Identification of divalent metal ion-dependent inhibition of activated ...

'rHE ~ O U R N A I .OF BIOLOGICAL CHEMISTRY IC 1991 hy The American Society for Biochemistry and Molecular B i o l o g y , Inc.

Vol. 266, No. 26, Issue of September 15, pp. 17606-17612,1991 Printed in U. S.A .

Identification of Divalent Metal Ion-dependentInhibition of Activated Protein C by cr2-Macroglobulin and cr2-Antiplasmin in Blood and Comparisons to Inhibition of Factor Xa, Thrombin, and Plasmin* (Received for publication, April 16, 1991)

Mary J. Heeb, Andras Gruber,and John H. Griffin$ From the Committee on Vascular Biolom and the DeDartment of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037

The half-life of activated protein C (APC) was 31 min in citrated blood and 18 min in whole blood. Immunoblotting analysis of citrated blood identified APC-protein C inhibitor (APC-PCI)and APC-al-antitrypsin complexes. Whole blood contained two additional APC-inhibitor complexes, one stimulated by Ca"+ andanother by Mg2+.The former was identified as APC-a2-macroglobulin(APC-a2M)while the latter was not identified. APC-a2-antiplasmin complexes (APC-a2AP) were identified, comigrating with APCPC1 complexes. Purified a2M and a2AP inhibited APC in the presence of Ca2+ (k2 = 99 and 100 M" s-', respectively. Inhibition of APC and Factor Xa by azM and inhibition of APC by azAP wasstimulated by Ca2+, Mn"+, andMg". Inhibition of thrombin bya2M and of plasmin by a2AP was not altered by EDTA or Ca2+, suggesting divalent metal ions affect APC and Factor Xa rather than the inhibitors. k2 values for the APC inhibitors and their plasma concentrations suggest that PC1 and al-antitrypsin are the more important APC inhibitors and that cr2M and a2AP are metal ion-dependent auxiliary inhibitors. Inhibitors can account for the in vivo half-life of APC.

Protein C is a vitamin K-dependent zymogen (1,2) which, when proteolytically cleaved to form activatedprotein C (APC),' serves as an anticoagulant regulator of coagulation pathways by inactivation of the cofactorsVa and VIIIa(3-5). Evidence for its physiologic importance stems from reportsof potentially fatal purpura fulminans in homozygous protein Cdeficient infants (6) and association in some families of venous thrombotic disease with heterozygous deficiency of protein C (7). Evidence of protein C activation during intravas-

* This study was supported in part by Grant HL-31950 from the National Institutes of Health and a fellowship (to A. G.) from the California Affiliate of The American Heart Association. A preliminary report has appeared in abstract form (Heeb, M. J., Gruber, A., and Griffin, J. H. (1990)Circulation 82, 111-305). 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 Dept. of Molecular and Experimental Medicine, BCR-5, Scripps Clinic and Research Foundation, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-8220; Fax: 619-554-6732. The abbreviations used are: APC, activated protein C; a2AP, 012antiplasmin; nlAT, a]-antitrypsin; n2M, a2-macroglobulin; 1-2581, N dansyl-(p-guanidine)-Phe-piperidine-HC1; kn, second order association rate constant; PCI, protein C inhibitor; S-2366, pyro-Glu-ProArg-p-nitrophenylanilide; TBS,Tris-bufferedsaline; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin.

cular coagulation has been presented (8). APC had an antithrombotic effect and prevented death due to septic shock whenadministeredin several animal models (9-12). The possibility of future therapeutic use of APC in humans has led to increased interest in its physiologic inhibition by protease inhibitors in blood. Recent work revealed that major inhibitors of APC in plasma include PC1 and alAT (13-16), both of which inhibit APC relatively slowly compared with inhibition of other coagulation enzymes by plasma inhibitors. The existence of other APC inhibitors has been suggested (16). Inadditiontothe twopreviouslyidentified plasma inhibitors, here we report that a 2 M , a,AP, and possibly another protein inhibit APC inwhole blood in a divalent metal ion-dependent manner, making thehalf-life of APC in whole blood withoutdivalentmetal ion chelators significantly shorterthanincitrated blood or plasma. To determine whether the unusual metal ion dependence was due to an effect on APC or to aneffect on azM and a2AP,we compared inhibition of APC to thatof Factor Xa, thrombin, and plasmin. EXPERIMENTALPROCEDURES

Reagents-Human protein C was purified and activated as described (9). A previously described monoclonal antibody to the light chain of protein C (C3) was used for immunoblotting and for solid phase assay of APC activity (9, 15, 17, 18). This antibody C3 recognizes with equalefficiency protein C, APC, orcomplexes of APC with PC1 or n,AT. For some immunoblots, goat polyclonal antibody to protein C was used (17). Rabbit antibody to PC1 was prepared as described (19, 20). Human anM wasa kind giftfrom Dr. Steven Gonias (University of Virginia Medical School). Human a2AP was obtainedfromAthensResearchand Technology. Recombinant Arg'"nlAT wasagenerousgiftfrom Drs. Michael Courtneyand Ranier Bischoff (Transgene, Strasbourg, France). Monoclonal antibody to a2M was obtained from Chemicon. Other antibodies were prepared in rabbits and obtained from Behring. Polyclonal antibody to a2APwas affinity-purified by absorption to a2APcoupled to AffiGel 10(Bio-Rad) according tomanufacturer'sinstructions.The bound antibody was eluted with 3 M NaSCN in Tris-buffered saline (TBS) andimmediately dialyzed against TBS. Cephalin andp-nitrophenylguanidinobenzoate were obtained from Sigma,protein ASepharose from Pharmacia LKB, Biotechnology Inc., heparin from Elkins-Sinn, and human Factor Xa, thrombin, and plasmin from Enzyme Research Laboratories. General Methods-Electrophoresis and immunoblotting were performed as previously described (17, 19). The concentrations of a2M and n,AP were determined from absorbance a t 280 nm, using E"" of 8.9 (21) and 6.7 (22), respectively. The n2M was determined to be 91% active by titration against active site-titrated thrombin (92% active), and the a,AP was determined to be 67% active by titration against active site-titrated plasmin (14% active), according to procedures previouslydescribed(23). The activity of thrombin titrated with n2M was determined by clotting of fibrinogen (Kabi) (24), and the amidolytic activity of plasmin titrated with a2AP was determined using the peptide substrate S-2366 (Kabi).

17606

Calcium Ion-dependent Inhibition Kinetics for Inhibition of APC and OtherProteases-For determi(k,) for inhibition nation of the second order association rate constant of APC by a2M, 0.5 p~ APC was incubated at 37 "C with 2.6 p~ n,M and 5 mM CaCI? or other additions as specified in TBS containing 0.05% bovine serum albumin. Aliquots of 4 pl were removed over time, and the residual APC anticoagulant activity was tested by its ability to prolong the activated partial thromboplastin time. The control curvewas obtained using dilutionsof APC incubated without tr,M. Clotting mixtures contained 100 pl of pooled normal plasma (George King Bio-Medical,Inc.) preincubated 200 s with the test sample and a cephalin-silica activator (100 p1 of Thrombosil, Ortho). Clotting was initiated by addition of 100 pl of 25 mM CaCI2. For inhibition of APC by a2AP, loss of amidolytic activity towardS-2366 was measured over time by assaying aliquots of mixtures containing . order rate APC (63 nM) and a,AP (from 4.6 to 14 p ~ )Pseudo-first constants (hi) were determined from the slopes of semilog plots of activity versus time, and k2 was calculated from hz = kl/[inhibitor]. The theoretical half-life of APC in blood was calculated from t,,, = 0.69/kz [inhibitor], using the plasma concentration of nnM, a2AP,or other inhibitor. For determination of the k, for inhibition of Factor Xa by a2M, 0.18 unit/ml Factor Xa was incubated a t 37 "C with 1.12 p~ a2M in TBS, 1% BSA containing 5 mM CaCI2 or otheradditiontobe were quenched specified. At selected times from5 to 15 min, reactions by dilution by addition of20 volumes of TBS-BSA, followed by immediate determination of residual Factor Xa activity ina Xa onestage clotting assayas follows. Normal plasma (100 pl) was preincubated with 100 pl of test sample and100 p1 of 0.2 mg/ml cephalin for 200 s. Clotting was initiated by addition of 100 pl of 30 mM CaCI,. Standard dilutions of Factor Xa were incubated without a2Mfor the same times as the test samples, and these clot times were used to construct a standard curve. For determination of the k? for inhibition of thrombin by a,M, 0.5 p M a,M was preincubated for 20 min a t 37 "C with 5 mM CaCI2 or other additions asspecified in TBS, 1% BSA. Thrombin (22.5 units/ ml) was added and incubation continued forselected times from 1 to 3 min. The reaction was quenched by addition of a 25-fold volume of 11% polyethylene glycol-8000, 20 mM CaCI2. An immediate test for clotting activity (24) was made using an equal volume of 4 mg/ml fibrinogen, and thrombin activitywas quantitated based on a standard curve constructed using control thrombin dilutions. For determination of the k, for inhibition of plasmin by a2AP, 2.8 pg/ml plasmin a t 5 "C was incubated with 7.8 pg/ml a,AP a t 5 "C in TBS, 1% BSA with 5 mM CaCI, or otheraddition as specified. Aliquots were removed a t 30-s intervals and immediately assayedfor amidolytic activity by diluting into 60 volumes of 0.8 mM S-2366,0.05 M Tris-HCI, 0.1 M NaC1, 0.1% BSA, p H 8.2. Inhibition of APC in Blood and Plasma-For half-life studies of APCactivityin blood, blood was collected from seven different normal donors into separate siliconized polypropylene tubes containing volume of 0.11 M trisodium citrate or TBS. APC was added to a final concentration of approximately 2 pg/ml in the fluid phase, and the mixture was incubated a t 37 "C. Aliquots were taken over time into tubes containing buffer with benzamidine (Kodak) to give a final concentration of 30 mM. Blood was centrifuged for 1 min in a Beckman Microfuge B (8,900 X g), and the supernatant plasma was assayed for APC activity in the wells of microtiter plates coated with monoclonal antibody C3 against protein C and blocked with TBS, 1% casein,0.02% NaN, (9,18). After incubation of aliquots containing APC in the wells and subsequent washing, the wells were incubated with 100 pl of 0.8 mM S-2366, the change in absorbance a t 405 nm was recorded over time using a Biotek 312 enzyme-linked immunosorbent assay plate reader, and the observed amidolytic activity was compared with APC standards. In parallel studies, aliquots of the incubation mixtures containingblood and APCwere taken over time without additionof benzamidine and centrifuged for immediate direct determination of APC amidolytic activity in0.8 mM S-2366 containing 20 p~ of the thrombin inhibitor 1-2581 (Kabi) (17, 19). Under these conditions 1-2581 does not inhibit APC but neutralizes any background activity dueto thrombin. In some experiments to compare inhibition of APC inblood uersus plasma, blood was collected into tubes with APC in TBS to give a final concentration of 16 pg/ml, prostaglandin E-1 at a final concentration of 1 pglml, and '/9 volume of either 0.11 M sodium citrate or TBS. Half of the sample containing citrate and half of the sample containing TBS were centrifuged 4 min a t 5,000 X g in a Dade microcentrifuge a t room temperature. The whole blood mixtures and the cell-free mixtures were incubated at 37 "C and aliquots takenover

of Activated Protein

C

17607

time for determination of APC anticoagulant activity, APC amidolytic activity, and APC antigen immunoblotting pattern as described above. Identification of APC-Inhibitor Complexes-In order to remove specific APC-inhibitor complexes from incubation mixtures of blood and APC by immunoabsorption, the mixtures were incubated for a specified time and then made30 mM in benzamidine usinga 500 mM benzamidine solution and centrifuged1 min in a Beckman Microfuge B. The supernatant plasma 7-pl in aliquots was mixed with 15pl each of IgG fractions of various antisera against known protease inhibitors. The sampleswere incubated 1h at 3'7 "C and2 h a t 4 "C. Each sample was then added to 20 pl of washed, packed protein A-Sepharose beads in a 1.5-ml conical microcentrifuge tube and mixed. The mixture was incubated 16 h a t 4 "C, with occasional mixing during the last hour of incubation. The samples were centrifuged 1 min, and 20 pl of each supernatant solutionwas subjected to immunoblottinganalysis. RESULTS

Effect of Metal Ions in Blood o n APC Half-life and Inhibitor Complexes-To study the influenceof divalent metal ions on the inhibition of APC by blood, APC was mixed with freshly drawn blood in the absence or presence of citrate, and the loss of APC activity was measured as a function of time. As calculated from the APC amidolytic activity data in Fig. 1 (upperpanel),the average half-lifeof APC amidolytic activity in whole blood from seven individuals was18& 2 min, whereas in blood containing 0.01 M trisodium citrate itwas 31 3 min ( p < 0.001). These data were determined in asolid-phase immunocapturemicrotiterplateassay for APC. Basedon direct amidolytic assaysof supernatants of centrifuged blood samples in substrate containing a thrombin inhibitor, the respective APC half-life values were 21 and 32 min. Separate experiments in which the blood cells were removed from the incubation mixture immediately after the addition of APC and prostaglandin E-1 gave half-life values in the presence and absence of citrate similar to those obtained in the presence of cells, showingthat the shorter half-life in whole blood,

*

Blood+APC Blood+APC+Citrate

0

5 15 30 60

I

0 5 15 30 60 TimelMinl

+Protein C

andAPC

FIG. 1. Inhibition and complex formation of APC added to human blood. The upper panel shows inhibition of APC activity over time during incubation with blood with and without the addition of citrate. The mean values from seven samples containingtrisodium citrate are indicatedby open squares, and the mean values from seven samples without citrate from the same donors are indicated by closed circles. Standard errors are indicated by error bars. The microplate assay for APC activity was described under "Experimental Procedures." The lower panel shows an immunoblot for protein C and APC in blood incubated with APC in the presence and absence of citrate. A representative individual sample from the experimentin the upper panelis shown. The immunoblot wasfroma 5% nondenaturing polyacrylamide gel and employed monoclonal antibody to protein C (C3) followed by "'I-protein C (15, 17).

Calcium Ion-dependent Inhibition

17608

of Activated I’rotpin C

or strontium ions but to a lesser extent than bv calcium ions. i.e. in the absence of citrate, was not due to any metal ionAddition of a metal ion mixture to give final concentrations dependent reactions involving blood cells under these condiof 1.8 mM MgCI,, and 0.1 mM each ZnCI,, MnCI,, CoCI?, and tions. Moreover, half-life determinations based on APC anCuCI,, led to the appearance of another new band, designated 20 minforAPCinwhole ticoagulant,activityassaysgave band .v (Fig. 2, lanr 3 ) . In other experiments appearance of blood without citrate, 33 min in blood with citrate, and in blood from which cells were removed, 17 min without citrat,e b a n d y was dependent on magnesium ions and not on anv of of a n d 28 min in the presence of citrate. Thus, the inhibitionof the other divalent ions tested (data not shown). Addition loss of phospholipids or protein S to citrated plasma did not change APC anticoagulant activity in blood coincided with amidolytic activit,y, was significantly influenced by divalent the overall pattern of complex formation (Fig. 2, lnncs 4 and by blood cells. -5).A diminution of the band ascribed toAI’C-PC1 complexes metal ions, and was not. significantly influenced was observed when calcium ions were added (Fig. 2, compare Otherexperimentsinwhichrecombinanthirudin(CibaGeigy, 2 pg/ml) was included in the blood mixtures suggested lanes 2, 3 , a n d 7 with lanes 1 and 6 ) , but not when calcium 2, ions were added in combination with phospholipids (Fig. that inhibition of APC activity in blood was not influenced lanes 4 , 5 , a n d 8). When APC was incubated with human by any thrombin that might be present. T h e lowerpand of Fig. 1 shows an immunoblotting analysis serum (Fig. 2, lane 9 ) . bands comigrating with hands x and .v were observed. Thus, the divalent metal ions, calcium and using a nondenaturing gel forprotein C antigeninblood samples incubated with APC. When APC was incubated with magnesium, stimulated the formation of APC complexes in blood in the presence of citrate (lanes 6-10), there were two blood, plasma, and serum. Idmtification of AI’C Cornplexed with tr,M and (r,AP in apparent major bands of AI’C-inhibitor complexes formed, Blood-Band x was identified as the complexAI’C-tr,M by its similar to previous reports (14, 15) for citrated plasma incubated with APC. Immunoblotting for PC1 or for trlAT (data removal from an incubation mixture of blood and AI’C using not shown) confirmed that these bands contained APC-PC1 monoclonal antibodyto n,M, followed by protein A-Sepharosc (Fig. 3, compare lanes 3 a n d 9 with lane 2 ) . Complexes of and APC-trIAT, as previously reported (14, 15). However, in the reaction mixhres using whole hlood, i e . , without citrate APC-cyIAT were removed in a similar manner by adsorption 3 , l a m 4 ) . Adsorption of (Fig. 1, louwpanel, lanes 1-.5), two additional bands contain- usinganti-trlATantibodies(Fig. reaction mixtures using antibodies to nl-antichvmotrypsin, ing APC antigen, designated x and y , were visible following C1 inhibitor, inter-tr,-protease inhihitor, and /jJ-glvcoprotein incubation of APC with blood. Other experiments (data not I, followed by protein A-Sepharose, did not remove band .v or shown) demonst.rated that addition of recombinant hirudin (2 pg/ml) to the mixt.uresof blood and APC had no effect on any of the other bands (Fig. 3 , lancs 5-8). In other experiments, adsorpt.ion using antibodies to plasminogen activator bands x and y or any of the other APC bands. To investigate whether formation of these bands was diva- inhibitor-1 did not remove any of the prominent bands conlent. metal ion-dependent, metal ions and other addkions weretaining APC antigen (data not shown). of bloodandAPCwithout When the reaction mixtures made to reactionmixtures of APC withcitratednormal to tr,AI’ citrate were adsorbed using affinity-purified antibodv plasma or citrated protein S-depleted plasma, and the mixfollowed by protein A-Sepharose, the band previously identitures were electrophoresed on nondenaturing gels and subfied as APC-PC1 was diminished (Fig.3 , compare lanc I 1 with jected to immunohlotting for protein C antigen. As seen in Fig. 2 (lanes 1 and 6), the only two apparent bands detected the control in lane IO). Moreover, adsorption using antibody to PC1 did not completely remove the hand in the region of intheabsence of addedmetalionsmigratedatpositions APC-PC1 complexes (Fig. 3, lane 12, compared with lanr 1 0 ) . previously assigned to APC-PC1 and APC-n,AT complexes. Addition of 12 mM calcium ions led to the appearance of a even though other immunoblots developed with antibodiest o PC1 new band (band x ) of very low electrophoretic mobility (Fig. PC1 showedthat all detectablecomplexescontaining 2, lanm 2 and 7 ) . Other experiment,s using protein S-depleted plasma revealed that. t.his low mobility band x seen in Fig. 2 Rlood. APC (lane 7 ) wasdependentoncalciumionsbutnotonthe presence of protein S. Inseparateexperiments(datanot shown), band x formation was also stimulated by magnesium

I

,

,

;

.

1

‘1

FIG. 2 . Effect of divrtlent metal ions, phospholipid, and protein S on AI’C complexes in plasma. I’ooled normal human plasma ( N H I ’ ) or protein S-depleted plnsma ( I ’ S d ) o r human serum I’rom a single donor ( 4 pl/lane) was incul);lted at X7 “C for 1 h with AI’C at a linal concentration o f 2 pg/ml. in a final volume of I O pl, with or without the additions indicated above the figure of 6.5 mM ( h ( ’ 1 2 , 1.8 mM MgCI:., 200 pg/mlcephalin,or 1 0 pg/ml protein S (final concentr;ltions).Where %n is indicated, a metal ion mixture was added t o the linal ronrmtr;ltions ofO.1 mM each of ZnCI,, MnCll, CoCI,, and Cu(’l,. Following inculxttion. the sampleswere electrophoresed on a 5”;’ nondennturing gel and immnnol)lotted for protein C nntigenusingmonoclonal antihodv t o protein (’ followed I)? ““Iprotein C.

Calcium Ion-dependent Inhibition of Activated Protein

C

17609

antigen had indeed been removed (data not shown). Control experiments showed that the affinity-purified antibody to EDTA A a2APdid not recognize PC1 (data not shown).Adsorption of the reaction mixture of blood and APC simultaneously using antibodies to both a2AP and PC1 completely removed the band(s) in this region (Fig. 3, lune 13),showing that both APC-PC1 and APC-a2AP complexes were originally present in the immunoblot band labeled APC-PCI (Fig. 2). This was further confirmed using a reaction mixture of PCI-depleted I 1 1 1 1 1 ~ I I I plasma with APC, calcium,and magnesium ions (Fig. 3, lunes 0 5 10 15 20 25 30 35 40 45 50 14 and 15).The PCI-depleted plasma formed complexes in the region of APC-PC1 (Fig. 3, lune 151,and it was devoid of Time (Mid PC1 antigen when analyzed by immunoblotting with antibodCa+ EDTACa EDTA, 084 4 4 ies to PC1 (data not shown). The identity of these complexes 0 120 5 15 30 60 9012060 90120 0 5 15 30960 5 15 30 960 1~nalMml -APC.o.M as APC-a,AP was established by their complete removalupon APC.o?Madsorption withaffinity-purified antibody to a2AP (Fig. 3, lune 14). Thus, APC complexes with both a2M and a2AP in whole blood. APC-APC An additional band containing APC antigen was seen on immunoblots, migrating just below bund y in some experiDenaturing Gel Nondenaturing Gel ments, e.g., in Fig. 3 (lanes 10 and 12). This band was faint FIG.4. Inhibition and complexation of APC by purified when samples were electrophoresed immediately after incu- ( Y ~ MUpper . panel: Pseudo-first order plot of inhibition of APC by bation of the reaction mixture containing APC, but it inpurified a?M. APC was incubated with n?M in the presenceof 5 mM creased in intensity when samples were frozen and thawedor CaC1, (closed circles) or 5 mM EDTA (open triangles), and aliquots handled for extended periods of time prior to immunoblottingwere tested for anticoagulant activityas described under “Experimental Procedures.” Lower panel, immunoblots for protein C antigen in analysis, suggestingthat this bandmay arise afterproteolysis samples upper panel.The left irnmunoblot from the experiment in the or degradation of one of the other species containing APC. is froma 4-1596 gradient nondenaturinggel, and theright blot is from This band may be related to APC-a2AP, since antibody to a 4-1576 gradient denaturing gel containing sodium dodecyl sulfate. a2APremoved this band inFig. 3, lanes I 1 and 13.Moreover, Immunoblotting was performed as in Fig. 1. as seen below in Fig. 6, a band of this mobility was detected on immunoblots of plasma/APC mixtures with antibodies a2Mfor 2 5 min in thepresence of calcium ions. Samples from against a2AP. incubation mixtures of blood and APC immunoblotted from Inhibition of APC by Purified a2M-Inhibition of APC by denaturing SDS gels had an identical pattern of immunoblot human a2M was studied using purified proteins in the presbandsrepresenting complexes of molecular weight over ence of 5 mM calcium ions or 5 mM EDTA. From the slopes 200,000 (data not shown). The molecular weight of these of pseudo-first order plots of APC activity against time(Fig. complexes on reduced denaturing SDS gels was 130,0004, upper panel), the second order association rate constant k2 150,000 (data not shown). In the nondenaturing gel blot (Fig. for inhibition of APC anticoagulant activity by a2M in the 4, lower panel), the total measured radioactivity per lane fell presence of calcium ions was 99 20 M-’ s” ( n = 3). by 33% for the 120-min incubation of APC and a2M in the Inhibition of APC bya2M in the presence of EDTA was presence of calcium ions, but the radioactivity per lane renegligible. Similar values for k2 were obtained by monitoring mained constant during120 min of incubation of APC in the the inhibition of APC amidolytic activity by a2M; however, presence of calcium ions with a2M and remained constant there was often a lag phase of up to 30 min before inhibition during 120 min of incubation of APC and a2M in presence the of APC amidolytic activity by a2M was observed, and maxi- of EDTA. This indicates that APC inAPC-azM the complexes mum inhibition did not exceed 80%. To see whether APC was underrepresentedinthe immunoblots, either because bound toa2M retained some activity against smallmolecules, APC was not fully accessible to the antibodyor because the but not macromolecules, incubation mixtures of APC and large APC-02M complexes did not transfer efficiently to the a2Mwere tested tomeasure how much of the remaining APC nitrocellulose paper, or both. In the denaturing SDS gel blot amidolytic activity was inhibited by Arg’”alAT. After 50 min (Fig. 4, lower panel), the total radioactivity per lane also of incubation of APC with a2M, 26% of the remaining APC decreased for the 30-min incubation with APC and a2M in amidolytic activity was protected from Arg’”alAT, after 70 the presence of calcium ions, but not in the presence of EDTA. min of incubation, 50% was protected, and after 16 h of Thus, although immunoblotting analysis qualitatively demincubation, 100% was protected. Thus, APC bound to a2M onstrates complexation of APC with purified a2M, itdoes not retained approximately 20% of its amidolytic activity. allow a quantitation of these complexes. Complex formation of APC with purified a2M was directly T o learn whether the unusual metal iondependence of demonstrated usingimmunoblotting. Fig. 4 (lowerpanel) inhibition of APC by a2M mightbe due to APC or to a2M,k2 shows an immunoblot for APC antigenfrom a nondenaturing values for inhibition by a2Mof APC, Factor Xa, and thrombin gel (left)and also an immunoblot froma denaturing gel (right) were obtained using kinetic studies and thencompared under of incubation mixtures of APC with purified human a2M. various conditions. The data in Table I show that 5 mM CaC12 Bands (Fig. 4, lower left) with the samemobility as bund x in stimulatedAPCinhibition at least 7-fold andFactorXa Figs. 1 and 2 and APC-aZM in Fig. 3 were apparent after 5 inhibition 4-fold when compared with 0.2 mM EDTA. Fig. 5 min of incubation in the presence of calcium ions but were shows that thek2 for inhibition of APC hada similar dependbarely detectable after 16 h of incubation in the presence of ence on calcium ion concentration as the k2 for inhibition of EDTA. The denaturing SDS gel immunoblot revealed a doub- the homologous vitaminK-dependentFactor Xa. The k2 let of heat-stable, detergent-stable bandsof molecular weight values obtained for inhibition of Factor Xa arein reasonable in excess of 200,000 formed after APC was incubated with agreement with those previously reported (25), which were +

+

+ -

Calcium Ion-dependent Inhibition

17610

of

TABLE I Second order rate constant (kJ for inhibition of nroteases bv a,M (M' s-')

100

~~

Addition

APC"

Factor Xa"

Activated Protein C

Thombin"

Citrate (11 mM) 22 f 15 343 f 140 6900 f 800 EDTA (0.2 mM) 190 f 68 7800 f 1200 4 2 480 f 21 30 f 18 TBS 7100 f 1600 86 k 14 770 f 240 CaCI2 (5.0 mM) 7200 f 1500 MgCln (5.0 mM) 70 f 19 643 f 46 ND MnC12 (5.0 mM) 63 f 3 2100 k 420 ND 910 f 230 83 f 24 MnCL (0.5 mM) ND Values for inhibition of APC or Factor Xa are the means of three or more determinations f standard deviations. Values for inhibition of thrombin are the means of five determinations f standard deviations. ND, not determined.

-

80

-g

60

0

40

>

.-

.u Q

20 Q

"0

20

40

I'

60

80

100 0 20 Time IMinl

40

60

80

100

FIG. 6. Inhibition of APC by purified a2AP. Leftpanel, pseudo-first order plots of inhibition of APC amidolytic activity by 14 I.LM a,AP in the presence of 5 mM CaC12 and 1.8 mM MgC12 or in the presence of 5 mM EDTA. Right panel, pseudo-first order plots of inhibition of APC amidolytic activity by 4.6 p~ azAP in thepresence of CaC12and/or MgClZ,with or without 1 unit/ml heparin. TABLEI1 kz for inhibition of APC and plasmin bya A P (M' s") Addition

APC"

Plasmin"

EDTA (0.1 mM) 36 f 12 1.5 f 0.2 x lo6 EDTA (5.0 mM) 28 f 1.5 2 f 0.2 x lo6 CaC12 0.4 mM 53 f 2 ND 0.6 mM 64 f 18 ND 1.0 mM 73 f 18 1.6 ? 0.2 X lo6 [ C a C l ~ (mM) l 5.0 mM 100 f 13 1.3 f 0.1 X lo6 FIG. 5. Dependence of inhibition of APC and Factor Xa by MgCIP a,M on concentration of calcium ions. k2 values were calculated 0.4 mM 40 f 5 ND from clotting assays as described under "Experimental Procedures." 1.0 mM 45 f 16 ND Values at "zero" calcium concentration were determined with the 5.0 mM 62 f 12 1.3 f 0.1 x lo6 addition of 0.2 mM EDTA (closed symbols) or with addition of TBS MnC12 (open symbols). 0.2 mM 53 f 5 ND 0.4 mM 62 f 13 ND 1.0 mM 78 f 16 1.4 f 0.2 X lo6 determined in the presence of 4 mM CaClZ.The patterns for 2.5 mM 71 f 13 ND calcium ion stimulation of a2M inhibition of APC and Factor a Values for inhibition of APC or plasmin are the means of three Xa were similar, although no final plateau for stimulation was or more determinations f standard deviations. ND, not determined.

observed for APC inhibition by a2M. Inhibition of both APC and Factor Xa by azM was stimulated by magnesium ions to a lesser extent than by calcium ions (Table I). Manganese a2APwas stimulated 3-4-fold by 5 mM CaC12,that itwas less ions were more stimulatory for Factor Xa inhibition thanfor strongly metal ion-dependent than inhibition of APC by a2M, and that inhibition was stimulated by other divalent metal APC inhibition. In clear contrast to APC and Factor Xa, inhibition of thrombin by a,M was unaffected by the divalent ions, including manganese and magnesium to a lesser extent. metal ionsor chelating agents thatwere tested (Table I). This In clear contrast to APC, inhibitionof plasmin by azAP was or chelating was true whether the ions tested were preincubated for 20 unaffected by any of the divalent metal ions min with a 2 Mor whether the additionwas made immediately agents thatwere tested (Table 11). prior to the thrombin(values in parentheses in Table I). The Complex formation of APC with a2AP was shown using k, values obtained for thrombin inhibition are in reasonable immunoblotting analysis. Purified azAP formed complexes with APC (Fig. 7, last lane) that comigrated with APC-PC1 agreement with thosepreviously reported (26). Inhibition of APC B y Purified aAP-Inhibition of APC by complexes and with the APC-a2AP complexes identified in purified a2APwas studied. Fig. 6 (left panel)is a pseudo-first incubation mixtures of plasma or blood with divalent metal ionsandAPC (Fig. 7, left panel). Inimmunoblotsfrom order plot for inhibition of APC amidolytic activity upon incubation with 14 p M a2AP in the presenceof calcium and denaturing 4-15% gradient gels, APC-a2AP complexes also magnesium ions or in the presence of EDTA. The rate of comigrated with APC-PC1 complexes (data not shown). Purified APC-a2AP and APC-a2AP complexes formed in plasma inhibition of APC by a2AP was four times higher in the presence of divalent metal ions than in the presence of EDTA. containingdivalentmetalionsreactedwithantibodiesto Fig. 6 (right panel)demonstrates thatcalcium ions were more azAP, and thesecomplexes were not formed in plasma from effective than magnesium ions in stimulating this inhibition a patient congenitally deficient in a2AP (27) (Fig. 7, right of APC. Heparin at 1 unit/ml diminished inhibition of APC panel, a A P d ) . The quantity of APC-a2AP complexes in the Fig. 7 (right panel)did not by a2AP. Kinetic analyses showed that the association rate 1-h incubation mixtures shown in or absence appear to be significantly different in the presence constant k, for inhibition of APC by a2AP was 100 k 9 M" s" in the presence of calcium and magnesium ions and 28 of divalent metal ions. However, in other experiments (not shown) divalent metal ions stimulated the formation of these M" s" in the presence of EDTA. complexes at incubation times of less than 30 min. Thus, T o determine whether this unusual metal ion dependence complexes identifiable of inhibition of APC by azAP was due to APC or to azAP, purified aZAP inhibits APC and forms purified inhibition of APC was compared with that of plasmin by on immunoblotsof reaction mixturesof APC with the a2AP. The data in TableI1 show that inhibition of APC by inhibitor or plasma.ImmunoblotsforPC1antigen(not

Calcium Ion-dependent Inhibition of Activated Protein C -. " cr,AP

..."

17611

(9). The discovery of divalent metal-ion dependent inhibition of APC by a2M, anAP, and possibly by one other inhibitorin - *APC blood helps to explain the differencesbetween the in vivo half-life of APC and thein vitro half-life of 31 min in citrated plasma. Thus, inhibition of APC by protease inhibitors in blood could entirely account for the regulation and removal of APC activity in vivo. Y An!, o,AP APC has approximately nine calcium ion binding sites (30), Oeleclmn A m Pwmn C FIG. 7. Immunoblots for purified APC-anAP and for APC- and calcium ions and phospholipids arerequired for optimal aaAP in various plasmas. Pooled normalplasma ( N H P ) , PCI- rates of inactivation of Factors Va and VIIIa (3, 5). Most of depleted plasma ( P C I d ) , or tr2AP-congenitally deficient plasma the calciumion bindingsites reside intheNHn-terminal (tr.JPd) were incubated for 1 h alone, with APC ( + A X ) , or with domaincontainingnine y-carboxyglutamyl (Gla) residues, APC and metal ions( + M e ) . The metal saltsused were 6.5 mM CaC12 but APC with theGla domain removed contains a t least one and 1.8 mM MgCl,, and the final APC concentration was 4 pg/ml. The samples were subjected to electrophoresis ona 5% nondenaturing high affinity binding sitefor calcium or manganese ions that gel and then immunoblotted for APC (left panel)or for n2AP (right may alter APC conformation (30, 31). Calcium ions are not required for inhibition of APC by PC1 (20), a l A T (14, 28), or panel). The last lune of each blot contains complexes of APC-n,AP formed by incubating purified n2AP a t 90 pg/ml with APC a t 140 pg/ plasminogenactivatorinhibitor-1 (32) or for inhibition of ml in TRS, 1% BSA in the presence of 5 mM CaCI, and 1.8 mM other coagulation serine proteasesby plasma protease inhibMgCI, at 37 "C for 6 h. The last lane in the left blot contains, in addition, complexes of purified APC-PC1 formed ina similar manner. itors. Consequently, findinga strong influenceof calcium ions on inhibition of APC by a2M and arAP was unexpected. T o assess whether the divalent metalion influences either shown) revealed that fewer APC-PC1 complexes were formed in blood or plasma in the presence than in the absence of the enzyme or the protease inhibitor in these reactions, the by a2M andof plasmin divalent metal ions a t all time points, even though these metalinhibition of Factor Xa and thrombin ions do not affect the rate of inhibition of APC by purified by arAP was studied. Inhibition of the vitamin K-dependent PC1 (20). The unidentifiedmagnesium-ion dependent bund y homologue, Factor Xa, is stimulated by divalent metal ions, of APC complexes was not related to either a2AP or to PCI, whereas inhibitionof thrombin by a2M orof plasmin by arAP since itwas formed in both PCI-depleted plasma and plasma- is not divalent metal ion-dependent. Thus, the unusualdivalent metal ion dependence of inhibition of APC and Factor deficient in a2AP (Fig. l , left panel). Xa is probably due to a property of the enzymes rather than due toa property of the inhibitors, anM and a2AP. DISCUSSION a2M, a tetramer of subunits of M , 180,000, inhibits a wide Inhibition of APC by the plasma inhibitorsaIAT andPC1 variety of proteases by a mechanism in which the protease is is not divalent metal ion-dependent in purified systems or in entrapped ina cagelike structure (33,34). The protease active plasma (14, 20, 28), and the inhibitionof plasma blood coagsite is not altered, and it may still exhibit partial enzymatic ulation enzymes by plasma protease inhibitors has not been activity toward small substrates. The protease usually cleaves previously shown to be stimulated by divalent metal ions. a "bait" region of the a2M, causinga change in conformation However, as described here, we found a difference in the halfwhich stimulatescovalentattachment between reactive life of APC in whole blood in the presence and absence of thioester groups of anM andNH, groups of the protease and citrate, a divalentmetalionchelator,andthis led us to which closes the cage. Such a mechanism for a2M and APC discover the existence of two additional types of apparent interactions is consistent with the observation here that APC APC-inhibitor complexes that areobserved in blood or serum be entirely inhibwhen citrate is absent. One of these complexes is identified amidolytic activity is diminished but cannot ited and that, over time, the remaining observed amidolytic here as APC-anM, and the other one has not yet been idenactivity becomes less susceptible to inhibition by Arg:'"alAT, tified or indeed proven to be associated with inhibition of APC. In addition, divalent metal ion-stimulated complexes of a very efficient macromolecular inhibitorthatneutralizes >98% of APC (28). APC in APC-a2M complexes is probably APC-anAP are shown here to form when APC is added to plasma, blood, or serum, and these complexes comigrate on less accessible to anti-protein C antibodies as well, since the total detectable anti-protein C antibody per lane on immuimmunoblots with APC-PC1 complexes. noblots decreased as APC-a2M complexes increased in incuThe immunoblotting observations prompted kinetic studies bation mixtures of APC and anM. Since APC-a2M complexes using purified proteins. Purified human a2M and a2AP inhibit APC in calcium ion-stimulated reactions with second order on immunoblots were stable to heat and detergent treatment s-I, respectively, of samples taken after5 min of incubation of APC with blood association rate constants of 99 and 100 M" compared with 11 M-' s-l for inhibition of APC by alAT and or with purified anM, it appears thatsome of the APC-a2M complexes were 6.0 X 10:' M" s" for inhibition of APC by PC1 (14, 20, 28). complexes are covalent. In fact, the APC-a2M SDS gel immunoblots as on equally intense on denaturing (1 Based on the plasma concentrations for a2M (3p ~ )a2AP , p ~ )alAT , (40 p ~ )and , PC1 (88 nM), the calculated half-life nondenaturing gel immunoblots. Theapparent molecular of APC in blood considering each inhibitor separately is 38, weight of APC-a2M complexes on reduced SDS gels of 110, 26, and 22 min, respectively. This kinetic estimate of 130,000-150,000 suggests that thelight or theheavy chain of APC is linked to fragments of a2M in the range of M , 90,000relative reactivity of inhibitors in plasma is consistent with the suggestion from the immunoblotting data that alAT and 120,000. Such fragment sizes have been reported for cleaved PC1 are primary inhibitors, whereas a2M and a2AP are aux-a n M (35), suggesting that APC cleaves a2M. a2M inhibits illary inhibitors of APC in blood. The observed half-life of thrombin (26) and Factor Xa (25, 36); however, we are not APC in whole human blood without citrate of 18-21 min is aware of any previous reports of divalent metal ion-dependent approximately what the combinationof calculated half-lives inhibition by a2M of these or any otherproteases. Investigawould imply and is similar to the reported half-life of 23 min tors previously found that only about 13% of "'I-Factor Xa for APC infused into humans (29) and is slightly longer than incubated with mouse or human citrated plasma bound to the in vivo half-life of 10-14 min for APC infused into baboons anM, whereas 90% of "'I-Factor Xa infused into mice was NHP

PCld

u,APd

" "

.APC

"

NHP

PCld e,AP

o,APd

" "

.APC ,piPC .APC .APC

J

.APC

"~

Calcium Ion-dependent Inhibition

17612

bound to aZMwithin 2 min (36). The difference between these in vitro and in vivo data could be explained as due to divalent metal ion stimulation of the inhibition of Factor Xa by a z M , as seen here. It was reported that purified a2M and azM in plasma do not inhibit APC, but theexperiments described by these authors (37) apparently did not include the addition of divalent metal ions to reaction mixtures. Perhaps thedivalent metal ion-dependent conformation of APC can fit into the to cleave the bait cagelike structure of a Z M inamanner region, or perhaps APC in thisconformation is more reactive with some macromolecular substrates. a2AP,a glycoprotein of M , 70,000, is a member of the serpin superfamily (38, 39) and is homologous to PC1 and alAT. It interacts very efficiently with plasmin with a kz of 4 x lo7 "' s-', forming 1:l complexes that are predominantly covalent. Inhibition of APC by a2AP is stimulated by divalent metal ions in a purified system and in blood. Interestingly, after 30 min of incubation of plasma with APC, complexes observed on immunoblots are more intense in theabsence of divalent metal ions than in their presence (Fig. 6, and data not shown). Proteolysis of a2AP or its complexes in plasma containing divalent metal ions may be responsible for this observation. We are unaware of previous reports of divalent metal ion stimulation of inhibition of proteases by a2AP. Complexes of APC with alAT and PC1 were identified in plasma of patients with disseminated intravascular coagulation (8), in baboons infused with APC (40),and in chimpanzees infused with phospholipid vessels containing Factor Xa (41). In the latter report,additional APC complexes were detected on immunoblots but notidentified. We have recently identified APC-a2M and APC-a2APcomplexes in plasma of baboons infused with APC (42). Thus, alAT, PCI, a2M, and a2AP each complex in vivo with infused APC, and each inhibitor can function physiologically to neutralize APC. In summary, divalentmetalions in blood significantly enhance the rateof inhibition of APC by the protease inhibitors aZM and a2AP, and the inhibition of APC by protease inhibitors in blood may adequately account for the clearance and i n vivo half-life of APC. Acknowledgments-We are grateful to Cecille Dalanon and Benjamin Montoya for skilled technicalassistance and Dr. Francisco Espaiia for preparation of antibody to PCI. We thank Dr. Steven Gonias for a generous gift of apM and for helpful comments, Drs. David Loskutoff and Raymond Schleef for antibodies to plasminogen activator inhibitor-1, and Drs. Rainer Bischoff and Michael Courtney for the gift of recombinant A r P q A T . REFERENCES 1. Stenflo, J. (1976) J . Biol. Chem. 251, 355-363 2. Kisiel, W . (1979) J . Clin. Invest. 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 59, 1067-1072 H., Katz, J.,Marble, R., and Griffin, J. H. (1983) Lancet " B;py?%-1168 7. Griffid, J. H., Evatt, B., Zimmerman, T. S., Kleiss, A. J., and Wid man, C. (1981) J . Clin. Invest. 68, 1370-1373 8. Heeb, J., Mosher, D., and Griffin, J. H. (1989) Blood 73,455461

b.

of Activated Protein C 9. Gruber, A., Griffin, J. H., Harker, L. A., and Hanson, S. R. (1989) Blood 73,639-642 10. Emekli, N. B., and Ulutin, 0. N. (1980) Haernatologica 65, 644 11. Emerick, S. C., Murayama, H., Yan, S. B., Long, G. L., Harms, C. S., Marks, C. A., Mattler, L. E., Huss, C. A., Comp, P. C., Esmon, N. L., Esmon, C. T., and Bang, N. U. (1987) in T h e Pharmacology and Toxicology of Proteins (Holcenberg, J. C., and Winkenhale, J., eds) pp.351-367, Alan R. Liss, New York 12. Taylor, F. B., Chang, A., Esmon, C. T., D'Angelo,A., ViganoD'Angelo, S., and Blick, K. E. (1987) J . Clin. Invest. 79, 918925 13. Suzuki, K., Nishioka, J., and Hashimoto, S. (1983)J . Biol. Chern. 258,163-168 14. Heeb, M. J., and Griffin, J. H. (1988) J . Biol. Chem. 263,1161311616 15. Heeb, M. J., Espaiia, F., and Griffin, J . H. (1989) Blood 73,446454 16. van der Meer, F. J. M., van Tilburg, N. H., van Wijngaarden, A., I. K., Briet, E., andBertina, R.M. (1989) vanderLinden, Thromb. Haemost. 62, 756-762 17. Heeb, M. J., Schwarz, H. P., White, T., Lammle, B., Berrettini, M., and Griffin, J. H. (1988) Thromb. Res. 52, 33-43 18. Geiger, M., White, T. M., and Griffin, J. H. (1989) Thrornb. Haemost. 6 1,86-92 19. Heeb, M. J., Espaiia, F., Geiger, M., Collen, D., Stump, D., and Griffin, J. H. (1987) J . Biol. Chem. 262, 15813-15816 20. Espaiia, F., Berrettini, M., and Griffin, J. H. (1989) Thromb. Res. 55,369-384 21. Gonias, S. L., Einarsson, M., and Pizzo, S. V. (1982) J . Clin. Invest. 70,412-423 22. Wiman, B., and Collen, D. (1977) Eur. J . Biochem. 78, 19-26 23. Chase, T., and Shaw, E. (1969) Biochemistry 8,2212-2224 24. Fenton, J. W., 11, and Fasco, M. J. (1974) Thromb. Res. 4, 809817 25. Meijers, J. C. M., Tijburg, P. N. M., and Bouma, B. N. (1987) Biochemistry 26,5932-5937 26. Feinman, R. D., Yuan, A. I., Windwer, S. R., and Wang, D. (1985) Biochem. J . 231, 417-423 27. Miles, L. A., Plow, E. F., Donnelly, K. J., Hougie, C., and Griffin, J. H. (1982) Blood 59, 1246-1251 28. Heeb, M. J., Bischoff, R., Courtney, M., and Griffin, J. H. (1990) J. Biol. Chem. 265, 2365-2369 29. Okajima, K., Koga, S., Kaji, M., Inoue, M., Nakagaki, T., Funatsu, A., Okabe, H., Takatsuke, K., and Aoki, N. (1990) Thromb. Haemost. 63, 48-53 30. AmDhlett. G. W.. Kisiel.. W.., and Castellino. F. J. (1981) . . Biochemis'try 20,215612161 31. Johnson. A.E.. Esmon. N. L.. Laue. T. M.. and Esmon. C. T. (1983)'J. Bioi Chem. 258, 5554-5560 ' 32. Gladson, C. L., Schleef, R. R., Binder, B. R., Loskutoff, D. J., and Griffin, J. H. (1989) Blood 74, 173-181 33. Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709724 34. Feldman, S. R., and Pizzo, S. V. (1986) Semin. Thromb. Hemost. 12,223-225 35. Barrett, A. J., Brown, M. A., and Sayers, C. A. (1979) Biochem. J . 181,401-418 36. Fuchs, H. E., and Pizzo, S. V. (1983) J . Clin. Invest. 72, 20412049 37. Marlar, R. A., and Kressin, D. C. (1989) Thrornb. Res. 54, 177186 38. Lijnen,H. R., and Collen, D. (1986) in ProteinaseInhibitors (Barrett. A. J.. and Salvesen. G.. eds). DD. 457-476. Elsevier Science Publishing Co., New York 39. Saito. H.. Goldsmith. G. H.. Moroi. M., and Aoki, N. (1979) Proc. Nat. Acad. Sci. U. S . A . 76,2013-2017 40. Espaiia, F., Gruber, A., Heeb, M. J., Hanson, S. R., Harker, L. A., and Griffin, J. H. (1991) Blood 77,1754-1760 41. Hooeendoorn. H.. Nesheim, M. E., and Giles, A. R. (1990) Blood 75,2164-2171 42. Heeb. M. J., Gruber, A., Espaiia, F., Hanson, S., Harker, L.A., and Griffin, J. H. (1990) Circulation 82,111-769 (abstr.) "