Time filter

Source Type

Gohda K.,Computer aided Molecular Modeling Research Center | Fujimori K.,Osaka University of Pharmaceutical Sciences | Teno N.,Hiroshima International University | Wanaka K.,Kobe Research Projects on Thrombosis and Haemostasis | Tsuda Y.,Kobe Gakuin University
Chemical Biology and Drug Design | Year: 2014

We here strove to overcome the limitations of expression analyses such as PCR and IHC, based on molecular recognition between target and probe molecules, by designing synthetic substrates specific to the target molecules to directly estimate the enzymatic functionality in situ. The specific substrate contains a probing unit, which is an organic fragment for specific enzyme binding, and a reactive unit, which is a natural peptide subject to catalysis. In this study, the activation of plasminogen to plasmin was examined in MDA-MB231 breast cancer cells using the plasmin-specific synthetic substrates designed from their inhibitors. The localization and function of the activated plasmin were successfully visualized by fluorophore combined with the specific substrate concurrently. This would be the first time for activated plasmin at work in situ by direct observation. Our concept to directly monitor the functionality of target enzymes can be used straightforwardly for other proteases such as cathepsins or caspases. Also, this substrate concept as a 'tailor-made substrate' would be utilized as a novel functional molecular probe in vivo with appropriate detectable probes. We designed synthetic substrates specific to target molecules to directly estimate enzymatic functionality in situ. It contains a probing unit, an organic fragment specific for enzyme-binding; and a reactive unit, a natural peptide subject to catalysis. In this study, activation of plasminogen to plasmin was examined in MDA-MB231 breast cancer cells, and the localization and function of plasmin were successfully visualized by fluorophore in the substrate. This would be the first time for activated plasmin at work in situ by direct observation. © 2013 John Wiley & Sons A/S.


Matsuo T.,Hyogo Prefectural Awaji Hospital | Motohashi S.,Yamanashi University | Wanaka K.,Kobe Research Projects on Thrombosis and Haemostasis | Walenga J.M.,Loyola University
Clinical and Applied Thrombosis/Hemostasis | Year: 2015

To study the production of anti-platelet factor 4 (anti-PF4)/heparin complex antibodies of Ig (immunoglobulin) G/IgA/IgM using enzyme-linked immunosorbent assay (ELISA; heparin-induced thrombocytopenia [HIT] antibodies) in 79 patients undergoing cardiovascular surgery, we employed Δoptical density (OD) as a marker of HIT-antibody production. The ΔODs were calculated from the differences in the ODs using ELISA. Patient were classified into 3 ΔOD ranges: ΔOD ≥ 1.0, ΔOD ≥ 0.4 to <1.0, and ΔOD < 0.4. The underlying disease, time course of the postoperative platelet count, d-dimer level, postoperative brain magnetic resonance imaging (MRI), use of cardiopulmonary bypass and postoperative thrombocytosis were not considered for the 3 ΔOD classifications. None of the 6 patients with ΔOD ≥1.0 and a positive functional assay was diagnosed with HIT due to the absence of HIT-derived thrombocytopenia. In conclusion, HIT-antibody production increased until day 7 after heparin cessation and reached a trace level on day 14. It was demonstrated that HIT-antibody production is in remission unless there is any evidence of a further increase during the second week postsurgery. © The Author(s) 2013


Teno N.,Hiroshima International University | Gohda K.,Computer Aided Molecular Modeling Research Center | Wanaka K.,Kobe Research Projects on Thrombosis and Haemostasis | Tsuda Y.,Kobe Gakuin University | And 3 more authors.
Bioorganic and Medicinal Chemistry | Year: 2014

In the development of plasmin inhibitors, a novel chemotype, pyrrolopyrimidine scaffold possessing two motifs, a hydantoin-containing P4 moiety and a warhead-containing P1 moiety, is uncovered. A unique feature of the new line of the plasmin inhibitors is that the interaction between the plasmin inhibitors and key subsites in plasmin can be controlled by a spacer like hydantoin. The application of the novel chemotype is demonstrated by 1n and provides further evidence on the importance of hydantoin as the spacer. © 2014 Elsevier Ltd. All rights reserved.


Teno N.,Hiroshima International University | Gohda K.,Computer aided Molecular Modeling Research Center | Wanaka K.,Kobe Research Projects on Thrombosis and Haemostasis | Sueda T.,Hiroshima International University | Tsuda Y.,Kobe Gakuin University
Bioorganic and Medicinal Chemistry Letters | Year: 2011

Lysine-nitrile derivatives having a trisubstituted benzene, which belongs to a new chemical class, were prepared and tested for inhibitory activities against plasmin and the highly homologous plasma kallikrein and urokinase. The use of the novel chemotype in the development of plasmin inhibitors has been demonstrated by derivatives of compound 9. © 2011 Elsevier Ltd. All rights reserved.


Teno N.,Hiroshima International University | Otsubo T.,Hiroshima International University | Gohda K.,Computer aided Molecular Modeling Research Center | Wanaka K.,Kobe Research Projects on Thrombosis and Haemostasis | And 4 more authors.
Journal of Peptide Science | Year: 2012

Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC50=7.7-11μM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin. © 2012 European Peptide Society and John Wiley & Sons, Ltd.


PubMed | Kobe Gakuin University, Kobe Research Projects on Thrombosis and Haemostasis, Hiroshima International University and Computer aided Molecular Modeling Research Center
Type: Journal Article | Journal: Bioorganic & medicinal chemistry letters | Year: 2016

In this letter we report the design and synthesis of a series of plasmin inhibitors, which share the amino acid-based linker with limited free rotation between the hydantoin moiety and the benzimidazole scaffold. Our studies led to potent plasmin inhibitors and yielded important new insights into their structure-activity relationship for binding to the active site of plasmin.


PubMed | Loyola University, Yamanashi University, Hyogo Prefectural Awaji Hospital and Kobe Research Projects on Thrombosis and Haemostasis
Type: Clinical Trial | Journal: Clinical and applied thrombosis/hemostasis : official journal of the International Academy of Clinical and Applied Thrombosis/Hemostasis | Year: 2015

To study the production of anti-platelet factor 4 (anti-PF4)/heparin complex antibodies of Ig (immunoglobulin) G/IgA/IgM using enzyme-linked immunosorbent assay (ELISA; heparin-induced thrombocytopenia [HIT] antibodies) in 79 patients undergoing cardiovascular surgery, we employed optical density (OD) as a marker of HIT-antibody production. The ODs were calculated from the differences in the ODs using ELISA. Patient were classified into 3 OD ranges: OD 1.0, OD 0.4 to <1.0, and OD < 0.4. The underlying disease, time course of the postoperative platelet count, D-dimer level, postoperative brain magnetic resonance imaging (MRI), use of cardiopulmonary bypass and postoperative thrombocytosis were not considered for the 3 OD classifications. None of the 6 patients with OD 1 .0 and a positive functional assay was diagnosed with HIT due to the absence of HIT-derived thrombocytopenia. In conclusion, HIT-antibody production increased until day 7 after heparin cessation and reached a trace level on day 14. It was demonstrated that HIT-antibody production is in remission unless there is any evidence of a further increase during the second week postsurgery.


PubMed | Kobe Gakuin University, Kobe Research Projects on Thrombosis and Haemostasis, Hiroshima International University and Computer Aided Molecular Modeling Research Center
Type: Journal Article | Journal: Bioorganic & medicinal chemistry | Year: 2015

Here we report a series of plasmin inhibitors which were originally derived from the parent structure of 1 and 2. Our efforts focused on the optimization of the P4 moiety of 2 and on the quest of alternative scaffold to pyrrolopyrimidine in the parent compounds. The results of the former gave us pivotal information on the further optimization of the P4 moiety in plasmin inhibitors and those of the latter revealed that appropriate moieties extending from the benzimidazole scaffold engaged with S4 pocket in the active site of plasmin.


Ishihara M.,Tokyo Medical University | Nishida C.,Tokyo Medical University | Tashiro Y.,Tokyo Medical University | Gritli I.,Tokyo Medical University | And 15 more authors.
Leukemia | Year: 2012

Activation of the fibrinolytic system during lymphoma progression is a well-documented clinical phenomenon. But the mechanism by which the fibrinolytic system can modulate lymphoma progression has been elusive. The main fibrinolytic enzyme, plasminogen (Plg)/plasmin (Plm), can activate matrix metalloproteinases (MMPs), such as MMP-9, which has been linked to various malignancies. Here we provide the evidence that blockade of Plg reduces T-cell lymphoma growth by inhibiting MMP-9-dependent recruitment of CD11b F4/80 myeloid cells locally within the lymphoma tissue. Genetic Plg deficiency and drug-mediated Plm blockade delayed T-cell lymphoma growth and diminished MMP-9-dependent CD11b F4/80 myeloid cell infiltration into lymphoma tissues. A neutralizing antibody against CD11b inhibited T-cell lymphoma growth in vivo, which indicates that CD11b myeloid cells have a role in T-cell lymphoma growth. Plg deficiency in T-cell lymphoma-bearing mice resulted in reduced plasma levels of the growth factors vascular endothelial growth-A and Kit ligand, both of which are known to enhance myeloid cell proliferation. Collectively, the data presented in this study demonstrate a previously undescribed role of Plm in lymphoproliferative disorders and provide strong evidence that specific blockade of Plg represents a promising approach for the regulation of T-cell lymphoma growth. © 2012 Macmillan Publishers Limited All rights reserved.


Matsuo T.,Hyogo Prefectural Awaji Hospital | Wanaka K.,Kobe Research Projects on Thrombosis and Haemostasis | Walenga J.M.,Loyola University
Clinical and Applied Thrombosis/Hemostasis | Year: 2013

A retrospective study was performed to elucidate the characteristics of heparin-induced thrombocytopenia (HIT) in newly treated hemodialysis (HD) patients who essentially required anticoagulation with unfractionated heparin (UFH). Seventy-eight patients suspected of having HIT within 3 months of starting HD with UFH were selected for this study. Their platelet counts were routinely followed, and anti-PF4/heparin complex antibodies (HIT antibodies) were measured with enzyme-linked immunosorbent assay (ELISA) until the titer became negative. The characteristics of thrombocytopenia were a platelet count of ≤150 × 109/L and/or decrease of ≥30% and as caused by the intermittent use (3 times/a week) of UFH during HD. Fifty-five patients showed unexpected clotting in the extracorporeal circuit and/or arteriovenous fistula (AVF) thrombosis, while 23 patients had neither of these complications. The patients were classified into HD-related and non-HD-related thrombus groups. The impact of various combinations of the 3 clinical factors (thrombocytopenia, timing, and HD-related thrombus) and the results of ELISA as a laboratory factor were examined. A combination of 2 platelet factors (thrombocytopenia and timing) and ELISA positivity did not reveal the presence of HIT, while a combination of the 3 clinical factors and a positive ELISA improved the accuracy of HIT diagnosis. The findings on the 4-factor combination were supported by high rates of seroconversion in a serotonin release assay. Combining appropriate clinical factors and a positive ELISA may lead to the proper management of HD patients suspected of having HIT. In conclusion, while HD patients showed a drop of ≤150 × 109/L or ≥30% on days 7 to 30, unexpected clotting in the circuit and/or AVF thrombosis was considered as a sign of HIT development. © 2012 The Author(s).

Loading Kobe Research Projects on Thrombosis and Haemostasis collaborators
Loading Kobe Research Projects on Thrombosis and Haemostasis collaborators