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doi:10.1182/blood-2003-12-4240Prepublished online January 29, 2004;2004 103: 3783-3788

Kyung N. Lee, Kenneth W. Jackson, Victoria J. Christiansen, Keun H. Chung and Patrick A. McKeedigestion

-antiplasmin inhibition of fibrin2A novel plasma proteinase potentiates

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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

A novel plasma proteinase potentiates 2-antiplasmin inhibition of fibrin digestion

Kyung N. Lee, Kenneth W. Jackson, Victoria J. Christiansen, Keun H. Chung, and Patrick A. McKee

Human 2-antiplasmin (2AP), alsoknown

as2-plasmin inhibitor, is the major inhib-itor of the proteolytic enzyme plasmin

that digests fibrin. There are 2 N-terminal

forms of 2AP that circulate in human

plasma: a 464-residue protein with Met as

the N-terminus, Met-2AP, and a 452-

residue version with Asn as the N-termi-

nus, Asn-2AP. We have discovered and

purified a proteinase from human plasma

that cleaves the Pro12-Asn13 bond of

Met-2AP to yield Asn-2AP and have

named it antiplasmin-cleaving enzyme

(APCE). APCE is similar in primary struc-

ture andcatalytic propertiesto membrane-bound fibroblast activation protein/se-

prase for which a physiologic substrate

has not been clearly defined. We found

that Asn-2AP becomes cross-linked to

fibrin by activated factor XIII approxi-

mately 13 times faster than native Met-

2AP during clot formation and that clot

lysis rates are slowed in direct proportion

to the ratio of Asn-2AP to Met-2AP in

human plasma. We conclude that APCE

cleaves Met-2AP to the derivative Asn-

2AP, which is more efficiently incorpo-

rated into fibrin and consequently makesit strikingly resistant to plasmin diges-

tion.APCE mayrepresent a new target for

pharmacologic inhibition, since less gen-

eration and incorporation of Asn-2AP

could result in a more rapid removal of

fibrin by plasmin during atherogenesis,

thrombosis, and inflammatory states.

(Blood. 2004;103:3783-3788)

2004 by The American Society of Hematology

Introduction

Human 2-antiplasmin (2AP), also known as 2-plasmin

inhibitor, is the main inhibitor of plasmin.1 Plasmin plays a

critical role in fibrin proteolysis and tissue remodeling. The

physiologic relevance of plasmin inhibition by 2AP to blood

clotting and fibrinolytic homeostasis is supported by the follow-

ing observations: (1) the rate of free plasmin inactivation by

circulating 2AP is much faster than fibrin(ogen) digestion by

plasmin,2 thereby eliminating the possibility of a systemic lytic

state and consequent bleeding; (2) 2AP is cross-linked to

forming fibrin by activated blood clotting factor XIII (FXIIIa)

and inhibits plasmin-mediated lysis in direct proportion to the

amount incorporated3-5; and (3) patients with homozygous 2AP

deficiency manifest serious hemorrhagic tendencies, while het-erozygotes tend to bleed only after major trauma or surgery.6

Human 2AP is synthesized primarily in the liver, and during

circulation in plasma, the secreted precursive Met-2AP form, a

464-residue protein having Met as the N-terminus, undergoes

proteolytic cleavage between Pro12 and Asn13 to yield Asn-

2AP, a 452-residue version with Asn as the N-terminus.7

Met-2AP accounts for approximately 30% of circulating 2AP,

and Asn-2AP, approximately 70%.7,8 While 3-fold more Asn-

2AP than recombinant Met-2AP was shown to cross-link to

fibrin,9 no data have been reported for native circulating

Met-2AP. Moreover, the effect of different ratios of the 2 2AP

forms on clot lysis has not been reported. Finally, the enzyme

responsible for converting Met-2AP to Asn-2AP has not been

identified, although it is presumed to be in plasma.7

We report here the purification and partial characterization of

antiplasmin-cleaving enzyme (APCE), a serine proteinase from

human plasma that cleaves the Pro12-Asn13 bond of Met-2AP to

yield Asn-2AP, and the comparison of these 2 circulating forms of

2AP for the ability to cross-link with fibrin and to inhibit

fibrinolysis.

Materials and methods

Isolation of 2AP

A mixture of Met-2AP andAsn-2AP was isolated using a modification of

a published procedure.10 Briefly, human plasma was precipitated using

polyethylene glycol 6000 (final concentration 5%), and the supernatant was

applied to a lysineSepharose 4B (Amersham Biosciences, Piscataway, NJ)

affinity column to remove plasminogen. The flow-through volume was

applied to an affinity column consisting of plasminogen kringles 1 to 3

bound to Sepharose 4B. After washing with 40 mM sodium phosphate0.5

M NaCl, pH 7.0, 2AP was eluted from this column using a 0- to 50-mM

linear gradient of -aminocaproic acid in the same buffer. To separate

Met-2AP andAsn-2AP, a goat antibody to the N-terminal 12amino acid

sequence unique to Met-2AP was prepared, using as the immunogen a

multiple antigenic peptide (MAP) that contained 8 copies of the N-terminal

peptide linked via their C-termini to a core peptide of 7 lysines. 11 The 2AP

mixture was applied to an immunoaffinity column made by conjugating the

purified goat antibody to Affigel-10 (Bio-Rad, Richmond, CA). After

washing with 40 mM Tris (tris(hydroxymethyl)aminomethane) HCl0.5

M NaCl, pH 7.5, Met-2AP was eluted with 0.1 M glycine, pH 2.5.

From the William K. Warren Medical Research Center and the Department of

Medicine,University of OklahomaHealth Sciences Center, OklahomaCity, OK.

Submitted December 12, 2003; accepted January 12, 2004. Prepublished online as

BloodFirst Edition Paper, January 29, 2004; DOI 10.1182/blood-2003-12-4240.

Supported by the William K. Warren Medical Research Center, the Oklahoma

Center for theAdvancementof Science and Technology, and NationalInstitutes

of Health (NIH) grant HL072995.

Presented in partas an abstractto the19thannualmeetingof theInternational Society

on Thrombosis and Haemostasis,Birmingham, United Kingdom,July12-18, 2003.31

An Inside Bloodanalysis of this article appears in the front of this issue.

Reprints: Kyung N. Lee or Patrick A. McKee, W. K. Warren Medical Research

Center, PO Box 26901, BSEB-306 Oklahoma City, OK 73190; e-mail: kyung-

[email protected] or [email protected].

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked advertisement in accordance with 18 U.S.C. section 1734.

2004 by The American Society of Hematology

3783BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10

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Fractions were immediately neutralized by collection into tubes containing

1.0 M Tris HCl, pH 9.0. Asn-2AP eluted in the flow-through fractions

from the immunoaffinity column. N-terminal amino acid sequence analyses

confirmed that unbound and bound fractions were Asn-2AP and Met-

2AP, respectively. FXIII was purified from fresh human plasma according

to published methods.12

Analysis of

2AP cross-linking to fibrin

Factor XIIIacatalyzed cross-linking reactions were performed, and free

2AP and 2AP-fibrin cross-linked complexes were detected by immuno-

staining with an antibody to 2AP as previously described.13 The rate of

formation of 2AP-fibrin cross-linked complexes (Figure 1A-B) was

determined by measuring the appearance of bands of2AP-fibrin com-

plexes at approximately 140 kDa and approximately 210 kDa and the

concomitant disappearance of the 70-kDa 2AP band from gel scans using a

Personal Densitometer SI (Molecular Dynamics, Piscataway, NJ) and

image analysis software (ImageQuant, version 5.2; Molecular Dynamics,

Piscataway, NJ). The percentage of 2AP-fibrin cross-linked complexes

was expressed as [(densitometric values for the 140 kDa and 210 kDa

bands)/(densitometric values for the 70 kDa, 140 kDa, and 210 kDa

bands)] 100 (Figure 1B).

Measurement of plasma clot lysis

After adding Met-2AP, Asn-2AP, or selected mixtures of the 2 forms (0.5

M) to 2AP-depleted plasma (Enzyme Research, South Bend, IN), a

mixture of 1 U/mL thrombin, 16 mM CaCl2, and 45 IU/mL urokinase (uPA)

(Abbott, Chicago, IL) was added to catalyze almost instant fibrin clot

formation and to initiate fibrinolysis. The rate of plasma clot lysis was

determined by a turbidimetric microtiter plate method.14-16

Purification of antiplasmin-cleaving enzyme (APCE)

APCE was isolated from 2.0 L of human citrated plasma (purchased from

Sylvan Goldman Blood Institute, Oklahoma City, OK) by sequential

chromatographic steps that followed ammonium sulfate fractionation

(15%-40%). The pellet was dissolved in 25 mM sodium phosphate 1 mM

EDTA (ethylenediaminetetraacetic acid) buffer, pH 7.5, containing 10%

ammonium sulfate and applied to a phenyl-Toyopearl hydrophobic interac-

tion chromatography column (Tosohaas, Montgomeryville, PA). After

washing with 10% ammonium sulfate25 mM sodium phosphate1 mM

EDTA, pH 7.5, bound proteins were eluted by a decreasing ammonium

sulfate gradient (linear, 5%-0%) in the same buffer. To monitor APCE

activity in chromatographic fractions, hydrolysis of the fluorogenic sub-

strate Z-Gly-Pro-AMC was measured at excitation/emission wavelengths

of 360/460 nm. Active fractions were pooled and further purified by elution

from a Q-Sepharose anion exchange column (Amersham Biosciences)

using a 0- to 0.5-M NaCl linear gradient in the buffer described above. The

activity peak was applied to a T-gel thiophilic column (Pierce, Rockford,

IL) equilibrated in 0.5 M ammonium sulfate in the same phosphate buffer,

and APCE activity was eluted by a decreasing ammonium sulfate gradient

(linear, 0.5-0 M). N-terminal sequence of the dominant protein in the activefractions, as judged by sodium dodecyl sulfatepolyacrylamide gel electro-

phoresis (SDS-PAGE), was determined by Edman sequence analysis. Based

upon the N-terminal sequence (IVLRPSRVHNSEENT) obtained, a mul-

tiple antigenic peptide (MAP) was prepared for immunizing a goat. The

linear form of the peptide was used to make an affinity column from which

the antibody was purified from goat serum. An immunoaffinity column

made with this antibody was used to purify the cleaving activity of

Z-Gly-Pro-AMC from the T-gel column. Following application and wash-

ing, the immunoaffinity column was eluted with 3 M sodium thiocyanate,

pH 7.5. The active fractions were pooled, dialyzed against 25 mM sodium

phosphate1 mM EDTA20% glycerol, pH 7.5, and stored at 80C. On

the basis of the Z-Gly-Pro-AMC assay, selected fractions were pooled from

each chromatographic step, and each pooled sample was also assayed for

Met-2AP cleaving activity (Figure 4A). Met-2AP (5 g) and each

chromatographic sample (54 g phenyl-Toyopearl chromatography frac-tion, 9 g Q-Sepharose chromatography fraction, 0.6 g T-gel thiophilic

chromatography fraction, or 0.2 g immunoaffinity chromatography frac-

tion) was incubated at 22C. At selected times, an aliquot of each reaction

mixture was subjected to SDS-PAGE and transferred to a nitrocellulose

membrane. Met-2AP was treated with antibody specific for its N-terminal

sequence and then detected by the horseradish-peroxidase system. Met-

2AP and Asn-2AP were detected by a monoclonal antibody (American

Diagnostica, Greenwich, CT), which was reactive to the C-terminal region

of either form of2AP (Figure 4B, right panel). Also, Met-2AP(5g) was

incubated with selected amounts of APCE (0.02-0.16 g) for 6 hours, and

Western blot analysis was performed using Met-2APspecific antibody

(Figure 4C).

Specificity of APCE activity in plasma

To assess APCE activity in human plasma and its recovery in each

purification step, we designed and synthesized a fluorescence resonance

energy transfer (FRET) peptide substrate that contained the APCE-sensitive

Pro12-Asn13 bond within the Thr9-Gln16 sequence of Met-2AP

as follows: Arg-Lys(DABCYL)-Thr-Ser-Gly-Pro-Asn-Gln-Glu-Gln-

Glu(EDANS)-Arg. Hydrolysis of the Pro-Asn bond separates the fluoro-

phore, EDANS (5-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid),from

the quenching group, DABCYL (4-(4-dimethylaminophenylazo)benzoyl),

to give an increase in fluorescence. APCE cleavage of the Pro-Asn bond inthe FRET peptide substrate was confirmed by analysis of products by liquid

chromatography mass spectrometry (LC/MS; data not shown). APCE was

selectively removed from normal human plasma by an antibody specific for

the N-terminal region of APCE (see above for purified goat antibody as

made for APCE purification). A nonspecific goat antibody was used as a

control. Each antibody was coupled to POROS EP20 beads (Applied

Biosystems, Framingham, MA), and the coupled beads were incubated

separately with plasma diluted 1:5 with 25 mM sodium phosphate 1 mM

EDTA, pH 7.5, on a rocker for 15 hours at 4C. Any APCE activity

remaining in the supernatant, and therefore not bound to the antibody-

POROS beads, was measured as a function of its ability to cleave the

APCE-specific FRET peptide (0.1 mM, 10 L), which was added to 200

mL of the supernatant and incubated at 22C. Fluorescence was monitored

with time at excitation and emission wavelengths of 360 and 460 nm, using

a BIO-TEK FL600 fluorescence plate reader (Winooski, VT).

Results and discussion

FXIIIa-catalyzed cross-linking of Met-2AP or Asn-2AP to fibrin

We determined the N-terminal sequence of purified 2AP from 52

normal plasma samples and found that Met-2AP is present as

33% 10% (mean SD) of the total 2AP, which is in accord with

previous values from 4 pools of human plasma.8 We isolated native

forms of both Met-2APandAsn-2APfrom human plasma and studied

these instead of recombinant forms.9 To compare cross-linking rates of

Met-2AP versus Asn-2AP to fibrin, mixtures of fibrinogen, Ca2,

FXIII, and Met-2AP or Asn-2AP were clotted by thrombin andallowedto cross-link for selected times; then solubilized in ureasodium

dodecyl sulfate (SDS)dithiothreitol (DTT); subjected to polyacryl-

amide gel electrophoresis (PAGE); analyzed by Western blots; and

quantified by densitometry. The content of2AP immunoreactivity in

high-molecular-weight cross-linked complexes of 2AP-fibrin in-

creased with time, and simultaneously immunoreactivity decreased in

the lower molecular-weight 2AP band (Figure 1A). Using essentially

physiologic concentrations, we found that the extent of purified native

Asn-2AP cross-linking to fibrin was approximately 13 times greater

than purified native Met-2AP during the initial 10 minutes as fibrin gel

formation achieved completion (Figure 1B). Our finding that native

Asn-2AP is far more rapidly incorporated into fibrin than its native

precursive form, Met-2AP, clarifies and extends a prior observationthat recombinant Met-2AP cross-linked to fibrin at one-third of the

3784 LEE et al BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10


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amount of purified plasmaAsn-2AP.9 Holmes et al17 reported a similar

finding in that recombinant Asn-2AP having an extension of 3

additional N-terminal amino acids could no longer be cross-linked to

fibrin. Our results can be explained by comparing the 2 purified plasma

forms, rather than plasma 2AP with recombinant Met-2AP as in the

previous report. Recombinant Met-2AP has been shown to have a bit

slower initial rate of free plasmin inhibition than plasma 2AP,9 raising

the question of whether slight structural differences might exist between

circulating 2AP and recombinant Met-2AP that could also impact

cross-linking. We could detect no differences between the 2 purified

circulating forms in this regard (Figure 1C). Important to note is that the

purified plasma 2AP used by Sumi et al9 was not characterized for

content of the 2 2AP forms, and no separation method was described.

Hence, if their plasma 2AP preparation were in fact a mixture of

Met-2AP and Asn-2AP, this could account for the much narrower

difference between the 2 forms for becoming cross-linked to fibrin than

we report here.9 Finally, the molar ratio of fibrinogen to 2AP used by

Sumi et al9 was around 70:1, which is significantly greater than the

physiologic ratio of 7:1 used in our work, and for that matter, even

higher than ratios in pathologic settings where fibrinogen concentrationis markedly elevated.

Effect of Met-2AP or Asn-2AP on plasma clot lysis

It has not been established whether Asn-2AP protects fibrin from

fibrinolysis more effectively than Met-2AP. Therefore we com-

pared the 2 forms by urokinase (uPA)induced plasma clot lysis.

After addition of selected ratios of purified Met-2AP and/or

Asn-2AP to give a total 2AP concentration of 0.50 M in

2AP-depleted plasma, a mixture of thrombin, Ca2, and uPA was

added to catalyze cross-linked fibrin formation and to initiate

fibrinolysis. As shown in Figure 2, turbidity was monitored at

405-nm wavelength, and plasma clot lysis times (PCLTs) were

defined as the midpoint between the highest and lowest absor-

bances. A control clot prepared from 2AP-depleted plasma was

rapidly digested by plasmin (PCLT 20 minutes). When only

Met-2AP was added, clot lysis was slightly prolonged (PCLT 23

minutes). As the proportion of Asn-2AP increased, clot lysis was

progressively delayed. When 100% Asn-2AP was added, clot lysis

was maximally delayed (PCLT 42 minutes), becoming about

2-fold longer than when only Met-2AP was present. These results

indicated a dose-response effect for ratios of the 2 forms of

circulating 2AP on fibrinolysis. It should be noted that normal

human plasma that had been depleted of2AP using immobilized

polyclonal antibodies to 2AP was used in these studies, thereby

negating any competitive impact that C-terminal shortened, non

plasminogen-binding forms of 2AP may exhibit for becoming

cross-linked to fibrin.18,19 The Met-2AP and Asn-2AP added to

2AP-depleted plasma were purified using their C-terminal affini-

ties for immobilized plasminogen kringles 1-3; hence, C-terminal

degraded forms of2AP would not bind. These studies demonstrate

clearly that Asn-2AP inhibits plasmin-mediated fibrin digestionmore effectively than Met-2AP.

Purification and identification of proteinase

that converts Met-2AP to Asn-2AP

Given the greater efficiency of Asn-2AP incorporation into fibrin,

we reasoned that the unidentified plasma proteinase responsible for

converting Met-2AP toAsn-2AP might be important in modulat-

ing the susceptibility offibrin to plasmin; we therefore attempted to

isolate the proteinase. The proteinase was purified from human

plasma using an initial ammonium sulfate fractionation (A),

followed by phenyl-Toyopearl hydrophobic interaction (P),

Q-Sepharose anion exchange (Q), T-gel thiophilic (T), and immu-

noaffinity (I) chromatographies. Through the successive purifica-tion steps prior to the immunoaffinity chromatography, the enzyme

activity increased concomitantly with the increase of the dominant

97-kDa protein band as detected by SDS-PAGE (Figure 3). This

band was subjected to protein sequencing following transfer to a

polyvinylidene difluoride membrane, and the identification of the

first 15 amino acids (IVLRPSRVHNSEENT) allowed the synthesis

of an identical peptide, which was used as an immunogen to

prepare a goat antibody. After purification, the antibody was linked

to a gel support for immunoaffinity chromatography (I). Through-

out the purification processes, proteolytic activity was monitored

by hydrolysis of either Z-Gly-Pro-AMC20 or the FRET peptide.

Active fractions in each purification step were pooled and analyzed

by SDS-PAGE (Figure 3). Cleavage of Met-2AP toAsn-2AP wasassessed by immunoblot analyses; the rate of Met-2AP conversion

Figure 1. Met-2AP and Asn-2AP cross-linking to fibrin and their plasmin-inhibitory activity. (A) Time-dependent cross-linking of Met-2AP andAsn-2AP to fibrin by

FXIIIa catalysis. Solutions of purified human 2AP, fibrinogen, and FXIII were clotted with thrombin and CaCl 2, and at the indicated times above each gel lane, clotting was

terminatedby solubilizing in urea-SDS-DTT. Noncross-linked 2APand 2AP-fibrin cross-linkedcomplexes weredetected by immunoblot analysis.(B) Densitometricanalysis

of the percent incorporation of 2APinto fibrin.Eachdatapointis themeanSD of 3 experiments.(C) Plasmin-inhibitoryactivityof Met-2APandAsn-2AP. In control sample,

2AP was replaced with reactionbuffer. Each2AP was incubated with an equimolaramount of plasmin for selected incubation periods, and thenusing a plasmin chromogenic

substrate (S-2251) residual plasmin activity was assayed by a published method.10 Each data point is the average of 2 experiments.

Figure 2. Plasma clot lysis times (PCLTs) determined with different Met-2AP/

Asn-2AP ratios. Each data point was determined in quadruplicate (mean SD).

PCLTs weredefined as midpointtimes () between thepeakof clot formation (highestabsorbance) and the maximal fibrinolysis (lowest absorbance).

ANTIPLASMIN-CLEAVING ENZYME 3785BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10


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by the proteinase clearly increased as its purification progressed

(Figure 4A). We termed this proteinase antiplasmin-cleaving

enzyme (APCE). Table 1 summarizes a typical purification of

APCE from human plasma.

Recently it was reported that phenyl hydrophobic interaction

chromatography could be used to separate 2 distinct Z-Gly-Pro-

AMChydrolyzing activities from bovine serum: one bound to the

phenyl column, while the other did not.21 We obtained a similar

result when ammonium sulfatefractionated human plasma was

subjected to phenyl-column chromatography, with each fraction

being assayed using Z-Gly-Pro-AMC. Importantly, however, only

the phenyl-bound fraction possessed proteinase activity that cleaved

the FRET peptide substrate and Met-2AP (Figure 4A, P). Table 1

shows a dramatic increase in purification with a high yield of

activity for this phenyl-Toyopearl step. To determine APCE

activity in human plasma, commercially available Z-Gly-Pro-AMC

cannot be used, since more than 1 Pro-Xaa cleaving enzyme ispresent in human plasma. Therefore, taking advantage of the FRET

peptide as a specific substrate, total APCE activity was assessed in

3 different pooled normal plasma samples, using the total activity

in each plasma sample and specific activity of purified APCE from

each to estimate the concentration of APCE in each plasma sample

as 560, 561, and 493 g/L.

When Met-2AP was incubated with APCE activity from the

final immunoaffinity step (Figure 4B), it was completely and

specifically cleaved at the Pro12-Asn13 bond as shown by the

following: (1) The 70-kDa Met-2AP band, which was easily

demonstrable at 0 time by antibody specific for Met-2AP, had

completely disappeared by 6 hours (Figure 4B, left panel). When

immunostained with an antibody to both 2AP forms, however, theintensities of the 70-kDa band at 0 hours and 6 hours did not change

(Figure 4B, right panel), but the Met-2AP band disappeared in

direct proportion to the concentration of APCE when analyzed with

an antibody that detected only Met-2AP (Figure 4C). (2) N-

terminal analyses of the 0-hour sample (Figure 4B) showed only

the Met-2AP sequence, MEPLGRQLTSGP, while the 6-hour

sample had only the Asn-2AP N-terminal sequence, NQEQVS-

PLTLLK. The proteinase isolated by the last purification step

showed a single 97-kDa band (Figure 3, I), with an N-terminal

sequence identical to that of the major protein band in sample T.

Interestingly, the sequence matched that deduced from the

cDNA sequence for Ile24-Thr38 of fibroblast activation protein

(FAP),22

a proline-specific serine proteinase also termed sepraseby another group.23

To determine additional sequence information from the protein,

the 97-kDa protein band from the SDS-PAGE gel was reduced,

alkylated, and digested with trypsin.24 The tryptic digest was

subjected to liquid chromatographymass spectrometrymass spec-

trometry (LC/MS/MS) to obtain molecular weights and MS/MS

fragment ion spectra and to use the MASCOT MS/MS ion search

engine to query the OWL nonidentical protein database (http://www.

matrixscience.com/cgi/search_form.pl?FORMVER2&SEARCHMIS). From our search results, 5 peptides had sequences identical with

FAP/seprase22,23: residues 210-219 (YALWWSPNGK); residues

247-254 (TINIPYPK); residues 487-499 (ILEENKELENALK); resi-

dues 500-509 (NIQLPKEEIK); and residues 522-530 (MILPPQFDR).

This degree of homology suggested that the 97-kDa APCE proteinase

had similarity to FAP/seprase; however, significant differences

remained. FAP/seprase is reported to be expressed on the cell

surface of reactive stromal fibroblasts of healing wounds and

epithelial cancers, but not on normal tissue or benign epithelial

tumors.25,26 It is believed to be particularly associated with an

invasive phenotype of human melanoma and carcinoma cells.23

Despite the plausibility that FAP/seprase may have a role in tissue

remodeling, a physiologic substrate has not been demonstrated forthe native enzyme, and hence a clearly defined biologic function

remains obscure. Although gelatin and collagen I appeared to serve

as substrates for recombinant FAP expressed on a human embry-

onic kidney epithelial cell line (293 cells), products of fragmenta-

tion or actual cleavage sites have not been identified.27 Impor-

tantly, a previous report indicated that neither soluble FAP nor its

cleavage products exist in the circulation.28 The APCE we describe

here was isolated from normal human plasma.

Figure 4. Immunoblot analysis of Met-2AP cleaving activity ofAPCE in pooled

fractions from each chromatographic step. (A) APCE activity increased at each

successive purification step. P, Q, T, or I represents APCE activity of each

chromatographic pool of samples with Z-Gly-Pro-AMC cleaving activity. The total

protein usedfrom eachchromatographicstep was as follows: P, 54g ; Q , 9g;T,0.6

g; or I, 0.2 g. Met-2AP cleavage was demonstrated by immunoblot analysis using

an antibody specific for its N-terminal peptide. (B) Incubation of Met-2AP with the

APCE activity pool from immunoaffinity chromatography (0.2 g) diminished in a

time-dependent manner when assessed by an antibody specific for the N-terminal

peptide of Met-2AP (left panel), but did not diminish2AP band intensity or generate

bands less than 70-kDa when assessed by a monoclonal antibody to the C-terminal

region of Met-2AP orAsn-2AP (right panel). (C) APCE (amount shown above each

gel lane) concentration-dependent cleavage of Met-2AP (5 g) detected by anantibody specific for Met-2AP.

Figure 3. Purification of APCE. Coomassie bluestained gel analysis of the activity

pool from each purification step. Proteins were analyzed on a 10% to 20% gradient

SDS-PAGE under reducingconditions. STD indicatesmolecular-weight standards;A,

ammonium sulfate fraction (15%-40%); P, phenyl-Toyopearl chromatography frac-

tion; Q, Q-Sepharose chromatography fraction; T, T-gel thiophilic chromatography

fraction; and I, immunoaffinity chromatography fraction.



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The possibility that human plasma APCE could be proteolyti-

cally derived from cell membranebound FAP/seprase is raised by

the following observations: (1) The N-terminal 15 amino acids of

APCE are identical to residues 24 to 38 of FAP/seprase, the latter

estimated to have its first 6 N-terminal amino acids in the fibroblast

cytoplasm, with another predicted 20-residue transmembrane do-

main, and then a 734-residue extracellular C-terminal catalytic

domain.22,23 (2) Internal sequences of APCE obtained from 5

tryptic peptides are identical to those deduced from the cDNA

sequence of a fairly long segment of FAP/seprase (amino acids 210through 530). (3) Nondenaturing gel filtration chromatography of

APCE indicates a native mass of approximately 180 kDa, and,

whether or not reduced, its denatured molecular weight by SDS-

PAGE is 97 kDa (Figure 3, I). Both values are consistent with the

reported dimeric structure of 190 kDa for FAP/seprase.27 (4)APCE

is inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl-

sulfonyl fluoride (PMSF), and AEBSF (4-(2-aminoethyl)-benzene-

sulfonyl fluoride), but not by iodoacetate, E-64, or EDTA, just as

reported for FAP/seprase.27 (5) Analogously, a closely related type

II transmembrane serine proteinase with prolyl specificity appar-

ently undergoes proteolytic cleavage of its N-terminal 38-residue

cytoplasmic/transmembrane domain to give rise to the circulating

human dipeptidyl peptidase IV (DPP IV, aka T-cell activationCD26, with 50% homology to FAP/seprase)29; however, DPP IV is

strictly a dipeptidyl aminopeptidase,20 and we found that it does not

cleave Pro12-Asn13 of Met-2AP as does APCE. If APCE indeed

results from proteolysis of the transmembrane domain of FAP/

seprase, a proteinase that might cleave the Cys23-Ile24 bond has

yet to be identified. The hydrophobic residue Ile24 as the P1

position of the bond could make this a preferred cleavage site for an

extracellular enzyme such as a matrix metalloproteinase.30

Alternatively, APCE may be a product of intracellular process-

ing. Conceivably, 2 pathways could exist in the same cell: one that

normally synthesizes, processes, and secretes APCE without a

transmembrane segment; and another that produces a membrane-

bound enzyme (FAP/seprase) when fibroblasts become activated

and proliferate in inflammatory states, embryogenesis, or metasta-ses of certain epithelial-derived malignancies.25,26 Finally, the

possibility remains that APCE is a product of a different gene or

that it results from alternative splicing. Our data indicate that APCE

is produced under normal physiologic states, and unlike the case of

FAP/seprase, pathologic disorders are not required to stimulate

gene expression. The fact that APCE is found in normal plasma and

cleaves Met-2AP suggests that one of its physiologic functions is

the regulation of Asn-2AP availability for plasmin inhibition

within cross-linked fibrin.

To determine if whole-plasma APCE is specifically responsible

for cleaving the Met-2AP sequence that generates Asn-2AP,

antibody to the N-terminal 15 residues of APCE was coupled to

beads and compared with a control antibody for the ability toremove significant amounts of APCE activity from human plasma

as described in Materials and methods. Control antibody did not

remove FRET peptide cleaving activity, whereas antibody specific

for APCE removed approximately 50%. Since the FRET peptide

substrate is specific for APCE activity, the remaining FRET peptide

cleaving activity in this experiment may be due to N-terminally

degraded forms of APCE that react poorly or not at all with the

antibody, particularly given that only the linear epitopes among the

first 15 residues of APCE would be recognized. Although it must

still be considered that human plasma may contain other protein-

ases that cleave the N-terminal 12-residue peptide of Met-2AP,our data indicate that this is unlikely because (1) only the 15% to

40% ammonium sulfate fraction was found to contain Met-2AP

cleaving activity; (2) when ammonium sulfatefractionated human

plasma was subjected to phenyl-column chromatography, phenyl-

bound and unbound cleaving activities toward Z-Gly-Pro-AMC

were indeed observed, but only the bound activity cleaved the

FRET peptide and Met-2AP; (3) only one peak of proteolytic

activity for the FRET peptide and Met-2AP was observed in

elution profiles from Q-Sepharose and T-gel chromatographies

(Figure 4A); and (4) on reduced SDS-PAGE, the peak from the

immunoaffinity chromatography contained only one band and it

had a single N-terminal sequence (Figure 3, I).

We conclude that human Met-2AP is a physiologically impor-tant substrate of APCE, since proteolytic cleavage between Pro12-

Asn13 yields the derivative Asn-2AP, which becomes cross-

linked significantly faster to fibrin by FXIIIa, and, as a consequence,

enhances the resistance of fibrin to digestion by plasmin. Since

human APCE augments the inhibition of fibrinolysis by increasing

the availability of the faster cross-linking form (ie, Asn-2AP),

potent and selective inhibitors of APCE might allow titration of

Asn-2AP production to lower in vivo levels, and thereby enhance

both endogenous and exogenous fibrin removal. Bleeding compli-

cations would be unlikely, based on the observation that persons

heterozygous for 2APdeficiency have minimal hemorrhagic risk.6

Hence, a window of safety may exist for lowering 2AP function in

healthy persons. Particularly in clinical situations where fibrin

formation is likely, the development of an agent that inhibits APCEmight result in decreased amount of Asn-2AP available for

cross-linking to fibrin as thrombi develop or inflammation

progresses. Then endogenous levels of generated plasmin, or

plasmin produced by administering small amounts of a plasmino-

gen activator, might be sufficient to effect fibrin removal so that

vessel patency and organ function are maintained and bleeding risk

is minimized. We believe that this hitherto unknown circulating

enzyme, APCE, may provide a valuable target for pharmacologic

modulation of the fibrinolytic system.

Acknowledgments

We thank C. S. Lee and J. G. Chun for technical assistance.

Table 1. Purification of APCE from human plasma

Purification step

Total activity,*

FI 10 3Total

protein, mg

Specific activity,

FI 10 3/mg

Purification,

fold Yield, %

Plasma, 2 L 58 950 185 650 0.3 1 100.0

Ammonium sulfate 41 520 48 840 0.9 3 70.4

Phenyl-Toyopearl 27 964 320 87.4 291 47.4

Q-Sepharose anion 17 687 32 552.7 1 842 30.0

T-gel thiophilic 8 576 0.45 19 057.8 63 526 14.5Immunoaffinity 4 738 0.09 52 644.4 175 481 8.0

*Total activity is defined as total fluorescence intensity (FI) produced by APCE-catalyzed FRET peptide cleavage during 2- hour incubation at 22C.

ANTIPLASMIN-CLEAVING ENZYME 3787BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10


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