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, less than 30 micrograms/ml (normoalbuminuria); group 2, 30-140 micrograms/ml (microalbuminuria); group 3, greater than 140 micrograms/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, n = 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, less than 20 micrograms/min (normoalbuminuria); group II, 20-200 micrograms/min (microalbuminuria); group III greater than 200 micrograms/min (clinical nephropathy).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of soluble thrombomodulin fragments in human urine.

The soluble thrombomodulin (TM) subspecies in human urine detected by polyclonal anti-human TM IgG were isolated and characterized. 105, 85, 80, 56, 33, 31 and 28 kDa subspecies under reducing conditions was comparable to 78, 66, 56, 200, 52, 30 and 25 kDa under non-reducing conditions, respectively, in the two-dimensional electrophoresis. Each subspecies under non-reducing conditions, except the 200 and 52 kDa molecules, was constituted of single subspecies, whereas the 200 and 52 kDa molecules were constituted of the tetramer of the 56 kDa subspecies of reducing conditions and a dimer of the 33 kDa subspecies, respectively. NH2-terminal amino acid sequences of the 105, 85 and 80 kDa subspecies maintained Ala1-Pro2-Ala3- of intact human TM, however, 56, 33, 31 and 28 kDa subspecies started from Glu137-Gln138-, Gln214-Gly215-, Ser228-Val229- and Ala240-Ile241-, respectively. All subspecies obtained under non-reducing conditions exhibited cofactor activity for thrombin-dependent protein C activation ranging from 58 to 162 pmol APC/min/nmol TM at 0.4 mM Ca2+ indicating that all of the subspecies maintained the fourth to sixth repeat of epidermal growth factor-like structure of intact TM. 85, 80, 56, 33, 31 and 28 kDa subspecies were suggested to lack both chondroitin sulfate glycosaminoglycan (CSGAG), transmembrane and cytoplasmic domains of intact TM, while 105 kDa subspecies lack only CSGAG from the results of kinetic properties and the interaction with phospholipid vesicles composed from phosphatidylcholine and phosphatidylethanolamine.

Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies.

Thrombomodulin, TM, is an endothelial cell membrane protein acting as a cofactor for the activation of plasma protein C. Soluble TM is present in plasma and urine of normal subjects. Enzyme immunoassay, EIA, for human TM was developed using mouse monoclonal antibodies against human placental TM in this paper. We obtained four types of the monoclonal antibodies against human placental TM. EIA sandwich method using three types of the monoclonal antibodies enabled us to measure almost all of 6 and 7 TM subspecies in plasma and urine, respectively, except 1 subspecies, 31 kDa TM. There was no interference from other components of plasma and urine in the assay conditions. Titration curves of purified TM in buffer or in normal plasma were linear within the range from 0.08 to 10 ng/ml. The coefficient of variation at 0.08 ng/ml TM was 4.7%. TM titer with buffer, assayed by this method, was reduced by the addition of thrombin at the final concentration of 20 U/ml, but the titer with plasma was not reduced even at 100 U/ml. These concentrations of thrombin are far larger than those which would be formed in circulation. TM levels in plasma and urine of normal subjects collected in the morning were 35.2 +/- 8.32 ng/ml (n = 346) and 111 +/- 31.6 ng/ml (n = 33), respectively. TM level in plasma did not differ from the level in serum. Circadian fluctuation of plasma TM was not significant in 10 normal adults, although a tendency of increase in TM excretion to urine was found rather in the day time than the other times.(ABSTRACT TRUNCATED AT 250 WORDS)

Noninvasive visualization of coronary arterial aneurysm in infants and young children with mucocutaneous lymph node syndrome with two dimensional echocardiography.

Real time two dimensional echocardiographic studies of the main coronary arteries were performed in 30 normal subjects and 58 patients with the mucocutaneous lymph node syndrome. Four echocardiographic coronary arterial patterns were observed: linear, dilated, fusiform or spherical. In normal subjects the left main coronary artery generally had a linear pattern but had a dilated pattern in two patients. The right coronary artery could not be visualized clearly. In 7 of the 58 patients with the mucocutaneous lymph node syndrome, a portion of the left main coronary artery had a fusiform or spherical pattern, or both, and the stem of the right coronary artery also had a spherical pattern in 2 patients. In all five patients who had coronary angiography, the procedure confirmed an aneurysm in the left main coronary artery or its branching point and the stem of the left anterior descending coronary artery or the stem of the right coronary artery, or both. In three of the five patients, coronary arterial aneurysms were predicted before coronary angiography was performed. Thus, two dimensional echocardiography is a reliable noninvasive method for evaluating coronary arterial aneurysms in infants and young children.

Two-dimensional echocardiographic assessment of left atrial size in children.

The ability of 2-dimensional echocardiography (2-D echo) to estimate end-systolic left atrial (LA) size and volume was assessed in 140 infants and children. These subjects were divided into 2 groups. Group A included 91 patients with normal LA volume and Group B included 49 patients with LA volume overload. Five echocardiographic views (left parasternal long-axis, left parasternal short-axis, apical 4-chamber, apical 2-chamber and subcostal 4-chamber) were used. From these views, the LA long-axis and minor-axis lengths were measured and the area was planimetered. These echocardiographically derived measurements were compared with angiographically calculated LA volume. Although all echocardiographic measurements correlated well with angiographic LA volume measurements, the echocardiographic area tracked better than length measurements. Echo LA volume was calculated using 5 single-plane and 3 biplane area-length methods. LA volume calculated from either single- or biplane methods correlated well with angiographically determined LA volume. The degree of correlation depended on the method used. Echocardiographic area and estimated LA volume measured from the parasternal long-axis and apical 2-chamber views best separated patients with LA volume overload from normal. Two-dimensional echo using these views accurately segregated all patients with a LA volume greater than 180% of normal and 15 of 21 patients (71%) with an LA volume between 138% and 179% of normal. Thus, 2-D echo is useful in the evaluation of LA size and volume in infants and children.

Clinical course of cardiovascular involvement in the mucocutaneous lymph node syndrome. Relation between clinical signs of carditis and development of coronary arterial aneurysm.

Seventy-nine patients with mucocutaneous lymph node syndrome were evaluated prospectively by clinical examination, electrocardiography, chest radiography, M mode and two dimensional echocardiography and thallium-201 myocardial scanning. Serial changes were categorized according to the duration of illness: stage I (1 to 10 days), stage II (11 to 20 days), stage III (21 to 30 days), stage IV (31 to 60 days) and stage V (61 days to 40 months). The presence of myocarditis in stages I and II was suggested in 40 of 79 patients (50.6 percent) by electrocardiographic, echocardiographic, radiographic and clinical abnormalities. Myocarditis was accompanied by pericarditis in six patients and by both endocarditis and pericarditis in one patient. These signs of inflammation were resolved by stage III in all but three patients with electrocardiographic abnormalities. In the active stage, large coronary arterial lesions were suspected only because of an abnormal spherical echo-free space in the region of the coronary arteries on two dimensional echocardiograph as well as electrocardiographic evidence of deep Q waves in leads II, III and aVF. One or more coronary aneurysms developed in 11 patients, primarily in stage II; regression of the aneurysm was noted in 5 of these patients during stages III, IV and V. Aneurysm regression demonstrated by angiography did not correlate with echocardiographic changes in aneurysm size in one patient. Moreover, the occurrence of coronary aneurysm did not correlate with the presence of signs of carditis, because the frequency of carditis was the same in patients with and without aneurysm.

Effect of suture closure of coronary artery fistula on aneurysmal coronary artery and myocardial ischemia.

This study indicates the importance of coronary angiography and myocardial scintigraphy on long-term follow-up of patients after surgery for coronary arterial fistula in view of the progression to coronary artery obstruction and myocardial ischemia.

Two-dimensional echocardiographic assessment of right ventricular volume in children with congenital heart disease.

The ability of 2-dimensional echocardiography to measure right ventricular (RV) volume and ejection fraction was assessed in 22 children with congenital heart disease. From the apical 4 chambers 2-dimensional echocardiographic image, the long-axis length of the right ventricle was measured and the area planimetered. On the anteroposterior and lateral cineangiocardiographic planes, the right ventricle was separated into 2 parts: RV sinus and outflow tract. The longest length, inflow tract length, and area of the sinus were measured from biplane cineangiographic views. The echographic long-axis length correlated well with the longest length of the RV sinus measured from both anteroposterior and lateral cineangiographic views at both end-systole and end-diastole. Moreover, the echographic area correlated well with the sinus area obtained from both cineangiographic views. From these regression analyses, the echographic long axis length and area were corrected to the angiographic longest length and area of the sinus. The new corrected echographic longest length and area were applied to 3 formulas (2 biplane and 1 uniplane) to calculate the sinus volume of the right ventricle. Total RV volume was then derived from the sinus volume. RV volumes and ejection fraction determined by 2-dimensional echocardiography were compared with those obtained from biplane cineangiography using Simpson's rule method. All formulas tested predicted RV volumes and ejection fraction with equal accuracy. Thus, 2-dimensional echocardiography can assess RV volume and ejection fraction in children with congenital heart disease.

Truncus arteriosus with interrupted aortic arch: successful correction using autologous flap.

A newborn baby with type II truncus arteriosus and type B interrupted aortic arch was successfully treated by creating a pulmonary tract using autologous flap made from truncal wall without excision of the pulmonary artery and by reconstructing the aortic arch with direct anastomosis. This method provided excellent hemodynamics with wide reconstruction of the pulmonary tract without conduit.

Anterior pulmonary translocation without conduit for the repair of truncus arteriosus.

A technique with autologous tissue for the correction of type III truncus arteriosus is described. The truncal root was excised as a cylinder that incorporated pulmonary arteries and that was translocated anterior to the ascending aorta. The proximal section of the cylinder was closed and the pulmonary tract was reconstructed with anastomosis of a widely opened distal section to the right ventricle. Autologous pericardium was sutured to the entire surface of the pulmonary tract.

Correction of truncus arteriosus with autologous arterial flap in neonates and small infants.

BACKGROUND: This study describes the results of techniques using the autologous truncal wall and part of the pulmonary artery for correction in anticipation of the growth of the pulmonary tract in patients with truncus arteriosus. METHODS: Seven consecutive patients with truncus arteriosus were reviewed. The posterior wall of the pulmonary tract was obtained by anastomosing the lower edge of the truncal arteriotomy to the upper corner of the ventriculotomy from the truncus in types I and II. Anterior translocation of the pulmonary artery was performed in a type III. A pericardial patch with or without a monocusp was placed to complete the right ventricular outflow tract. RESULTS: There were two hospital deaths, one of which was unrelated to a cardiac problem. Postoperative right-to-left ventricular peak pressure ratio was less than 0.55. There was one left pulmonary stenosis due to monocusp adherence in the late postoperative period. The sizes of the pulmonary tract at anastomosis were between 107% and 166% of the normal value between 7 months and 3.8 years of follow-up. CONCLUSIONS: The use of autologous arterial wall instead of a conduit is recommended for the repair of truncus arteriosus to expect growth of the pulmonary tract.

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 cow's 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.

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.

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.

[Thrombomodulin in diabetic retinopathy].

Thrombomodulin is a glycoprotein located on the surface membrane of vascular endothelial cells. Recently the presence of thrombomodulin antigens was detected in circulating blood plasma. The thrombomodulin antigens are thought to have been released from injured endothelial cells, and thus plasma thrombomodulin has been attracting attention as a new molecular marker indicating injuries to vascular endothelial cells. We investigated plasma thrombomodulin levels in relation to the severity of diabetic retinopathy, and found that they increased as retinopathy progressed. The Mann-Whitney U test revealed significant differences between no retinopathy and proliferative retinopathy (p = 0.002), between simple retinopathy and proliferative retinopathy (p = 0.01), and between preproliferative retinopathy and proliferative retinopathy (p = 0.03). However, ocular vascular injuries may correlate with systemic vascular injuries. Therefore, the progress of retinopathy may be predicted by monitoring plasma thrombomodulin levels in selected patients.

[Echocardiographic assessment of cardiac function in the children of chronic renal failure with cardiomegary].

Five children (four boys and one girl) with chronic renal failure (CRF) developed congestive heart failure 0.5 to 11 years after the onset of the disease. Their ages were from 4 to 13 years old. They noticed tachypnea, tachycardia, cough, chest anxiety, general fatigue and their chest X-rays showed cardiomegaly with cardio-thoracic ratio (CTR) of from 55 to 63% and pulmonary congestion. Their echocardiograms showed no cardiomuscular hypertrophy, but the dilatation of left ventricular diastolic diameter (LVDd), and the decreased ejection fraction (EF) were observed. They were treated with water restriction, antihypertensive agents, cardiotonics and dialysis. Their clinical symptoms improved promptly, but their cardiomegary and echocardiographic findings improved gradually. The causes of heart failure in these patients seemed to be due to uremia, fluid overload and hypertension. The echocardiographic examination was useful for the management of the children with CRF in heart failure.

[Surgical treatments of nonconfluent pulmonary arteries with congenital cardiac defects].

Pulmonary artery angioplasty or reconstruction was performed in seven patients with nonconfluent pulmonary arteries and congenital cardiac defects. Age of these patients were ranged from 6 months to 41 years old. Five of them had pulmonary truncal atresia and complex cardiac anomalies. Two of these five patients demonstrated nonconfluent pulmonary arteries due to deformities at ductal insertion of pulmonary arteries. Three patients had had previous systemic to pulmonary artery shunt operations which caused pulmonary artery distortions. Other two patients had intrapulmonary arterial obstructions due to pulmonary artery thrombosis. Patch pulmonary artery plasty was carried out in three patients, dilatation of severe stenotic pulmonary artery was done in one patient simultaneously with pulmonary valvotomy. Central shunt operation was added in one patient with the pulmonary artery which was unable to be reconstructed. Last two patients underwent intrapulmonary artery reconstruction with the rolled pericardial graft. Hospital death occurred in one patient with unproperly increased pulmonary blood flow by central shunt. Average follow-up period of these six survivors after operation was 1.4 +/- 0.8 years. As definite repairs, two patients had Fontan operation, two patients had right ventricle to pulmonary artery reconstruction. And remaining two patients are still to be followed until sufficient growth of pulmonary artery suitable for Fontan operation.