SummaryThrombomodulin, 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. It was concluded that this EIA is reliable for TM assay in human plasma and urine, which will reflect activation or injury of endothelial cells.

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.

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)

To study the significance of plasma thrombomodulin (TM) values in diabetes mellitus, we determined plasma TM in 34 patients with non-insulin-dependent diabetes mellitus (NIDDM) men, mean age 54 (SE 2) years. Plasma TM was determined by an enzyme immunoassay with anti-TM monoclonal antibodies. The plasma TM values were significantly greater in NIDDM patients with nephropathy than in patients without nephropathy (P less than 0.001). Also, a significant positive correlation was noted between the concentration of plasma TM and serum creatinine (r = 0.55, P less than 0.001). The plasma TM values of the patients with retinopathy were significantly greater than the values of those without it (P less than 0.002). Furthermore, we noted a significant positive correlation (r = 0.78, P less than 0.001) between plasma TM and the severity of diabetic retinopathy as graded by Scott's classification. These results suggest a close relationship between TM and diabetic microangiopathy.

We have examined whether oxidized low-density lipoprotein (ox-LDL) affects the function of tissue-factor-pathway inhibitor (TFPI), an anti-coagulant regulator in the extrinsic pathway of coagulation, in cultured human umbilical vein endothelial cells (HUVEC). Treatment of culture medium of HUVEC with ox-LDL, but not with native or acetylated LDLs, drastically decreased the reactivity of TFPI to its antibody specific for Kunitz domain 1 or one specific for the conformation between Kunitz 1 and 2 of TFPI, and caused a rapid, concentration-dependent decrease in the functional activity of TFPI to inhibit Factor X activation. When 5 ng of recombinant TFPI (rTFPI) was mixed with 10 microg of ox-LDL for 30 min, almost all of the rTFPI was detected in the ox-LDL fraction and no free rTFPI was observed on non-denaturing PAGE, in contrast with the virtual absence of rTFPI in the native LDL fraction. Ox-LDL decreased the antigen level of TFPI in the lysate of HUVEC in a time-dependent manner. It did not affect the mRNA level, but ox-LDL-dependent reduction of the TFPI antigen level in HUVEC was reversed by the simultaneous treatment of ox-LDL with bafilomycin A1, an inhibitor of the lysosomal proton pump. These results indicate that ox-LDL lessens the anti-coagulant function of TFPI through both oxidative modification and accelerated degradation of the molecule outside and inside HUVEC respectively.