitor was from HOChldd Pharm.. Tokvo. Jaoan.

Rabblt antisera for human whole serum, 0 -antrtrypsin a -antrchpotrypsm,

bromlde sepharase 48 according to the instructions of the manufacturer.

The protein C was activated with thrombin or Russel‘s Y~per venom Factor Xa - H u m ” protein C was purified by the method of Suzukz et al.117).

activator. whxh was purified according to Pyxie and Fvrie (18). The activated protein C was isolated by SP-Sephadex c-50 and then OM-sephadex A-50 Chromato- graphy according to COmp and Esmn ( 1 0 ) . Hman thrombin was prepared by the method of Lundblad et al. 119). Specific aetlvzty of the thrombin was 2,800 N I H unlt/mg protein. Antithrombin I11 was purlfled according to Kurachz et al. ( 1 4 ) from human plasma. Protein concentration& were deteminedlfy Bpectrophoto- metrically at 280 m with the use of extinction coefficient (E ) of 13.7 and 18.3 fnr activated prcqgm c ( 4 ) and thrombin 120), respecti%ly. ~n extrnctlan coefficient (E at 280 MI) of 10.0 was assumed for antlthrombln 111.

Dextran aulfate-agaroae was prepared from dextran sulfate and cyanogen . .

Methods

Electrophoretic Technlcs - Analytlcal polyacrylamide disc gel electropho- resxs was carried out employing an anionlc buffer system suitable for basrc

pelyacrylamlde gel electrophoresis was performed employmg 10 9 polyacrylamide proteins according to Tamura and UI (21) uslng 7 P polyacrylamide gels. SDS-

gel6 as described ( 2 2 ) . Standard proteins for the M estimation COnsi6ted of phosphorylase b (M =94 000) b e n n e serum albumin (nr=67 000) ovalbumin (M =

and o-lactalbumzn (Mr=14,4000)f Aiaros; gel electrophoresis was carrfed out on 43,000), carbonic anhy&ase‘(M =30 000) Soybean try6sm’m.hibitor In =20,060) 1 9 agarose gels in 0.05 M barbltal buffer. pH 8.6, contalnlng 2.0 mM CaC12 or 2.0 mM EDTA by the method of Johansson (23). Analytical eleCtrOfoCUsmg on polyacrylamide gels was performed using PAGplate [pH range from 3.5 to 9.5) according to the inatructmns Of manufacturez.

procedure ( 2 4 ) employing the Hltachi a m m o acid analyzer, model 835 (Hitachr Amno ACld AnalyBes - Amlno acid analyses yere carried out by Standard

Seisakusho, Tokyo, Japan). Protein samples for the analyses were hydrolyzed at llO°C for 24. 48 and 72 h In 6 N HC1 under vacuum. The m o u n t Of tryptophan was determmed 9peCt=OphotOmet=1Cally (251.

by immunizing with a purlfied inhlbltor Of the first peak on DEAE-Sephacel Immunochemlcal Experiments - Antiserum against purlfied PC1 was prepared

chromatography. ImunOglobUlin for the mhlbltor was isolated from the antiserum by using proteln &-Sepharose CL-48 by the method described in the lnstruo-

...~ ~ ..

’ The abbreviations used are: PCI, protein C inhibitor; PMSF, phenylmethylsulfonyl fluoride; DFP, diisopropyl fluorophosphate; Boc, t-butyloxycarbonyl; MCA, 4-methylcoumaryl-7-amide; AMC, 7- amino-4-methylcoumarin; SDS, sodium dodecyl sulfate; APTT, activated partial thromboplastin time; PEG-6000, polyethyleneglycol- 6000; PCMB, p-chloromercuribenzoic acid.

Portions of this paper (including “Experimental Procedures,” Tables I and 11, and Figs 1-3) are presented in miniprint as prepared by the authors. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-1350, cite authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

163

164 Protein C Inhibitor

I 1 0.9 6 0.8

5 ,

- 0.7 .- 5 - 0.6

- 0.5 9

3 E - 0.4 -

Y

- Y

.- - 2 0.3

2 % -1 0.2 j 0.1

0

pooled

tmns of manufacturer. Double Imunodlffusmn was carried out in 0.8 8 agarose

cally by the method Of Scheideqger 126). I" 0.075 U barbltal buffer. pH 8.6. Imunoelectr~ph~res~~ was performed has1-

Assay for PC1 ACtiVlt - The actlvlty of PC1 wag measured basically by the following procedure: Firs?, 50 p l Of actlvated proteln C in the buffer COns~st- l n g of 0.05 PI TTIS-HCI, pH 8.0. 0.15 W NaC1, 2 mn CaC12. 0.1 U bovine serum albumin. and 50 p l of PC1 wlth varylnq ConcentratiOnS ~n the Same buffer were mlxed and Incubated at 37-c I" polystyrene tubes. After this mlxture had been

Boc-leU-Ser-ThT-Arg-MCA. called peptlde-MCA. dissolved in 0.05 M Tris-HC1. pH incubated for 60 mln, 20 p1 aliquot was removed and added to 2 . 5 ml Of 100 YM

8.0. 0.15 M NdCl and 2 mM CaC12, kept at 37''. Changes of the intensity Of fluarrsence of the 7-amlno-4-methylcoumarin (AMCI lrberated from the substrate were then measured at 440 m by Inltlal-rate method as described 1 2 7 ) . The actlvlty of activated protem c was expressed rn terns of moles AMC l~berated per m r n . The a c t l v ~ t y of PC1 was expressed in terms of unlt whlch was the ac- tlvzty found In 1 m l of pooled normal human plasma.

determined by the ordlnary method uslng Auto-FI (Dade Corp.,Mlami, ~ 1 . ) as follows. 50 pl actxvated proteln C and 50 p l of varying concentrations of PCI

Clottzng Test In Activated Partla1 Thromboplastin Time (APTT) - APTT was

were incubated in polystyrene tubes at 37-C far 60 m l n , after which 15 p l s m -

human plasm in the reaction vessel for mTT assay. To mitiate the plasma plea of the Incubation mlXtUPe were withdrawn and mlxed with 100 ul Of noma1

clotting process, a 100 pl of phospholLpid aolution containing elladic acid

to the vessel. (ACTIN. Dade Corp.) was added. and 2 min later, 100 p1 of 25 mM CllCl2 was added

Purificatron of human PC1

The procedure far the purlfication of human PC1 ~8 s m r i z e d ln Table I,

rage nvmber of several preparations. A11 steps in the purification procedure ~n which the speclfic activity and percentage of yreld are lndlcated as an ave-

were performed at I T .

from the local blood bank. Benzamldine cblorlde (10 m), DFP 11 M I , Pl l sF I 1 mnl and soybean trypsin inhibitor 150 mg/liter) were added to the plasma.

Starting Materlal - Fleshly frozen h m n plasma ( 4 liter) was Obtalned

ter plasma stirring end the mixture stirred for one mre hour. The barium Clt- rate was then rewved by centrifugatlon at 5 . 0 0 0 z p for 30 mi". The barium Citrate pellet was stored at -70-c as d source of protein C preparation.

Barium Cltrate Adsorption - EO ml Of 1 M BaClZ was added dropwise per 11-

supernatant resulting frao the previous step, and stirred for 1 h. The preci- PEG-6,000 Fractmndtion - Solid PEG-6,000 was added 160 g/llterl to the

pitate was removed by centrifugation at 5,000 rpm for 15 min. 60 g of solid PEG-6.000 was further added to each liter of the supernatant. After the mixture had been stirred for 1 h . the Drecioitate was collected by centrifwation st 5 , 0 0 0 rpm for 30 nin.

. .

substrate for activated protein C. the precipitate of PEG fractionation from DE--Sepharose Treatment - In order to r-ve the coagulant Factor V, a

6 to 12 8 was passed through a column of DE--Sepharose CL-68 128). The PEG- precipitated material was dissolved in 500 ml chilled 0.05 M Tris-HC1, 0.1 M NX@, pH 7.5. contaming 10 mn benzamidine chloride, 1 mM DPP and 1 mX PIISF. The solutim was apphed to DE=-Sepharose CL-6B colymn ( 5 x 25 c m l equilibra- ted with the mame buffer used for the dissolution of the PEG-precipitate, and the madsorbed material W ~ B collected.

of concentrating the wadsorbed fraction of the prev~ous step and removing the PEG-6,000 from the fraction. Solzd -onium sulfate was added to the unadaorb- ed fraction to make a 50 8 Saturation. After this mixture had been stirred for 1 h, the supernatant of 5 0 0 m n i u m sulfate was obtained by centrifugatmn at 8,000 rpm for 15 min. Solid m n i u m sulfate was then added to the supernatant to make a 7 0 % saturation. The Solution was then stirred for 1 h and the precipitate was collected by centrifugetlon at 8,000 rpm for 30 mln.

Amxon~um Sulfate Fractionation - This step was performed for the purpose

Step was dissolved in 0 . 0 5 M Tris-HC1, pH .O. contalning 0.1 M NaCl, 1 mM ben- Dextran Sulfate-Agarose Chromatograph - The precipltate from the prevlOus

zamldine chloride, 0.1 mn DFP and 0.1 mi PllsF and dlalyred overnight against the Same buffer. The dialysate was applled to a dextran sulfate-agarose column

tione containlng the Inhibitory activity were precipxtated by solid ammonium (Fq. 1). Actlvity of the PC1 was measured and the proteins in pooled frac-

sulfate I80 8 saturatlonl.

was collected by centrifugation at 10.000 rpm for 15 m ~ n and dissolved in a Gel Flltration on UltrOqel AcA-44 - The preclpltate from the previous step

mlnimal volume Of 0.05 M Tris-HCI, 0.15 M NaCl, pH 7 . 5 . The sample was applled to a column of ACA-44 (Fig. 2 ) . The fractions contaming PC1 activity were pooled and dlalyred overnight against 0.05 M Tris-HCl, pH 9.0.

DEAE-Sephacel Chromatography - Dialysate f m m the previous step was ap- lled to a column af DERE-Sephacel (Fig. 3 ) . The mhlhitory aCtlVltY was eluted in the first two peaks. The averaged speclfic actlviries of the inhibitors ln the first and second peak in several Preparations were 220 unlts/mg and 167 unlts/mg protein, respectl~ely. The fractions containing PC1 dctlvity were pooled as lndlcated ~n the figure and used for Subsequent experiments.

Protein C Inhibitor 165

RESULTS

Physicochemical Characterization of P C I Gel Electrophoresis-Purified PCI, in the first and second

peak from DEAE-Sephacel chromatography, migrated as a single band on analytical polyacrylamide disc gel electrophoresis (Fig. 4). On SDS-polyacrylamide gel electrophoresis, the inhibitor, both unreduced and reduced, in the first peak showed a single band with M, = 57,000. The protein in the second peak showed another minor band with M , = 54,000. The minor band was probably an inhibitor protein degraded by proteolysis during the purification procedure, since when the preparation of the inhibitor from plasma was carried out without any serine protease inhibitors, the low M , band was seen to increase slightly in the final product. When lyophilization was performed during the preparation of the inhibitor, a dimerized inhibitor protein was observed on SDS-polyacrylamide gels. Both PC1 in the first and second peak migrated to the late a,-globulin region on agarose gel electrophoresis in the presence of CaC12 or EDTA (data not shown).

Immunochemical Studies-The purified PC1 in both peaks did not react with antisera against al-antitrypsin, al-antichy- motrypsin, antithrombin 111, inter-a-trypsin inhibitor, m-plasmin inhibitor, a*-macroglobulin, and CI-inactivator on double immunodiffusion. Both PC1 in the first and second peak showed immunological monospecificity on double immunodiffusion (Fig. 5a) and immunoelectrophoresis (Fig. 56), determined with antiserum produced with the prepared inhibitor protein. Ten p1 of plasma did not produce a precipitin arc against anti-PC1 serum on immunoelectrophoresis under the same condition (data not shown).

Isoelectric Points-PC1 displays microheterogeneity on isoelectric focusing. PC1 in the first peak had PI values of 8.6, 8.4, 8.0, and 7.9. The second peak PC1 had PI values of 8.0, 7.9, 7.6, and 7.4 (data not shown).

Ultraviolet Spectra-Ultraviolet spectral analysis of purified PC1 showed a typical protein spectrum. Amax and Ami. of the inhibitor proteins in both peaks were 278 and 253 nm, respectively.

Extinction Coefficient-The extinction coefficient (E!':.,,, at 280 nm in 0.05 M Tris-HC1, pH 7.5) was 14.1 as determined by the dye-binding method of Bradford (29).

Amino Acid Composition-The amino acid composition is shown in Table 11. The overall composition of the PC1 in the

A B C D E F FIG. 4. Polyacrylamide gel electrophoresis of purified PCI.

Analytical polyacrylamide disc gel (7%) electrophoresis of PC1 (10 pg) in A and B was carried out at 1 mA/tube at pH 10.0. SDS-polyacrylamide gel (10%) electrophoresis of PC1 in the first peak, unreduced (0 and reduced (D) , in the second peak, unreduced ( E ) and reduced (F), was done at 2 mA/tube, pH 8.9. Reduction was in 5% 2-mercap- toethanol. M, of proteins was estimated by comparison with five standard proteins.

A

B

1

AS

P

FIG. 5. Double immunodiffusion test and immunoelectrophoresis of PCI. In the center well in a, 5 pl antiserum prepared against purified PC1 was added. Wells I and 4 contained 1 pg of purified PC1 in the fmt and second peak, respectively. Wells 2 and 5 contained 30 p1 of plasma, and wells 3 and 6 contained 10 pl of plasma. The agarose plate was incubated in a humid chamber overnight, and was washed with saline and dried. The immunoprecipitate was stained with Coomassie blue. b, immunoelectrophoresis of purified PCI. An- ode is to the right. In wells PCI-I, PCI-2, and P were placed 2 pg of purified PC1 from the fmt peak, from the second, and 5 p1 of human plasma, respectively. Electrophoresis was carried out at 3 mA/cm width for 2 h. Troughs APCI and AS were filled with antiserum against purified PC1 and antiserum against human serum, respectively. Diffusion, washing, and staining were carried out in the same manner as indicated in a.

first peak was very similar to that in the second peak. This inhibitor is a relatively basic amino acid-rich protein, suggest- ing that this property results in the high isoelectric points of the inhibitor. Compared with other identified protease inhibitors of similar M,, including heparin cofactor I1 (30), the PC1 contained relatively larger amounts of arginine, lysine, leucine and serine, but smaller amounts of methionine, glycine, and aspartic acid.

Stability-The inhibitor protein can be made susceptible to serine proteases during purification. However, for several months, the purified inhibitor was rather stable a t both high (1 M NaCl) and low (0.05 M Tris) concentration of salts a t neutral pH and -20 "C. Lyophilization markedly decreased the solubility of the inhibitor and destroyed the activity of the purified material.

Biologic Characterization of PCI As noted previously, the specific activity of the purified PC1

in the first peak on DEAE-Sephacel chromatography was a little higher than that in the second peak. The PC1 in the first peak was used for the subsequent biologic studies. In the following experiments, 0.05 M Tris-HC1, pH 8.0, containing 0.15 M NaCl, 2 mM CaC12, 0.1% bovine serum albumin was used except when noted.


-t E 0 100

10 20 30 60 Reaction time (mid

FIG. 6. Time-dependent inactivation of amidolytic activity of activated protein C by purified PCI. 50 pl of activated protein C (675 ng) in 0.05 M Tris-HC1, pH 8.0, containing 0.15 M NaCl, 2 mM CaClz, 0.1% bovine serum albumin were incubated with 50 p1 of PC1 containing 315 ng (A), 625 ng (O), or 1250 ng (0) in the buffer at 37 "C, respectively. At intervals, 20-4 aliquots were withdrawn from each incubation mixture and added to 2.5 ml of 100 p~ peptide-MCA solution in a cuvette kept at 37 "C to measure the remaining activity of activated protein C.

40 120 200 300 400 Amount of PC1 (ng)

FIG. 7. Stoichiometric inhibition of activated protein C by purified PCI. 10 pl of activated protein C (135 ng) and 20 pl of PC1 a t varying concentrations were mixed and incubated at 37 "C for 60 min. To the incubation mixture, 2.5 ml of 100 p~ peptide-MCA solution were added, and the remaining activity of activated protein C was measured at 37 "C.

As shown in Fig. 6, the inhibitor inactivated the amidolytic activity of activated protein C toward a synthetic fluorogenic peptide substrate, not instantaneously, but gradually according to the length of the period of incubation. We also con- fiimed that antithrombin I11 does not block activated protein C, not even with heparin (data not shown).

In the experiment shown in Fig. 7 , activated protein C was treated with increasing amounts of PCI. The amount of PC1 necessary for complete inhibition of the activated protein C (135 ng) was extrapolated to be 125 ng. Therefore, if 61,000 is taken as the M, of activated protein C, the molar ratio of PC1 and the enzyme in this interaction is calculated to be nearly 1:l.

Fig. 8 illustrates a Dixon plot of PC1 against activated protein C at two different substrate concentrations. From this figure, it followed that PC1 inhibits activated protein C as a noncompetitive inhibitor and the inhibition constant (K t ) is calculated to be 5.8 X lo-' M.

The influence of PC1 on activated protein C was further studied by measuring APTT. The clotting time of normal human plasma in the absence of activated protein C, as control, was 47.2 s according to the assay method described in this text. As shown in Fig. 9, the prolonged APTT due to activated protein C was shortened to the control level in proportion to the amount of the inhibitor added.

In order to determine the concentration of PC1 in plasma, the usual immunological assay (rocket method) was per-

formed. However, the results were inconclusive, probably by reason of the low concentration of PC1 in plasma. Thus, the concentration of PC1 was tentatively estimated with the use of the specific activity of purified PCI, calculated to be about 5 mg/liter of plasma.

I t 10.

._ c -

0 73 146 220 292 PC1 (nM)

FIG. 8. Dixon plot of the purified PC1 for activated protein C. 10 p1 of activated protein C (270 ng) was incubated with 20 p1 of PC1 a t different concentrations a t 37 "C. After incubation for 15 min, 2.5 ml of 100 p~ (0) or 67 p~ (0) peptide-MCA solution was added to the incubation mixture, and the remaining activity of activated protein C was measured at 37 "C.

401, , , , , , , I 4

35 70 141 281 563 1125 Amount of PC1 (ng)

FIG. 9. Effect of the purified PC1 on activated protein C as determined by APlT assay. 50 p1 of activated protein C (700 ng) and 50 p l of PC1 at varying concentrations were mixed and incubated at 37 "C for 60 min. The remaining activity of activated protein C was measured by APTT assay, as described under "Experimental Procedures."

Amount of anti-PCI-immunoglobulin (mg)

FIG. 10. Removal of plasma inhibitory activity against activated protein C by anti-PC1 immunoglobulin. To 150 pl of normal human plasma, 50 pl of anti-PC1 immunoglobulin at varying concentrations were added and incubated a t 37 "C for 60 min. Thereafter, the incubation mixture was centrifuged at 10,OOO rpm for 15 min, and then the inhibitory activity against activated protein C in 150 p1 of the supernatant was measured according to the determination of PC1 activity, as described under "Experimental Procedures."

Protein C Inhibitor 167

TO find out whether PC1 acts on activated protein c as an enzyme, the influences of DFP, PCMB, and EDTA on PC1 activity were studied according to the inhibitor assay system. These tests showed that the inhibitor activity of PC1 was not blocked by prior treatment with 25 mM DFP, 2.5 mM PCMB, or by the presence of 10 mM EDTA (data not shown).

In order to find out whether our prepared PC1 protein was the only plasma inhibitor acting on activated protein C, the effect of anti-PC1 immunoglobulin on the inhibitory activity in normal human plasma was investigated. The method of carrying out the experiment was as follows. First, varying amounts of the immunoglobulin were added and incubated in a fixed amount of plasma at 37 "C. After 1 h, the inhibitory activity in the supernatant of the incubation mixture against activated protein C was measured. As shown in Fig. 10, the immunoglobulin removed the inhibitory activity present in plasma completely in proportion to the amount of immunoglobulin. This means that our PC1 is the only plasma inhibitor against activated protein C.

DISCUSSION

From the results obtained so far by several investigators, activated protein C not only can stimulate fibrinolysis but also appears to be a major regulatory protein acting on activated procoagulant cofactors Factor Va and Factor VIIIa. A congenital deficiency in protein C may result in recurring thrombotic disease (32). On the other hand, with activated procoagulant plasma serine proteases, antithrombin 111 seems to be a major regulatory protein, because inherited deficien- cies in antithrombin I11 also appear to cause thrombophilia (33, 34). Thus, both inhibitors, activated protein C and antithrombin 111, may function complementarily on the coagulation system as negative regulatory proteins. In addition to the above observations, the recent finding that the presence of a new plasma inhibitor for activated protein C in normal human beings, and the lack of this inhibitor in plasma deficient in both Factor V and VI11 (15) firmly supports the theory that protein C functions physiologically at least as a regulator of Factor V and Factor VIII, and also that the inhibitor has a role in regulating the protein C activity in normal human plasma. Thus, in order to understand the exact function of protein C in regard to the coagulation and fibrinolytic systems, it is essential to know the basic properties of the inhibitor protein.

The present paper is the first article of its kind to describe the purification procedure for PC1 from human plasma. It is also the first to describe the characterization of the inhibitor protein. From the fact that the inhibitor activity was not incorporated into the barium citrate precipitate, it is thought that y-carboxyglutamic acid is not present in the inhibitor protein. In the purification process, the first four steps only slightly raised the purity of PCI. However, in the first step, the vitamin K-dependent protease zymogens including protein C were removed. In the second step, high M , proteins, such as fibrinogen, were removed into a 6% PEG-6000 precipitate, and the proteases which destroy the PC1 activity were separated from the PC1 by fractionation with 12% PEG-6000. The third step was performed to remove procoagulant Factor V, a substrate for activated protein C. The most important step is to utilize dextran sulfate-agarose to separate the inhibitor from other major plasma proteins. Although only 25% of the inhibitor in the fraction from the previous step (50-70% ammonium sulfate precipitate) was recovered on dextran sulfate-agarose, the specific activity of the inhibitor appeared to be increased markedly (520-fold) by this chromatography. After this step, it was noted that the inhibitor protein became stable enough to be stored for a long period.

Marlar and Griffin (15) obtained an inhibitor fraction from plasma by using DEAE-Sephadex a t low ionic strength, pH 8.4. They suggested that PC1 is a high M, protein because the inhibitor activity was digested by trypsin and not dialyzable through the membrane. The PC1 which we prepared is probably the same as that they had intended to purify. We believe that the presence of species with different isoelectric points may be due to heterogeneity in the carbohydrate moiety and/ or microheterogeneity in the primary structure of the inhibitor. PC1 appears to differ immunologically and chemically from the seven previously identified plasma inhibitors. In particular, antithrombin 111 is distinguishable from PC1 because antithrombin I11 does not inactivate activated protein C (14). PC1 seems to be unlike heparin cofactor 11, isolated recently as a new thrombin inhibitor by Tollefsen et al. (30), because several physicochemical properties of the cofactor are distinguishably different from those of the PC1 which we purified.

The concentration of the PC1 in human plasma could not be determined exactly, because an immunologic assay was not available. However, it was estimated to be about 5 mg/liter of plasma from the specific activity of purified PCI. Compared with the concentration of other plasma protease inhibitors, the PC1 concentration appears to be considerably lower.

In respect to the interaction of the inhibitor with activated protein C, the inhibitor was found to reduce the amidolytic activity of activated protein C, in a noncompetitive manner, by forming a 1:l complex with the enzyme. From the rate at which complexes are formed by enzyme and inhibitor, PC1 appears to belong in the category of progressive inhibitors, such as antithrombin 111 in the absence of heparin. The following inhibition mechanisms are possible: ( a ) PC1 forms a noncovalent complex with activated protein C; (b ) PC1 makes a covalent linkage with the serine-OH in the active center of activated protein C; and/or ( c ) limited proteolysis of PC1 by activated protein C requires formation of the complex (34). PC1 also shortens the clotting time of prolonged APTT by activated protein C. This suggests that PC1 protects Factor V (Va) and/or Factor VI11 (VIIIa) from activated protein C.

The finding that monospecific immunoglobulin produced with our prepared inhibitor removed all of the inhibitory activity in plasma against activated protein C shows that the inhibitor which we purified is the only inhibitor protein against activated protein C in plasma.

In preliminary experiments, PC1 appears to inhibit thrombin and trypsin (36). The specificity of PC1 is currently under investigation. There still are many unsolved problems to clarify, such as how PC1 regulates the Factor V (Va) and/or Factor VI11 (VIIIa) level in plasma when coagulation is trig- gered, how the inhibitor affects a fibrinolysis stimulated by activated protein C, and so on. It appears that PC1 is lacking in plasma of patients with a combined deficiency of Factor V and Factor VI11 (15,35). However, in such cases, it is not clear whether this is because the amount of inhibitor protein is substantially lower or whether this depends on abnormalities of the inhibitor molecule.

Acknowledgment-We wish to thank Dr. Johan Stenflo (Depart- ment of Clinical Chemistry, Malmo General Hospital, Malmo, Swe- den) for his guidance and for his continuing interest in this project.

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3. Kisiel, W. (1979) J. Clin. Inuest. 64, 761-769 4. Kisiel, W., Canfield, W., Ericsson, L., and Davie, E. W. (1977)

J. Biol. Chem. 251,3052-3056

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