Sayuri Hiraishi

Elevation of Plasma Thrombomodulin Level in Diabetic Patients With Early Diabetic Nephropathy

Thrombomodulin (TM) is a membrane protein in the vascular endothelium, and it plays an important role as a cofactor in the thrombin-catalyzed activation of protein C. It has also been found in human plasma; however, its clinical significance is not known. In this study, fasting plasma TM concentrations in 67 diabetic patients with different degrees of albuminuria (39 men aged 57 ± 8 yr, 28 women aged 57 ± 11 yr; means ± SD) and 34 age- and sex-matched healthy subjects were investigated by use of a one-step sandwich enzyme immunoassay, a new method developed by H.I. and others. As a screening, the patients were divided into three groups according to the first morning urinary concentrations of albumin: group 1, <30 μg/ml (normoalbuminuria); group 2, 30–140 μg/ml (microalbuminuria); group 3, >140 μg/ml (clinical nephropathy). There was no significant difference in plasma TM level between the control group (17.7 ± 3.7 ng/ml, n = 34) and group 1 (16.9 ± 3.4 ng/ml, ± = 30); however, plasma TM concentrations in group 2 (22.8 ± 3.4 ng/ml, n = 22) and group 3 (29.6 ± 6.1 ng/ml, n = 15) increased significantly compared with those in the control group and group 1, respectively. As a further investigation, three timed overnight urine collections were made. The patients were allocated to three groups according to their rates of albumin excretion: group I, <20 μg/min (normoalbuminuria); group II, 20–200 μg/min (microalbuminuria); group III >200 μg/min (clinical nephropathy). No significant difference was found in plasma TM level between the control group (17.8 ± 3.8 ng/ml, n = 16) and group I (17.8 ± 3.0 ng/ml, n = 17); however, plasma concentrations in group II (22.1 ± 2.8 ng/ml, n = 18) and group III (29.2 ± 6.0 ng/ml, n = 13) increased significantly compared with those in the control group and group I, respectively. No significant differences were found in blood pressure between the control group, group I, and group II. The vascular endothelium could be injured by various metabolic derangements because of diabetes. Accordingly, it is supposed that an injury in the vascular endothelial cell may progress with the advance of diabetic angiopathy, and TM existing on the endothelial membrane surface may be released into the plasma. The vascular permeability that permits glomerular leakage of albumin may also be found in other vessels. Thus, our findings suggest that an increased influx of TM to the plasma may be caused by generalized endothelial damage in patients with early diabetic nephropathy.

Oxidized phospholipids in oxidized low-density lipoprotein reduce the activity of tissue factor pathway inhibitor through association with its carboxy-terminal region.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits the initial reactions of blood coagulation. In this study, we explored the nature of active components that reduce the anticoagulant activity of TFPI in oxidized low-density lipoprotein (ox-LDL). The organic solvent-soluble fraction obtained from ox-LDL was fractionated by normal-phase HPLC. The binding profile of each fraction to TFPI showed a single peak eluting near purified oxidized phospholipid. To explore further the components in oxidized phospholipid that inhibit TFPI activity, we used oxidized phospholipids that mimic the biological activity of ox-LDL. The oxidation products of 1- and/or 2-oleoyl phosphatidylcholine or phosphatidylethanolamine were the most potent inhibitors of TFPI activity, whereas those of arachidonyl phosphatidylcholine possessed only a weak inhibitory effect on the TFPI activity. These oxidized phospholipids mainly associated with the C-terminal basic region of the TFPI molecule. The results indicate that oxidation products of delta-9 unsaturated phospholipids are candidate active components of ox-LDL that impair the function of TFPI through specific association with its C-terminal basic region.

Changes in plasma thrombomodulin antigen in rabbit developing endotoxin-induced disseminated intravascular coagulation and the effect of heparin

Soluble thrombomodulin (TM) antigen level was 1.64 +/- 0.64 microgram/ml (n = 18, mean +/- S.D.) in plasma of normal male rabbits as measured by enzyme immunoassay, and the antigen consisted of subspecies of 94, 83 and 51 kd. When disseminated intravascular coagulation (DIC) was induced by intravenous infusion of endotoxin into rabbits, the TM antigen level in plasma was elevated to about 1.5 times of the control value, and an increase in the 83 kd subspecies as well as the appearance of new subspecies of 76 and 48 kd was observed concomitantly with disappearance of the 94 kd subspecies in plasma. Elevation of the antigen level and disappearance of the 94 kd subspecies caused by infusion of endotoxin were reduced by simultaneous infusion of heparin. Addition of leukocytes stimulated with endotoxin plus FMLP to cultured endothelial cells induced release of TM antigen to the medium accompanying cell injury as measured by 51Cr release, which was prevented by treatment with heparin. It was suggested that the increase in plasma TM antigen level in parallel with the generation of DIC reflected endothelial injury of rabbits, and that the elevation of TM antigen and the endothelial cell injury were prevented by heparin treatment.

Thiolprotease inhibitor, EST, can inhibit thrombin-induced platelet activation

Participation of thiolprotease in platelet activation was investigated. When platelets were treated with EST (L-trans-epoxysuccinyl-leucylamide (3-methyl)butane-ethyl ester, a membrane-permeable thiolprotease inhibitor) for 30 min, thrombin-induced aggregation and secretion were inhibited, and remained so even when the platelets were washed and resuspended in EST-free medium after the pretreatment. The inhibitory action of EST on thrombin-induced platelet aggregation and secretion was both dose-dependent and incubation-time-dependent. The inhibitory action of EST on platelet aggregation and secretion was shown not only in response to thrombin but also to platelet activating factor (PAF). Pretreatment of platelets with 1 mM EST for 30 min inhibited the 65% of calpain (thiolprotease) activity in platelets. The phosphorylation of 40 kDa and 20 kDa proteins of platelets caused by stimulation with thrombin was blocked by the pretreatment with 1 mM EST. Phosphatidylinositol hydrolysis and inositol-1-phosphate production, which appear after stimulation of platelets with thrombin, were also inhibited by the pretreatment with 1 mM EST. The results suggest that EST was incorporated to inside of platelets, and inhibited activation of platelet through inhibition of thiolprotease.

The effect of plasma on platelet function in hypercholesterolemic rabbits and the changes in fatty acid composition of the plasma

Rabbits were fed with 1% cholesterol-containing standard diet for 1 to 3 months. The arachidonic acid (AA)-induced aggregation of the platelet-rich plasma (PRP) of the control rabbits was accelerated by substitution of hypercholesterolemic plasma. The incorporation of 14C-AA into thromboxane B2 in platelets was increased approximately 1.6 times with PRP and 1.2 times with the washed platelet suspension (WPS) in hypercholesterolemic rabbits as compared with those of the control. Analysis of the fatty acid compositions of phospholipids and total lipids of hypercholesterolemic rabbits revealed an increase in AA of platelets and plasma, and a decrease in docosahexaenoic acid (DHA) in plasma. The AA/DHA ratio of plasma increased dependently on the period of feeding with the high cholesterol diet, and the increase in the ratio was parallel with the acceleration of platelet aggregation by AA in PRP.

A platelet aggregating substance contained in xanthine oxidase preparation from cow's milk.

Superoxide-generating system (xanthine-xanthine oxidase system) induced the platelet aggregation and the release of 3H-serotonin from washed human platelet. However, the aggregation was induced even when xanthine substrate was excluded from the system or when the activity of xanthine oxidase was completely abolished by the addition of 1.6 mM allopurinol to the system. Xanthine oxidase preparation from cows milk was chromatographed on Sephadex G-200 column, and it was found that the platelet aggregating substance was separated from xanthine oxidase activity by this procedure. The aggregating activity of the substance was destroyed by boiling it for 3 min. Its molecular weight was estimated to be about 17,000. The levels of malondialdehyde and thromboxane B2 production in the platelets were increased during the aggregating 9 and 4 times, respectively, compared to those of resting cells.

Oxidation products of phospholipid-containing δ-9 fatty acids specifically impair the activity of tissue factor pathway inhibitor

In the present study, we explored the active components in oxidized low-density lipoprotein (ox-LDL) that reduce the catalytic activity of tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor of the extrinsic blood coagulation pathway. The active fraction was extracted from the phospholipid fraction of ox-LDL and separated. The oxidation products of 1- and/or 2-oleoyl phosphatidylcholine (PC) or phosphatidylethanolamine were the most potent compounds, while those of arachidonyl PC possessed only a weak inhibitory effect on the TFPI activity. These oxidized phospholipids associated strongly with rTFPI containing the carboxyl-terminal domain. When rTFPI was incubated with purified oxononanoyl PC (9CHO-PC) and its carboxylic form (9COOH-PC), the catalytic activity was specifically impaired, though neither oxovaleroyl PC (5CHO-PC) nor lyso-phospholipids reduced the TFPI activity. We conclude that the oxidation products of delta-9 unsaturated phospholipid in the lipoproteins are the active components that impair the anti-coagulation activity of TFPI.

Characterization of a new type of variant of rat basophilic leukemia 2H3 cells presenting a different pattern of calcium signal

We selected a new type of variant (designated 3C7) derived spontaneously from parental RBL-2H3 cells. 3C7 cells showed lower contact inhibition, anchorage dependency, and serotonin release activity than those of RBL-2H3 cells. We conclude that 3C7 cells are a transformant of RBL-2H3 cell with greater malignancy. The production of inositol bisphosphate and the release of Ca2+ from intracellular stores induced by IgE-antigen stimulation were enhanced in 3C7. Oscillation of [Ca2+]i in individual 3C7 cells was observed by a digital imaging microscopic technique. We propose that 3C7 cells are a useful model system for studies on the mechanisms of stimulus-secretion coupling and the relationships between malignant alterations and disorders of signal transduction.

Abnormal expression by cytokines of pro- and anti-thrombotic factors produced in endothelial cells and a remedy for the abnormality.

Influence of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin 1-β (IL-1β) on the expression of thrombomodulin (TM, anticoagulant factor) and tissue factor-like procoagulant activity (PA, procoagulant factor) on cultured human umbilical vein endothelial cells and the effect of retinoic acid (RA, vitamin A acid) on the behavior of TM and PA in the cells caused by the cytokines were investigated. TNF-α and IL-1β induced the reduction of TM expression and elevation of PA in the cells in a dose-dependent manner in a range between 1 and 100U/ml. The reduction of TM expression by the cytokines accompanied with the decrease in TM mRNA levels, and the elevation of PA was inhibited by treatment with cycloheximide, inhibitor for protein synthesis. The current results indicate that the cytokines markedly increase procoagulant properties on the cell membrance through both the reduction of anticoagulant factor and the elevation of procoagulant factor, and suggest that the behaviors of anti- and pro-coagulant factor caused by the cytokines results from changes in protein synthesis. Treatment of the cells with RA prevented both the reduction of TM expression and the elevation of PA on the cell membrane caused by TNF-α treatment, in a dose-dependent manner in a range between 0.01 and 10μM. RA increased TM mRNA levels in the cells. It was suggested that RA regulates the balance between the expression of anti- and pro-coagulant factors on the cell membrance through regulation of gene transcription levels.