The serpin alpha2-antiplasmin (a2AP) has a major role in regulating fibrinolysis. This proposal's goal is to (1) define interactions that determine how much a2AP becomes clot-bound for inhibiting plasmin; and (2) identify means of regulating a2AP so that plasmin activity might be enhanced long-term in diseases where fibrin deposition is a major factor. a2AP is secreted mainly from liver into blood as a polypeptide of 464 amino acids. While circulating, the secreted form (""""""""pro""""""""-a2AP) is cleaved at Pro12-Asn13 to give """"""""activated""""""""- a2AP (a2APact) which is crosslinked to fibrin by FXlIla approximately 5-fold quicker than a2APpro and provides about 80% of fibrin's resistance to plasmin. The proteinase that makes this cleavage is unknown, but our findings suggest it is a proline-specific serine proteinase similar to cell membrane fibroblast activating proteinase (mFAP), which has no known physiologic substrate and does not circulate in blood, whereas the one we have found does, and is termed cFAP by us. Studies will be done to determine if cFAP, which lacks the N-terminal 23 residues of mFAP, is a proteolytic derivative of mFAP. mFAP and cFAP will be purified from human plasma or recombinant cell cultures and their kinetic efficiencies assessed with a2APpro or a fluorogenic peptide as substrate. Substrate specificity and subsite preferences of cFAP will be sought using a synthetic peptide library of sequences surrounding the Pro12-Asn13 bond cleaved by cFAP. A phage display library of random peptide sequences will be used to determine kinetic efficiency of cFAP for each peptide and those with high efficiency will be used in database searches to identify other physiological substrates. Efforts will be made to enhance cFAP inhibitory properties of selected peptides by substituting specific residues based on subsite preferences. Selected domains of cFAP recombinant variants and FAP-inhibitor complex(es) will be subjected to crystallization for x-ray structural analyses. Our pilot data indicate that a2APpro has an Arg6Trp polymorphism, and that Arg6-form is cleaved by cFAP approximately 8x faster than Trp6-form. This fits with our initial findings that a2APact/a2APpro ratios are higher in human plasma containing Arg6-form. Comparisons will be made between a2APpro(Arg6), a2APpro(Trp6) and a2APact as factor XIIla substrates and plasmin inhibitors. Lastly, allelic frequencies of the CGG(Arg6) and TGG(Trp6) polymorphisms will be examined in a normal population for correlation with plasma ratios of a2APact/a2APpro and endogenous fibrinolytic activity.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
Program Officer
Hasan, Ahmed AK
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University of Oklahoma Health Sciences Center
Internal Medicine/Medicine
Schools of Medicine
Oklahoma City
United States
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Christiansen, Victoria J; Jackson, Kenneth W; Lee, Kyung N et al. (2013) Targeting inhibition of fibroblast activation protein-? and prolyl oligopeptidase activities on cells common to metastatic tumor microenvironments. Neoplasia 15:348-58
Lee, K N; Jackson, K W; Christiansen, V J et al. (2011) Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor ?2-antiplasmin. J Thromb Haemost 9:987-96
Tsurupa, Galina; Yakovlev, Sergiy; McKee, Patrick et al. (2010) Noncovalent interaction of alpha(2)-antiplasmin with fibrin(ogen): localization of alpha(2)-antiplasmin-binding sites. Biochemistry 49:7643-51
Lee, Kyung N; Jackson, Kenneth W; Terzyan, Simon et al. (2009) Using substrate specificity of antiplasmin-cleaving enzyme for fibroblast activation protein inhibitor design. Biochemistry 48:5149-58
Mosesson, M W; Siebenlist, K R; Hernandez, I et al. (2008) Evidence that alpha2-antiplasmin becomes covalently ligated to plasma fibrinogen in the circulation: a new role for plasma factor XIII in fibrinolysis regulation. J Thromb Haemost 6:1565-70
Lee, K N; Jackson, K W; Christiansen, V J et al. (2007) Why alpha-antiplasmin must be converted to a derivative form for optimal function. J Thromb Haemost 5:2095-104
Christiansen, Victoria J; Jackson, Kenneth W; Lee, Kyung N et al. (2007) The effect of a single nucleotide polymorphism on human alpha 2-antiplasmin activity. Blood 109:5286-92
Christiansen, Victoria J; Jackson, Kenneth W; Lee, Kyung N et al. (2007) Effect of fibroblast activation protein and alpha2-antiplasmin cleaving enzyme on collagen types I, III, and IV. Arch Biochem Biophys 457:177-86
Narra, Kalyani; Mullins, Stefanie R; Lee, Hyung-Ok et al. (2007) Phase II trial of single agent Val-boroPro (Talabostat) inhibiting Fibroblast Activation Protein in patients with metastatic colorectal cancer. Cancer Biol Ther 6:1691-9
Lee, Kyung N; Jackson, Kenneth W; Christiansen, Victoria J et al. (2004) Alpha2-antiplasmin: potential therapeutic roles in fibrin survival and removal. Curr Med Chem Cardiovasc Hematol Agents 2:303-10

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