Targeting inhibition of fibroblast activation protein-α and prolyl oligopeptidase activities on cells common to metastatic tumor microenvironments.

Fibroblast activation protein (FAP), a membrane prolyl-specific proteinase with both dipeptidase and endopeptidase activities, is overexpressed by reactive stromal fibroblasts during epithelial-derived cancer growth. FAP digests extracellular matrix as tissue is remodeled during cancer expansion and may also promote an immunotolerant tumor microenvironment. Recent studies suggest that nonspecific FAP inhibitors suppress human cancer xenografts in mouse models. Prolyl oligopeptidase (POP), another prolyl-specific serine proteinase, is also elevated in many cancers and may have a regulatory role in angiogenesis promotion. FAP and POP cell-associated activities may be targets for diagnosis and treatment of various cancers, but their accessibilities to highly effective specific inhibitors have not been shown for cells important to cancer growth. Despite their frequent simultaneous expression in many cancers and their overlapping activities toward commonly used substrates, precise, separate measurement of FAP or POP activity has largely been ignored. To distinguish each of the two activities, we synthesized highly specific substrates and inhibitors for FAP or POP based on amino acid sequences surrounding the scissile bonds of their respective putative substrates. We found varying amounts of FAP and POP protein and activities on activated fibroblasts, mesenchymal cells, normal breast cells, and one breast cancer cell line, with some cells exhibiting more POP than FAP activity. Replicating endothelial cells (ECs) expressed POP but not FAP until tubulogenesis began. Targeting FAP-positive cells, especially mesenchymal stem cells and cancer-associated fibroblasts for inactivation or destruction, and inhibiting POP-producing EC may abrogate stromal invasion and angiogenesis simultaneously and thereby diminish cancer growth.

Using substrate specificity of antiplasmin-cleaving enzyme for fibroblast activation protein inhibitor design.

Circulating antiplasmin-cleaving enzyme (APCE), a prolyl-specific serine proteinase, is essentially identical to membrane-inserted fibroblast activation protein (FAP) that is transiently expressed during epithelial-derived cancer growth. Human precursive alpha(2)-antiplasmin (Met-alpha(2)AP), the only known physiologic substrate for APCE, is cleaved N-terminally to Asn-alpha(2)AP that is rapidly cross-linked to fibrin and protects it from digestion by plasmin. Identifying a specific inhibitor of APCE/FAP continues to be intensely pursued. Recombinant FAP cleavage of peptide libraries of short amino acid sequences surrounding the scissile bond, -Pro(12)-Asn(13)-, indicated that P2 Gly and P1 Pro are required, just as we found for APCE. We examined cleavage of P4-P4' peptides, using 19 amino acid substitutions at each position and selected ones in P8-P5. K(m) values determined for peptide substrates showed that P7 Arg has the highest affinity for APCE. Peptide cleavage rate increased with Arg in P6 rather than P5 or native P7. Placing Arg in P4 or P8 reduced cleavage rates dramatically. Cleavage of substrates with extended peptide sequences before or after the scissile bond showed endopeptidase to be superior to dipeptidase activity for APCE. A substrate analogue inhibitor, Phe-Arg-(8-amino-3,6-dioxaoctanoic acid)-Gly-[r]-fluoropyrrolidide, inhibited APCE with a K(i) of 54 microM but not dipeptidyl peptidase IV even at 2 mM. The inhibitor also blocked cleavage of Met-alpha(2)AP with an IC(50) of 91 microM. Replacing Arg with Gly at the same distance from fluoropyrrolidide as P7 Arg is from P1 Pro reduced its inhibition of APCE approximately 10-fold. Results indicate that Arg at P5, P6, or P7 distances from P1 enhances affinity and efficiency of substrates or inhibitors toward APCE or FAP.

The effect of a single nucleotide polymorphism on human alpha 2-antiplasmin activity.

The primary inhibitor of plasmin, alpha(2)-antiplasmin (alpha(2)AP), is secreted by the liver into plasma with Met as the amino-terminus. During circulation, Met-alpha(2)AP is cleaved by antiplasmin-cleaving enzyme (APCE), yielding Asn-alpha(2)AP, which is crosslinked into fibrin approximately 13 times faster than Met-alpha(2)AP. The Met-alpha(2)AP gene codes for either Arg or Trp as the sixth amino acid, with both polymorphic forms found in human plasma samples. We determined the Arg6Trp genotype frequency in a healthy population and its effects on Met-alpha(2)AP cleavage and fibrinolysis. Genotype frequencies were RR 62.5%, RW 34.0%, and WW 3.5%. The polymorphism related to the percentage of Met-alpha(2)AP in plasma was WW (56.4%), RW (40.6%), and RR (23.6%). WW plasma tended to have shorter lysis times than RR and RW plasmas. APCE cleaved purified Met-alpha(2)AP(Arg6) approximately 8-fold faster than Met-alpha(2)AP(Trp6), which is reflected in Asn-alpha(2)AP/Met-alpha(2)AP ratios with time in RR, RW, and WW plasmas. Removal of APCE from plasma abrogated cleavage of Met-alpha(2)AP. We conclude that the Arg6Trp polymorphism is functionally significant, as it clearly affects conversion of Met-alpha(2)AP to Asn-alpha(2)AP, and thereby, the rate of alpha(2)AP incorporation into fibrin. Therefore, the Arg6Trp polymorphism may play a significant role in governing the long-term deposition/removal of intravascular fibrin.

Effect of fibroblast activation protein and alpha2-antiplasmin cleaving enzyme on collagen types I, III, and IV.

The circulating enzyme, alpha2-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 alpha2-antiplasmin to a derivative more easily cross-linked 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. Amino-terminal 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.

Antiplasmin-cleaving enzyme is a soluble form of fibroblast activation protein.

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 (in(280 nm)(1%)) of 20.2 and 20.5, common sequences of tryptic peptides, and cross-reactivity with FAP antibody. APCE and rFAP are homodimers with monomeric subunits of 97 and 93 kDa. Only homodimers appear to have enzymatic activity, with essentially identical kinetics toward Met-alpha2-antiplasmin (Met-alpha2AP) and peptide substrates. APCE and rFAP cleave both Pro3-Leu4 and Pro12-Asn13 bonds of Met-alpha2AP, but relative kcat/Km values for Pro12-Asn13 are about 16-fold higher than for Pro3-Leu4. APCE and rFAP demonstrate higher kcat/Km values toward a peptide modeled on P4-P4' sequence surrounding the Pro12-Asn13 primary cleavage site than for Z-Gly-Pro-AMC and Ala-Pro-AFC substrates. These data support APCE as a soluble derivative of FAP and Met-alpha2AP as its physiologic substrate. Conversion of Met-alpha2AP by membrane or soluble FAP to the more easily fibrin-incorporable form, Asn-alpha2AP, may increase plasmin inhibition within fibrin surrounding certain neoplasms and have an impact on growth and therapeutic susceptibility.

Alpha2-antiplasmin: potential therapeutic roles in fibrin survival and removal.

Alpha2-antiplasmin (alpha2AP) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Two forms of alpha2AP circulate in human plasma: a 464-residue protein with methionine as the amino-terminus (Met-alpha2AP) and an N-terminally-shortened 452-residue form with asparagine as the amino-terminus (Asn-alpha2AP). Human plasma alpha2AP concentration is 1 micro M and consists of approximately 30% Met-alpha2AP and approximately 70% Asn-alpha2AP. The major form (Asn-alpha2AP) is rapidly crosslinked to fibrin during blood clotting by activated coagulation factor XIII and as a consequence, fibrin becomes more resistant to fibrinolysis. It is apparent that alpha2AP is important in modulating the effectiveness and persistence of fibrin with respect to its susceptibility to digestion and removal by plasmin. Hence, the physiologic role of alpha2AP suggests that it may be a useful target for developing more effective treatment of thrombotic diseases. Research on alpha2AP appears to be moving in two main directions: (1) efforts to use variant forms of alpha2AP to reduce bleeding secondary to thrombolytic therapy while not slowing thrombolysis; and (2) efforts to use variant forms to diminish the activity of alpha2AP as a plasmin inhibitor so that fibrinolysis becomes enhanced. Methods to accomplish these two goals mostly involve manipulation of defined functional domains within the molecular structure of alpha2AP, or inhibition of a newly described novel plasma proteinase, termed antiplasmin-cleaving enzyme, that generates the more favorable form of alpha2AP, Asn-alpha2AP, for crosslinking to fibrin. The antiplasmin-cleaving enzyme has similarity in primary structure and catalytic properties to fibroblast activation protein/seprase. This review summarizes recent studies that may hold promise for modulating alpha2AP activity and its interactions with certain proteins as new therapeutic strategies for preventing and treating thrombotic disorders.

A novel plasma proteinase potentiates alpha2-antiplasmin inhibition of fibrin digestion.

Human alpha2-antiplasmin (alpha2AP), also known as alpha2-plasmin inhibitor, is the major inhibitor of the proteolytic enzyme plasmin that digests fibrin. There are 2 N-terminal forms of alpha2AP that circulate in human plasma: a 464-residue protein with Met as the N-terminus, Met-alpha2AP, and a 452-residue version with Asn as the N-terminus, Asn-alpha2AP. We have discovered and purified a proteinase from human plasma that cleaves the Pro12-Asn13 bond of Met-alpha2AP to yield Asn-alpha2AP and have named it antiplasmin-cleaving enzyme (APCE). APCE is similar in primary structure and catalytic properties to membrane-bound fibroblast activation protein/seprase for which a physiologic substrate has not been clearly defined. We found that Asn-alpha2AP becomes cross-linked to fibrin by activated factor XIII approximately 13 times faster than native Met-alpha2AP during clot formation and that clot lysis rates are slowed in direct proportion to the ratio of Asn-alpha2AP to Met-alpha2AP in human plasma. We conclude that APCE cleaves Met-alpha2AP to the derivative Asn-alpha2AP, 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-alpha2AP could result in a more rapid removal of fibrin by plasmin during atherogenesis, thrombosis, and inflammatory states.

Effect of a synthetic carboxy-terminal peptide of alpha(2)-antiplasmin on urokinase-induced fibrinolysis.

alpha(2)-Antiplasmin (alpha(2)AP) interferes with the binding of plasminogen to fibrin because lysine residues in its carboxy-terminal region compete with those in fibrin, presumably the same way that free lysine or epsilon-aminocaproic acid (EACA) inhibits plasminogen binding to fibrin. While this overall process causes an inhibition of fibrinolysis, the converse was observed with a 26-residue synthetic peptide (AP26) corresponding to the carboxy-terminal region of alpha(2)AP. The AP26 peptide, in fact, accelerated urokinase-induced lysis of (1) fully crosslinked fibrin with complete gamma-dimer and alpha-polymer formation; (2) partially crosslinked fibrin that had undergone only gamma-dimerization; and (3) noncrosslinked fibrin. The AP26 peptide also inhibited factor XIIIa-catalyzed crosslinking of fibrin alpha-chains, and this also accelerated lysis of fibrin. EACA had no effect. In the presence of noncrosslinked fibrin, AP26 promoted plasminogen activation by urokinase and fibrinolysis. EACA only slightly increased the rate of plasminogen activation, and as expected, it inhibited fibrinolysis. Since AP26 peptide enhanced the lysis of partially crosslinked and noncrosslinked fibrin, our results indicate that inhibition of factor XIIIa-catalyzed alpha-polymer formation by AP26, although associated with accelerated fibrinolysis, is not the primary mechanism. Instead, our data support the conclusion that AP26 enhances the conversion of plasminogen to plasmin approximately 5-fold, probably by inducing a conformational change in plasminogen structure just as occurs with low concentrations of lysine or EACA. At higher concentrations, however, AP26 apparently does not approach the avidity or affinity of lysine or EACA for the kringle structures of plasminogen or plasmin so that their binding to fibrin is blocked. Whether AP26 alone, or as part of another molecule, could have potential for enhancing thrombolysis will require further study.