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F Sekiya

Publications and source records attributed to F Sekiya.

35 records · Page 2Linked to original sources
PubMed1991

Immunological detection of propolypeptide of von Willebrand factor on platelet surface.

Recently we have found that propolypeptide of von Willebrand factor (pp-vWF) obtained from platelets binds to type I collagen. It is known that pp-vWF is present in platelet alpha-granules and is secreted upon activation. In this paper, we demonstrate the two following evidences to show that it is also present on the surface of resting platelets. [1] The antibody against pp-vWF bound to the surface of platelets. [2] The antibody induced aggregation of platelets. The binding of the antibody and the antibody-induced aggregation of platelets were inhibited in a dose-dependent manner by Fab fragment of the antibody. Platelets from von Willebrand disease patients bound less of the antibody and responded weakly to the antibody.

Antibodies↗
PubMed1991

Monoclonal antibodies that inhibit binding of propolypeptide of von Willebrand factor to collagen. Localization of epitopes.

We reported previously that bovine propolypeptide of von Willebrand factor (pp-vWF) binds to type I collagen. To determine the collagen-binding sites of pp-vWF we generated monoclonal antibodies (mAbs) against bovine pp-vWF. One mAb, designated TC8, very strongly inhibited the binding of pp-vWF to type I collagen; three other mAbs, designated TC2, TC6 and TC7, exhibited moderate inhibition. Competition between the mAbs for binding to intact pp-vWF revealed that the epitope for TC8 was structurally independent of that for TC6 and TC7. To determine directly the location of the epitope for each mAb on the bovine pp-vWF molecule, we tested the reactivity of mAbs by immunoblotting toward peptide fragments obtained by digestion with lysylendopeptidase. TC2 and TC8 recognized a fragment of mass 21 kDa, while TC6 and TC7 recognized a distinct fragment of 18 kDa. These two fragments were purified to homogeneity and their N-terminal amino acid sequences were determined. Comparing these sequences with the sequence of human pp-vWF, the locations of these fragments in the primary structure were estimated to be Phe570--Lys682 for the 21-kDa fragment and Glu281--Lys375 for the 18-kDa fragment. These data suggest that pp-vWF contains at least two collagen-binding sites which lie within or close to the regions between Phe570--Lys682 and Glu281--Lys375.

Amino Acid Sequence↗
PubMed1991

Collagen-binding domain within bovine propolypeptide of von Willebrand factor.

Two reduced/alkylated fragments of bovine propolypeptide of von Willebrand factor (pp-vWF) that inhibit pp-vWF binding to collagen were isolated. One is a tryptic fragment of molecular mass of about 30 kDa and inhibits the binding at a molar concentration about 20 times higher than the intact pp-vWF. Amino acid sequence of this fragment was determined almost completely, and it was revealed that this fragment corresponded to the carboxyl-terminal region of pp-vWF molecule beginning with Phe557. The other active fragment was obtained by lysyl endopeptidase digestion. This migrated as a 21.5/21-kDa doublet in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but deglycosylation of this doublet resulted in production of single species of 19 kDa. The difference between the doublet constituents, therefore, was of carbohydrate composition. The extent of inhibition of collagen-binding by this 21.5/21-kDa fragment was comparable to that of the 30-kDa fragment, and furthermore, location of this fragment in the molecule was established to be between Phe570 and Lys682. These were the only fragments among those obtained by proteolytic digestions that had significant competitive effect on the binding of intact pp-vWF to collagen. These results strongly suggest that at least one collagen-binding site should be present in the carboxyl-terminal region of bovine pp-vWF extending from residue 570 to 682.

Amino Acid Sequence↗
PubMed1990

Metabolism of 3-deoxyglucosone, an intermediate compound in the Maillard reaction, administered orally or intravenously to rats.

The Amadori rearrangement compound, the product in the early step of the Maillard reaction of proteins with glucose, is known to be degraded into 3-deoxyglucosone (3DG), a 2-oxoaldehyde. In order to elucidate the metabolic pathway of 3DG, [14C]3DG was synthesized from [14C]-glucose and administered to rats orally and intravenously. 2 h after oral administration of [14C]3DG, the percentages of radioactivity (RaI%) in stomach, small intestine and urine were 3.9, 60 and 6.4%, respectively, while RaI% in liver, kidney, spleen, blood and CO2 were less than 0.5%. The absorption rate of 3DG was obviously lower in comparison with that of glucose. 3 h after intravenous administration of [14C]3DG, the RaI% in urine was 72% and those in liver, kidney, spleen, blood and CO2 were less than 1%. It therefore appeared that the absorbed 3DG was not biologically utilized by the rats, but was rapidly excreted in the urine. Some metabolites of [14C]3DG were detected in urine by TLC-autoradiography. The main metabolite was purified and identified as 3-deoxyfructose by FD-MS and 13C-NMR spectroscopy, indicating that the aldehyde group of 3DG was reduced to an alcohol.

Absorption↗
PubMed1990

Cooperativity between platelet-activating factor and collagen in aggregation of bovine platelets III.

In cooperative aggregation of bovine platelets induced by simultaneous addition of PAF and collagen at subthreshold concentrations the following observations were made. (i) Formation of phosphatidic acid and arachidonic acid metabolites, which characterize PAF and collagen alone-induced aggregation, respectively, was observed very obviously. (ii) A thromboxane antagonist did not completely block the cooperative aggregation. The extent of residual aggregation activity was dependent on concentration of collagen used in the simultaneous administration with PAF. These results suggest that both signal transduction pathways activated by PAF and collagen alone at high concentrations are attained by simultaneous addition of both agonists at subthreshold concentrations through unknown mechanisms.

Animals↗
PubMed1990

Cooperativity between platelet-activating factor and collagen in aggregation of bovine platelets, II.

When subthreshold amounts of platelet-activating factor (PAF) and collagen were added simultaneously, strong aggregation of platelets was induced. However, each agonist alone at these concentrations could not induce aggregation at all (S. Kojima et al, (1987) Biochem. Biophys. Res. Commun. 145, 915-920). This cooperativity was observed not only in aggregation but also in changes of intracellular Ca2+ concentration, 47 kDa protein phosphorylation, and formation of thromboxane. These findings suggest that various steps of signal transduction pathways are enhanced during their cooperativity.

Animals↗
PubMed1990

Feedback regulation of platelet function by 12S-hydroxyeicosatetraenoic acid: inhibition of arachidonic acid liberation from phospholipids.

We have proposed a mechanism that platelet aggregation is regulated by its 12-lipoxygenase product, 12S-hydroxyeicosatetraenoic acid (12-HETE) (Sekiya, F., Takagi, J. and Saito, Y. (1989) Thrombos. Res. 56, 407-415). Inhibition of endogenous 12-HETE production by 15-HETE, a specific inhibitor of 12-lipoxygenase, accelerated aggregation of bovine platelets in response to collagen and arachidonic acid liberation from phospholipids was enhanced. Exogenously added 12-HETE suppressed collagen-induced liberation of arachidonic acid and the aggregation was also inhibited. On the other hand, 12-HETE did not interfere with thromboxane synthesis from free arachidonic acid in a cell-free system. These observations suggest that 12-HETE exerts a negative feedback to prevent excess aggregation through interference with arachidonic acid liberation from membrane phospholipids.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

Elucidation of a role of plasma albumin during collagen-induced aggregation of bovine platelets.

Bovine platelets of which phospholipids were labeled with [14C] arachidonate were stimulated by collagen. Omitting albumin in suspending medium suppressed aggregation and arachidonate liberation from phospholipids. Analysis of [14C] arachidonate metabolites released into medium revealed that release of 12S-hydroxyeicosatetraenoic acid (12-HETE) from stimulated platelets was dependent on the existence of albumin, while thromboxane B2 release was not. Moreover, exogenously added 12-HETE inhibited collagen-induced aggregation. These observations suggest that albumin potentiates aggregation by preventing intracellular accumulation of 12-HETE, and strengthen our previous finding that albumin in plasma is very essential for collagen-induced aggregation of platelets (Sekiya, F., Takagi, J., Kasahara, K., Inada, Y. and Saito, Y. (1988) Thrombos. Res. 50, 837-846.).

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

A collagen-binding glycoprotein from bovine platelets is identical to propolypeptide of von Willebrand factor.

Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose. Among these proteins, a protein of about 100,000 daltons was purified to homogeneity by two additional affinity chromatographies, an organomercurial-agarose and a lentil lectin-agarose. This protein consisted of a single polypeptide chain which contains carbohydrate moiety and many intrapolypeptide disulfide bridges. In addition to platelets, this protein was present in plasma and cultured endothelial cells but not in red blood cells, leukocytes, and smooth muscle cells. Furthermore, it was released from platelets upon stimulation by various agonists. The purified 100-kDa protein was labeled with 125I to quantitate its binding to fibrillar type I collagen. The protein specifically bound to fibrillar collagen with the apparent dissociation constant of 5.6 x 10(-8) M for the high affinity site and 5.5 x 10(-7) M for the low affinity site. Analyses of amino acid sequences of both intact and tryptic fragments of this protein revealed that it had strong homology to the propolypeptide of human von Willebrand factor, which is also known as von Willebrand antigen II. Various properties of this protein listed above also strongly suggest that it was indeed the propolypeptide of bovine von Willebrand factor.

Amino Acid Sequence↗
PubMed1989

Inhibition of platelet-collagen interaction by propolypeptide of von Willebrand factor.

A collagen-binding glycoprotein was isolated from human platelets using affinity chromatography of immobilized collagen. Based upon characterizations of this protein we confirmed that it was identical to the propolypeptide of von Willebrand factor (pp-vWF), which is also called von Willebrand antigen II. The characteristics we have investigated are molecular weight, existence of carbohydrate chains, and the NH2-terminal amino acid sequence. pp-vWF has strong affinity to collagen and inhibits collagen-induced aggregation of human platelets at a concentration as low as 2 micrograms/ml even in the presence of plasma. This inhibitory effect is specific for collagen-induced aggregation since it does not inhibit aggregation of platelets induced by other agonists such as ADP, arachidonic acid, platelet-activating factor, ionophore A23187, and ristocetin. As pp-vWF is quickly released from platelets upon activation by various agonists, it is possible that pp-vWF functions as a repressor for excess platelet aggregation induced by collagen and constitutes a negative feed-back mechanism. Considering the fact that mature vWF supports platelet adhesion to subendothelium, present observations suggest that the propeptide portion and the mature protein could have opposing effects on hemostasis.

Amino Acid Sequence↗
PubMed1988

Subunit B of factor XIII is present in bovine platelets.

Distribution of B subunit of coagulation factor XIII(FXIII B) in bovine platelets was determined. Intracellular location of FXIII B in platelets was confirmed by fluorescent antibody technique. Positive staining of FXIII B was observed with Triton-permeabilized platelets but not with non-permeabilized platelets. Immunoblots of plasma and platelet lysate revealed that the antibody was specific for FXIII B and this antigen existed in the supernatant of sonication-solubilized platelets. The amount of FXIII B in platelets was determined to be 17.3 +/- 6.7 ng/10(8) platelets by radioimmunoassay. This is the first visual and quantitative demonstration of intracellular FXIII B.

Animals↗
PubMed1988

Plasma albumin is essential for collagen-induced platelet aggregation.

We found that platelets must have albumin on the surface to respond to collagen and aggregate. Albumin, however, was not absolutely necessary for ADP-, platelet activating factor-, serotonin- or thrombin-induced aggregation, while fibrinogen was required for ADP- or serotonin-induced aggregation. Immunofluorescent microscopy revealed that albumin was retained on gel-filtrated platelets but not on washed platelets. Albumin was not required for platelet adhesion to immobilized collagen. Without albumin thromboxane formation upon collagen-stimulation was diminished. These data suggest that albumin is essential in some step(s) that results in production of thromboxane A2.

Animals↗
PubMed1988

"A" subunit of factor XIII is present on bovine platelet membrane and mediates collagen-induced platelet activation.

The Fab fragment of a polyclonal antibody against platelet factor XIII inhibited the collagen-induced platelet aggregation in a dose-dependent manner. This inhibitory effect was specific for collagen, and it had no effect on arachidonic acid-, ADP-, and serotonin-induced aggregations. This finding strengthens our notion that platelet factor XIII is involved in collagen-induced platelet aggregation. (Saito, Y., Imada, T., Takagi, J., Kikuchi, T. and Inada, Y. J. Biol. Chem. 261, 1355-1358, 1986). We have investigated membrane localization of bovine platelet factor XIII using immunological techniques. Immunofluorescent visualization revealed that the factor XIII was expressed on the surface of non-permeabilized bovine platelets, where we detected neither lactate dehydrogenase, a cytoplasmic enzyme marker, nor B subunit of factor XIII, which is present in plasma. Cell surface iodination and immunoprecipitation also confirmed that it existed on the surface of platelets.

Animals↗
PubMed1988

Venom from southern copperhead snake (Agkistrodon contortrix contortrix). II. A unique phospholipase A2 that induces platelet aggregation.

A platelet aggregation factor was purified from the venom of southern copperhead snake (Agkistrodon contortrix contortrix) by DEAE-cellulose ion-exchange chromatography, precipitation with ammonium sulfate, affinity chromatography using bovine serum albumin as ligand, and gel filtration on Cellulofine GCL-2000. It had molecular weights of 11,000 and 14,000, as determined by gel filtration chromatography and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), respectively. It consists of a single polypeptide, and was identified as a phospholipase A2. It was quite resistant to heat and various denaturing reagents including urea and SDS. It lost both phospholipase A2 activity and platelet aggregating activity upon modification of histidine residue(s) with p-bromophenacyl bromide. Its specificity towards the beta-position of phospholipid in esterolytic reaction was confirmed by gas-liquid chromatography using a pure synthetic phosphatidylcholine. Platelet aggregation by this phospholipase A2 was completely inhibited by prostacyclin, but was little inhibited by aspirin which indicates almost no direct participation of released arachidonic acid in the aggregation mechanism.

Animals↗
PubMed1987

Cooperativity between platelet-activating factor and collagen in platelet aggregation.

Cell lysate obtained from cultured vascular endothelial cells contained a substance which induced platelet aggregation. This substance was identified as a phospholipid, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor; PAF), by thin-layer chromatography, phospholipase A2 digestion, inhibition by a specific antagonist, CV-3988, and agonist-specific refractory state. It was further found that PAF and collagen together induced extensive aggregation of platelets even with the concentrations by which each agonist alone could not induce aggregation of platelets at all.

Animals↗
PubMed1987

Analysis of distribution of receptors among platelets by flow cytometry.

We have investigated the binding of fluorescein isothiocyanate (FITC)-labeled fibrinogen to platelets using a fluorescence spectrophotometer and a flow cytometer. The amount of fibrinogen bound by stimulation of adenosine diphosphate (ADP) and 5-hydroxytryptamine (5HT) increased in a dose-dependent manner. Stimulation of one agonist was independent of that of another agonist. Fibrinogen could bind to platelets in response to ADP, regardless of whether they were desensitized by 5HT, and vice versa. It was found by flow cytometric analysis that individual platelets responded uniformly to both ADP and 5HT, indicating that individual platelets have receptors for both ADP and 5HT on their surfaces.

Adenosine Diphosphate↗