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Biomedical subjects

M Okuma

Publications and source records attributed to M Okuma.

At least 235 records · Page 13Linked to original sources
PubMed1988

Lipoxygenase activities of human platelets and their subcellular fractions: comparison between lipoxygenase-deficient platelets and normal platelets.

Lipoxygenase activities were estimated in washed platelets (intact platelets) and their subcellular fractions obtained from 7 patients with deficient platelet lipoxygenase activities and 9 normal subjects. From sonicated platelet preparations, 12,000 g supernatant (F-I), cytosol (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. When 12-hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with intact platelets or each of their subcellular fractions from normal subjects was measured by reversed-phase high-performance liquid chromatography analysis, the lipoxygenase activities of F-I, F-II and F-III were 87%, 31% and 17%, respectively, of the enzyme activity of intact platelets. One of the patients showed no detectable lipoxygenase activity in any preparation, while the other patients showed reduced enzyme activities in all preparations. The addition of CaCl2 significantly increased 12-HETE synthesis solely by F-I from these patients. In most of these patients, contrary to normal subjects, it appeared that the lipoxygenase activity was not fully expressed in intact platelets, since the F-I produced more 12-HETE than the intact platelets.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

Hemorrhagic thrombocytopathy with platelet thromboxane A2 receptor abnormality: defective signal transduction with normal binding activity.

Subnormal platelet responses to thromboxane A2 (TXA2) were found in a patient with polycythemia vera, and the mechanism of this dysfunction was analyzed. The patient's platelets showed defective aggregation and release reaction to arachidonic acid, enzymatically generated TXA2 and synthetic TXA2 mimetics (STA2, U-46619). In contrast, they showed normal responses to thrombin. When the platelet TXA2 receptor was examined with both a 125I-labelled derivative of a TXA2 receptor antagonist ([125I]-PTA-OH) and a 3H-labelled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (Kd) and the maximal concentrations of binding sites (Bmax) of the patient's platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 failed to induce normal elevation in the cytoplasmic free calcium ion concentration, phosphatidic acid formation and 40 kD protein phosphorylation in the patient's platelets, whereas these responses to thrombin were within normal ranges. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) also evoked normal response in the 40 kD protein phosphorylation in the patient's platelets. These results suggested that the patient's platelets had TXA2 receptor abnormalities which were characterized by defective transduction of the binding signal to postreceptor reactions after normal TXA2 binding.

Adenine Nucleotides↗
PubMed1987

[Prostaglandin].

Explore the source record for details and available documents.

Arachidonic Acids↗
PubMed1987

A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia.

We found a novel platelet aggregating factor in a patient with steroid-responsive immune thrombocytopenic purpura that is associated with defective collagen-induced platelet functions. The aggregating factor and platelet functions were analyzed. The patient, a 58-year-old female, had purpura and prolonged bleeding time despite adequate platelet counts (greater than 140,000/microL) after steroid therapy. The patient's platelets responded normally to all agonists except collagen. Platelet adhesion to collagen fibrils was decreased. The patient's plasma induced irreversible aggregation and ATP release in normal platelet-rich plasma (PRP). This platelet aggregating factor was found in F(ab')2 fragments of the patient's IgG, which caused thromboxane B2 synthesis, elevation of cytoplasmic Ca2+ levels, and phosphorylation of 40 kDa protein in normal platelets. Platelet aggregation by the patient's IgG was inhibited by prostacyclin, dibutyryl cAMP, diltiazem, disodium ethylenediaminetetraacetate, and antimycin A plus iodoacetate, but ADP scavengers, cyclo-oxygenase inhibitors, and heparin had little or no effect. The aggregating activity of the patient's IgG absorbed to and eluted from normal platelets. The patient's Fab fragments did not induce platelet aggregation in eight of ten normal PRP but specifically inhibited aggregation induced by collagen and by the patient's IgG. The major component of an immunoprecipitate made with the patient's IgG from radiolabeled membrane proteins of normal platelet extract had a 62 kDa mol wt, while no such precipitate appeared in extracts of the patient's platelets. These results indicated that platelet aggregation by the patient's IgG was induced by the reaction of an antibody with a specific antigen on the normal platelet membrane through stimulus-response coupling. This antigen may be a collagen receptor on the platelet, most likely a polypeptide of 62 kDa under reducing condition. The defect of collagen-induced aggregation of the patient's platelets seemed to be due to alteration of the membrane protein related to this putative collagen receptor.

Autoimmune Diseases↗
PubMed1986

Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12-hydroperoxyeicosatetraenoic acid-stimulated neutrophils.

Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S, 12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance liquid chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with 12-HPETE. In experiments using [1-14C]arachidonate-labeled neutrophils, little radioactivity was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous.

Arachidonic Acid↗
PubMed1986

Platelet prostaglandin D2 dehydrogenase in patients with myeloproliferative disorders.

NADP-linked 15-hydroxyprostaglandin dehydrogenase for prostaglandin D2 (PGD2DH) transforms prostaglandin D2 (PGD2) to inactive 15-keto-PGD2. This enzyme activity was spectrophotometrically determined in the cytosol of platelets and platelet sensitivities to PGD2 were studied in patients with myeloproliferative disorders (MPD) as well as in normal subjects. Platelet sensitivities to exogenous and endogenous PGD2 were estimated by IC50 of added PGD2 for platelet aggregation and by the inhibitory effect of a specific thromboxane synthetase inhibitor (OKY-046) on collagen-induced aggregation, respectively. PGD2DH activities of MPD patients were significantly higher than those of normal subjects (p less than 0.01). Although decreased sensitivity to exogenous PGD2 was detected in some MPD patients, they were not always associated with the increased enzyme activities. Furthermore, no specific correlation was found between PGD2DH activities and the inhibitory effects of OKY-046. Thus, PGD2DH seems to have little effect on the action of PGD2 against platelet aggregation in MPD patients and normal subjects.

Adult↗
PubMed1986

Binding of a radioiodinated 13-azapinane thromboxane antagonist to platelets: correlation with antiaggregatory activity in different species.

Binding of a 125I-labelled derivative of the 13-azapinane thromboxane antagonist (ONO-11120), [125I]-9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyp hen yl)-13, 14-dihydro-13-aza-15-beta-omega-tetranor-thromboxane A2 ([125I]-PTA-OH), to washed platelets of human, dog and rabbit was studied. Results were compared with the in vitro inhibitory potency of ONO-11120 on platelet aggregation induced by arachidonate and a thromboxane agonist, 9,11-epithio-11,12-methano-thromboxane A2 (STA2). [125I]-PTA-OH bound to washed human platelets in a reversible, saturable and temperature-dependent manner, and specific binding displaced by 20 microM ONO-11120 constituted about 40% of the total binding. Scatchard analyses revealed a single class of specific binding and the equilibrium dissociation constant (KD) and maximal concentration of binding sites (Bmax) were 22 nM and 390 fmol per 10(8) platelets (about 2,300 sites per platelet), respectively. In addition to ONO-11120, STA2 and another thromboxane receptor agonist, (15S)-hydroxy-11,9-epoxymethano-prosta-5Z,13E-dienoic acid (U-46619), effectively displaced the binding with IC50 values of 44 and 125 nM respectively. Prostaglandin D2 (PGD2) partially displaced the binding only at a concentration above 1 microM. PGE1 and thromboxane B2 (TXB2) were without effect up to 100 microM. Similar binding of [125I]-PTA-OH was observed on dog platelets. The KD and Bmax were 12 nM and 110 fmol per 10(8) platelets (about 680 sites per platelet), respectively, and these values did not change significantly after adrenaline treatment which potentiated arachidonate-induced aggregation of platelets in this species. On the other hand, no specific binding of ['251]-PTA-OH was found on rabbit platelets. 4 Consistent with the results from binding studies, ONO-11120, 0.5 microM, completely suppressed arachidonate-induced aggregation of human platelets, whereas, at concentrations up to 5 microM, this agent did not significantly inhibit aggregation of rabbit platelets induced by the same stimulus. STA2- induced aggregation of rabbit platelets also showed less sensitivity to ONO-I 1120. When a similar extent ofirreversible aggregation was induced by STA2 and the inhibitory potency ofONO-1 1120 was compared in human and rabbit platelets, about one hundred times greater concentration of ONO- 11120 was required to suppress aggregation of rabbit platelets than that of human platelets. 5 These results suggest that [1251]-PTA-OH binds to a platelet thromboxane receptor, and that the structure of the binding site(s) on the receptor may vary between species.

Animals↗
PubMed1986

Deficient induction of leukotriene synthesis in human neutrophils by lipoxygenase-deficient platelets.

The effect of human platelets with deficient lipoxygenase activities on leukotriene B4 (LTB4) synthesis by neutrophils was studied. When arachidonic acid (AA) metabolites obtained from the incubation of washed normal neutrophils and platelets with N-formylmethionylleucylphenylalanine (FMLP), cytochalasin B, and AA were analyzed by reversed-phase high-performance liquid chromatography, the synthesis of 5-lipoxygenase products, including LTB4, was remarkably stimulated by platelets, with their maximal effect at a ratio of platelets to neutrophils of 15:1. However, the use of lipoxygenase-deficient platelets obtained from four patients with myeloproliferative disorders instead of normal platelets showed the deficient production of 5-lipoxygenase-derived products, whereas platelets with normal lipoxygenase activities obtained from MPD patients stimulated the 5-lipoxygenase pathway similarly to the way in which normal platelets did. The addition of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), a labile AA metabolite via the platelet lipoxygenase pathway, could activate the 5-lipoxygenase pathway in neutrophils incubated with FMLP, cytochalasin B and AA, but its stable end product, 12-hydroxyeicosatetraenoic acid, could not. Thus, it is suggested that lipoxygenase-deficient platelets did not sufficiently stimulate LTB4 synthesis during platelet-neutrophil interactions because of defective formation of 12-HPETE. This altered interaction between platelets and neutrophils through the lipoxygenase pathway might result in deficient responses at sites of thrombosis or inflammation in patients with deficient platelet lipoxygenase activities.

Adult↗