Mutsuyoshi Kazama

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.

Heparin-like glycosaminoglycan is a receptor for Antithrombin III-dependent but not for thrombin-dependent prostacyclin production in human endothelial cells

Antithrombin III (ATIII) induced a marked increase in prostacyclin (PGI2) release from cultured human umbilical vein endothelial cells (HUVEC) after incubation for more than 2 hr and the induction continued for 8 hr, while thrombin induced the increase within 10 min. ATIII-dependent production of PGI2 was abolished by addition of heparin, but pretreatment of HUVEC with polyclonal antibody against thrombomodulin could not prevent the PGI2 productions by ATIII and thrombin. ATIII-dependent PGI2 production was significantly inhibited by pretreatment of HUVEC with beta-D-xylosides or heparitinase, though neither pretreatment affected thrombin-induced PGI2 production. After treatment of HUVEC with 1 micrograms/ml cycloheximide. ATIII-dependent PGI2 production was completely abolished. These results indicate that the mechanism of the induction of PGI2 production by ATIII involves heparin-like glycosaminoglycans on HUVEC and the stimulation of synthesis of a protein related to PGI2 production. The ATIII-induced PGI2 production is very different from that induced by thrombin.

Nafamostat Mesilate: A Regional Anticoagulant for Hemodialysis in Patients at High Risk for Bleeding

107 hemodialysis patients at high risk for intradialytic bleeding due to previous surgery or active bleeding from other sites were treated with nafamostat mesilate (FUT-175; FUT) as hemodialysis anticoagulant for 2 weeks. In contrast to heparin. FUT prolonged clotting times only in the extracorporeal circuit. Clotting times were not prolonged even at the conclusion of the treatment, and bleeding from the puncture site after removal of the needle was shorter than with heparin. The exacerbation of bleeding by hemodialysis was noted in only 21 out of 573 hemodialysis procedures (3.7%), and 134 of 145 hemodialysis procedures (92.4%) with active bleeding were successfully completed without increasing the bleeding. Adverse effects of FUT were noted in only 6 cases (5.6%) or 1.2% of HD procedures. These results indicate that FUT is a very useful anticoagulant for HD, especially in patients with high risk of bleeding.

Procalpain is activated on the plasma membrane and the calpain acts on the membrane.

The mechanism of activation of human erythrocyte calpain was investigated using the immunoblotting technique with anticalpain monoclonal antibody. The purified calpain underwent a Ca2+-induced fragmentation of the 80 kDa subunit to 76 kDa and 36 kDa fragments. The behavior of the 76 kDa fragment in electrophoresis corresponded to the proteinase activity of calpain, whereas the behavior of the 80 kDa subunit and the 36 kDa fragment did not. When inside-out membrane vesicles were added to the reaction mixture of calpain and Ca2+ and the vesicles were separated from the supernatant solution by centrifugation, the 80 kDa subunit and 76 kDa fragment were found in the vesicle fraction. No other fragments were found in this fraction. On the other hand, the 80 kDa subunit and 36 kDa fragment were found in the supernatant fraction. When right-side-out membrane vesicles were added to the reaction mixture and the vesicles were separated from the supernatant fraction, no fragment was found in the vesicle fraction, while only the 36 kDa fragment was found in the supernatant fraction. These results indicate that the 80 kDa subunit of procalpain was bound in a Ca2+-dependent manner to the cytosolic surface of the plasma membrane and then underwent fragmentation to produce the 76 kDa fragment (active form) and that it expressed its proteinase activity at the surface of the membrane.

Effect of Total-Body Cold Exposure on Plasma Concentrations of von Willebrand Factor, Endothelin-1 and Thrombomodulin in Systemic Lupus erythematosus Patients with or without Raynaud’s Phenomenon

The effect of total-body cold exposure on plasma concentrations of von Willebrand factor (vWF), endothelin-1 (ET) and thrombomodulin (TM), all of which are considered to be generated from the endothelium, was studied in systemic lupus erythematosus (SLE) patients with and without Raynauds phenomenon. The plasma levels of vWF, ET and TM in SLE patients, irrespective of the presence of Raynauds phenomenon, were significantly higher than in normal controls even before the cold provocation test. After the cold provocation test, plasma levels of vWF and ET were significantly higher in SLE patients with Raynauds phenomenon than in those without and in normal controls. No significant increase in TM was observed in either the SLE patients or the controls. These results suggest that SLE patients, regardless of the presence of Raynauds phenomenon, are in a hypercoagulable state and that this state may be further intensified by cold exposure. Hence, it is concluded that we should consider antithrombotic therapy for SLE patients, especially those with Raynauds phenomenon, to prevent unwanted activation of the coagulation system and possible endothelial damage.

Cyclic AMP increases thrombomodulin expression on membrane surface of cultured human umbilical vein endothelial cells

Dibutyryl-cyclic AMP (Bt2cAMP; final concentration 1-5 mM) or beraprost sodium (synthetic prostacyclin, 100 nM) enhanced the expression of thrombomodulin (TM; an anticoagulant factor of endothelial cells) on the membrane surface of cultured human umbilical vein endothelial cells up to 1.4 times over the control within 9 hrs after the treatment, while the expression fell below the control level at 12 hrs and thereafter. 8-Bromo-cAMP (final concentration 1-5 mM) or 3-isobutyl-1-methylxanthine (IBMX; an inhibitor of phosphodiesterase; final concentration 10-1000 microM) enhanced the expression of TM on the cell surface at 12 hrs after the treatment. The enhancement of TM expression caused by Bt2cAMP was inhibited by incubation with phorbol 12-myristate 13-acetate. These results suggest that cAMP stimulates expression of TM in the endothelial cells.

High-performance affinity chromatography of plasmin and plasminogen on a hydrophilic vinyl-polymer gel coupled with p-aminobenzamidine

p-Aminobenzamidine was covalently attached via a spacer moiety to a microparticulate hydrophilic vinyl-polymer gel (Toyopearl HW65S) and this affinity adsorbent was used for the separation of plasmin and plasminogen by high-performance affinity chromatography. Toyopearl HW65S was alkylated with chloroacetylglycylglycine in dimethyl sulphoxide using methylsulphinyl carbanion as a catalyst, then p-aminobenzamidine was coupled to the carboxyl group of glycylglycine to form an acid amide bond. A column packed with the adsorbent retained both plasmin and plasminogen. Plasminogen was eluted with 6-aminohexanoic acid, a haptenic compound for the lysine-binding sites of plasminogen. For the elution of plasmin, the coexistence of 6-aminohexanoic acid and leupeptin (a competitive inhibitor for plasmin) was necessary. The results indicate a two-site interaction of plasmin with the immobilized ligand, i.e., at the lysine-binding sites and the catalytic site. Fluorometric detection of eluted protein and on-line assay of plasmin activity using a fluorogenic substrate, peptidylmethylcoumarylamide, revealed that effective chromatographic separation of the enzyme could be achieved with high sensitivity (10 micrograms) within 1 h.

Immunocytochemical localization of thrombomodulin in the aqueous humor passage of the rat eye.

Abstract This report describes the distribution and localization of thrombomodulin (TM) in the rat eye by light and electron microscopic immunocytochemistry. In addition to the endothelium of the entire vasculature, TM was found on the non-vascular structures lining the cavities of the posterior and anterior chambers and the limbus. TM was localized on the basal, lateral, and apical plasma membranes of the inner and outer ciliary epithelium, and the posterior iris epithelium in which there was no polarized expression of TM. In the anterior chamber, TM was localized on the luminal surface of the corneal endothelium, but was negative on the anterior border layer of the iris, which is composed of a discontinuous layer of fibroblasts and collagen fibers. Thus, TM was present at sites of cell-to-cell contact. TM was also present on the endothelia of the trabecular meshwork and the Schlemm’s canal in the limbus. TM was localized not only on the luminal plasma membrane, but also on the cytoplasmic giant vacuoles in the endothelial cells of the Schlemm’s canal. These findings extend the importance of anticoagulant mechanisms to the systems of secretion, circulation, and drainage of the aqueous humor.

Procalpain I in cytoplasm is translocated onto plasma and granule membranes during platelet stimulation with thrombin and then activated on the membranes

Thrombin stimulation of platelets resulted in changes in the subcellular localization of calpain I, with a concomitant alteration of its molecular weight as measured by immunoblotting. Calpain I in resting platelets was distributed as procalpain I, an 80 kDa form which does not exhibit the enzyme activity, and 83% of the total antigen was localized in the cytosol fraction. When platelets were stimulated with thrombin, the total content of calpain I antigen was not significantly changed as compared with that of the resting platelets, though a decrease in the cytosolic distribution of 80 kDa form (from 83% to 47% of the total antigen) was observed with concomitant appearance of the active 76 kDa and intermediate 78 kDa forms of calpain I and increase in the 80 kDa form in the granule and membrane fractions. These results indicated that calpain I was translocated from the cytosol to both the plasma and granule membranes as procalpain I and then activated on the membranes during platelet stimulation with thrombin.

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.