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.
BackgroundTo develop a new molecular targeted treatment for brain (AVMs), identification of membrane proteins that are localised on the AVM endothelium is crucial. Current treatment methods are surgery and radiosurgery. However, complete occlusion post radiosurgery are achieved within 3 years, while patient remain at risk of haemorrhage. This study aims to identify potential protein targets in AVM endothelial cells that discriminate these vessels from normal vessels; these proteins targets will be investigated for the molecular therapy of brain AVMs to promote rapid thrombosis after radiosurgery.MethodsWe employed in vitro biotinylation that we developed, and mass spectrometry to detect cell surface-exposed proteins in cultures of murine cerebral endothelial cells (bEnd.3). Two forms of mass spectrometry were applied (iTRAQ-MS and MSE) to identify and quantify membrane protein expression at various time-points following irradiation which simulates a radiosurgical treatment approach. Immunocytochemistry was used to confirm the expression of selected membrane proteins. ProteinPilot V4.0 software was used to analyse the iTRAQ-MS data and the MSE data was analysed using ProteinLynx Global Server version 2.5 software.ResultsThe proteomics data revealed several differentially expressed membrane proteins between irradiated and non-irradiated cells at specific time points, e.g. PECAM-1, cadherin-5, PDI, EPCR and integrins. Immunocytochemistry data confirmed the expression of these proteins.ConclusionCell surface protein biotinylation and proteomics analysis successfully identified membrane proteins from murine brain endothelial cells in response to irradiation. This work suggests potential target protein molecules for evaluation in animal models of brain-AVM.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-017-9151-3) contains supplementary material, which is available to authorized users.
855 Previously, we demonstrated that changing residue Leu17 (BPTI/Aprotinin numbering) to Arg in Kunitz domain 1 (73-residue KD1-L17R) of TFPI-2 abolishes its anticoagulant functions and enhances its plasmin inhibition (Bajaj et al., J Biol Chem 286, 4329–4340, 2011). In that study we used the entire KD1 domain, which in addition to the core structural homologous region of BPTI (58 residues) included 9 residues on the N-terminal and 6 residues on the C-terminal side of the protein. Conformation of these 15 residues may be different in the isolated KD1-domain as compared to the complete TFPI-2 molecule. Thus, these residues could be potentially immunogenic. To address these concerns, we investigated weather N- and C-terminal regions of 73-residue KD1-L17R could be cleaved upon prolonged incubation with thrombin (IIa). Incubation of 73-residue KD1-L17R with IIa for 72 hrs yielded smaller version(s) of KD1-L17R as analyzed by SDS-PAGE. N-terminal sequence and MALDI-TOF/ESI mass spectrometry analyses revealed three closely related species present in the truncated KD1-L17R preparations (Fig. 1). Species 1 has Gly-Asn-Asn as the amino terminus and Val-Pro-Lys as the C-terminus. Species 2 and 3 are similar to species 1 except species 2 is produced after losing Gly and Asn from the N-terminus, whereas species 3 is produced after losing Val-Pro-Lys from the C-terminus. Thus, all three species have the intact core Kunitz domain with minor variations at the N- and C-terminus regions. Further, these species are cleaved at the viable albeit very slow IIa-cleavage sites; herein, these species are collectively referred to as truncated KD1-L17R. A plausible mechanism for proteolysis at these cleavage sites is shown in Fig. 2. Similar to the 73-residue KD1-L17R, the truncated preparations did not inhibit (Ki > 3 μM) plasma kallikrein, factor (F) XIa, FVIIa/soluble tissue factor, FXa, activated protein C, tissue plasminogen activator (tPA), IIa and IIa/soluble thrombomodulin. Importantly, the truncated KD1-L17R preparations inhibited plasmin with Ki ∼1.2 nM. Further, the truncated KD1-L17R inhibited tPA-induced plasma clot fibrinolysis with an apparent IC50 of ∼0.37 μM, a value similar to that obtained with the 73-residue KD1-L17R and BPTI. Two lysine analogues, Epsilon amino caproic acid (EACA) and tranexamic acid (TE) inhibited tPA-induced plasma clot fibrinolysis with an apparent IC50 of ∼80 μM and ∼20 μM, respectively. Further, efficacy of truncated KD1-L17R was tested in a mouse liver laceration model of bleeding. As compared to saline, the amount of blood loss was reduced by ∼65% by truncated KD1-L17R (N=6, p 0.001), ∼70% by BPTI (N=10, p 0.003), ∼52% by TE (N=10, p 0.019) and ∼25% by EACA (N=16, p 0.03). We also observed seizures in four (25%) of the animals treated with a single dose of EACA. In conclusion, truncated KD1-L17R is an effective antifibrinolytic agent similar to the 73-residue KD1-L17R and BPTI/Aprotinin. Although lysine analogues are relatively effective in reducing blood loss, EACA caused seizures in our studies. These observations are consistent with recent reports that one of the major side effects of lysine analogues is seizures (Martin et al., J Cardiothorac Vasc Anesth 25, 20–25, 2011; Koster and Schirmer, Curr Opin Anaesthesiol 24, 92–97, 2011). We conclude that truncated KD1-L17R may serve as an excellent alternative to BPTI and lysine analogues in preventing blood loss during major surgeries including coronary artery bypass graft (CABG) surgery. We are currently expressing the 60-residue KD1-L17R (NH2Asn-Ala-Glu······Ile-Glu-Lys) protein for further efficacy studies. We are also generating additional mutant(s) on the 60-residue KD1-L17R molecule for achieving increased plasmin potency without provoking anticoagulant functions. Supported By HL89661 and HL36365. Disclosures: No relevant conflicts of interest to declare.
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