Differential inhibition of platelet function by cilostazol in combination with clopidogrel.

PURPOSE: To assess the antiplatelet effect of cilostazol clinically, we compared the effects of cilostazol in combination with clopidogrel on various platelet function tests. METHODS: We recruited patients with ischemic stroke at high risk of recurrence who were treated with clopidogrel alone within 180 days after stroke onset. Subjects underwent baseline platelet function tests, and were then randomly assigned to receive dual antiplatelet therapy (DAPT) comprising clopidogrel and cilostazol or clopidogrel monotherapy (SAPT). After 6 months, platelet function was measured again and compared to that at baseline in each group, and the rate of change was compared between groups. RESULTS: Thirty-four patients were enrolled, but 4 patients were excluded for various reasons. In total, 30 subjects (13 in DAPT and 17 in SAPT group) were analyzed. Adenosine diphosphate- and collagen-induced aggregation, VerifyNow P2Y12 reaction units, vasodilator-stimulated phosphoprotein (platelet reactivity index: PRI) and plasma p-selectin concentration were significantly lower (P = 0.004, 0.042, 0.049, 0.003 and 0.006 respectively), while VerifyNow % inhibition was significantly higher at 6 months compared to baseline (P = 0.003) in the DAPT group only. Comparison of the rate of change in each parameter from baseline to 6 months showed that while PRI decreased at a greater rate (P = 0.012), VerifyNow % inhibition increased at a greater rate (P = 0.003) in the DAPT group than the SAPT group. CONCLUSIONS: The inhibitory effects of adjunctive cilostazol added to clopidogrel on platelet function differed by type of platelet function test. VerifyNow % inhibition and PRI were more inhibited than the other platelet function tests. TRIAL REGISTRATION: CSPS.com substudy in TWMU (UMIN000026672), registered on April 1, 2017. This study was performed as a substudy of CSPS.com (UMIN000012180, registered on October 31, 2013) and was retrospectively registered.

A modified microchip-based flow chamber system for evaluating thrombogenicity in patients with thrombocytopenia.

BACKGROUND: In the intensive care unit (ICU), patients with thrombocytopenia are at high risk for bleeding and should be assessed for their thrombogenic potential. However, the analytical conditions of conventional hemostatic tests are unsuitable for the evaluation of low-platelet samples. Here we aimed to establish suitable analytical conditions with the Total Thrombus-formation Analysis System (T-TAS) for quantitative assessment of thrombogenic potential in patients with thrombocytopenia and to investigate how T-TAS values relate to bleeding symptoms and the effects of platelet transfusion. METHODS: Modified chips with a different chamber depth were developed for the analysis of low-platelet samples in the T-TAS. We included 10 adult patients admitted to the ICU of Kagoshima University Hospital who required platelet transfusion. Patients were divided into major and minor bleeding groups according to their bleeding scale before platelet transfusion. The thrombogenic potential of these patients before and after platelet transfusion was assessed with hemostatic function tests, including rotational thromboelastometry, multiplate aggregometry, and the T-TAS. RESULTS: Analysis of low-platelet samples revealed that, compared with the conventional chip (80-µm-deep chamber), the modified chip (50-µm-deep chamber) achieved higher sensitivity in detecting elevation of flow pressure caused by growth of an occlusive thrombus in the T-TAS analytical chamber. All patients in the minor bleeding group retained thrombogenic potential that occluded the modified chip (occlusion time 16.3 ± 3.3 min), whereas most patients in the major bleeding group were unable to occlude the modified chip during the 30-min measurement (P <  0.01). The recovery of thrombogenic potential after platelet transfusion was confirmed with the T-TAS and correlated with the function, rather than the count, of transfused platelets. Among all evaluated parameters in hemostatic function tests, only the T-TAS showed significant differences in occlusion time and area under the curve both between the minor and major bleeding groups and between pre- and post-platelet transfusion. CONCLUSIONS: We developed a modified microchip-based flow chamber system that reflects the hemostatic function of patients with thrombocytopenia.

Proximity proteomics identifies cancer cell membrane cis-molecular complex as a potential cancer target.

Cancer-specific antigens expressed in the cell membrane have been used as targets for several molecular targeted strategies in the last 20 years with remarkable success. To develop more effective cancer treatments, novel targets and strategies for targeted therapies are needed. Here, we examined the cancer cell membrane-resident "cis-bimolecular complex" as a possible cancer target (cis-bimolecular cancer target: BiCAT) using proximity proteomics, a technique that has attracted attention in the last 10 years. BiCAT were detected using a previously developed method termed the enzyme-mediated activation of radical source (EMARS), to label the components proximal to a given cell membrane molecule. EMARS analysis identified some BiCAT, such as close homolog of L1 (CHL1), fibroblast growth factor 3 (FGFR3) and α2 integrin, which are commonly expressed in mouse primary lung cancer cells and human lung squamous cell carcinoma cells. Analysis of cancer specimens from 55 lung cancer patients revealed that CHL1 and α2 integrin were highly co-expressed in almost all cancer tissues compared with normal lung tissues. As an example of BiCAT application, in vitro simulation of effective drug combinations used for multiple drug treatment strategies was performed using reagents targeted to BiCAT molecules. The combination treatment based on BiCAT information moderately suppressed cancer cell proliferation compared with single administration, suggesting that the information about BiCAT in cancer cells is useful for the appropriate selection of the combination among molecular targeted reagents. Thus, BiCAT has the potential to contribute to several molecular targeted strategies in future.

Five decades of research on mitochondrial NADH-quinone oxidoreductase (complex I).

NADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson's and Alzheimer's, and it is proposed to be involved in aging. Complex I has one non-covalently bound FMN, eight to 10 iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membrane in situ analysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone (SQ) state. In the aerobic steady state of respiration the semi-ubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, SQ signals have been resolved which were designated SQNf and SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the SQ radicals and their possible part in proton pumping.

Comparative evaluation of direct thrombin and factor Xa inhibitors with antiplatelet agents under flow and static conditions: an in vitro flow chamber model.

Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s(-1). Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy.

Antithrombotic effects of PAR1 and PAR4 antagonists evaluated under flow and static conditions.

INTRODUCTION: Thrombin-mediated activation of human platelets involves the G-protein-coupled protease-activated receptors PAR1 and PAR4. Inhibition of PAR1 and/or PAR4 is thought to modulate platelet activation and subsequent procoagulant reactions. However, the antithrombotic effects of PAR1 and PAR4 antagonism have not been fully elucidated, particularly under flow conditions. MATERIALS AND METHODS: A microchip-based flow chamber system was used to evaluate the influence of SCH79797 (PAR1 antagonist) and YD-3 (PAR4 antagonist) on thrombus formation mediated by collagen and tissue thromboplastin at shear rates simulating those experienced in small- to medium-sized arteries (600s(-1)) and large arteries and small veins (240s(-1)). RESULTS: At a shear rate of 600s(-1), SCH79797 (10µM) efficiently reduced fibrin-rich platelet thrombi and significantly delayed occlusion of the flow chamber capillary (1.44 fold of control; P<0.001). The inhibitory activity of SCH79797 was diminished at 240s(-1). YD-3 (20µM) had no significant effect at either shear rate. The antithrombotic effects of SCH79797 were significantly augmented when combined with aspirin and AR-C66096 (P2Y12 antagonist), but not with YD-3. In contrast, no significant inhibition of tissue factor-induced clot formation under static conditions was observed in blood treated with SCH79797 and YD-3, although thrombin generation in platelet-rich plasma was weakly delayed by these antagonists. CONCLUSIONS: Our results suggest that the antithrombotic activities of PAR1 and/or PAR4 antagonism is influenced by shear conditions as well as by combined platelet inhibition with aspirin and a P2Y12-antagonist.

Studies of a microchip flow-chamber system to characterize whole blood thrombogenicity in healthy individuals.

INTRODUCTION: A whole blood flow-chamber system, the Total Thrombus-formation Analysis System (T-TAS), was developed for quantitative analysis of platelet thrombus formation (PTF) using microchips with thrombogenic surfaces (collagen, PL chip; collagen plus tissue thromboplastin, AR chip) under shear stress conditions. We evaluated the usefulness of the T-TAS for assessing individual thrombogenicity compared with other platelet function tests. MATERIALS AND METHODS: Blood samples from 31 healthy volunteers were applied to the T-TAS to measure PTF starting time (T10: time to reach 10 kPa), occlusion time (T60 for PL chip; T80 for AR chip), and area under the curve (AUC10, area under curve until 10 min for PL chip; AUC30, 30 min for AR chip) under various shear rates (1000, 1500, 2000s(-1) for PL chip; 300 s(-1) for AR chip). Platelet functions were also tested using platelet aggregometry, the PFA-100 (collagen and epinephrine [C/EPI], collagen and adenosine diphosphate [C/ADP]), and the VerifyNow P2Y12 assay. RESULTS: Individual pressure waveforms, including PTF starting and ending points, varied among healthy subjects. In the PL chip, T10 and AUC10 showed a shear-dependent correlation with C/EPI or C/ADP. VerifyNow P2Y12 values were not significantly associated with the parameters of the T-TAS. Platelet counts were correlated with all AR measurements, and mostly with PL measurements. CONCLUSION: The results of the T-TAS were associated with those of the PFA-100 in many respects, indicating that its characteristics are related to shear-induced PTF. The T-TAS showed few correlations with platelet aggregometry and the VerifyNow P2Y12 assay. The T-TAS may allow for the measurement of comprehensive parameters of individual thrombogenicity under whole blood flow conditions.

A new trend in the complex I research field.

To date, the 17th European Bioenergetics Conference (EBEC) in Freiburg, Germany (September 15-20, 2012) was the most exciting conference for the complex I (NADH-Q oxidoreductase) research field. A whole day of oral presentations was dedicated to complex I, including three plenary lectures and 11 symposium talks. In addition, 43 posters were dedicated to complex I projects. Here is a brief summary of an exciting paradigm shift from 'structure major' to 'structure plus function', clearly observed in the complex I research field.

Analysing responses to aspirin and clopidogrel by measuring platelet thrombus formation under arterial flow conditions.

High residual platelet aggregability and circulating platelet-monocyte aggregates in patients administered aspirin and clopidogrel are associated with ischaemic vascular events. To determine the relevance of these factors with residual thrombogenicity, we measured platelet thrombus formation using a microchip-based flow-chamber system in cardiac patients receiving aspirin and/or clopidogrel, and evaluated its correlation with agonist-inducible platelet aggregation and platelet-monocyte aggregates. Platelet thrombus formation was analysed by measuring flow pressure changes due to the occlusion of micro-capillaries and was quantified by calculating AUC 10 (area under the flow pressure curve. The growth and stability of platelet thrombi that formed inside microchips at shear rates of 1000, 1500, and 2000 s⁻¹ were markedly reduced in patients receiving aspirin and/or thienopyridine compared to healthy controls (n=33). AUC 10 values of aspirin therapy patients (n=20) were significantly lower and higher than those of healthy controls and dual antiplatelet therapy patients (n=19), respectively, and showed relatively good correlations with collagen-induced platelet aggregation and platelet-monocyte aggregates at 1000 and 1500 s⁻¹ (rs >0.59, p<0.01). In contrast, AUC 10 in dual antiplatelet therapy patients was significantly correlated with ADP-induced platelet aggregation at all examined shear rates (rs >0.59, p<0.01), but did not correlate with collagen-induced aggregation. Aspirin monotherapy patients with high residual platelet thrombogenicity also exhibited significant elevations in both collagen-induced platelet aggregation and platelet-monocyte aggregates. Our results, although preliminary, suggest that residual platelet thrombogenicity in aspirin-treated patients is associated with either collagen-induced platelet aggregation or circulating platelet-monocyte aggregates, but it is predominantly dependent on ADP-induced platelet aggregation in patients receiving dual antiplatelet therapy.

Fibroblast growth factor receptor 3 (FGFR3) associated with the CD20 antigen regulates the rituximab-induced proliferation inhibition in B-cell lymphoma cells.

Rituximab is reported to inhibit the proliferation of lymphoma cells through an unknown CD20-mediated signal transduction pathway. Herein, we investigated cell surface molecules involved in the CD20-mediated signal transduction pathway by using a recently developed technique named enzyme-mediated activation of radical sources. Using this method, we found that under stimulation with rituximab and another anti-CD20 antibody B-Ly1, CD20 was physically associated with fibroblast growth factor receptor 3 (FGFR3) as well as some other receptor tyrosine kinases in Raji cells. However, under stimulation with a noncytotoxic anti-CD20 antibody 2H7, CD20 was not associated with FGFR3 but with the PDGF receptor ß. When the tyrosine kinase activity of FGFR3 was inhibited by the chemical inhibitor PD173074 or an siRNA knockdown strategy, the proliferation inhibition by rituximab was attenuated, indicating that FGFR3 participates in the rituximab-dependent signal transduction pathway leading to proliferation inhibition. These observations raise the possibility that concomitant targeted therapy toward FGFR3 might improve the efficacy and safety of the rituximab therapy.

Electron transfer in subunit NuoI (TYKY) of Escherichia coli NADH:quinone oxidoreductase (NDH-1).

Bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1) consists of a peripheral and a membrane domain. The peripheral domain catalyzes the electron transfer from NADH to quinone through a chain of seven iron-sulfur (Fe/S) clusters. Subunit NuoI in the peripheral domain contains two [4Fe-4S] clusters (N6a and N6b) and plays a role in bridging the electron transfer from cluster N5 to the terminal cluster N2. We constructed mutants for eight individual Cys-coordinating Fe/S clusters. With the exception of C63S, all mutants had damaged architecture of NDH-1, suggesting that Cys-coordinating Fe/S clusters help maintain the NDH-1 structure. Studies of three mutants (C63S-coordinating N6a, P110A located near N6a, and P71A in the vicinity of N6b) were carried out using EPR measurement. These three mutations did not affect the EPR signals from [2Fe-2S] clusters and retained electron transfer activities. Signals at g(z) = 2.09 disappeared in C63S and P110A but not in P71A. Considering our data together with the available information, g(z,x) = 2.09, 1.88 signals are assigned to cluster N6a. It is of interest that, in terms of g(z,x) values, cluster N6a is similar to cluster N4. In addition, we investigated the residues (Ile-94 and Ile-100) that are predicted to serve as electron wires between N6a and N6b and between N6b and N2, respectively. Replacement of Ile-100 and Ile-94 with Ala/Gly did not affect the electron transfer activity significantly. It is concluded that conserved Ile-100 and Ile-94 are not essential for the electron transfer.

EPR detection of two protein-associated ubiquinone components (SQ(Nf) and SQ(Ns)) in the membrane in situ and in proteoliposomes of isolated bovine heart complex I.

The success of Sazanov's group in determining the X-ray structure of the whole bacterial complex I is a great contribution to the progress of complex I research. In this mini-review of 35years' history of my laboratory and collaborators, we characterized the function of protein-associated semiquinone molecules in the proton-pumping mechanism in complex I (NADH-quinone oxidoreductase). We have constructed most of the frame work of our hypothesis, utilizing EPR techniques before the X-ray structures of complex I were reported by Sazanov's and Brandt's groups. One of the semiquinones (SQ(Nf)) is extremely sensitive to a proton motive force imposed on the energy-transducing membrane, while the other (SQ(Ns)) is insensitive. Their sensitivity to rotenone inhibition also differs. These differences were exploited using tightly coupled bovine heart submitochondrial particles with a high respiratory control ratio (>8). We determined the distance between SQ(Nf) and iron-sulfur cluster N2 on the basis of their direct spin-spin interaction. We are extending this line of work using reconstituted bovine heart complex I proteoliposomes which shows a respiratory control ratio >5. Two frontier research groups support our view point based on their mutagenesis studies. High frequency (33.9GHz; Q-band) EPR experiments appear to favor our two-semiquinone model. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction.

We previously reported a simple method to analyze the interaction of cell-surface molecules in living cells. This method termed enzyme-mediated activation of radical sources (EMARS) is featured by radical formation of the labeling reagent by horseradish peroxidase (HRP). Herein, we propose an approach to the cell-surface molecular interactome by using combination of this EMARS reaction and MS-based proteomics techniques. In the current study, we employed a novel labeling reagent, fluorescein-conjugated arylazide. The fluorescein-tagged proteins resulting from the EMARS reaction were directly detected in the electrophoresis gels with a fluorescence image analyzer. These products were also purified and concentrated by immunoaffinity chromatography with anti-fluorescein antibody-immobilized resins. The purified fluorescein-tagged proteins were subsequently subjected to an MS-based proteomics analysis. Analysis using HRP-conjugated cholera toxin subunit B, which recognizes a lipid raft marker, ganglioside GM1, revealed 30 membrane and secreted proteins that were candidates for the cell-surface molecules coclustering with GM1. The proposed approach will provide a clue to study functional molecular interactions in a variety of biological events on the cell surface.

A microchip flow-chamber system for quantitative assessment of the platelet thrombus formation process.

As the pathogenesis of arterial thrombosis often includes platelet thrombus formation (PTF), antiplatelet agents are commonly used for the prevention of thromboembolic events. Here, using a novel microchip flow-chamber system we developed to quantitatively analyze the PTF process, we evaluated the pharmacological efficacies of antiplatelet agents under different arterial shear rates. Hirudin-anticoagulated whole blood was perfused over a collagen-coated microchip at shear rates of 1000, 1500, and 2000s(-1), and PTF in the absence and presence of various antiplatelet agents was observed microscopically and quantified by measuring flow-pressure changes. The onset of PTF was measured as T(10) (time to reach 10 kPa), and AUC(10) (area under the flow pressure curve for the first 10 min) was calculated to quantify the overall stability of the formed thrombus. Aspirin and AR-C66096 (P2Y(12)-antagonist) at high concentrations (50 µM and 1000 nM, respectively) prolonged T(10) only modestly (AR-C66096>aspirin), but effectively decreased AUC(10), resulting in unstable PTF at all examined shear rates. With dual inhibition using both aspirin (25 µM) and ARC-66096 (250 nM), AUC(10) was drastically reduced. Nearly complete suppression of AUC(10) was also observed with abciximab (2 µg ml(-1)) and beraprost (PGI(2)-analog; 4 nM). Although OS-1 (GPIbα-antagonist; 100 nM) prevented complete capillary occlusion, significant amounts of microscopic thrombi were observed on the collagen surface. In contrast to abciximab and beraprost, OS-1 differentially affected PTF under higher shear conditions. Our novel analytical system is capable of distinguishing the pharmacological effects of various antiplatelet agents under physiological shear rates, suggesting that this system may aid in the determination of the appropriate type and dose of antiplatelet agent in the clinical setting.

A comparative study of prothrombin complex concentrates and fresh-frozen plasma for warfarin reversal under static and flow conditions.

Prothrombin complex concentrates (PCCs) and fresh-frozen plasma (FFP) have been clinically used for acute warfarin reversal. The recovery of prothrombin time (PT) or international normalised ratio (INR) is often reported as an endpoint, but haemostatic efficacies of PCCs and FFP may not be fully reflected in static clotting test in platelet-poor plasma. Using various in vitro assays, we compared the effects of two PCC preparations (3-factor PCC; Bebulin and 4-factor PCC; Beriplex) and FFP on warfarin reversal under static and flow conditions. First, we added an aliquot of either PCC (0.3 or 0.72 U/ml) or 20% FFP (v/v) to commercial warfarin plasma (INR 3.2, or 10.3), and then measured PT, factor II, factor VII, and thrombin generation. Subsequently, we collected whole blood samples from six consented warfarin-treated patients with mean INR 3.0 ± 0.5 (range 2.5-3.7), and compared clot formation under flow conditions at 280 s-1 before and after addition of either PCC preparation (0.3 and 0.6 U/ml) or 20% of FFP (v/v). PT/INR were restored by either PCC in plasma with INR 3.0, but they were more effectively corrected by 4-factor PCC than 3-factor PCC in plasma with INR 10.3. Effects of FFP were similar to 0.3 U/ml of PCCs in terms of PT, but FFP was less efficacious than PCCs in recovering thrombin generation or factor II levels. In flow experiments, the onset of thrombus formation was shortened by either PCC, but not by FFP, contrary to shortened PT values. For warfarin reversal 20% volume replacement with FFP is inferior to PCCs.

Celebrating Professor Britton Chance (1913-2010), a founding father of redox sciences.

Renowned great scientist and redox pioneer, Dr. Britton Chance, closed his 97 years of legendary life on November 16, 2010. He was the Eldridge Reeves Johnson emeritus professor of biophysics, physical chemistry, and radiologic physics at the University of Pennsylvania. He achieved fame as a prominent biophysicist and developer of highly innovative biomedical instrumentation. His scientific career stretched over almost one century and he achieved many scientific and engineering breakthroughs throughout his long prolific career. The advances that he and his colleagues achieved led to great strides in our understanding of biology and disease. He was among the first scientists to recognize the importance of free radicals and reactive oxygen species in mitochondrial metabolism and cells as well as to map pathways of redox biology and signaling. Dr. Chance served as a pioneer and inspiration to generations of researchers in the fields of redox biochemistry, metabolism, and disease. He will be missed by all of us in the research community but will live on through his monumental scientific accomplishments, the novel instrumentation he developed, as well as the many scientists whom he trained and influenced.

A new hypothesis on the simultaneous direct and indirect proton pump mechanisms in NADH-quinone oxidoreductase (complex I).

Recently, Sazanov's group reported the X-ray structure of whole complex I [Nature, 465, 441 (2010)], which presented a strong clue for a "piston-like" structure as a key element in an "indirect" proton pump. We have studied the NuoL subunit which has a high sequence similarity to Na(+)/H(+) antiporters, as do the NuoM and N subunits. We constructed 27 site-directed NuoL mutants. Our data suggest that the H(+)/e(-) stoichiometry seems to have decreased from (4H(+)/2e(-)) in the wild-type to approximately (3H(+)/2e(-)) in NuoL mutants. We propose a revised hypothesis that each of the "direct" and the "indirect" proton pumps transports 2H(+) per 2e(-).

M-AAA-thrombin: potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1.

BACKGROUND: Thrombosis is a major cause of morbidity and mortality, and thrombin is a major inducer of thrombus formation. Thus, several antithrombotic agents targeting thrombin have been developed. We previously reported on a thrombin derivative prepared by dual chemical modifications designated as 'M-anhydrothrombin', which possessed both anticoagulant and antiplatelet properties. In order to obtain a more potent antithrombotic thrombin derivative, we prepared a recombinant thrombin mutant and its chemically-modified derivative, and examined their antithrombotic efficacies. METHODS AND RESULTS: We prepared a thrombin mutant, 65A43A205A-Th (designated as "AAA-Th") in which Lys65(70)(1), and His43(57) and Ser205(195) were replaced by Ala, and its chemically-modified derivative at the carboxyl groups (designated as "M-AAA-Th"). M-AAA-Th possessed no enzymatic activity, but retained high affinity and specificity for factor VIII, and prolonged the APTT with a slight effect on PT and no effect on TT. Platelet aggregation induced by PAR1 activation was also suppressed by M-AAA-Th. In contrast, conventional thrombin inhibitors, argatroban and hirulog, substantially prolonged the TT rather than the APTT and the PT. In thromboelastgraph assays, M-AAA-Th suppressed whole blood clotting in a dose-dependent manner, and its effect was synergistically enhanced in the presence of soluble thrombomodulin (s-TM). M-AAA-Th also demonstrated a potent antithrombotic property in the FeCl(3)-induced carotid arterial thrombosis model in guinea pigs with minimum effects on the APTT and PT and no prolongation of the TT. CONCLUSION: M-AAA-Th may be a potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1.

The membrane subunit NuoL(ND5) is involved in the indirect proton pumping mechanism of Escherichia coli complex I.

Complex I pumps protons across the membrane by using downhill redox energy. Here, to investigate the proton pumping mechanism by complex I, we focused on the largest transmembrane subunit NuoL (Escherichia coli ND5 homolog). NuoL/ND5 is believed to have H(+) translocation site(s), because of a high sequence similarity to multi-subunit Na(+)/H(+) antiporters. We mutated thirteen highly conserved residues between NuoL/ND5 and MrpA of Na(+)/H(+) antiporters in the chromosomal nuoL gene. The dNADH oxidase activities in mutant membranes were mostly at the control level or modestly reduced, except mutants of Glu-144, Lys-229, and Lys-399. In contrast, the peripheral dNADH-K(3)Fe(CN)(6) reductase activities basically remained unchanged in all the NuoL mutants, suggesting that the peripheral arm of complex I was not affected by point mutations in NuoL. The proton pumping efficiency (the ratio of H(+)/e(-)), however, was decreased in most NuoL mutants by 30-50%, while the IC(50) values for asimicin (a potent complex I inhibitor) remained unchanged. This suggests that the H(+)/e(-) stoichiometry has changed from 4H(+)/2e(-) to 3H(+) or 2H(+)/2e(-) without affecting the direct coupling site. Furthermore, 50 µm of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific inhibitor for Na(+)/H(+) antiporters, caused a 38 ± 5% decrease in the initial H(+) pump activity in the wild type, while no change was observed in D178N, D303A, and D400A mutants where the H(+) pumping efficiency had already been significantly decreased. The electron transfer activities were basically unaffected by EIPA in both control and mutants. Taken together, our data strongly indicate that the NuoL subunit is involved in the indirect coupling mechanism.

An adder-mixer for adding a few microliters of reagent into an electron paramagnetic resonance quartz tube.

In electron paramagnetic resonance (EPR) experiments, it is very difficult to add a few microliters of reagent into the solution in an EPR tube and to mix it efficiently. This report explains how we could overcome this problem.