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Y Komiyama

Publications and source records attributed to Y Komiyama.

At least 37 records · Page 2Linked to original sources
PubMed1998

[Atherosclerosis and activation of platelet and blood coagulation].

It is widely accepted that atherosclerosis and thrombus formation are closely associated. Recent progress in the development of molecular markers of blood coagulation and platelet enabled us to estimate even the slight activation of blood coagulation and platelet activation systems. We investigated a contract phase-activate coagulation and factor -inhibitor complex (factor XIa-alpha 1 antitrypsin) and expression of CD62P on platelet surface, a specific antigen on platelet, in patients with coronary arterial diseases. We compared these activation markers after stratifying patients according to the number of significant coronary artery stenoses. Patients with three-vessel disease had significantly increased levels of factor XIa-alpha 1 antitrypsin, fibrinogen, and CD62P expression compared to those in other subgroups. Although it is unknown whether these abnormalities are the cause or the result of the vascular lesion, these findings may be important to understand the pathophysiology underlying atherosclerosis or propagation of the atherosclerotic process itself.

Aged↗
PubMed1997

Tyrosine phosphorylation and p72syk activation by an anti-glycoprotein Ib monoclonal antibody.

NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ib alpha, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-Fc gamma II receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab' fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab' fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the Fc gamma II receptor. Cross-linking of the receptor-bound Fab' fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5-induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.

Antibodies, Monoclonal↗
PubMed1997

Daidzein stimulation of bone resorption in pit formation assay.

We found that daidzein stimulated bone resorption in the pit formation assay at the concentration of 10(-8)-10(-10) M. On the other hand, genistein and ipriflavone at these concentrations did not have any effect on pit formation. We speculated that daidzein had the ability to induce osteoclasts directly or indirectly from their progenitors and might be a tool by study osteoclast differentiation.

Animals↗
PubMed1997

Vasodepressor effects of exercise are accompanied by reduced circulating ouabainlike immunoreactivity and normalization of nitric oxide synthesis.

Our object was to evaluate the effects of regular mild exercise on blood pressure and on circulating level of ouabainlike factors (OLF) and of nitrate anion, an endproduct of nitric oxide (NO) in humans. We measured plasma ouabainlike immunoreactivity (OLI) and nitrate ions (NO3.) before and after mild exercise for 3 months' duration in 16 patients with essential hypertension, diabetes mellitus, obesity, or hyperlipidemia. Plasma OLI was measured using an amplified ELISA system with anti-ouabain antibody and biotinyl-tyramide. Serum NO3. was measured with high-performance liquid chromatography (HPLC) with an anion-exchange column. With the reverse phase HPLC system with an octa decylsilyl silicagel column, the elution volume of plasma OLI of a healthy volunteer matched that of authentic ouabain in a gradient elution system of acetonitrile/H2O. Plasma OLI levels decreased significantly by about 34% after mild exercise, and NO3. levels tended to be within the reference interval in normal volunteers. Body weight, diastolic and systolic blood pressure, serum triglyceride and acetylcholine esterase (a marker of the fatty liver) were significantly decreased (p < 0.01) after 3 months of regular mild exercise. The plasma OLI level was significantly correlated with plasma NO3., there was a trend toward a correlation with diastolic blood pressure (p = 0.06) before and after regular exercise. Regular mild exercise led to a decrease in plasma levels of OLI, and acetylcholine esterase activity and blood pressure in adult patients. Results suggest that changes in OLF production contribute to the blood pressure regulation seen in patients who exercise regularly.

Adult↗
PubMed1997

Effect of a new monoclonal anti-glycoprotein IX antibody, KMP-9, on high shear-induced platelet aggregation.

Human platelet glycoprotein Ib/IX complex acts as a receptor for von Willebrand factor. It is widely accepted that glycoprotein Ib is the essential receptor component, but the role of glycoprotein IX is still unclear. We produced a new monoclonal anti-glycoprotein IX antibody (KMP-9) by the hybridoma technique using platelets from a patient with Glanzmann's thrombasthenia. The epitope of KMP-9 was localized to the C-terminal 8 kD fragment of glycoprotein IX using ELISA analysis of polyethylene-pin-synthesized peptides, as well as Western blot analysis of platelets after digestion with N-glycosidase and Staphylococcus aureus V8 protease. KMP-9 partially inhibited high shear stress-induced platelet aggregation, but had no effect on aggregation induced by ristocetin or low shear stress. Its inhibitory effect on high shear stress-induced aggregation was weaker than that of anti-glycoprotein Ib or anti-glycoprotein IIb/IIIa monoclonal antibodies. A 21-mer synthetic peptide (glycoprotein IX L110-G130) inhibited the binding of KMP-9 to platelets. It also competively inhibited the suppression of high shear stress-induced platelet aggregation by KMP-9, but had no direct effect on this aggregation. KMP-9 may be useful to clarify the physiological role of GPIX.

Amino Acid Sequence↗
PubMed1997

[Shear stress and platelet-derived microparticles].

One of the responses of activated platelets to certain stimuli is the shedding of microparticles. Many studies have attempted to characterize the role of microparticles under various clinical situations or experimental conditions. Pathological levels of fluid shear stress may occur in diseased small arteries and arterioles partially obstructed by atherosclerosis or vasospasm and such shear stress may induce the activation and aggregation of circulating platelets. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A cone-plate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and remnant platelets showed procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor to platelet glycoprotein Ib, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C promoted shear-dependent microparticle formation. These findings suggest that local generation of microparticles in atherosclerotic arteries, the site at which pathological levels of shear stress could occur, contributes to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.

Arteriosclerosis↗
PubMed1997

[A case of osteopetrosis with an abnormal CK-MB/T-CK ratio].

Creatine kinase (CK)-MB subunit has been recognized as a useful marker for acute myocardial infarction (AMI). However, we recently experienced one case of osteopetrosis with moderately high CK-MB and an abnormal (more than 100%) CK-MB/total (T)-CK ratio without evidence of AMI in a medical examination. We have already experienced 17 cases with an abnormal CK-MB/T-CK ratios in addition to the present case. Those cases were patients with malignant tumor with metastasis (n = 13), leukemia (2), liver cirrhosis (1), and cerebral death (1), and thereby the band of macro-CK was found in the electrophoresis. However, we detected neither the band of macro-CK nor the abnormal levels of tumor markers such as CEA, alpha-fetoprotein, CA-19-9 in the present case. Instead of the macro CK, the high level of CK-BB was detected in electrophoresis. In the medical examination, especially in screening tests, the CK-MB was generally assayed with use of the immunoinhibition method in automated analyzers. The method principle was based on the absence of CK-BB in the patient serum. Since the patient had the past history of pathological fracture in his boyhood, this patient was diagnosed as osteopetrosis. These results suggest that we must consider the possibility of osteopetrosis when an abnormal CK-MB and CK-MB/T-CK ratio without evidence of serious diseases were found. This is simply because of the assay method of immunoinhibition for CK-MB activity.

Adult↗
PubMed1996

High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles.

Previous studies have demonstrated that a high level of shear stress can produce platelet aggregation without the addition of any agonist. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A coneplate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and the remnant platelets showed the exposure of procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor (vWF) to platelet glycoprotein lb, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C (PKC) promoted shear-dependent microparticle formation. Epinephrine did not influence microparticle formation, although it enhanced platelet aggregation by high shear stress. These findings suggest the possibility that local generation of microparticles in atherosclerotic arteries, the site that pathologically high shear stress could occur, may contribute to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.

Blood Platelets↗
PubMed1996

Association of tissue factor with a gamma chain homodimer of the IgE receptor type I in cultured human monocytes.

Tissue factor (TF) is a high-affinity receptor for coagulation factors VII (F VII) and VIIa. The F VII/VIIa/TF complex is the major cellular initiator of the extrinsic coagulation cascade. We found that the occupancy of TF by its ligand, F VIIa, is involved with signal transduction and that TF is associated with the gamma chain homodimer identified as a component of IgE receptor type I (Fc epsilon RI). When 4-day cultured human monocytes were incubated with F VIIa, several polypeptides, especially a 70-kDa polypeptide, were transiently phosphorylated on tyrosine, residues. These phosphorylation events were inhibited by prior binding of anti-TF monoclonal antibody (mAb) HTF-K14, but not anti-TF mAb HTF-K180 to intact cultured monocytes. HTF-K14 blocked the binding of F VII/ VIIa to cell surface TF, whereas HTF-K180 did not. Anti-TF immunoprecipitates prepared from 1% digitonin lysates of cultured human monocytes incorporated phosphate in a gamma chain homodimer when incubated with [gamma-32P] ATP. The identity of the T-associated structures as gamma chains was established by immunoblot analysis of anti-TF mAb immunoprecipitates with anti-gamma chain mAb. In addition, anti-TF immunoblot analysis showed that TF co-precipitated with anti-gamma chain mAb. Our data suggest that gamma chains may play an important role in signaling via TF in human monocytes/macrophages.

Cells, Cultured↗
PubMed1996

High-density lipoprotein inhibits the synthesis of platelet-activating factor in human vascular endothelial cells.

The regulation of platelet-activating factor (PAF) synthesis by serum lipoproteins was investigated in human umbilical vein endothelial cells. High-density lipoprotein (HDL) inhibited PAF synthesis in agonist (thrombin, histamine, and A23187)-stimulated endothelial cells, that was determined by incorporation of [3H]acetate into PAF and by bioassay. The inhibition by HDL was increased in a concentration-dependent manner, but was reversed as the concentration of thrombin increased. HDL did not affect the time course of PAF production. HDL lipids suppressed the PAF production to a lesser extent than HDL. The reduction of PAF accumulation in HDL, did not result from degradation of PAF but inhibition of PAF synthesis, which was mainly mediated via the blockade of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activation. HDL did not prevent the release of [3H]arachidonic acid in thrombin-stimulated endothelial cells. The binding of 125I-HDL to endothelial cells and its uptake were not enhanced by thrombin stimulation. These results demonstrate that HDL may inhibit the activation of acetyltransferase by thrombin at the cell surface. This observation may explain a part of mechanism of HDL action.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Flow cytometric analysis of platelet activation markers CD62P and CD63 in patients with coronary artery disease.

We investigated whether platelet activation could be quantitatively detected in patients with coronary artery disease using flow cytometric analysis of the expression of CD62P and CD63, which are activation-specific antigens on the platelet surface. Platelet samples were obtained from 16 healthy control subjects and 65 patients, of whom 25 had angiographically normal coronary arteries and 40 had at least one major coronary artery stenosis (> or = 50% narrowing). In both patient groups, CD62P expression was significantly higher than in the control group, but the difference between the two patient groups was not significant. In contrast, CD63 expression did not differ among the three groups. We also compared expression of these antigens after stratifying the patients according to the number of significant coronary artery stenoses. Patients with three-vessel disease had significantly increased CD62P and CD63 expression compared with the other subgroups. Our findings indicate that platelet activation occurs in patients with severe coronary artery stenosis.

Adult↗
PubMed1996

Effects of intracerebroventricular administration of 6-hydroxydopamine on ouabain-like immunoreactivity in plasma and the hypothalamo-pituitary axis in rats.

OBJECTIVE: To examine the role of central mechanisms on the production and release of an ouabain-like factor, the effects of intracerebroventricular injections of 6-hydroxydopamine on the tissue content and on the plasma level of the ouabain-like factor were determined in rats. METHODS: The vehicle (0.1% ascorbic acid in 0.9% saline) and 6- hydroxydopamine (250 micrograms/rat) were injected into the left lateral ventricle in ether-anaesthetized Wistar rats. Hypothalamus, pituitary, adrenal and venous blood was sampled 24h and 7 days later. The procedure was repeated using another rat group 7 days later. Characteristics of immunoreactive ouabain-like factor were determined by a combination of high-performance liquid chromatography and a highly sensitive enzyme-linked immunosorbent assay for ouabain. The level of the ouabain-like factor in these tissues and in plasma extracts measured by the enzyme-linked immunosorbent assay was compared between the two groups receiving 6-hydroxydopamine and the vehicle. RESULTS: Twenty-four hours after the intracerebroventricular injections of 6-hydroxydopamine, the ouabain-like factor level in the pituitary, hypothalamus and plasma had decreased significantly, whereas the ouabain-like factor level in the adrenal had not changed. The content of noradrenaline in the hypothalamus was also decreased markedly 7 days later and the content of ouabain-like factor in the pituitary remained low. On liquid chromatography the elution pattern of the ouabain-like factor in plasma and in tissue extracts coincided with that of authentic ouabain. CONCLUSIONS: Intracerebroventricular treatments with 6-hydroxydopamine elicited decreases in ouabain-like factor contents in the pituitary, the hypothalamus and the plasma. These results suggest that the production and release of ouabain-like factor are closely associated with the brain, particularly the hypothalamus-pituitary axis, and that noradrenergic or dopaminergic neurons, or both, play a key role in this mechanism.

Animals↗
PubMed1996

[Evaluation of the factor XIIa assay kit].

The assay for activated coagulation factors has recently been considered to be a useful tool for detecting hypercoagulable state. Measurement of activated factor XI and alpha 1 antitrypsin complex developed by us is the only one measure for detecting contact phase hypercoagulation. The ACTIVATED FACTOR XII kit (Shield Diagnostics) has recently been commercially available, and the reference range was reported in the instruction manual. However, because the recovery rate of purified factor XIIa (XIIa) in normal pooled plasma by this assay kit was strikingly decreased, the effects of inhibitors in plasma, such as low molecular weight serine proteinase inhibitor (diisopropyl fluoro-phosphate, DFP), the specific inhibitor for XIIa (corn trypsin inhibitor, CTI), the main inhibitor for XIIa (C1 inhibitor, C1I) were examined. These three inhibitors and XIIa were incubated for 18 hours, then the XIIa levels of these complexes and the recovery rate in the normal pooled plasma were assayed by this assay kit. The recovery ratios in the normal pooled plasma were; DFP-XIIa 82%, CTI-XIIa 63%, and C1I-XIIa 0%, XIIa without inhibitor 7%. These results suggest that this XIIa kit does not reflect the levels of complexed form of XIIa and inhibitors in plasma samples.

Adult↗
PubMed1995

Expression of prothrombinase activity and CD9 antigen on the surface of small vesicles from stimulated human endothelial cells.

We employed flow cytometry and monoclonal antibodies (MoAb) to study the surface membrane protein of shed particles (small vesicles, SV) that were released from vascular endothelial cells (EC) by agonists such as a Ca ionophore (A23187) and thrombin. After stimulation of EC by A23187, CD9 antigens disappeared entirely from the EC surface in a time- and concentration-dependent manner; they subsequently moved onto the SV surface. Von Willebrand factor (vWF) and P-selectin from Weibel-Palade (W-P) bodies were expressed rapidly on the EC surface after thrombin stimulation, but not on the SV surface. P-selectin may have some effect on maintenance of hemostasis on the EC surface. We demonstrated that the surfaces of SV and EC significantly supported prothrombinase activity and confirmed that A23187-induced SV from EC express binding sites for factors IXa and Xa. These results suggest that the SV are an important factor in a novel controlling mechanism of the coagulation system on the EC surface.

Antibodies, Monoclonal↗
PubMed1995

Regulation of the tertiary structure and function of coagulation factor IX by magnesium (II) ions.

The indispensable role of Ca2+ ions in the maintenance of the functional tertiary structures of vitamin K-dependent coagulation factors has been definitively established but the participation of Mg2+ ions, another alkaline-earth metal that is present abundantly in blood plasma, in such a process is not yet understood. We show here that the Ca(2+)-stabilized conformation of coagulation factor IX undergoes a further conformational change upon binding of Mg2+ ions using three independent structural probes. The probes we used were (i) IX/X-bp, a snake venom anticoagulant that recognizes the Gla domains in coagulation factors IX and X, (ii) conformation-specific polyclonal antibodies against bovine factor IX, and (iii) monoclonal antibodies against the Gla domain of human factor IX. The binding of all these probes had an absolute requirement for Ca2+ ions, and Mg2+ ions alone were ineffective. However, when added together with Ca2+ ions, Mg2+ ions at physiological concentrations greatly augmented the binding of these probes to factor IX; the required concentration of Ca2+ ions was much reduced, and the affinity of each probe for factor IX was increased even in the presence of an excess of Ca2+ ions. These results suggest the presence of a Mg(2+)-specific binding site that does not interact with Ca2+ ions in factor IX. Furthermore, Mg2+ ions potentiated the susceptibility of factor IX to activation by factor XIa, concomitant with their effect on the conformation. Similarly, the required Ca2+ concentration was reduced by Mg2+ ions, and the rate of conversion to factor IXa was increased by Mg2+ ions in the presence of an excess of Ca2+ ions. At a saturating concentration of Ca2+ ions (5 mM), addition of 1 mM Mg2+ reduced the apparent Km value for factor IX from 0.31 to 0.18 microM, and in the presence of a physiological concentration of Ca2+ ions (1 mM), the reduction in Km by Mg2+ ions was far more striking (from 0.91 to 0.24 microM). The apparent Vmax values were hardly affected by Mg2+ ions. Our present data reveal a hitherto novel physiological role of the Mg2+ ions in plasma. Not only Ca2+ ions but also Mg2+ ions are important regulators of the stabilization of the native conformation of factor IX as well as of its efficient activation.

Animals↗
PubMed1995

Activation of hepatocyte growth factor by two homologous proteases, blood-coagulation factor XIIa and hepatocyte growth factor activator.

Hepatocyte growth factor (HGF) is secreted as an inactive single-chain precursor from the producing cells, and normally remains in this form associated with the extracellular matrix. In response to tissue injury, the single-chain precursor is converted to a biologically active heterodimer by a serine protease, the activity of which is induced in the injured tissue. We have previously identified HGF activator, a serum serine protease that activates single-chain HGF. The sequence of HGF activator cDNA revealed that the HGF activator is homologous to blood-coagulation factor XIIa. In this study, we found that coagulation factor XIIa has an ability to activate single-chain HGF. Factor XIIa exhibited a significant level of HGF-converting activity in the presence of dextran sulfate, although the specific activity of factor XIIa was slightly lower than that of the HGF activator. Since factor XIIa is activated during the initiation of contact activation induced by tissue injury, factor XIIa may function as an HGF-converting enzyme together with HGF activator in the injured tissue. C1-inhibitor, antithrombin III and alpha 2-antiplasmin, that regulate the blood-clotting activity of factor XIIa, were also effective against the HGF-converting activity of factor XIIa. Furthermore, factor XIIa was not active in the HGF-converting activity in serum. Thus, the HGF-converting activity of factor XIIa may be regulated by these serum inhibitors.

Factor XIIa↗