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Biomedical subjects

T Sugo

Publications and source records attributed to T Sugo.

At least 55 records · Page 3Linked to original sources
PubMed1993

Molecular defect in factor IX Tokyo: substitution of valine-182 by alanine at position P2' in the second cleavage site by factor XIa resulting in impaired activation.

Utilizing polymerase chain reaction and directly sequencing the amplified exon 6 of the factor IX gene derived from a mild hemophilia Bm patient, we have identified a T to C mutation at nucleotide 20,525. This point mutation predicted a Val182 to Ala substitution in the abnormal factor IX molecule, designated as factor IX Tokyo. The patient manifested a low factor IX activity and a moderately prolonged ox-brain prothrombin time but a normal factor IX antigen level in plasma. Immunopurified factor IX derived from the patient was found to have a normal molecular weight but a reduced specific activity (23% of normal). Limited proteolysis by activated factor XI or by a snake venom-derived factor X-activating enzyme was considerably delayed, indicating the presence of structural alteration(s) most probably at or near the second enzyme-cleavage site. Once activated, however, factor IXa Tokyo was able to activate factor X normally and was inactivated by antithrombin III also in a normal fashion. The structural model of factor IXa and a docking model of factor IX and activated factor VII (factor VIIa) suggested that the Val182 to Ala substitution would not affect the local conformation of the catalytic domain. This mutation would rather loosen the fitness of the molecule into the substrate-binding pocket of factor VIIa due to a shorter side chain of the Ala substitution at the P2' position of the second cleavage site.

Adolescent↗
PubMed1993

Role of sugar chains in the in-vitro activity of recombinant human interleukin 5.

The structures of O-linked sugar chains of recombinant human interleukin 5 (rhIL-5) produced by Chinese hamster ovary cells were determined employing high-performance anion-exchange column chromatography under high pH conditions. The core sequence was elucidated to be Gal beta 1-->3-GalNAc by its susceptibility to endo-alpha-N-acetylgalactosaminidase and sialic acid linkages were determined using sialidases of different specificities. To investigate the role of sugar chains in the in-vitro activity of rhIL-5, it was digested with various glycosidases. While removal of N-linked sugar chains resulted in 2.8-fold increase of the activity, de-O-glycosylated rhIL-5 showed 10-fold higher activity than intact rhIL-5, suggesting that the presence of O-linked sugar chains suppresses the activity more effectively than that of N-linked chains. While incubation of de-N-glycosylated rhIL-5 at 70 degrees C for 30 min decreased the activity, intact and de-O-glycosylated rhIL-5 lost little activity, suggesting that N-linked sugar chains contributed to the thermostability of the molecule.

Borohydrides↗
PubMed1993

Flow cytometric analysis of surface membrane proteins on activated platelets and platelet-derived microparticles from healthy and thrombasthenic individuals.

We used flow cytometry to investigate surface membrane protein expression by platelets and platelet-derived microparticles from normal individuals and a patient with Glanzmann's thrombasthenia. Microparticles were detected by both forward scatter and side scatter using FACScan. The binding of coagulation factors on microparticles was investigated by using monoclonal anti-Factor IX (IXa) and anti-Factor X (Xa) antibodies. Furthermore, the procoagulant activity of microparticles was measured with a chromogenic substrate (S-2222) using a microtiter enzyme-linked immunosorbent assay. Both types of platelets showed similar release of microparticles. Microparticles released from platelets after activation with the calcium ionophore A23187 did not bind factors IXa and Xa, but when purified factors Va and Xa were added to the incubation buffer, factor Xa binding increased markedly in both normal and thrombasthenic platelets. Both normal and thrombasthenic platelets showed a similar time-dependent release of microparticles when activated with A23187. However, the binding of an antibody to granule membrane protein-140 also increased time-dependently in normal microparticles, but was little increased in thrombasthenic microparticles. These findings suggest that glycoprotein IIb/IIIa does not participate in the expression of prothrombinase activity on the surface of activated platelets and microparticles, whereas this glycoprotein appears to have an important role in the movement of granule membrane protein-140 from platelets to microparticles.

Adult↗
PubMed1991

Adsorption and elution of bovine gamma-globulin using an affinity membrane containing hydrophobic amino acids as ligands.

A hollow-fibre affinity membrane containing hydrophobic amino acids as ligands was prepared by the radiation-induced grafting of glycidyl methacrylate onto a porous polyethylene hollow fibre and subsequent phenylalanine (Phe) or tryptophan (Trp). The densities of the Phe and Trp ligand of the resulting affinity membrane were 0.4 and 0.4 mol/kg, respectively. The Trp-containing affinity membrane exhibited a higher amount of adsorbed bovine gamma-globulin (BGG) than the Phe-containing membrane. To evaluate the adsorption behaviour of the membrane, the BGG-containing buffer solution was permeated from the inside to the outside of the Trp-containing hollow-fibre affinity membrane through the ligand-immobilized pores. The breakthrough curves as a function of effluent volume coincided irrespective of the flow-rate, i.e. the residence time (55-220 s) of the solution across the membrane (thickness 0.83 mm), as a result of negligible mass transfer resistance. A series of chromatographic procedures, (adsorption-washing-elution) was repeated twice and a satisfactory quantitative elution was attained. The reproducible profile of the flux and the protein concentration assured a quantitative cycle of chromatography using the affinity membrane containing Trp as a ligand.

Animals↗
PubMed1991

Synthesis of protein C in human umbilical vein endothelial cells.

By monitoring the activation of protein C and the regulation of factor Xa-catalyzed thrombin formation by the activated protein C (APC) on the surface of human umbilical vein endothelial cells (HUVEC), we found that functional protein C was synthesized in cultured HUVEC and expressed thereon in the presence of vitamin K. Furthermore, without exogenously added protein S, time-dependent and saturable accumulation of APC (20 fmol APC/10(5) cells) on the surface of HUVEC was observed. During prothrombin activation by the complex of membrane-bound factor Xa and endogenous factor Va formed on the surface of HUVEC, APC was generated, and the rate of thrombin formation decreased. Treatment of HUVEC with an antibody that inhibits the APC-catalyzed inactivation of endogenous factor Va clearly quenched the activity of surface-associated APC. Immunostaining of HUVEC with a horseradish peroxidase (HRP)-conjugated antibody that solely recognizes human protein C confirmed the presence of protein C on the surface of HUVEC. Northern blot analysis revealed that an about 1.8 kb mRNA species derived from HUVEC was hybridized with 32P-labeled protein C cDNA, as in the case of those from HepG2, which are known to synthesize normal protein C. The increase in the amount of protein C mRNA in HUVEC in parallel with cell growth provided supporting evidence for the synthesis of protein C during the culture of HUVEC. These results indicate that blood coagulation is regulated by endogenously generated and activated protein C, together with or without protein S, through inactivation of factor Va on the surface of endothelial cells.

Antibodies, Monoclonal↗
PubMed1990

Anti-human factor IX monoclonal antibodies specific for calcium ion-induced conformations.

Four monoclonal antibodies have been produced, which are specific for the Ca2+ or Sr2(+)-induced conformation of human factor IX. Certain, but not all, gamma-carboxy-glutamic acid residues in factor IX are involved in the epitope expression together with the conformation stabilized by the adjacent region of Gla-domain and a disulfide bridge. All the antibodies interfered with the binding of factor IX to phospholipids and inhibited the procoagulant activity of factors IX and IXa beta.

Antibodies, Monoclonal↗
PubMed1990

Chemical modification of gamma-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (des-gamma-carboxy)prothrombin.

Chemical modification of gamma-carboxyglutamic acid (Gla) residues in human prothrombin to gamma-methyleneglutamic acid (gamma-MGlu) residues elicited a conformation similar, if not identical, to that of des-gamma-carboxy prothrombin or PIVKA-II, i.e., prothrombin molecules induced by vitamin K antagonists or vitamin K deficiency states. The reaction seems to proceed sequentially by preferentially modifying a Gla at residue 32 that is located innermost among 10 Gla residues of human prothrombin. The initial modification resulted in nearly 50% losses of barium salt adsorption, the procoagulant activity and thrombin generation by the prothrombinase complex. The subsequent modification of two Gla residues at positions 6 and 16 gave rise to the immunoreactivity to an established monoclonal antibody that specifically recognizes the des-gamma-carboxy prothrombin. Further modification of Gla residues increased the reactivity to the antibody, indicating that the conformation recognized by the antibody was stabilized so as to more readily fit the recognition site of the antibody. The appearance of the immunoreactivity was obviously related to the modification of Gla residues in prothrombin, since all other similarly treated derivatives of prothrombin lacking the Gla-domain failed to react with the antibody. Such chemically modified prothrombins may serve as models for studying abnormal des-gamma-carboxy prothrombin produced in vitamin K deficiency states.

1-Carboxyglutamic Acid↗
PubMed1986

Calcium binding of bovine protein S. Effect of thrombin cleavage and removal of the gamma-carboxyglutamic acid-containing region.

Thrombin cleaves protein S at arginine residues 52 and 70 resulting in loss of cofactor activity and reduced Ca2+ ion binding. After thrombin cleavage the NH2-terminal region containing gamma-carboxyglutamic acid (Gla) is linked to the large COOH-terminal fragment by a disulfide bond. Measurements of the rate of disulfide bond reduction by thioredoxin in intact protein S showed that the disulfide bonds are largely inaccessible to thioredoxin in the presence of Ca2+ ions, whereas in the presence of EDTA apparently all of the disulfide bonds are rapidly reduced. Probing the reactivity of the disulfide bonds in thrombin-modified proteins indicated that the thrombin cleavage induces a conformational change in the protein. After thrombin cleavage of protein S, the domain containing gamma-carboxyglutamic acid could be removed by selective reduction with thioredoxin followed by alkylation of the sulfhydryl groups. Ca2+ ion binding was compared in intact protein S, thrombin-modified protein S, and Gla domainless protein S. The intact protein S bound several Ca2+ ions, and the binding was not saturable. Thrombin-modified protein S, whether intact or with the Gla domain removed by selective reduction, bound two to three Ca2+ ions with a KD of 15-20 microM. The Gla domain in thrombin-modified protein S thus does not contribute significantly to the high affinity Ca2+ ion binding. Thrombin cleavage of protein S may be of physiological importance in the regulation of blood coagulation.

1-Carboxyglutamic Acid↗
PubMed1985

Protein C in bovine plasma after warfarin treatment. Purification, partial characterization, and beta-hydroxyaspartic acid content.

Protein C was purified from the plasma of a cow treated with the vitamin K antagonist warfarin. The purified protein appeared not to bind Ca2+ ions in contrast to protein C from an untreated animal. The gamma-carboxyglutamic acid content of the abnormal protein C was reduced to approximately 10% of normal, whereas the beta-hydroxyaspartic acid content was only slightly decreased, suggesting that vitamin K is not involved in the postribosomal hydroxylation of the aspartic acid residue in position 71 of the light chain of protein C. The abnormal and normal proteins were activated at the same rates by thrombin, but normal protein C was more rapidly activated by the thrombin-thrombomodulin complex. Compared to normal protein C, the abnormal one had virtually no anticoagulant activity.

Animals↗
PubMed1985

Kinetic studies on surface-mediated activation of bovine factor XII and prekallikrein. Effects of kaolin and high-Mr kininogen on the activation reactions.

The kaolin-mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high-Mr kininogen and kaolin surface on the kinetics of these activation reactions were studied. High-Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high-Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 X 10(7) s-1 X M-1, respectively. The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high-Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 microM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 X 10(4) to 1.1 X 10(6) s-1 X M-1). These results suggest that high-Mr kininogen accelerates the surface-mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.

Amidohydrolases↗
PubMed1985

Activation of factor XII and prekallikrein with polysaccharide sulfates and sulfatides: comparison with kaolin-mediated activation.

The activation of Factor XII and prekallikrein by polysaccharide sulfates and sulfatides in the presence of high-molecular-weight (HMW) kininogen was studied, and compared with the kaolin-mediated activation reaction. Among a variety of artificially-sulfated polysaccharides and native polysaccharide sulfates, amylose sulfate (M.W.= 380,000 and sulfur content, 19.1%) and sulfatide were found to have the most efficient ability to trigger the activation of prekallikrein by Factor XII. The effects of these two kinds of negatively-charged surfaces on the following three activation reactions were compared; the activation of prekallikrein by Factor XII (reaction 1), the activation of Factor XII by kallikrein (reaction 2) and the activation of prekallikrein by Factor XIIa (reaction 3). All three reactions mediated by the selected surfaces were strongly accelerated by HMW kininogen and its derivatives, kinin-free protein and fragment 1.2-linked light chain, like the kaolin-mediated activation. However, this accelerating effect of HMW kininogen on the amylose sulfate- and sulfatide-mediated activations (reaction 1) was diminished after treatment with fluorescein iso-thiocyanate, whereas the effect on the kaolin-mediated activation was not influenced by fluorescein-labeling. In addition, reaction 2 mediated by amylose sulfate and sulfatide was extremely slow even in the presence of HMW kininogen, and the results also differed from those with kaolin. The sulfatide-mediated activation of reaction 1 was not inhibited by fragment 1.2 (His-rich fragment), which is released from HMW kininogen by the action of kallikrein, and is known to be a potent inhibitor of the kaolin-dependent activation. These results indicate that the mechanisms responsible for surface activation triggered by soluble amylose sulfate, sulfatide micelles and kaolin differ from each other as regards the molecular interaction with the contact factors.

Animals↗
PubMed1984

Calcium-binding properties of bovine factor X lacking the gamma-carboxyglutamic acid-containing region.

In bovine protein C normal activation by the thrombin-thrombomodulin complex requires binding of calcium to one high affinity binding site, contained in a protein fragment that lacks the gamma-carboxyglutamic acid (Gla) region (Esmon, N. L., De Bault, L. E., and Esmon, C. T. (1983) J. Biol. Chem. 258, 5548-5553). In this work, the calcium binding to and the conformational change induced by calcium in the corresponding Gla-domainless fragment of bovine factor X, prepared by limited proteolysis by chymotrypsin, were compared with the calcium-binding properties of Gla-domainless protein C. Equilibrium dialysis experiments demonstrated that the proteolytically modified factor X has one high affinity calcium ion-binding site with Kd = 180 microM, a value almost identical to the Kd for the binding of calcium to proteolytically modified protein C. Measurements of the rate of disulfide bond reduction by thioredoxin showed that the disulfide bonds of both factor X and protein C lacking the Gla domains were more rapidly reduced in the absence than in the presence of calcium. Thus, calcium binding induces a conformational change in both proteolytically modified proteins. Calcium binding to Gla-domainless protein C is accompanied by a quenching of the intrinsic tryptophan fluorescence and by changes in the CD spectrum, indicative of perturbation of the environment of aromatic amino acids by the metal ion. However, no such changes were observed with the proteolytically modified factor X. This difference may be due to the fact that one tryptophan residue (in position 84) is present in the light chain of the proteolytically modified protein C but none in the light chain of the modified factor X. The light chain of factor X has beta-hydroxyaspartic acid in position 64 which is homologous to the beta-hydroxyaspartic acid in position 71 in the light chain of protein C. Our results are compatible with the hypothesis that beta-hydroxyaspartic acid is involved in the Ca2+ ion binding.

1-Carboxyglutamic Acid↗
PubMed1984

erythro-beta-Hydroxyaspartic acid in bovine factor IX and factor X.

To localize beta-hydroxyaspartic acid in factor IX and factor X the two proteins were cleaved with cyanogen bromide and trypsin, respectively. Peptides containing beta-hydroxyaspartic acid were isolated and subjected to Edman degradation. The phenylthiohydantoin derivative of beta-hydroxyaspartic acid was identified by HPLC in position 3 in the factor IX fragment and in position 1 in the factor X fragment. This corresponds to position 64 in factor IX and position 63 in the light chain of factor X. The assignments were confirmed by subtractive Edman degradation and with the dansyl method.

Amino Acid Sequence↗
PubMed1983

Role of HMW kininogen in surface-mediated activation of Factor XII.

We have shown that bovine HMW kininogen remarkably accelerates the activation of Factor XII and prekallikrein in the presence of kaolin, adsorbing on kaolin through the fragment 1.2 region and forming a complex with prekallikrein through the light chain region (Sugo et al., 1980; Ikari et al., 1981). The present study was undertaken to examine the role of HMW kininogen in the activation of Factor XII and prekallikrein with other negatively-charged surfaces. The activation system used here was as follows; (1) Activation of prekallikrein by Factor XII, (2) Activation of Factor XII by plasma kallikrein and (3) Activation of prekallikrein by Factor XIIa. Among a variety of foreign surfaces, amylose sulfate and sulfatide were the most efficient in the activation reaction of Factor XII and prekallikrein. Bovene HMW kininogen accelerated all the three reactions triggered by these surfaces. However, the accelerating effect of HMW kininogen on the activation of Factor XII by plasma kallikrein was very weak, when amylose sulfate or sulfatide was used as surface. The three reactions were highly dependent on the amounts of HMW kininogen and surfaces contained in the reaction mixtures. Excess amount of them inhibited these reactions. Among the various fragments, which were prepared from HMW kininogen digests with plasma and urinary kallikreins (Sugo et al., 1980), a large fragment consisting of fragment 1.2 and light chain accelerated the reactions. Thus both fragment 1.2 and the light chain region in HMW kininogen were essential for these activation reactions.

Amylose↗
PubMed1983

Mechanism of surface-mediated activation of bovine Factor XII and prekallikrein.

The mechanism of kaolin-mediated activation of bovine Factor XII was studied in the presence of prekallikrein and HMW kininogen. The activated enzymes were assayed using fluorogenic peptides, Boc-Glu (OBzl)-Gly-Arg-4-methylcoumaryl-7-amide (MCA) for Factor XIIa and Z-Phe-Arg-MCA for plasma kallikrein. The rates of activation of the zymogens were separately measured by blocking either of the active enzymes with specific inhibitors, corn inhibitor for Factor XIIa (Ki = 6.7 nM) and Trasylol for plasma kallikrein (Ki = 3.9 nM). The result was as follows: (1) At the early stage of the activation reaction, kallikrein activity was first generated after short lag time, and then Factor XIIa activity was generated with a sigmoidal curve. In the presence of corn inhibitor, the activation of prekallikrein was observed, but in the presence of Trasylol, the activation of Factor XII was not observed. In the presence of high concentration of Ala-Phe-Arg-Ch2Cl, which inactivates immediately both of the active enzymes, the cleavage of a single chain prekallikrein into the two chain form by Factor XII was shown by SDS-PAGE, using nonlabelled and tritiated prekallikrein. (2) The incubation of Factor XII alone in a quartz cuvette or in the presence of kaolin and HMW kininogen did not result in the activation of Factor XII. The concave upward curve due to an autocatalytic activation was not observed even after the addition of Factor XIIa to Factor XII preparation. Moreover, no structural change of Factor XII during the incubation with kaolin and HMW kininogen was shown by SDS-PAGE, using 3H-Factor XII. (3) The rates of activation of prekallikrein by Factor XII and by Factor XIIa were approximately the same at higher concentration of prekallikrein. However, at lower concentration of prekallikrein the rate of activation of prekallikrein by Factor XII was shown to be a sigmoidal curve and slower than that by Factor XIIa. These results indicate that the activation of bovine Factor XII is initiated by the attack of Factor XII on prekallikrein, followed by the reciprocal activation of Factor XII by plasma kallikrein generated. The autocatalytic activation of bovine Factor XII by Factor XIIa was not demonstrated.

Amino Acid Chloromethyl Ketones↗