title: Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain.
abstract: Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2' residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin-KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin-KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes.
authors.affiliation: Department of Orthopaedic Surgery, UCLA School of Medicine , Los Angeles, California 90095, United States.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Kumar
authors.forename: Kanagasabai
authors.initials: K
authors.lastname: Vadivel
authors.forename: Amy E
authors.initials: AE
authors.lastname: Schmidt
authors.forename: Godwin I
authors.initials: GI
authors.lastname: Ogueli
authors.forename: Sathya M
authors.initials: SM
authors.lastname: Ponnuraj
authors.forename: Nalaka
authors.initials: N
authors.lastname: Rannulu
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Bajaj
authors.forename: S Paul
authors.initials: SP
authors.lastname: Bajaj
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005341:Fibrinolysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D018082:Kringles / Q000187:drug effects / Q000502:physiology*; D022125:Lacerations / Q000188:drug therapy; D008099:Liver / Q000293:injuries; D051379:Mice; D010958:Plasminogen / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors; D020298:U937 Cells
chemical_list: D006023:Glycoproteins; D011956:Receptors, Cell Surface; C021240:tissue-factor-pathway inhibitor 2; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin