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T Nakagaki

Publications and source records attributed to T Nakagaki.

At least 19 recordsLinked to original sources
PubMed2003

Large-scale production and properties of human plasma-derived activated Factor VII concentrate.

BACKGROUND AND OBJECTIVES: An activated Factor VII (FVIIa) concentrate, prepared from human plasma on a large scale, has to date not been available for clinical use for haemophiliacs with antibodies against FVIII and FIX. In the present study, we attempted to establish a large-scale manufacturing process to obtain plasma-derived FVIIa concentrate with high recovery and safety, and to characterize its biochemical and biological properties. MATERIALS AND METHODS: FVII was purified from human cryoprecipitate-poor plasma, by a combination of anion exchange and immunoaffinity chromatography, using Ca2+-dependent anti-FVII monoclonal antibody. To activate FVII, a FVII preparation that was nanofiltered using a Bemberg Microporous Membrane-15 nm was partially converted to FVIIa by autoactivation on an anion-exchange resin. The residual FVII in the FVII and FVIIa mixture was completely activated by further incubating the mixture in the presence of Ca2+ for 18 h at 10 degrees C, without any additional activators. For preparation of the FVIIa concentrate, after dialysis of FVIIa against 20 mm citrate, pH 6.9, containing 13 mm glycine and 240 mm NaCl, the FVIIa preparation was supplemented with 2.5% human albumin (which was first pasteurized at 60 degrees C for 10 h) and lyophilized in vials. To inactivate viruses contaminating the FVIIa concentrate, the lyophilized product was further heated at 65 degrees C for 96 h in a water bath. RESULTS: Total recovery of FVII from 15 000 l of plasma was approximately 40%, and the FVII preparation was fully converted to FVIIa with trace amounts of degraded products (FVIIabeta and FVIIagamma). The specific activity of the FVIIa was approximately 40 U/ micro g. Furthermore, virus-spiking tests demonstrated that immunoaffinity chromatography, nanofiltration and dry-heating effectively removed and inactivated the spiked viruses in the FVIIa. These results indicated that the FVIIa concentrate had both high specific activity and safety. CONCLUSIONS: We established a large-scale manufacturing process of human plasma-derived FVIIa concentrate with a high yield, making it possible to provide sufficient FVIIa concentrate for use in haemophiliacs with inhibitory antibodies.

Blood Component Transfusion↗
PubMed2001

Path finding by tube morphogenesis in an amoeboid organism.

We have studied how the plasmodium of Physarum polycephalum, a large amoeboid cell, is able to track the shortest path between two selected points in a labyrinth. When nutrients are supplied at these points to a sheet-like plasmodium extended fully in a maze, the organism forms a single tube which connects the two sites via the shortest route. During the path finding, plasmodial parts in dead ends of the maze shrink and finally the tube with the minimum-length is selected from the existing possibilities. A simple cellular mechanism based on interacting cellular rhythms may describe the experimental observations.

Animals↗
PubMed2001

Spatiotemporal symmetry in rings of coupled biological oscillators of Physarum plasmodial slime mold.

Spatiotemporal patterns in rings of coupled biological oscillators of the plasmodial slime mold, Physarum polycephalum, were investigated by comparing with results analyzed by the symmetric Hopf bifurcation theory based on group theory. In three-, four-, and five-oscillator systems, all types of oscillation modes predicted by the theory were observed including a novel oscillation mode, a half period oscillation, which has not been reported anywhere in practical systems. Our results support the effectiveness of the symmetric Hopf bifurcation theory in practical systems.

Animals↗
PubMed2001

Induction of acquired factor IX inhibitors in cynomolgus monkey (Macaca fascicularis): a new primate model of hemophilia B.

Inherited hemophilia dog and other transient hemophilic animal models have been used for evaluation of hemostatic agents for use in treatment of hemophilia. We established the first nonhuman primate hemophilic model by immunizing cynomolgus monkeys with human FIX (hFIX) in adjuvants. FIX activities of all three hFIX-immunized monkeys decreased transiently to less than 10% in accordance with prolongation of activated partial thromboplastin time (APTT). Forty micrograms of human factor VIIa (hFVIIa) per kilogram body weight (that was reported to be clinically effective) was administered to the monkey with the highest inhibitor titer to evaluate its usefulness as a hemophilia inhibitor model. Results of thromboelastography (TEG) after the injection demonstrated that the hemostatic effect of FVIIa in this model would be similar to that in hemophiliacs with inhibitors. The antibodies purified from the monkey's plasma by hFIX-immobilized gel were composed of two types: Ca(2+)-dependent and -independent antibodies, with features of IgG(1) and IgG(4). Both types of antibodies reacted to cynomolgus FIX, and only Ca(2+)-dependent antibodies also expressed inhibitory activity against cynomolgus FIX. Immunoblotting analyses of Ca(2+)-dependent antibodies using hFIX and its derivatives suggested that they recognized the Ca(2+)-dependent conformation related to the gamma-carboxyglutamic acid (Gla) domain. Comparison of FIX cDNA from human, cynomolgus monkey, and other species, and the results of immunization of various animals (goats, beagle dogs, rabbits, and rats) with hFIX in adjuvants strongly suggested that the development of acquired FIX inhibitors in the monkeys might be due to high cross-reactivity of the antibodies to molecular mimic antigens, hFIX, and cynomolgus FIX.

Animals↗
PubMed2001

Factor VIIa modified in the 170 loop shows enhanced catalytic activity but does not change the zymogen-like property.

Factor VIIa (VIIa) is an unusual trypsin-type serine proteinase that appears to exist in an equilibrium between minor active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is assumed to shift the equilibrium into the active state. The proteinase domain of VIIa contains a unique structure: a loop formed by a disulfide bond between Cys310 and Cys329, which is five residues longer than those of other trypsin types. To examine the functional role of the loop region, we prepared two mutants of VIIa. One of the mutants, named VII-11, had five extra corresponding residues 316-320 of VII deleted. The other mutant, VII-31, had all of the residues in its loop replaced with those of trypsin. Functional analysis of the two mutants showed that VIIa-11 (Kd = 41 nm) and VIIa-31 (Kd = 160 nm) had lower affinities for soluble tissue factor as compared with the wild-type VIIa (Kd = 11 nm). The magnitude of tissue factor-mediated acceleration of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31 (2-fold) were also smaller than that of wild-type VIIa (30-fold). In the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced activity; the catalytic efficiencies of VIIa-31 toward various chromogenic substrates were 2-18-fold greater than those of the wild-type VIIa. Susceptibility of the alpha-amino group of Ile-153 of VIIa-31 to carbamylation was almost the same as that of wild-type VIIa, suggesting that VIIa-31 as well as wild-type VIIa exist predominantly in the zymogen-like state. Therefore, the tested modifications in the loop region had adverse effects on affinity for tissue factor, disturbed the tissue factor-induced conformational transition, and changed the catalytic efficiency of VIIa, but they did not affect the equilibrium between active and zymogen-like conformational states.

Amino Acid Sequence↗
PubMed2001

Smart behavior of true slime mold in a labyrinth.

Even for humans it is not easy to solve a maze. But the plasmodium of true slime mold, an amoeba-like unicellular organism, has shown an amazing ability to do so. This implies that an algorithm and a high computing capacity are included in the unicellular organism. In this report, we discuss information processing in the microorganism to focus on the issue as to whether the maze-solving behavior is akin to primitive intelligence.

Animals↗
PubMed2001

A novel human metalloprotease synthesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease?

We identified a novel metalloprotease, which could be responsible for cleaving the Tyr842-Met843 peptide bond of von Willebrand factor (vWF). This metalloprotease was purified from Cohn Fraction-I precipitate of human pooled plasma by the combination of gel filtration, DEAE chromatography, and preparative polyacrylamide gel electrophoresis in the presence of SDS. The NH2-terminal amino acid sequence of the isolated protein was: AAGGILHLELLVAVGPDVFQAHQEDTRRY. Based on this sequence, we searched human genomic and EST databases, and identified compatible nucleotide sequences. These results suggested that this protein is a novel metalloprotease, a member of the family of a disintegrin and metalloprotease with thrombospondin type-1 motifs (ADAMTS), and its genomic DNA was mapped to human chromosome 9q34. Multiple human tissue northern blotting analysis indicated that the mRNA encoding this protease spanned approximately 5 kilobases and was uniquely expressed in the liver. Furthermore, we determined the cDNA sequence encoding this protease, and found that this protease was comprised of a signal peptide, a proregion followed by the putative furin cleavage site, a reprolysin-type zinc-metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (TSP1) motif, a cysteine-rich region, a spacer domain, and COOH-terminal TSP1 motif repeats.

ADAM Proteins↗
PubMed2000

Interaction between cell shape and contraction pattern in the Physarum plasmodium.

The relationship between cell shape and rhythmic contractile activity in the large amoeboid organism Physarum polycephalum was studied. The organism develops intricate networks of veins in which protoplasmic sol moved to and fro very regularly. When migrating on plain agar, the plasmodium extends like a sheet and develops dendritic veins toward the rear. After a particular stimulation, the vein organization changes into veinless or vein-network structures. In both structures, the mixing rate of the protoplasm, which is related to communication among contraction oscillators, decreased compared with that of the dendritic one. Accompanying these changes in vein structure, the spatio-temporal pattern of the rhythmic contraction changed into a small-structured pattern from a synchronized one. In the above process, cell shape affects the contraction pattern, but, conversely, the contraction pattern effects the cell shape. To demonstrate this, a phase difference in the rhythmic contraction was induced artificially by entraining the intrinsic rhythm to external temperature oscillations. New veins then formed along the direction parallel to the phase difference of the rhythm. Consequently, the vein organization of the cell interacts with the contractile activity to form a feedback loop in a mechanism of contraction pattern formation.

Animals↗
PubMed2000

Effect of activated human protein C on disseminated intravascular coagulation induced by lipopolysaccharide in rats.

Protein C is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating coagulation and fibrinolysis by inactivating not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). The aim of the present study was to examine the effect of a human APC product (designated as CTC-111), compared with that of heparin, on the disseminated intravascular coagulation (DIC) induced by lipopolysaccharide (LPS) in rats. LPS (1 mg/kg/h) infusion was performed through a femoral vein for 4 h. One-fifth amount of the total dosage of CTC-111 or heparin was injected into the other femoral vein, followed by a 4-h infusion of the remainder. Both CTC-111 (10,000-100,000 U/kg) and heparin (400-800 IU/kg) inhibited the decrease in platelet count and fibrinogen level equally. The prolonged activated partial thromboplastin time and prothrombin time observed in DIC rats were further elongated in both CTC-111- and heparin-treated rats. But, this prolongation was less in CTC-111-treated rats than in the heparin-treated ones. Heparin inhibited the increase in fibrin and fibrinogen degradation products more prominently than CTC-111. On the other hand, CTC-111 strongly inhibited the increase in PAI-1 activity but heparin did not. These results suggest that CTC-111 may enhance fibrinolysis through its direct inhibitory effect on PAI-1. The parameters for liver or renal damage, i.e., plasma glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), creatinine (Cre) and blood urea nitrogen (BUN), were significantly increased by LPS infusion. Both CTC-111 (100,000 U/kg) and heparin (800 IU/kg) decreased the increase in GOT and GPT levels significantly, whereas neither affected the increase in Cre or BUN. From these results, the activation of the blood coagulation system might partially contribute to the progression of liver damage caused by LPS, and might be less involved in the progression of renal damage in this model. In conclusion, CTC-111 showed both anticoagulant and profibrinolytic activity in the LPS-induced DIC model without excessive prolongation of coagulation time. From these results, CTC-111 is expected to be a useful remedy for DIC without the risk of bleeding.

Animals↗
PubMed1999

Reaction-diffusion-advection model for pattern formation of rhythmic contraction in a giant amoeboid cell of the physarum plasmodium

The plasmodium of Physarum polycephalum is a large amoeboid organism showing rhythmic contraction everywhere within an organism, and moves by forming spatio-temporal patterns of the rhythmic contraction. We propose a reaction-diffusion-advection model for the pattern formation. This model is constructed under physiological suggestions that the chemical oscillator acts as a clock regulating the rhythmic contraction and interacts spatially not only by diffusion but also by advection of protoplasm. Behavior of the model is studied by numerical calculation, especially the effects of the advection term on a simple reaction-diffusion system. The advection effect reproduces experimentally observed phenomena of fluctuating propagation of the contraction wave. Concept of the reaction-diffusion-advection system is promising for modeling the mechanism of amoeboid behaviour in the Physarum plasmodium. Copyright 1999 Academic Press.

Journal Article↗
PubMed1998

Large-scale preparation of human thrombin: polyethylene glycol potentiates the factor Xa-mediated activation of prothrombin.

We investigated the ability of polyethylene glycol 4000 to accelerate thrombin generation in a mixture of prothrombin and factor X at concentrations of 1-30%. In the presence of 5 mM of CaCl2, polyethylene glycol 4000 promoted prothrombin activation at concentrations above 1%. The peak of activation was seen at levels of 14 and 20% of polyethylene glycol 4000. The effect of the polyethylene glycol was remarkably dependent on its molecular weight; molecular weights greater than 2000 were required for accelerating thrombin generation. Under optimal conditions, polyethylene glycol 4000, in the presence of CaCl2, promoted conversion of all of the prothrombin into thrombin and its derivatives. We conclude that polyethylene glycol 4000, at concentrations ranging from 14 to 20%, effectively accelerates thrombin generation in the presence of 5 mM of CaCl2. This new method for preparing thrombin is based on the use of polyethylene glycol 4000 and CaCl2 and is applicable to the manufacture of thrombin.

Calcium Chloride↗
PubMed1998

A kinetic analysis of the interaction of human recombinant tissue factor pathway inhibitor with factor Xa utilizing and immunoassay and the effect of antithrombin III/heparin on the complex formation.

We have recently shown that a complex formation of tissue factor pathway inhibitor (TFPI) and factor Xa (Xa) promotes a clearance of proteoglycans-associated TFPI. In the current studies, the interaction between human recombinant TFPI (h-rTFPI) and Xa were kinetically analyzed by utilizing both a protease inhibitor, p-(amidophenyl) methanesulfonyl fluoride hydrochloride, and a specific enzyme-linked immunosorbent assay for the complex of h-rTFPI with Xa. We further investigated the effect of antithrombin III on the complex formation between h-rTFPI and Xa. We found that the h-rTFPI/Xa complex formed in a time-dependent manner: the second-order rate constant (K1) for the complex formation was calculated to be 0.86x10(6) M(-1)s(-1). The addition of antithrombin III to the h-rTFPI solution modestly reduced the rate of the complex formation between h-rTFPI and Xa. Heparin strikingly enhanced antithrombin III's inhibition of Xa and resulted in complete abrogation of the complex formation between h-rTFPI and Xa in the absence or presence of acidic phospholipids. Furthermore, antithrombin III induced dissociation of the preformed h-rTFPI/Xa complex in the presence of heparin. These results suggest that in the presence of heparin, antithrombin III interferes with the catabolism of TFPI mediated via Xa.

Antithrombin III↗
PubMed1997

Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production.

We investigated the effect of activated protein C (APC) on pulmonary vascular injury and the increase in tumor necrosis factor (TNF) levels in lipopolysaccharide (LPS)-treated rats to determine whether APC reduces LPS-induced endothelial damage by inhibiting cytokine production. Intravenously administered LPS (5 mg/kg) induced pulmonary vascular injury, as indicated by an increase in the lung wet-to-dry weight ratio. LPS-induced pulmonary vascular injury was prevented by APC but not by active site-blocked factor Xa [dansyl glutamyl-glycyl-arginyl chloromethyl detone-treated activated factor X (DEGR-Xa)], a selective inhibitor of thrombin generation, or inactivated APC [diisopropyl fluorophosphate-treated APC (DIP-APC)]. APC, but not DEGR-Xa or DIP-APC, significantly inhibited the LPS-induced increase in the plasma level of TNF. APC significantly inhibited the production of TNF by LPS-stimulated monocytes in a dose-dependent fashion in vitro, but DIP-APC did not. APC did not inhibit the functions of activated neutrophils in vitro. These findings suggest that APC prevented LPS-induced pulmonary vascular injury by inhibiting TNF production by monocytes and not via its anticoagulant activity. The serine protease activity of APC appears to be essential for inhibition of TNF production.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Species specificity of anticoagulant activity of activated human protein C: involvement of factor V as well as protein S.

Activated protein C (APC) possesses species specificity in its anticoagulant activity. Human APC exerts only weak activity in rat plasma compared with that in human plasma. The present study was undertaken to estimate the difference in interaction of human and rat factors with human APC and to assess the cause of the species specificity. Human or rat protein S (PS), factor V, or factor VIII was used to supplement human plasma depleted of each respective factor, and the anticoagulant activity of human APC was measured in term of the elongation of activated partial thromboplastin time (APTT). The activity of human APC in rat PS- or factor V-supplemented plasma was weaker than that in the human PS- or factor V-supplemented plasma. Furthermore, using purified human and rat factor V, human APC showed weaker inactivation of rat factor V than human factor V. Equal anticoagulant activity was observed in human or rat factor VIII-supplemented plasma. And there was a little difference in the interaction of APC with its inhibitors in human or rat plasma during a few minutes of incubation as judged by measurement of residual activity by an enzyme capture assay. From these results factor V as well as PS seems to play a major role in the species specificity of APC.

Animals↗
PubMed1996

Phase switching of oscillatory contraction in relation to the regulation of amoeboid behavior by the plasmodium of Physarum polycephalum.

The plasmodium of the true slime mould Physarum polycephalum is a large aggregate of protoplasm and behaves like an amoeboid cell, exhibiting rhythmic contraction everywhere within the organism. Phase dynamics of these oscillations were studied in relation to the global organization of amoeboid behavior, by analysing the thickness oscillation, isotonic tension and the motive force of the streaming. Usually the plasmodium showed synchrony, the phase of the oscillation being the same everywhere excepting the peripheral part. We found several situations where this in-phase relationship switched to anti-phase. This occurred either at the early stages of the plasmodial coalescence, or when a single plasmodium was nearly separated by partition, or when the streaming of the protoplasm was hindered by applying the hydrostatic pressure. Furthermore, the motive force of the protoplasmic streaming increased once the anti-phase relationship was established. In this way, the weak interactions among plasmodial parts induce the switching of phase relationship from in-phase to anti-phase, and this transition in turn acts to increase the interaction by promoting a rapid mixing of the protoplasm. This global feedback mechanism by phase switching should help maintain a large single plasmodium without separating into parts. The possible mechanism of phase switching is discussed in terms of coupled nonlinear oscillators.

Animals↗
PubMed1996

Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats.

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS-induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.

Amino Acid Chloromethyl Ketones↗