John Curd and Bruce Zuraw, Scripps Clinic, La Jolla, CA. Polyacrylamide gel ..... Walker, F. J., Sexton, P. W., and Esmon, C. T. (1979) Biochim. 5. Marlar, R. A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263 No. 24 Issue of August 25, pp, 11613-11616,1988 0 1988 by The American Sdciety fo; Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Communication Physiologic Inhibition of Human Activated Protein C by al-Antitrypsin* (Received for publication, May 18, 1988)
Mary J. Heeb and John H. Griffin$ From the Research Institute of Scripps Clinic, La Jolla, California 92037
plasma inhibitor of APC is heparin-dependent,has been characterized (9, lo), and is known as protein C inhibitor (PCI). Recent workshowed that there is a second major plasma inhibitor of APC which is heparin-independent (11, 12). Here we describe the isolation of this inhibitor, its identity to al-antitrypsin, and appearance its as APC-al-antitrypsin complexes in plasmas from patients with DIC. MATERIALS ANDMETHODS
The heparin-independent inhibitor of APC was monitored by its The plasma antithrombotic enzyme activated protein inhibition of APC amidolytic activity and its ability to form comC (APC) hastwo major plasma inhibitors. One is hep- plexes with APC as determined by immunoblotting, and was isolated arin-dependent, has been characterized, and is known as follows. The supernatantof fresh barium-adsorbed plasma3 (1)was as protein C inhibitor. The second inhibitor was iso- made 12% inpolyethylene glycol 6000 with stirring at 4 “C,and after lated based on its heparin-independent ability to in- 30 min was centrifuged for 15 min at 5000 X g. The precipitate contained >95% of the heparin-dependent PCI? The supernatant hibit and complex with APC.The purified inhibitor containing the heparin-independent inhibitor was made 20% in polhad the amino acid composition and NH2 terminus of yethylene glycol and centrifuged as described above. The precipitate cyl-antitrypsin and reacted with monoclonal antibodies was resuspended and dialyzed against the starting buffer for DEAEto a,-antitrypsin. Theinhibitor was >95% pure a1- Sephadex chromatography performed as described elsewhere (13). antitrypsin as judged by electroimmunoassay, inacti- For the DEAE-Sephadex chromatography, 10 mgof protein/ml of vation of trypsin, and electrophoresis in two gel sys- column bedwere applied, and for the remaining chromatography tems. Toidentify the secondmajor plasma inhibitor of steps, a maximum of 2 mg of protein was applied per ml of column bed. Elution gradient total volumes were seven times the column bed APC,immunoblot studies ofenzyme-inhibitorcomplexes were made to compare APC addition to normal volumes. The inhibitor pool eluting from DEAE-Sephadex a t a conof 7-9 mmho was concentrated by precipitation with 65% plasma and to plasma deficient in protein C inhibitor ductivity saturated ammonium sulfate followed by resuspension and dialysis or a,-antitrypsin. The results showed thatal-antitryp- against starting buffer for hydroxylapatite chromatography. For sin is the second major plasma APC inhibitor. Given hydroxylapatite chromatography a gradient of 0.01-0.3 M potassium the associationrate constant ofa,-antitrypsin for APC phosphate containing 1 mM benzamidine, 0.02% NaN3, pH 7.5, was of 10 M“ s-‘ and its plasma concentration of -40 WM, employed, and the inhibitor eluted at approximately 0.04 M phosphate. For heparin-Sepharose chromatography, a gradient of 0-0.5 M it accounts for approximately half of the heparin-independent APC inhibitory activity of plasma. Based on NaCl in 0.05 M Tris-HCI, 1 mM benzamidine, 0.02%NaN3, 1 mM immunoblot analysis plasmas of15 patients with intra- EDTA, pH 7.4, was employed, and the inhibitor eluted at about 0.05 M NaCl. For the first hexyl agarose column (ICN), a gradient of 30 vascularcoagulationcontainedAPC-a,-antitrypsin to 0% saturated ammonium sulfate in 0.05 M Tris-HC1,0.02% NaN3, complexes suggesting that this inhibition reaction oc- pH 7.4, was employed, and theinhibitor eluted at approximately 20% curs in vivo. Thus, al-antitrypsin is a major physiologic saturated ammonium sulfate. The second hexyl agarose chromatoginhibitor ofAPC. raphy step was performed in the samemanner, except that the gradient was modified to 30 to 10% saturated ammonium sulfate. The purified inhibitor was dialyzed against 0.05 M Tris-HC1, 0.1 M NaC1,0.02% NaNa3, pH 7.4 (TBS) andstored a t -70 “C. Amino acid analysis of the purified inhibitor was performed on a Protein C (1, 2) is a vitamin K-dependent anticoagulant Beckman model 6300 high performance analyzer after a 24-h acid regulatory serineprotease zymogen. Activated proteinC hydrolysis at 110 “C, and NHz-terminal amino acid sequencing was l(APC)’ inactivates the procoagulant factors V. and VIII. (3- performed on an Applied Biosystems model 470A gas phase sequen5). Activated protein C is amajor physiologic antithrombotic cer. agent in man since heterozygous deficiency of protein C is Protein C was purified and activated as described (5). The antiassociated with venous thrombotic disease in some families protein C monoclonal antibody (C3), directed against the light chain (6) and homozygous deficiency of protein C leads to poten- of protein C, recognizes protein C, APC, and APC-inhibitor comin immunoblots with the same efficiency? PC1 was purified, tially fatal purpura fulminans at birth (7). Evidence for pro- plexes rabbit antibodies against it were prepared, and plasma immunodetein C activationand complexation with plasma protease pleted of PC1 was prepared as described3(11).Monoclonal antibodies inhibitors was found in studies of the plasma of many patients to al-antitrypsin were obtained from Chemicon. Purified @‘-antiwith disseminated intravascular coagulation (DIC) (S).2 One trypsin and rabbit antiserum against it were obtained from Calbiochem. Protein was estimated using A% = 14.5 for protein C (2) and * This work wassupported in part by National Institutes of Health A% = 4.36 for al-antitrypsin (14). Individual normal plasmas, pooled Grant HL-24891 and by a National Institutes of Health training normal human plasma (NHP), andplasma of patients with DIC were grant. The costs of publication of this article were defrayed in part collected as previously described’ (8, 15). Plasma of a patient homoby the payment of page charges. This article musttherefore be hereby zygous for the z variant of al-antitrypsin was obtained form Drs. marked “aduertisement” in accordance with 18 U.S.C. Section 1734 John Curd and Bruce Zuraw, Scripps Clinic, La Jolla, CA. Polyacrylamide gel and immunoblotting techniques and Iz5I-labelsolely to indicate this fact. ing techniques were previously described (11,15). $To whom correspondence should be addressed. The amidolytic activity of APC toward the peptide substrate S‘The abbreviations used are: APC, activatedprotein C; DIC, disseminated intravascular coagulation; NHP, pooled normal human 2366 (Kabi) was measured as described (5,15). Inhibition of APC was plasma; PCI,proteinCinhibitor; S-2366, pyro-Glu-Pro-Arg-p-ni- measured by reference to a standard curve of the APC inhibitory troanilide; TBS, Tris-buffered saline; SDS, sodium dodecyl sulfate. Heeb, M. J., Mosher, D., and Griffin, J. H.; and Heeb, M. J., Espaiia, F., Berrettini, M., and Griffin, J. H., submitted for Espaiia, F., and Griffin, J. H., submitted for publication. publication.
11613
11614
al-Antitrypsin Protein Activated Inhibits
activity of various N H P dilutions, where 1 ml of N H P contains one unit of inhibitory activity. N H P values were corrected for base-line amidolytic activity in the absence of APC. Porcine trypsin (Sigma) was active site-titrated and used to titrate the activity of as-antitrypsin as described (16). The second order rate constant for inhibition of APC by as-antitrypsin was determined by incubating 12 nM APC with 36 or 18 PM as-antitrypsin at 37 “C in TBS. At various times, aliquots were removed and the APC amidolytic activity was determined and compared to a control of APC incubated in the same manner with TBS 0.1% bovine albumin in placeof al-antitrypsin.
C
peak A was used for somestudies described below. Its specific inhibitory activity of 0.45 units/mg suggests that itis present in plasma at -2.2 mg/ml. The amino acid composition (except cysteine and tryptophan)of the isolated heparin-independent APC inhibitor (Fig. 1, peak A ) was compared to that of aIantitrypsin (17) and found to be indistinguishable with the coefficient of variation for the molar ratio of individual amino acids averaging 0.09. The NHz-terminal sequence of the purified inhibitor (Fig. 1, peak A ) was Glu-Asp-Pro-Gln-Gly, identical to that of al-antitrypsin (18). RESULTS Electroimmunoassays (6) made of the isolated APC inhibThe heparin-independent plasma inhibitor of APC was itor (Fig. 1peak A ) uersw NHP dilutions using al-antitrypsin purified based onassays of its inhibition and complexation of antibodies suggested that the inhibitor was purified al-antiAPC using the steps listed in Table I and the precipitations trypsin, assuming that the normal plasma level is approxiunder “Materials and Methods.” Ouchterlony analysis of the mately 1.7 mg/ml (18). DEAE-Sephadex pool of inhibitor detected antithrombin 111, Immunoblotting analyses of reaction mixtures (90 min, al-antitrypsin, andalbumin, but not PCI, al-antichymotryp- 37 “C) containing purified reference al-antitrypsin, APC, sin, C1-inhibitor, az-macroglobulin,&glycoprotein I, or C4b- NHP, and plasmas deficient in PC1 or al-antitrypsin were binding protein. The pools of inhibitor from heparin-Sepha- made to study complex formation of APC with PC1 and alrose and the first hexyl-agarose column contained al-anti- antitrypsin (Fig. 2). When al-antitrypsin was incubated with trypsin and albumin antigens, but not antithrombin 111. The APC, some APC was complexed with the inhibitor (Fig. 2, elution profile of the second hexyl-agarose columnconsisted lanes 6 and 6’,as compared to APC alone in lanes 9 and 9’ or of three protein peaks (Fig. 1).The protein in the first peak the inhibitor alone in lanes 5 and 5 ’ ) . The antibodies to al( A ) appeared to be electrophoretically homogeneous on both antitrypsin recognized complexedantigen with much greater SDS-denaturing (Fig. 1, left inset, middle lane) and alkaline- efficiency than free antigen. In data not shown, very similar nondenaturing gels (data not shown), comigrating with al- results were obtained with the isolated APC inhibitor as with antitrypsin in both gel systems. Peak A protein reacted with commercial al-antitrypsin. As previously reported’ (19), two monoclonal antibodies to ax-antitrypsin. Peak B contained major bands of APC-inhibitor complexes are formed when primarily al-antitrypsin of slightly different electrophoretic APCis incubated with normal plasma in the absence of mobility than peak A on nondenaturing gels and also albumin. heparin (Fig. 2, lane 4). One band (designated APC:PCI in The third peak contained primarily albumin. The inhibitor in Fig. 2) corresponds to APC:PCI complexes since this band reacts with antibodies to PC1 (11)and since this band is very TABLE I weak in plasma immunodepleted of PC1 (Ref. 11and see Fig. Purification of heoarin-indewendent inhibitorAPC of 2, lane 8).The other band of APC-inhibitor complexes formed in normal plasma (Fig. 2, lane 4 ) comigrated with the band of purified APC-al-antitrypsin and reacted with antibodies to al-antitrypsin (Fig. 2, lane 4‘, compared to a comigrating DEAE-Sephadex 14 35.8 11.6 0.58 500 0.023 band in lane 4). Plasma of a patient homozygous for the z Hydroxyapatite 3.2 95 9.6 0.32 304 0.032 variant of al-antitrypsin formed apparently normal levels of Heparin-Sepharose 305.0 7.50.25 150 0.050 APC:PCI complexes upon incubation with APC (Fig. 2, lane Hexyl-agarose I 50 0.18 0.21 9.042 1.0 Hexyl-agarose I1 2) but very low levels of the complex of APC-al-antitrypsin A 18 0.18 3.2 0.40 7.2 0.45 (Fig. 2, lane 2 compared to lanes 4 and 6,and lane 2’ compared B 13 7.7 0.39 0.15 3.0 0.59 to lanes 4’ and 6’).A normal level of the bands ascribed to APC-ax-antitrypsin complexes wasseen when APC was added to PCI-deficient plasma (Fig. 2, lanes 8 and 8’). 0.5 In a previous study, plasmas of 16 patients with DIC were found to contain >20% of protein C antigen in complexed 0.4 form with the heparin-independent inhibitor designated PCI2 prior to its identification as al-antitrypsin’ (8).Plasmas of 0.3 15 of these patients were immunoblotted for al-antitrypsin. Two immunoblots from nondenaturing gels are shown in Fig. 2 0.2 3. All of the 15 patient plasmas tested had a band comigrating with complexes formed when APC was incubated with normal 0.1 plasma (Fig. 3, lanes 6-15,compared to lanes 2-5). In other data not shown, 12 individual normal plasmas did not have 0.0 0 20 40 60 80 100 120 140 160 detectable levels of APC-ax-antitrypsin complexes and had an immunoblotting profile similar to the NHP profile seen Volume Eluted(mll FIG.1. Hexyl-agarose chromatography I1 of heparin-inde- here in lane 1. Compared to NHP(Fig. 3, lane I), the patient pendent inhibitor of APC. Chromatography was performed as plasmas in Fig. 3, lanes 6-15,had apparently higher levels of described under “Materials andMethods.” A precipitin line based on al-antitrypsin including forms of low electrophoretic mobility Ouchterlony analysis of al-antitrypsin and albumin is indicated by whichmay represent heterodimers with IgA (20). This is +. Aliquots of selectedfractions weredialyzed against TBS and consistent with reports that al-antitrypsin is an acute-phase assayed for inhibition of APC activity (open circles). PeaksA and B reactant (18). werepooled as indicated. The inset shows a silver-stainedSDS To assess the covalent bond nature and the apparent moelectrophoresis gel (6% gel) of pooled peak A (lane 2) compared to reference purified al-antitrypsin (alAT) ( l a n e I ) and molecular weight lecular weight of APC-al-antitrypsin complexes, immunostandards (lane 3 ) . blotting studies were made using SDS-denaturing gels and
I
D
C
aI-Antitrypsin Protein Activated Inhibits FIG.2. Immunoblots of APC-inhibitor complexes and of ax-antitrypsin (alAT) using nondenaturing gels. Development was made with monoclonal antibody (C3) to protein C followed by "'1-protein C on the left gel, andwithrabbitantibodiestoal-antitrypsin followed by '*'I-a1-antitrypsin on the right gel. All samples were incubated a t 37 "C for 90 min prior to electrophoresis. In the samples on the left, 4 pl of the indicated plasma or 4 pl of 2 mg/ml a,-antitrypsin in TBS wasincubated with 4 pl of 4 pg/ml APC (indicated above lanes as + APC) in TBS/O.l% bovine albumin or with 4 p1 of TBS/ 0.1% bovine albumin. In the samples on the right, 1 pl of the indicated plasmaor 1 pl of 2 mg/ml al-antitrypsin was incubated with 7 pl of 140 pg/ml APC in TBS/O.l% bovine albumin.
a,AT Dalicwnl Plasma
Normal
hrifi
Plasma
e,AT
PC1 bpbld Plasma
APC
APC AFT
11615 PC1
o,AT Dalicmnl Plasma
Nmal Plasma
APC AFT APC
APCAPC
~~-~
APC
AFT
APC:PCI+ -APCa,AT
APCor Protein
-a,AT
c+ 1
2
3
4
5
,
7
6
9
8
1'
2'
Anti-Protein C +'251.PC
2
6'
7'
8' 9'
Anti~alAT+1251~orAT
DIC Patient Plasmas
a
u~AT Deficient Plasma
" "
APC APC
5'
c
4
+
4'
3'
"
treated plasma samples (designated by numbering P-24, P-25, etc.) were 1 pl. N H P was incubatedwith APC at 300 pg/ml for 2 h a t 37 "C.The equivalent of 1 pl of plasma was electrophoresed in lane 2, and 1-pl dilutions of 1:3, l:lO, and 1:30 in TBS/O.l%bovine albumin of the incubation mixture as indicated in lanes 3-5. In lane 16, 1 pl of NHP was incubated for 2 h with 5 pl of 40 pg/ml APC in TBS/O.l%bovine albumin.
AT
Plasma
APC:a,AT"
FIG.3. Immunoblot for al-antitrypsin (azAT) antigenin normal plasma incubated with APC and in untreated patient plasmas. All un-
Normal Plasma
Dapkled
"~~
Normal Plasma
Purified
Purilid a,AT
+ A k
E
5 MAx 10.31
-200 APC:Q~AT-
-97 -68
Q 1 AT-
1 2 3 4 5 6 7 8 9 FIG.4. Immunoblot for al-antitrypsin (alAT) antigen using an SDS-denaturing 6% gel. Sample incubation and immunoblotting were performed as in Fig. 2, right panel, except that an SDS gel system was used. P-24 is from the patient sample also analyzed in Fig. 3, lane 6.Purified PC1 (2 pg) in 6 pl of TBS/O.l%bovine albumin was analyzed in lane 8.
antibodies to at-antitrypsin (Fig. 4). The data in Fig. 4 indicate that the molecular weight of the complex of APC-atantitrypsinis approximately 110,000,whichwouldbe expected for a 1:1 complex of enzyme with inhibitorunder nonreducing conditions (lanes 2, 4, and 7). As seen with antibodies to al-antitrypsin, complexes formed between purified APC and at-antitrypsin comigrate with the major band
5
6
7
8
9
,
10
11
12 13
14 1615
of complexes detected when APC is incubated with plasma (Fig. 4, lane 2 compared to lane 4), although some unidentified minor forms of inhibitor antigen at higher molecular weight were seen in plasma. It is possible that these are aggregates or complexes involving IgA (20). The plasma of the al-antitrypsin-deficient patient, containing -15% of the z variant, formed much lower than normal levels of APC-al-antitrypsin complex upon incubation with APC (Fig. 4, lane 7 compared to lane 4). Plasma from one patient (P-24) with DIC is seen in Fig. 4, lane 5 and showed a band of cul-antitrypsin antigen comigrating with APC-al-antitrypsin complexes (lanes 2 and 4). These data suggest that at least some of the APC-atantitrypsin complexes formed in uiuo and in vitro are covalent and withstand electrophoresis in SDS under denaturingconditions. Kinetic studies of the inactivation of APC amidolytic activity by purified al-antitrypsin were performed. To quantitate the at-antitrypsin activity of the isolated APC inhibitor (Fig. 1,peak A ) porcine trypsin that was 81%active based on active site titration was used. The purified al-antitrypsin (Fig. 1, peak A ) was found to be 64% active in inactivating trypsinon a molar basis. The second order rate constant for the inactivation of APC by the purified inhibitor was 10 M-' s-', and heparin did not stimulate this inhibition. DISCUSSION
A heparin-independent inhibitorof APC was isolated from plasma by virtue of its ability to inhibit APC amidolytic
11616
al-Antitrypsin Inhibits
activity and complex with APC in the absence of heparin. The protein was found to be identical to a>-antitrypsin by a number of criteria including amino acid analysis, NHz-terminal amino acid sequence, comigration with al-antitrypsin in two polyacrylamide gel electrophoresis systems, electroimmunoassay, immunoblotting with monoclonal and polyclonal antibodies to al-antitrypsin, and immunoblotting of its APC complexes with monoclonal antibody to protein C and polyclonal antibody to al-antitrypsin. Thepurity estimates of the inhibitor by these various criteria ranged from 91 to 100%. The purification procedure employed here (Table I) was not efficient. Once the APC inhibitor was identified as a well studied protease inhibitor, al-antitrypsin,no further attempts were made to optimize the purification steps or procedures used. Evidence that al-antitrypsin is a physiologic inhibitor of APC was obtained. When normal plasma is incubated with APC in the absence of heparin, two bands of APC-protease inhibitor complexes arise at approximately the same rate and at about the same levels as seen on nondenaturing immunoblots' (19). One of these bands is APC:PCI' (ll),and the other is here identified as APC-al-antitrypsin. This is based on thefact that theband formed in plasma comigrates in two gel systems with bands of purified APC complexed with purified al-antitrypsin (Figs. 2-4). Furthermore, formation of thisband is deficient when APC is added to a1antitrypsin-deficient plasma (Figs. 2 and 4). Evidence of in vivo formation of APC-al-antitrypsin complexes was found upon analyzing the plasmas of patients with DIC. All 15 patient plasmas tested from a previous study that had been found to have high levels of APC in complexed form were also found here to have detectable amounts of al-antitrypsin antigen comigrating with APC-al-antitrypsin complexes (Figs. 3 and 4), whereas 12 individual normal plasmas did not have detectable antigenic forms. Thus, cul-antitrypsin appears to inhibit APC i n vivo. The second order rate constant for association of APC and al-antitrypsin is low (10 M- s-'). However, the high plasma concentration (40 PM) of al-antitrypsin makes the calculated half-life of APC in plasma approximately 30 min. This is similar to the observed half-life (30 min) of APC in whole plasma without heparin (11). The calculated half-life of APC in plasma containing PC1 at 88 nM, with a second order rate constant3 in the absence of heparin of 0.6 X lo4 M-' s-', is approximately 22 min. These calculations would suggest that PC1 is slightly more effective in plasma in inhibition of APC.
Activated Protein C However, the observed data suggest that both inhibitors are physiologically important in the absence of heparin, since both complex in vitro with APC to approximately the same extent and since in vivo formed complexes of both inhibitors,' especially of al-antitrypsin, are found in plasmas of patients with DIC. Acknowledgements-We are grateful for the excellent technical assistance of Anthony Potente andsecretarial work of Leslie Sherry. We thank Dr. Francisco Espaiia for the preparation of PC1 and PCIdepleted plasma and helpful discussions. REFERENCES 1. Stenflo, J. (1976) J. Biol. Chem. 2 5 1 , 355-363 2. Kisiel, W. (1979) J. Ctin. Znuest. 64, 761-769 3. Kisiel, W., Canfield, W.M., Ericsson, L. H., and Davie, E. W. (1977) Biochemistry 16,5824-5831 4. Walker, F. J., Sexton, P. W., and Esmon, C. T. (1979) Biochim. Biophys. Acta 571,333-342 5. Marlar, R. A., Kleiss, A. J., and Griffin, J. H. (1982) Blood 5 9 , 1067-1072 6. Griffin, J. H., Evatt, B., Zimmerman, T. S., Kleiss, A. J., and Wideman, C. (1981) J. Clin. Znuest. 6 8 , 1370-1373 7. Branson, H., Katz, J., Marble, R., and Griffin, J . H. (1983) Lancet 2,1165-1168 8. Heeb, M. J., Mosher, D. J., and Griffin, J. H. (1987) Fed. Proc. 4 6 , 716 (abstr.) 9. Suzuki, K., Nishioka, J., and Hashimoto, S. (1983) J. Biol. Chem. 258,163-168 10. Suzuki, K., Deyashiki, Y., Nishioka, J., Kurachi, K., Akira, M., Yamamoto, S., and Hashimoto, S. (1987) J. Biol. Chem. 262, 611-616 11. Heeb, M. J., Espaiia, F., Geiger, M., Collen, D., Stump, D., and Griffin, J . H. (1987) J. Biol. Chem. 262,15813-15816 12. van der Meer, F. J. M., van Tilburg, N. H., van der Linden, I. K., Briet, E., and Bertina, R. M. (1987) Thromb. Huemostasis 5 8 , 277 (abstr.) 13. Griffin, J. H., and Cochrane, C. (1976) Methods Enzymol. 4 5 , 56-65 14. Crawford, I. P. (1973) Arch. Biochem. Biophys. 156,215-219 15. Heeb, M. J., Schwarz, H. P., White, T., Lammle, B., Berrettini, M., and Griffin, J. H. (1988) Thromb. Res., in press 16. Beatty, K., Bieth, J., and Travis, J. (1980) J. Biol. Chem. 2 5 5 , 3931-3934 17. Pannell, R., Johnson, D., and Travis, J. (1974) Biochemistry 2 6 , 5439-5444 18. Carrell, R. W., Jeppsson, J. O., Laurell, C.B., Brennan, S.O., Owen, M.C., Vaughan, L., and Boswell, D. R. (1982) Nature 298,329-333 19. Heeb, M. J., Schwarz, H. P., White, T., and Griffin, J. H. (1986) Circulation 74,II-234 (abstr.) 20. Dawes, P. T.,Jackson, R., Shadforth, M. F., Lewin, I.V., and Stanworth, D. R. (1987) Br. J. Rheumatol. 26,351-353
