Circulating antiplasmin-cleaving enzyme (APCE) has a role in fibrinolysis and appears structurally similar to fibroblast activation protein (FAP), a cell-surface proteinase that promotes invasiveness of certain epithelial cancers. To explore this potential relationship, we performed comparative structure/function analyses of the 2 enzymes. APCE from human plasma and recombinant FAP (rFAP) exhibited identical pH optima of 7.5, extinction coefficients (

Human ␣ 2 -antiplasmin (␣ 2 AP), the main inhibitor of plasmin-mediated fibrinolysis, is a substrate for plasma transglutaminase, also termed activated factor XIII (FXIIIa). Of 452 amino acids in ␣ 2 AP, only Gln 2 is believed to be a fibrin-cross-linking (or FXIIIa-reactive) site. Kinetic efficiencies (k cat /K m(app) ) of FXIIIa and the guinea pig liver tissue transglutaminase (tTG) and reactivities of Gln substrate sites were compared for recombinant wild-type ␣ 2 AP (WT-␣ 2 AP) and Q2A mutant ␣ 2 AP (Q2A-␣ 2 AP). Human ␣ 2 -antiplasmin (␣ 2 AP) 1 contains 452 amino acid residues and has three functional domains that contribute to protecting fibrin clots from plasmin-mediated lysis (1-3). Initially, Gln 2 in the N-terminal domain of ␣ 2 AP becomes crosslinked to each fibrin ␣-chain by plasma transglutaminase, which is also termed FXIIIa. Then, the C-terminal domain of ␣ 2 AP interacts with the kringle structures of plasmin so that the reactive-site Arg 364 in the third domain aligns and forms a covalent bond with the active-site Ser in plasmin to give an inactive protease-inhibitor complex. Chemical modification (4) or mutation (5) of the reactive-site Arg 364 in ␣ 2 AP results in complete loss of plasmin inhibitory activity, but either form still competes very effectively with native ␣ 2 AP for becoming cross-linked to fibrin by FXIIIa catalysis; and as a consequence, fibrinolysis occurs significantly faster (4, 5).Transglutaminases are Ca 2ϩ -dependent enzymes that catalyze post-translational modification of proteins through the formation of ␥-glutamyl-⑀-lysine cross-links between polypeptide chains (6). FXIII is a heterotetramer proenzyme composed of paired A and B chains and is activated to FXIIIa by thrombin plus Ca 2ϩ . Tissue transglutaminase (tTG), a single-chain enzyme that is widely distributed in cells and tissues, does not require activation by proteolysis. The substrate specificity of FXIIIa for ␤-casein, fibrin/fibrinogen, or their peptide derivatives differs from that of tTG (7-9). Among the plasma proteins that have Gln substrate site(s) for FXIIIa, ␣ 2 AP is recognized as having the highest kinetic efficiency (k cat /K m(app) ), with its substrate site being the second Gln from the N terminus (10, 11). However, the kinetic efficiency of tTG for ␣ 2 AP and the identity of the substrate site(s) involved have not been established. tTG is believed to catalyze the cross-linking of ␣ 2 AP to fibrinogen in vitro (12) and the cross-link formation in fibrinfibrinogen complexes in human atherosclerotic plaques (13). In addition, ␣ 2 AP has been found in fibrin-rich matrix and granulation tissue of human skin wounds (14), and tTG has been suggested to promote wound healing (15). Given these observations, we compared the Gln cross-linking sites in ␣ 2 AP for the tTG isolated from guinea pig liver versus FXIIIa using both wild-type ␣ 2 AP (WT-␣ 2 AP) and mutant ␣ 2 AP in which Gln 2 was replaced with Ala (Q2A-␣ 2 AP).Although a previous report suggested that only the Gln 2 site in ␣ 2 AP participates in c...

Human α2‐antiplasmin (α2AP) is the primary inhibitor of plasmin‐mediated fibrinolysis and is an efficient substrate of activated factor XIII (FXI‐IIa). Among 452 amino acid residues in α2AP, Gln2 is believed to be the sole FXI‐IIa‐reactive site that participates in crosslinking α2AP to fibrin. We studied the effect of mutating Gln2 on the ability of FXIIIa to catalyze crosslinking of α2AP to fibrin. By FXIIIa catalysis, [14C]methylamine was incorporated into a Q2A‐α2AP mutant in which Gln2 (Q) was replaced by Ala (A), thereby indicating that wildtype α2AP has more than one FXIIIa‐reactive site. To identify the FXIIIa‐reactive sites in α2AP, wildtype α2AP and Q2A‐α2AP were labeled with 5‐(biotinamido)pentylamine by FXIIIa. Each labeled α2AP was digested with trypsin and applied to an avidin affinity column to capture labeled peptides. Edman sequencing and mass analysis of each labeled peptide showed that out of 35 Gln residues in wildtype α2AP, four were labeled with the following order of efficiency: Gln2 > Gln21 > Gln419 > Gln447. Q2A‐α2AP was also labeled at the three minor sites, Gln21 > Gln419 > Gln447. Q2A‐α2AP became crosslinked to fibirin(ogen) by FXIIIa catalysis at approximately one‐tenth the rate of wt‐α2AP. These results demonstrate that α2AP has one primary (Gln2) and three minor substrate sites for FXIIIa and that the three minor sites identified in this study can also participate in crosslink formation between α2AP and fibrin, but at a much lower efficiency than the Gln2 site.

Human α2-antiplasmin (α2AP), the primary inhibitor of plasmin, is secreted from liver to plasma as a 464-residue protein with Met as the N-terminus (Met-α2AP). As it circulates, antiplasmin-cleaving enzyme, a possible derivative of fibroblast activation protein, cleaves the Pro12-Asn13 bond of Met-α2AP to yield Asn-α2AP. The Asn-α2AP form becomes crosslinked to fibrin by activated factor XIII (FXIIIa) ~13X faster than Met-α2AP, and consequently, fibrin clot stability increases in direct proportion to the ratio of Asn-α2AP/Met-α2AP in plasma. FXIIIa, a transglutaminase catalyzes isopeptide bond crosslinking between a primary amine donor Lys in fibrin and an acceptor Gln in α2AP. Using recombinant Asn-α2AP mutants, we reported multiple FXIIIa-catalyzed fibrin crosslinking sites in Asn-α2AP: the previously reported Gln14 (Gln2 in Asn-α2AP) site and our discovery of three other Gln residues. This current study was performed to determine whether the major crosslinking site in Met-α2AP is also Gln14 or whether steric hindrance shifts the predominant crosslinking site to one of the other three identified Gln residues. Native Asn-α2AP and two different forms of Met-α2AP defined by an Arg6Trp polymorphism were labeled with 5-(biotinamido)pentylamine (BPA) by FXIIIa catalysis. To identify FXIIIa-reactive sites in Met-α2AP(Arg6), Met-α2AP(Trp6) and Asn-α2AP, each BPA-labeled α2AP was reduced, alkylated, and digested by trypsin. The BPA-labeled peptides were isolated from the tryptic digest mixture by avidin affinity chromatography, and further separated by reverse-phase HPLC. Using N-terminal sequence analysis of BPA-labeled peptides, we identified only the Gln14 residue as a FXIIIa-reactive site in all three forms of α2AP. Although four Gln sites in recombinant Asn-α2AP were involved in crosslinking, the Gln14 site appears to function as the only significant crosslinking site in the native form of the protein, whether either polymorphic form of precursive Met-α2AP, or the derivative, Asn-α2AP. The finding of Gln14 as the significant crosslinking site in native Asn-α2AP, as opposed to the four sites in recombinant Asn-α2AP, is likely related to differences in glycosylation, since the native protein is glycosylated while the recombinant protein is not. Using liquid chromatography/mass spectrometry analysis of tryptic digested BPA-labeled Asn-α2AP, another FXIIIa-reactive site (Gln33; Gln21 in Asn-α2AP) was identified, but the reactivity of this residue was ~200-fold less than the Gln14 residue. Finally, the BPA-labeling efficiencies of α2AP were determined by avidin blot analysis. BPA was incorporated into all three forms of α2AP by FXIIIa catalysis, but Met-α2AP(Arg6) and Met-α2AP(Trp6) became labeled by BPA far more slowly than Asn-α2AP, which is consistent with the difference in fibrin-crosslinking rates. Met-α2AP(Arg6) and Met-α2AP(Trp6) became labeled by BPA at a similar rate despite the Arg6Trp polymorphism being located close to the Gln14 used for crosslinking to fibrin. These data suggest that the Gln14 site is cryptic in Met-α2AP, being sheltered by the 12-residue N-terminal peptide, which when cleaved, yields Asn-α2AP that becomes crosslinked to fibrin more quickly to provide greater protection from plasmin.

The involvement of nucleus in the maturation processes of Dengue-2 virus in a mosquito cell line, C6/36 cells, has been identified by the electron microscopy and immunocytochemistry. The C6/36 cells were obtained from ATCC and maintained in MEM culture medium containing 10% fetal bovine serum at 28°C. The cell suspensions or cells grown on teflon-coated coverslips were infected with Dengue-2 virus (107/ml) for various time periods of 2 hours, 3, 6, 8, and 10 days. The cells were then fixed in buffered 1.5% glutaraldehyde, and washed in acetone before immunolabeled with monoclonal antibody. An indirect immunocytochemical labeling method of avidin-biotin complex (ABC) conjugated with peroxidase or gold particles (20 nm in diameter) and a flat embedding technique were used to localize the virus particles.At early stages of infections (before 3 days), there were no virion particles detected. After 6 days and on of infections, cytopathic effect (CPE) was observed and showed positive immuno-peroxidase reactions under the light and electron microscopies.