Decoy Plasminogen Receptor Containing a Selective Kunitz-Inhibitory Domain

• Published in: Biochemistry (Easton) 2014-01, Vol.53 (3), p.505-517
• Main Authors: Kumar, Yogesh, Vadivel, Kanagasabai, Schmidt, Amy E, Ogueli, Godwin I, Ponnuraj, Sathya M, Rannulu, Nalaka, Loo, Joseph A, Bajaj, Madhu S, Bajaj, S. Paul
• Format: Article
• Language: English
• Subjects: active sites; Amino Acid Sequence; Animals; blood; cattle; Fibrinolysin - antagonists & inhibitors; Fibrinolysin - metabolism; fibrinolysis; Fibrinolysis - drug effects; Glycoproteins - genetics; Glycoproteins - pharmacology; Humans; Kringles - drug effects; Kringles - physiology; Lacerations - drug therapy; Liver - injuries; lysine; Mice; mutants; plasmin; plasminogen; Plasminogen - metabolism; Receptors, Cell Surface - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; surface plasmon resonance; t-plasminogen activator; thrombin; Tissue Plasminogen Activator - antagonists & inhibitors; trypsin inhibitors; U937 Cells; valine
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/bi401584b

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 (K d ∼ 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.