Cross-linking of Plasminogen Activator Inhibitor 2 and α2-Antiplasmin to Fibrin(ogen)*

In this study, we identified lysine residues in the fibrinogen Aα chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with α2-antiplasmin (α2-AP), which is known to cross-link to lysine 303 of the Aα chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to α2-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-αC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aα chain, respectively. PAI-2 or α2-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aα chain. α2-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with α2-AP, and the two proteins utilized different lysines in the Aα chain. Therefore, PAI-2 and α2-AP can cross-link simultaneously to the α polymers of a fibrin clot and promote resistance to lysis.

Fibrinogen is a 340-kDa plasma protein and is a symmetrical dimer containing 29 disulfide bonds. Fibrinogen consists of 2 copies of A␣, B␤, and ␥ polypeptide chains, which have molecular masses of 63.5, 56, and 47 kDa, respectively. Release of fibrinopeptides A and B by thrombin initiates rearrangement of the molecule to form fibrin fibrils (reviewed in Ref. 1). Crosslinking of fibrin(ogen) by transglutaminases is responsible for generation of an insoluble, stable fibrin clot (1). Transglutaminases catalyze formation of covalent ⑀-(␥-glutamyl) lysine bonds. An acyl transfer reaction occurs where the ␥ carboxyamide group of a glutamine residue acts as the donor and the ⑀-amino group serves as an acyl acceptor (reviewed in Ref. 2). Cross-linking occurs only between the A␣ and ␥ chains of aligned fibrin fibrils; the B␤ chain is not involved in crosslinking (1). Initially, adjacent ␥ chains cross-link to form ␥-␥ dimers. This reaction occurs between glutamine 398 or 399 of one chain and lysine 406 of another fibrin fibril (3). A␣ chains then polymerize to form higher molecular mass polymers; this reaction occurs more slowly than ␥-␥ dimer formation (4). Many lysine residues have been implicated as substrates for crosslinking and subsequent generation of high molecular mass oligomers of A␣ chain (5,6). It has been found that multimerization of ␥-␥ dimers can occur over extended time periods (7).
Factor XIII (FXIII) 1 and tissue transglutaminase (tTG) are two major members of the TG family and differ with respect to location and regulation (2). Plasma FXIII is found as a zymogen that consists of two A and two B subunits. The A subunit contains the catalytic domain and shows homology to tTG, which is a monomer. Thrombin is required for activation of FXIII, which is associated with fibrinogen in the plasma. This ensures that generation of fibrin is accompanied by FXIIImediated cross-linking of fibrin to form a strong insoluble clot. Tissue transglutaminase has a broad distribution in tissues, where cells such as endothelial cells and macrophages have been found to synthesize tTG (2). Both FXIII and tTG can cross-link fibrinogen, but the pattern of cross-linking differs. FXIII generates ␥-␥ dimers, whereas tTG cross-links hybrids of an A␣ and ␥ chain (8). Cross-linked A␣-␥ heterodimers were detected in plasma fibrinogen, indicative of tissue transglutaminase activity (9).
Cross-linking that occurs between the chains of fibrinogen is not thought to contribute in a major way to resistance of a fibrin clot to lysis by plasmin. However, there has been controversy on this point. It has been shown that formation of ␥-␥ dimers did not affect fibrin lysis (10), but formation of complex ␣-polymers increased resistance to fibrin lysis (11). Other studies have found that neither formation of ␥-␥ dimers nor of ␣ polymers contributed to resistance of fibrin to lysis (12). Recent studies demonstrate that multimerization of ␥-dimers after prolonged incubation of a fibrin clot decreased the rate of fibrin lysis (7).
The major way in which cross-linking affects resistance of a fibrin clot to lysis is when inhibitors of fibrinolysis are crosslinked to fibrin(ogen) (13,14). The serine protease inhibitor (serpin), ␣ 2 -AP, is the major physiological inhibitor of plasmin, which degrades fibrin. Localization of ␣ 2 -AP along the fibrin strands by FXIII-mediated cross-linking decreased lysis of fibrin by plasmin (14). ␣ 2 -AP has been shown to be cross-linked to fibrinogen via glutamine 2 of ␣ 2 -AP and lysine 303 of the A␣ chain (15,16).
We have shown that PAI-2, an inhibitor of plasmin generation, was cross-linked to fibrinogen (17). PAI-2 inhibits fibrinolysis by neutralizing urokinase and tissue-type plasminogen activator. This inhibits plasmin generation and subsequent fibrin degradation and/or extracellular matrix remodeling (18). Both tTG and FXIIIa were found to catalyze cross-linking of PAI-2 to fibrinogen (17). The glutamines on PAI-2 were identified as residues 83 and 86, which are found on an exposed loop between helices C and D. This unique 33-residue loop is distinct from the active site of PAI-2, and cross-linked PAI-2 remains active as an inhibitor of plasminogen activators (17,19). Other studies have demonstrated that PAI-2 can be crosslinked to trophoblast membranes and to extracellular matrix, again via the exposed loop (20). Intracellular PAI-2 has been shown to inhibit programmed cell death, and this process was also dependent on the interhelical loop (21).
This study investigated the lysine residues in fibrinogen that were involved in transglutaminase-catalyzed cross-linking of PAI-2. ␣ 2 -AP is known to be cross-linked to fibrin(ogen) via lysine 303 in the A␣ chain; therefore, cross-linking of ␣ 2 -AP was compared with that of PAI-2. Interestingly, we have found that PAI-2 was not cross-linked via lysine 303 but utilized many other lysine residues in the A␣ chain of fibrinogen.

EXPERIMENTAL PROCEDURES
Transglutaminase-mediated Cross-linking of PAI-2 and ␣ 2 -AP-Fibrinogen for routine cross-linking reactions was purchased from Kabi (Sweden). Peak 1 fibrinogen was produced from fraction I-2 fibrinogen by anion exchange chromatography on DEAE-cellulose (22). This material contained Ͼ80% intact A␣ chains and was free of other protein contaminants. Fraction I-9 fibrinogen was prepared by subfractionation of the glycine precipitate of plasma (23) and lacked C-terminal A␣ segments of ϳ100 residues (24,25). Des-␣C fibrinogen (fraction I-9D) was produced from intact fibrinogen by limited plasmin digestion (25) and lacked C-terminal A␣ segments of ϳ390 residues (26,27). Recombinant 47-kDa non-glycosylated PAI-2 was kindly provided by Biotech Australia Ltd.
Fibrinogen ( Peptides were synthesized in-house using a Pioneer peptide synthesizer (PE Biosystems) and were confirmed by mass spectrometry. These peptides were incorporated into cross-linking reactions at a final concentration of 1 mM. The sequence of the peptides corresponded to residues 290 to 319 of fibrinogen A␣ chain and were as follows.
K303, NPGSSGTGGTATWKPGSSGPGSTGSWNSGS R303, NPGSSGTGGTATWRPGSSGPGSTGSWNSGS Samples were prepared for SDS-polyacrylamide gel electrophoresis by solubilization in an equal volume of 8 M urea, 200 mM Tris, 4% w/v SDS, and 40 mM dithiothreitol. Samples were electrophoresed on 8% Prosieve (Flowgen, UK) acrylamide gels, which mimic a 4 -12% acrylamide gradient, at a constant voltage of 125 V. Proteins were transferred overnight to nitrocellulose (Bio-Blot-NC, Costar, UK) in 25 mM Tris, 192 mM glycine, 20% v/v methanol. Transfer of protein was monitored by staining of the nitrocellulose with 1% w/v Ponceau S in 3% v/v trichloroacetic acid and destaining with distilled H 2 0. Membranes were blocked with 50 mM bicarbonate/carbonate buffer, pH 9.6, containing 3% w/v bovine serum albumin. Antigen was detected by binding of a mouse monoclonal antibody to PAI-2 (Biopool, 0.2 g/ml) or a rabbit IgG preparation to ␣ 2 -AP (Dako, 6 g/ml). A rabbit antiserum (CS004) raised to a peptide corresponding to residues 601-608 of the fibrinogen A␣ chain was provided by Drs. C. Stirk and W. T. Melvin, Dept. of Molecular & Cell Biology, University of Aberdeen and was used at a 1:1000 dilution. Detection was by either anti-mouse or anti-rabbit IgG conjugated to alkaline phosphatase and 0.6 mM 5Ј-bromo 4Ј-chloro 3-indolyl-phosphate and 0.5 mM nitro blue tetrazolium in 200 mM Tris/ HCl, 10 mM MgCl 2 , pH 9.5.
Enzymatic Digestion of Cross-linked Samples under Denaturing Conditions-Fibrinogen (Kabi, 2 mg) was cross-linked using 300 g of guinea pig liver transglutaminase and 2.5 mM CaCl 2 in the absence or presence of 800 g PAI-2 or ␣ 2 -AP for 2 h at 37°C. Samples were reduced and carboxymethylated (16,29,30) and taken up in 0.1 M Tris/HCl, 1 mM EDTA, pH 6.8, containing 6 M urea. Dithiothreitol was added to a final concentration of 50 mM under a nitrogen atmosphere and incubated for 30 min at room temperature followed by a further 30-min incubation in the presence of 500 mM iodoacetamide. Samples were separated by gel filtration on Sephacryl S-200 in the presence of 8 M urea. PAI-2/␣ 2 -AP that was cross-linked to fibrinogen was detected in fractions by immunoblotting using either a monoclonal antibody to PAI-2, polyclonal antibody to ␣ 2 -AP or polyclonal antibody to fibrinogen (Dako). Three fractions were identified as containing cross-linked material and were pooled, dialyzed, and lyophilized. Alternatively, 6 M fibrinogen was cross-linked to 6 M PAI-2 or ␣ 2 -AP in the presence of 2.5 mM CaCl 2 , 600 nM tTG or 600 nM FXIIIa, and 0.4 units/ml thrombin for 2 h before electrophoresis on a 4 -12% NuPage acrylamide gel (Novex) under reducing conditions. Cross-linked products were stained with Coomassie Blue, and individual cross-linked bands were excised from the gel and digested.
Cross-linked samples were digested overnight with 0.5 g of porcine trypsin (Promega, UK) in 20 mM ammonium bicarbonate or with 0.5 g endoproteinase Glu-C from Staphylococcus aureus (V8, Promega) in 50 mM sodium phosphate, pH 7.8, vacuum-dried, and dissolved in 500 l of 10% formic acid. The peptides obtained were desalted on a reverse phase high performance liquid chromatography microcolumn. Masses of the peptides were determined on a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (PE Biosystems Voyager STR). Samples were prepared in either sinapinic or ␣-cyano-4-hydroxy cinnamic acid matrices. Predicted masses of tryptic and endopeptidase Glu-C peptides of fibrinogen, PAI-2, and ␣ 2 -AP were calculated using the PeptideMass software package available from the ExPASy web site. All experiments were carried out at least five times except the gel filtration and trypsin digestion analysis of cross-linked samples, which was performed on two separate occasions.

Cross-linked PAI-2 Co-localized with the A␣ Chains of Fi-
brinogen-Cross-linking of PAI-2 to the ␣ chains of fibrinogen was examined by immunoblotting for the A␣ chain and for PAI-2. Free A␣ chains were seen at ϳ63.5 kDa (Fig. 1, lane a). Cross-linking of fibrinogen by tTG resulted in the formation of ␣ polymers at approximately 100, 210, 240, 270 kDa (lane b). Immunoblotting of the same samples for PAI-2 demonstrated free PAI-2 at ϳ47 kDa and a degradation product (ϳ37 kDa, lane c). The addition of tTG generated higher molecular mass cross-linked PAI-2 at 210, 240, and 270 kDa (lane d), which was consistent with PAI-2 that was cross-linked to ␣ polymers. Some cross-linked PAI-2 was detected at approximately 110 kDa, and this is likely to represent PAI-2 cross-linked to one A␣ chain.
The time dependence for cross-linking of PAI-2 ( Fig. 2A) and ␣ 2 -AP (Fig. 2B) to fibrinogen was compared (0 -60 min). Both these inhibitors were cross-linked rapidly to fibrinogen, and high molecular cross-linked products (Ͼ200 kDa) were identified within 5 min of adding tTG. Cross-linked products of PAI-2 and ␣ 2 -AP increased from 5 to 60 min in a time-dependent manner, but most cross-linking occurred within 30 min. Free ␣ 2 -AP was seen at ϳ65 kDa. Cross-linked products of ␣ 2 -AP were similar to those of PAI-2 and were detected at ϳ140 kDa, Fibrinogen A␣ chains (a and b) and PAI-2 (c and d) were detected by immunoblotting. PAI-2 was incubated in the presence of fibrinogen and CaCl 2 . Cross-linking of PAI-2 to fibrinogen was initiated by the addition of tTG (b and d). Samples were analyzed by SDS-PAGE on 8% Prosieve acrylamide gels under reducing conditions. which was consistent with ␣ 2 -AP cross-linked to one A␣ chain and at Ͼ200 kDa; this represented ␣ 2 -AP cross-linked to A␣ polymers.
PAI-2 and ␣ 2 -AP Cross-linked to Different Lysines on the Fibrinogen A␣ Chain-The role of lysine 303 of the fibrinogen A␣ chain in cross-linking to PAI-2 was investigated by incorporation of peptides corresponding to residues 290 to 319 of the A␣ chain of fibrinogen. Peptide K303 contained lysine 303, and the control peptide, R303, had lysine 303 replaced with an arginine residue. The effect of the peptides on cross-linking of fibrinogen to PAI-2 and ␣ 2 -AP was studied by immunoblotting. Cross-linking of PAI-2 to fibrinogen was not affected by either peptide K303 or R303 (Fig. 3A); higher molecular mass crosslinked PAI-2 (Ͼ200 kDa) was still evident in the presence of either peptide (Fig. 3A). This was in contrast to cross-linking of ␣ 2 -AP, which was inhibited by peptide K303, whereas peptide R303 had no effect (Fig. 3B). Neither peptide affected crosslinking of fibrinogen chains as assessed by protein stain of fibrinogen (data not shown).
Cross-linking of PAI-2 to fibrinogen was investigated in the presence of excess ␣ 2 -AP or PAI-1, an inhibitor of fibrinolysis that is not cross-linked to fibrinogen (17). Neither ␣ 2 -AP nor PAI-1 competed with cross-linking of PAI-2 to fibrinogen, even when present in 10-fold molar excess (Fig. 4). In addition, cross-linking of PAI-2 or ␣ 2 -AP to fibrinogen was not affected by equimolar concentrations of ␣ 2 -AP or PAI-2, respectively (data not shown). These data show that ␣ 2 -AP and PAI-2 could be cross-linked simultaneously to fibrinogen.
Cross-linking of PAI-2 and ␣ 2 -AP to Truncated Fibrinogens-The regions of the A␣ chain involved in cross-linking of PAI-2 and ␣ 2 -AP to fibrinogen were studied using preparations of truncated fibrinogens (22)(23)(24)(25)(26)(27). Peak I fibrinogen is intact, highly purified fibrinogen. I-9 fibrinogen lacks ϳ100 amino acids from the C terminus of the A␣ chain and has lysine 303. Des-␣C fibrinogen lacks the final ϳ390 residues of the A␣ chain and, therefore, lacks lysine 303. PAI-2 was cross-linked to all three fibrinogens (Fig. 5A), and high molecular mass PAI-2 (Ͼ200 kDa) was generated following the addition of tTG (Fig.  5A, lanes b, d, and f)). The degree of PAI-2 cross-linking was dependent on the length of the A␣ chain in that most cross-linking was seen with Peak I fibrinogen. Cross-linking of ␣ 2 -AP to des-␣C fibrinogen was not evident (Fig. 5B), consistent with cross-linking of ␣ 2 -AP to lysine 303. Similar degrees of crosslinked ␣ 2 -AP were evident with Peak I and I-9 fibrinogen.
Identification of the Acceptor Lysine Residue in A␣ Chain-The lysine residues in fibrinogen that were involved in transglutaminase-mediated cross-linking to PAI-2 were identified by mass spectrometry of peptides digested by either trypsin or endopeptidase Glu-C. Cross-linked products were separated, identified on the basis of immunoblotting, and were digested for analysis by mass spectrometry. Cross-linking of fibrinogen to ␣ 2 -AP was used for comparison, whereas that of fibrinogen alone served as a control; peptides found in this sample and common to all three reactions were omitted from the analysis. Only peptides specific to cross-linked PAI-2 or cross-linked  Analysis of tryptic peptides of cross-linked PAI-2 is summarized in Table IA and was consistent with cross-linking of PAI-2 to numerous peptides of the A␣ chain. These peptides contained lysine residues 183 or 457, 148, 176 or 224, and 230. Peptides (4093-4410 Da) corresponded to one tryptic peptide of PAI-2 (residues 57-87) containing glutamines 83-86 (3486.6 Da), which was cross-linked to one tryptic peptide of the A␣ chain. Larger tryptic peptides were also detected (5729 -6258 Da), and these represented PAI-2 cross-linked to two A␣ chain peptides. Both of these larger peptides corresponded to region 142-148, which contained lysine 148 and glutamine 143, which participates in further cross-linking between A␣ chains. Endopeptidase Glu-C (V8 from S. aureus) digestion of PAI-2 crosslinked to fibrinogen by tTG yielded peptides of 2426.1, 2521.4, 2727.5, 2824.1 Da (Table IA), which corresponded to residues 75-92 of PAI-2 (2050.9 Da) cross-linked to peptides of 375.2, 470.5, 676.6, and 773.2 Da. These peptides of the A␣ chain contain lysines 176, 457, 148, and 183, respectively (Table IA). No larger peptides were detected on digestion with endopeptidase Glu-C. Therefore, digestion of cross-linked PAI-2 with trypsin and endopeptidase Glu-C has demonstrated that lysines 148, 176, 183, and 457 on the A␣ chain of fibrinogen mediate cross-linking to PAI-2 by tTG.
FXIIIa-mediated cross-linking of PAI-2 to the A␣ chain of fibrinogen was also analyzed using enzymatic digestion and mass spectrometry. Detection of tryptic peptides revealed the presence of residues 57-87 of PAI-2 (3486.6 Da), which was cross-linked to regions 142-148, 225-230, and 408 -413 of the A␣ chain of fibrinogen (Table II) (Table IB) were consistent with cross-linking by tTG via glutamine 2 of ␣ 2 -AP and lysine 303 of the A␣ chain of fibrinogen. The donor glutamine is found within a peptide from residues 1 to 12 (1369.8 Da). Lysine 303 is contained within a 5534.5 Da peptide, corresponding to residues 290 to 348 of the A␣ chain. Tryptic peptides that were detected corresponded to ␣ 2 -AP cross-linked either to two or three A␣ chains for tTG-mediated cross-linking (Table IB). Cross-linking was seen between Gln-2 of ␣ 2 -AP and lysine 303 of A␣ with further polymerization to another A␣ chain between glutamine 328 and lysine 78. Gln-328 has pre-  cross-linked PAI-2 and/or ␣ 2 -AP Trypsin or endopeptidase Glu-C-digested peptides specific to crosslinked PAI-2 (A) and tryptic peptides specific to cross-linked ␣ 2 -AP (B) were compared with predicted peptide masses. Peptides of PAI-2 or ␣ 2 -AP are highlighted in bold, and the A␣ chain peptides that were cross-linked to PAI-2/␣ 2 -AP are shown in regular text. Peptides of the A␣ chain that were involved in cross-linking of the A␣-A␣ chain are shown in italics.  viously been shown to act as a glutamine donor during polymerization of the A␣ chains (31). The tryptic peptide of the A␣ chain from residues 290 to 348, which contains both Gln-328 and Lys-303, can therefore direct cross-linking between A␣ chains and between ␣ 2 -AP and A␣ chains. Some larger peptides (8287-8441 Da) were also detected, which suggested crosslinking of ␣ 2 -AP to polymers containing three A␣ chains. This implies that additional glutamine residues can direct crosslinking of A␣-A␣ chains as well as glutamine 328. Digestion of cross-linked ␣ 2 -AP with endopeptidase Glu-C did not yield any specific peptides, which probably reflects the large size of the ␣ 2 -AP peptide (residues 1-115, 12232.4 Da) and the peptide containing lysine 303 (residues 266 -347, 7663.4 Da), neither of which was detected in the mass spectrometer. FXIIIa-mediated cross-linking of ␣ 2 -AP to fibrinogen was also analyzed, and a single specific tryptic peptide of 6902.5 Da was detected, corresponding to ␣ 2 -AP (residues 1-12, 1369.8 Da) cross-linked to lysine 303 (residues 290 -348, 5534.5 Da), which agreed with previously published results (16).

DISCUSSION
Transglutaminases are responsible for the introduction of post-translational covalent bonds that confer strength and stability in a variety of physiological and pathological situations, including formation of a fibrin clot (2). We have shown that cross-linking of the serpin, PAI-2, to fibrin(ogen) by transglutaminases allows localization of PAI-2 activity along the fibrin strands, which can contribute to persistence of deposited fibrin (17). In this study, we have shown that lysines 148, 176, 183, 230, 413, and 457 in the A␣ chain of fibrinogen were crosslinked to glutamines 83 and 86 in PAI-2 during cross-linking by tissue transglutaminase (lysines 148, 176, 183, 457) and by FXIIIa (148, 176, 230, 413).
Digesting cross-linked products with two enzymes and analysis of peptides by mass spectrometry identified lysine residues that were involved in cross-linking. This approach was successful despite the large sizes of the proteins involved and was in good agreement with other unequivocal experiments using peptide competitors and truncated fibrinogens. PAI-2 cross-linking to fibrinogen was not competed out by a peptide containing lysine 303 and was still cross-linked to fibrinogen in the presence of excess ␣ 2 -AP. PAI-2 was also cross-linked to I-9 and des-␣C fibrinogens, which lack regions from the C terminus of the A␣ chain and lack lysine 457. Less cross-linked PAI-2 was evident with the truncated fibrinogens compared with intact fibrinogen. This was consistent with cross-linking of PAI-2 to a number of lysines distributed along the A␣ chain and not including lysine 303.
PAI-2 did not interfere with cross-linking of the A␣ chains, which is not surprising since no lysines were common to crosslinking of A␣ chains and cross-linking to PAI-2. One study shows that lysines 508 and 580 were involved in polymerization of the A␣ chain (31). A separate study identifies lysine residues 556, 580, 539, 508, 418, 448, 601, 606, 427, 429, 208, 224, and 219 of the A␣ chain as substrates for cross-linking of the A␣ chains to form polymers (6). That study used a peptide corresponding to the N terminus of ␣ 2 -AP, which had glutamine 2 as the donor residue (6). The A␣ lysines involved in cross-linking are listed in order of preference, and lysines 556 and 580 accounted for 50% of ␣ chain cross-linking (6). However, full length ␣ 2 -AP was found to be cross-linked only to lysine 303 and demonstrates the importance of the structure of the target protein in determining which glutamine or lysine can act as substrates for transglutaminases. It is thought that transglutaminase enzymes show greater specificity for glutamines than for lysines (2,32), and this is certainly the case for cross-linking of PAI-2, where only glutamines 83 and 86 are active in cross-linking. The factors that determine whether a glutamine or a lysine can be cross-linked are unclear, but structure of the surrounding sequence is important (33,34). Accessibility on a solvent-exposed region may also play a role (33), as is the case for glutamines 83 and 86 of PAI-2, which are located on an exposed loop.
Comparison of PAI-2 and ␣ 2 -AP has shown that these two serpins utilized different lysines in the A␣ chain for crosslinking to fibrinogen and that both inhibitors could be crosslinked simultaneously to fibrin(ogen). The most interesting finding was that PAI-2 was cross-linked to numerous lysines, unlike ␣ 2 -AP, which was cross-linked to one lysine residue, Lys-303. Our findings agree with previous literature, which studied cross-linking of ␣ 2 -AP via glutamine 2 to fibrin(ogen) via lysine 303 by FXIIIa (16). The crystal structures of fragment D (35), which contains residues 111-197 of the A␣ chain and modified bovine fibrinogen (lacking residues 391-580 of the A␣ chain) have recently been elucidated (36). A schematic diagram showing the triglobular nature of fibrinogen with ␣-helical coiled coils and the location of fragments D and E is shown in Fig. 6A. The potential location of the cross-linking lysines in the A␣ chain is shown in more detail in Fig. 6B. It is known that the A␣ chain initially forms a triple helix with the ␤ and ␥ chains until residue 160, where the A␣ chain reverses due to a disulfide ring and forms a four helix coiled coil. This four helix coiled coil continues until residue 220, which is aligned to residue 92 of the A␣ chain. Therefore, lysines 148, 176, and 183 involved in cross-linking of PAI-2 are located along the four helix coiled coil. A hinge region is seen at residue 220, which is plasmin-sensitive, and the remainder of the A␣ chain forms the highly flexible C-terminal domains. Region 220 -390 was not seen in the crystal structure of bovine fibrinogen, consistent with a flexible region. Lysine 230 and lysine 303 involved in cross-linking of PAI-2 and ␣ 2 -AP, respectively, are located on the flexible C-terminal domain, as are lysines 413 and 457. This flexible C-terminal domain of the A␣ chain is the primary target for proteolysis of fibrinogen.
PAI-2 is a member of the ov-serpin subfamily (37), and the crystal structure of PAI-2 has recently been elucidated, but the structure was determined using a mutant PAI-2 that lacked residues 66 -98 (38). The structure of other serpins, such as ovalbumin, is known (39), and a schematic diagram of PAI-2 with its exposed loop (residues 66 -98) is shown (Fig. 6B). It is clearly demonstrated that glutamines 83 and 86 are located on a solvent-exposed region. The crystal structure of ␣ 2 -AP has not been elucidated, and several members of the family whose structures have been solved lack the extended N terminus seen in this serpin. Antithrombin has a similar N-terminal extension but the reported structures do not provide information about this region, except in one case, where Pro 12 in antithrombin, which corresponds to Gln 2 in ␣ 2 -AP, is visible (40). It suggests that Gln 2 may be in a surface pocket that is not totally exposed, as hypothesized in Fig. 6B. It is possible that the exposed cross-linking loop of PAI-2 may essentially act as a peptide that can access many lysines on the fibrinogen surface. In contrast, the donor glutamine of ␣ 2 -AP, which is possibly less exposed, may target only specific lysine residues on the A␣ chain of fibrinogen.
Cross-linked PAI-2 retains its activity (17), and cross-linking of PAI-2 allows the molecule to be directed to the fibrin strands where PAI-2 can function as an inhibitor of fibrinolysis. The unique cross-linking loop that is inserted between helices C and D of PAI-2 also contains a potential glycosylation site (Asn-75), and it has been speculated that glycosylation may inhibit cross-linking of PAI-2 (18). We have shown that PAI-2 secreted by monocytes was not glycosylated and did not enter the ER-Golgi, consistent with the lack of a signal peptide that is the characteristic feature of ov-serpins (41). Therefore, PAI-2 released by activated monocytes would be available for crosslinking in inflammatory lesions.
Local expression of PAI-2 by activated cells is an important phenomenon. PAI-2 is not detected in normal plasma, but PAI-2 has been demonstrated to be synthesized by activated monocytes in inflamed tissues, and PAI-2 is produced by keratinocytes and is present in the skin (42). The relevance and importance of cross-linking of PAI-2 by tTG or FXIIIa in tissues is unknown. However, PAI-2, tTG, and FXIIIa are all present in the atherosclerotic plaque along with fibrin(ogen), which is a major constituent of the diseased vessel wall (43)(44)(45)(46). The extent of cross-linking of PAI-2 is difficult to analyze in vivo, since cross-linking renders the molecule insoluble and therefore unextractable from tissue. However, it has been shown that keratinocytes stained positively for PAI-2, but PAI-2 could not be extracted from this tissue; this was suggested to be due to cross-linking (47). In addition, PAI-2 has been detected in human thrombi, and PAI-2 was found to align along fibrin strands (17), suggesting cross-linking of PAI-2 to fibrin(ogen). Cross-linking of PAI-2 and ␣ 2 -AP to the A␣ chain of fibrinogen, by tissue transglutaminase or factor XIIIa allows these two serpins to localize on the surface of fibrin strands, where efficient inhibition of plasmin generation can occur.