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

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

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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

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

Kyung N. Lee, Kenneth W. Jackson, Victoria J. Christiansen, Keun H. Chung, and Patrick A. McKee Human 2-antiplasmin (2AP), also known as 2-plasmin inhibitor, is the major inhibitor of the proteolytic enzyme plasmin that digests brin. 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 452residue version with Asn as the N-terminus, Asn-2AP. We have discovered and puried 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 structure and catalytic properties to membranebound broblast activation protein/seprase for which a physiologic substrate has not been clearly dened. We found that Asn-2AP becomes cross-linked to brin by activated factor XIII approximately 13 times faster than native Met2AP 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 Asn2AP, which is more efciently incorporated into brin and consequently makes it strikingly resistant to plasmin digestion. APCE may represent a new target for pharmacologic inhibition, since less generation and incorporation of Asn-2AP could result in a more rapid removal of brin by plasmin during atherogenesis, thrombosis, and inammatory 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 brin proteolysis and tissue remodeling. The physiologic relevance of plasmin inhibition by 2AP to blood clotting and brinolytic homeostasis is supported by the following observations: (1) the rate of free plasmin inactivation by circulating 2AP is much faster than brin(ogen) digestion by plasmin,2 thereby eliminating the possibility of a systemic lytic state and consequent bleeding; (2) 2AP is cross-linked to forming brin 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 deciency manifest serious hemorrhagic tendencies, while heterozygotes 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 Asn2AP, 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 Asn2AP than recombinant Met-2AP was shown to cross-link to brin,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 identied, although it is presumed to be in plasma.7 We report here the purication 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 brin and to inhibit brinolysis.

Materials and methods

Isolation of 2AP A mixture of Met-2AP and Asn-2AP was isolated using a modication of a published procedure.10 Briey, human plasma was precipitated using polyethylene glycol 6000 (nal concentration 5%), and the supernatant was applied to a lysineSepharose 4B (Amersham Biosciences, Piscataway, NJ) afnity column to remove plasminogen. The ow-through volume was applied to an afnity 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 and Asn-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 immunoafnity column made by conjugating the puried goat antibody to Afgel-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 Oklahoma Health Sciences Center, Oklahoma City, OK. Submitted December 12, 2003; accepted January 12, 2004. Prepublished online as Blood First 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 the Advancement of Science and Technology, and National Institutes of Health (NIH) grant HL072995. Presented in part as an abstract to the 19th annual meeting of the International Society on Thrombosis and Haemostasis, Birmingham, United Kingdom, July 12-18, 2003.31

An Inside Blood analysis 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: kyunglee@ouhsc.edu or patrick-mckee@ouhsc.edu. 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

BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10

3783

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

Fractions were immediately neutralized by collection into tubes containing 1.0 M Tris HCl, pH 9.0. Asn-2AP eluted in the ow-through fractions from the immunoafnity column. N-terminal amino acid sequence analyses conrmed that unbound and bound fractions were Asn-2AP and Met2AP, respectively. FXIII was puried from fresh human plasma according to published methods.12 Analysis of 2AP cross-linking to brin Factor XIIIacatalyzed cross-linking reactions were performed, and free 2AP and 2AP-brin cross-linked complexes were detected by immunostaining with an antibody to 2AP as previously described.13 The rate of formation of 2AP-brin cross-linked complexes (Figure 1A-B) was determined by measuring the appearance of bands of 2AP-brin complexes 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-brin 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 brin clot formation and to initiate brinolysis. The rate of plasma clot lysis was determined by a turbidimetric microtiter plate method.14-16 Purication 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 phosphate1 mM EDTA (ethylenediaminetetraacetic acid) buffer, pH 7.5, containing 10% ammonium sulfate and applied to a phenyl-Toyopearl hydrophobic interaction 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 uorogenic substrate Z-Gly-Pro-AMC was measured at excitation/emission wavelengths of 360/460 nm. Active fractions were pooled and further puried 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 active fractions, as judged by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), was determined by Edman sequence analysis. Based upon the N-terminal sequence (IVLRPSRVHNSEENT) obtained, a multiple antigenic peptide (MAP) was prepared for immunizing a goat. The linear form of the peptide was used to make an afnity column from which the antibody was puried from goat serum. An immunoafnity 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 washing, the immunoafnity 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 fraction, 9 g Q-Sepharose chromatography fraction, 0.6 g T-gel thiophilic

chromatography fraction, or 0.2 g immunoafnity chromatography fraction) 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 specic for its N-terminal sequence and then detected by the horseradish-peroxidase system. Met2AP and Asn-2AP were detected by a monoclonal antibody (American Diagnostica, Greenwich, CT), which was reactive to the C-terminal region of either form of 2AP (Figure 4B, right panel). Also, Met-2AP (5 g) was incubated with selected amounts of APCE (0.02-0.16 g) for 6 hours, and Western blot analysis was performed using Met-2APspecic antibody (Figure 4C). Specicity of APCE activity in plasma To assess APCE activity in human plasma and its recovery in each purication step, we designed and synthesized a uorescence 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-GlnGlu(EDANS)-Arg. Hydrolysis of the Pro-Asn bond separates the uorophore, EDANS (5-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid), from the quenching group, DABCYL (4-(4-dimethylaminophenylazo)benzoyl), to give an increase in uorescence. APCE cleavage of the Pro-Asn bond in the FRET peptide substrate was conrmed by analysis of products by liquid chromatographymass spectrometry (LC/MS; data not shown). APCE was selectively removed from normal human plasma by an antibody specic for the N-terminal region of APCE (see above for puried goat antibody as made for APCE purication). A nonspecic 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 phosphate1 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 antibodyPOROS beads, was measured as a function of its ability to cleave the APCE-specic 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 uorescence plate reader (Winooski, VT).

Results and discussion

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

We determined the N-terminal sequence of puried 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-2AP andAsn-2AP from human plasma and studied these instead of recombinant forms.9 To compare cross-linking rates of Met-2AP versus Asn-2AP to brin, mixtures of brinogen, Ca2, FXIII, and Met-2AP or Asn-2AP were clotted by thrombin and allowed to cross-link for selected times; then solubilized in ureasodium dodecyl sulfate (SDS)dithiothreitol (DTT); subjected to polyacrylamide gel electrophoresis (PAGE); analyzed by Western blots; and quantied by densitometry. The content of 2AP immunoreactivity in high-molecular-weight cross-linked complexes of 2AP-brin increased 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 puried native Asn-2AP cross-linking to brin was approximately 13 times greater than puried native Met-2AP during the initial 10 minutes as brin gel formation achieved completion (Figure 1B). Our nding that native Asn-2AP is far more rapidly incorporated into brin than its native precursive form, Met-2AP, claries and extends a prior observation that recombinant Met-2AP cross-linked to brin at one-third of the

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

Figure 1. Met-2AP and Asn-2AP cross-linking to brin and their plasmin-inhibitory activity. (A) Time-dependent cross-linking of Met-2AP and Asn-2AP to brin by FXIIIa catalysis. Solutions of puried human 2AP, brinogen, and FXIII were clotted with thrombin and CaCl2, and at the indicated times above each gel lane, clotting was terminated by solubilizing in urea-SDS-DTT. Noncross-linked 2AP and 2AP-brin cross-linked complexes were detected by immunoblot analysis. (B) Densitometric analysis of the percent incorporation of 2AP into brin. Each data point is the mean SD of 3 experiments. (C) Plasmin-inhibitory activity of Met-2AP and Asn-2AP. In control sample, 2AP was replaced with reaction buffer. Each 2AP was incubated with an equimolar amount of plasmin for selected incubation periods, and then using 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.

amount of puried plasma Asn-2AP.9 Holmes et al17 reported a similar nding in that recombinant Asn-2AP having an extension of 3 additional N-terminal amino acids could no longer be cross-linked to brin. Our results can be explained by comparing the 2 puried 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 puried circulating forms in this regard (Figure 1C). Important to note is that the puried 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 brin than we report here.9 Finally, the molar ratio of brinogen to 2AP used by Sumi et al9 was around 70:1, which is signicantly greater than the physiologic ratio of 7:1 used in our work, and for that matter, even higher than ratios in pathologic settings where brinogen concentration is markedly elevated.
Effect of Met-2AP or Asn-2AP on plasma clot lysis

405-nm wavelength, and plasma clot lysis times (PCLTs) were dened as the midpoint between the highest and lowest absorbances. 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 brinolysis. It should be noted that normal human plasma that had been depleted of 2AP 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 brin.18,19 The Met-2AP and Asn-2AP added to 2AP-depleted plasma were puried using their C-terminal afnities for immobilized plasminogen kringles 1-3; hence, C-terminal degraded forms of 2AP would not bind. These studies demonstrate clearly that Asn-2AP inhibits plasmin-mediated brin digestion more effectively than Met-2AP.
Purication and identication of proteinase that converts Met-2AP to Asn-2AP

It has not been established whether Asn-2AP protects brin from brinolysis more effectively than Met-2AP. Therefore we compared the 2 forms by urokinase (uPA)induced plasma clot lysis. After addition of selected ratios of puried 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 brin formation and to initiate brinolysis. As shown in Figure 2, turbidity was monitored at

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 were dened as midpoint times () between the peak of clot formation (highest absorbance) and the maximal brinolysis (lowest absorbance).

Given the greater efciency of Asn-2AP incorporation into brin, we reasoned that the unidentied plasma proteinase responsible for converting Met-2AP to Asn-2AP might be important in modulating the susceptibility of brin to plasmin; we therefore attempted to isolate the proteinase. The proteinase was puried 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 immunoafnity (I) chromatographies. Through the successive purication steps prior to the immunoafnity 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 diuoride membrane, and the identication of the rst 15 amino acids (IVLRPSRVHNSEENT) allowed the synthesis of an identical peptide, which was used as an immunogen to prepare a goat antibody. After purication, the antibody was linked to a gel support for immunoafnity chromatography (I). Throughout the purication processes, proteolytic activity was monitored by hydrolysis of either Z-Gly-Pro-AMC20 or the FRET peptide. Active fractions in each purication step were pooled and analyzed by SDS-PAGE (Figure 3). Cleavage of Met-2AP to Asn-2AP was assessed by immunoblot analyses; the rate of Met-2AP conversion

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3786 LEE et al BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10

Figure 3. Purication of APCE. Coomassie bluestained gel analysis of the activity pool from each purication step. Proteins were analyzed on a 10% to 20% gradient SDS-PAGE under reducing conditions. STD indicates molecular-weight standards; A, ammonium sulfate fraction (15%-40%); P, phenyl-Toyopearl chromatography fraction; Q, Q-Sepharose chromatography fraction; T, T-gel thiophilic chromatography fraction; and I, immunoafnity chromatography fraction.

by the proteinase clearly increased as its purication progressed (Figure 4A). We termed this proteinase antiplasmin-cleaving enzyme (APCE). Table 1 summarizes a typical purication of APCE from human plasma. Recently it was reported that phenyl hydrophobic interaction chromatography could be used to separate 2 distinct Z-Gly-ProAMChydrolyzing 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 purication 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 is present in human plasma. Therefore, taking advantage of the FRET peptide as a specic substrate, total APCE activity was assessed in 3 different pooled normal plasma samples, using the total activity in each plasma sample and specic activity of puried 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 nal immunoafnity step (Figure 4B), it was completely and specically 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 specic for Met-2AP, had completely disappeared by 6 hours (Figure 4B, left panel). When immunostained with an antibody to both 2AP forms, however, the intensities 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) Nterminal 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, NQEQVSPLTLLK. The proteinase isolated by the last purication 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 broblast activation protein (FAP),22 a proline-specic serine proteinase also termed seprase by 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 spectrometry (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&SEARCH MIS). 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); residues 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, signicant differences remained. FAP/seprase is reported to be expressed on the cell surface of reactive stromal broblasts 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 for the native enzyme, and hence a clearly dened biologic function remains obscure. Although gelatin and collagen I appeared to serve as substrates for recombinant FAP expressed on a human embryonic kidney epithelial cell line (293 cells), products of fragmentation or actual cleavage sites have not been identied.27 Importantly, 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 of APCE in pooled fractions from each chromatographic step. (A) APCE activity increased at each successive purication 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 used from each chromatographic step was as follows: P, 54 g; Q, 9 g; T, 0.6 g; or I, 0.2 g. Met-2AP cleavage was demonstrated by immunoblot analysis using an antibody specic for its N-terminal peptide. (B) Incubation of Met-2AP with the APCE activity pool from immunoafnity chromatography (0.2 g) diminished in a time-dependent manner when assessed by an antibody specic for the N-terminal peptide of Met-2AP (left panel), but did not diminish 2AP band intensity or generate bands less than 70-kDa when assessed by a monoclonal antibody to the C-terminal region of Met-2AP or Asn-2AP (right panel). (C) APCE (amount shown above each gel lane) concentration-dependent cleavage of Met-2AP (5 g) detected by an antibody specic for Met-2AP.

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

Table 1. Purication of APCE from human plasma

Purication step Plasma, 2 L Ammonium sulfate Phenyl-Toyopearl Q-Sepharose anion T-gel thiophilic Immunoafnity Total activity,* FI 103 58 950 41 520 27 964 17 687 8 576 4 738 Total protein, mg 185 650 48 840 320 32 0.45 0.09 Specic activity, FI 103/mg 0.3 0.9 87.4 552.7 19 057.8 52 644.4 Purication, fold 1 3 291 1 842 63 526 175 481 Yield, % 100.0 70.4 47.4 30.0 14.5 8.0

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

The possibility that human plasma APCE could be proteolytically 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 rst 6 N-terminal amino acids in the broblast cytoplasm, with another predicted 20-residue transmembrane domain, 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 210 through 530). (3) Nondenaturing gel ltration chromatography of APCE indicates a native mass of approximately 180 kDa, and, whether or not reduced, its denatured molecular weight by SDSPAGE 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 uorophosphate (DFP), phenylmethylsulfonyl uoride (PMSF), and AEBSF (4-(2-aminoethyl)-benzenesulfonyl uoride), 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 specicity apparently 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 activation CD26, 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 identied. 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 processing. 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 membranebound enzyme (FAP/seprase) when broblasts become activated and proliferate in inammatory states, embryogenesis, or metastases 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 brin. To determine if whole-plasma APCE is specically 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 to remove signicant amounts of APCE activity from human plasma

as described in Materials and methods. Control antibody did not remove FRET peptide cleaving activity, whereas antibody specic for APCE removed approximately 50%. Since the FRET peptide substrate is specic 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 rst 15 residues of APCE would be recognized. Although it must still be considered that human plasma may contain other proteinases 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, phenylbound 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 proles from Q-Sepharose and T-gel chromatographies (Figure 4A); and (4) on reduced SDS-PAGE, the peak from the immunoafnity 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 important substrate of APCE, since proteolytic cleavage between Pro12Asn13 yields the derivative Asn-2AP, which becomes crosslinked signicantly faster to brin by FXIIIa, and, as a consequence, enhances the resistance of brin to digestion by plasmin. Since human APCE augments the inhibition of brinolysis 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 brin removal. Bleeding complications would be unlikely, based on the observation that persons heterozygous for 2AP deciency have minimal hemorrhagic risk.6 Hence, a window of safety may exist for lowering 2AP function in healthy persons. Particularly in clinical situations where brin formation is likely, the development of an agent that inhibits APCE might result in decreased amount of Asn-2AP available for cross-linking to brin as thrombi develop or inammation progresses. Then endogenous levels of generated plasmin, or plasmin produced by administering small amounts of a plasminogen activator, might be sufcient to effect brin 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 brinolytic system.

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

From bloodjournal.hematologylibrary.org by guest on March 16, 2013. For personal use only.
3788 LEE et al BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10

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