Human ␣ 2 -antiplasmin (␣ 2 AP), also known as ␣ 2 -plasmin inhibitor, is the major inhibitor of the proteolytic enzyme plasmin that digests fibrin. There are 2 N-terminal forms of ␣ 2 AP that circulate in human plasma: a 464-residue protein with Met as the N-terminus, Met-␣ 2 AP, and a 452-residue version with Asn as the N-terminus, Asn-␣ 2 AP. We have discovered and purified a proteinase from human plasma that cleaves the Pro12-Asn13 bond of Met-␣ 2 AP to yield Asn-␣ 2 AP and have named it antiplasmin-cleaving enzyme (APCE). APCE is similar in primary structure and catalytic properties to membranebound fibroblast activation protein/seprase for which a physiologic substrate has not been clearly defined. We found that Asn-␣ 2 AP becomes cross-linked to fibrin by activated factor XIII approximately 13 times faster than native Met-␣ 2 AP during clot formation and that clot lysis rates are slowed in direct proportion to the ratio of Asn-␣ 2 AP to Met-␣ 2 AP in human plasma. We conclude that APCE cleaves Met-␣ 2 AP to the derivative Asn-␣ 2 AP, which is more efficiently incorporated into fibrin 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-␣ 2 AP could result in a more rapid removal of fibrin by plasmin during atherogenesis, thrombosis, and inflammatory states. IntroductionHuman ␣ 2 -antiplasmin (␣ 2 AP), 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 ␣ 2 AP to blood clotting and fibrinolytic homeostasis is supported by the following observations: (1) the rate of free plasmin inactivation by circulating ␣ 2 AP is much faster than fibrin(ogen) digestion by plasmin, 2 thereby eliminating the possibility of a systemic lytic state and consequent bleeding; (2) ␣ 2 AP is cross-linked to forming fibrin by activated blood clotting factor XIII (FXIIIa) and inhibits plasmin-mediated lysis in direct proportion to the amount incorporated 3-5 ; and (3) patients with homozygous ␣ 2 AP deficiency manifest serious hemorrhagic tendencies, while heterozygotes tend to bleed only after major trauma or surgery. 6 Human ␣ 2 AP is synthesized primarily in the liver, and during circulation in plasma, the secreted precursive Met-␣ 2 AP form, a 464-residue protein having Met as the N-terminus, undergoes proteolytic cleavage between Pro12 and Asn13 to yield Asn-␣ 2 AP, a 452-residue version with Asn as the N-terminus. 7 Met-␣ 2 AP accounts for approximately 30% of circulating ␣ 2 AP, and Asn-␣ 2 AP, approximately 70%. 7,8 While 3-fold more Asn-␣ 2 AP than recombinant Met-␣ 2 AP was shown to cross-link to fibrin, 9 no data have been reported for native circulating Met-␣ 2 AP. Moreover, the effect of different ratios of the 2 ␣ 2 AP forms on clot lysis has not been reported. Finally, the enzyme responsible for converting Met-␣ 2 AP to Asn-␣ 2 AP has not been ident...

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 (

The circulating enzyme, α 2 -antiplasmin cleaving enzyme (APCE), has very similar sequence homology and proteolytic specificity as fibroblast activation protein (FAP), a membrane-bound proteinase. FAP is expressed on activated fibroblasts associated with rapid tissue growth as in embryogenesis, wound healing, and epithelial-derived malignancies, but not in normal tissues. Its presence on stroma suggests that FAP functions to remodel extracellular matrix (ECM) during neoplastic growth. Precise biologic substrates have not been defined for FAP, although like APCE, it cleaves α 2 -antiplasmin to a derivative more easily crosslinked to fibrin. While FAP has been shown to cleave gelatin, evidence for cleavage of native collagen, the major ECM component, remains indistinct. We examined the potential proteolytic effects of FAP or APCE alone and in concert with selected matrix metalloproteinases (MMPs) on collagens I, III and IV. SDS-PAGE analyses demonstrated that neither FAP nor APCE cleaves collagen I. Following collagen I cleavage by MMP-1, however, FAP or APCE digested collagen I into smaller peptides. These peptides were analogous to, yet different from, those produced by MMP-9 following MMP-1 cleavage. Aminoterminal sequencing and mass spectrometry analyses of digestion mixtures identified several peptide fragments within the sequences of the two collagen chains. The proteolytic synergy of APCE in the cleavage of collagen I and III was not observed with collagen IV. We conclude that FAP works in synchrony with other proteinases to cleave partially degraded or denatured collagen I and III as ECM is excavated, and that derivative peptides might function to regulate malignant cell growth and motility.

Science 264, 1593-1596). Here, we evaluated the ability of G h as compared with G q to mediate receptor-stimulated inositol phosphate turnover by the three ␣ 1 -subtypes (␣ 1A , ␣ 1B , and ␣ 1D ). In addition, we questioned if the transglutaminase function of G h is involved in its receptor signaling activity. A mutant form of a human TGase II cDNA in which the codon for the active site cysteine (Cys 277 ) was replaced by serine was cloned into the mammalian expression vector pMT2. Compared with wild-type TGase II, no transglutaminase activity was observed with transient transfection of this Cys3 Ser mutant in COS-1 cells. However, like wild-type TGase, the Cys3 Ser mutant mediated receptor-stimulated inositol phosphate turnover when cotransfected with an ␣ 1B -AR cDNA. G ␣q supported ␣ 1 -AR-mediated inositol phosphate turnover by all three receptor subtypes. By contrast, although both the wild-type and Cys3 Ser construct mediated receptor signaling by the ␣ 1B AR and ␣ 1D AR, the ␣ 1A -AR was unable to interact with G h . However, a G h -dependent signaling phenotype could be rescued by a chimeric ␣ 1A construct in which the third intracellular loop of the ␣ 1A -AR was replaced by that of the ␣ 1B -AR. Thus, the signaling function of G h is independent of its transglutaminase activity and is ␣ 1 -AR subtype specific. This subtype specificity of the interaction between ␣ 1 ARs and G h involves important determinants in their third intracellular loops.

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