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.
SummaryPrevious studies have shown that protein kinase C (PKC) activators and dibutyryl cyclic AMP (Bt2cAMP) synergistically increase the antigen level of plasminogen activator inhibitor type-2 (PAI-2) in a human myeloid leukemia cell line PL-21. To clarify the mechanism, PAI-2 gene expression induced by phorbol myristate acetate (PMA), a PKC activator, and Bt2cAMP was investigated by Northern blot hybridization using a PAI-2 cDNA probe cloned from a human placental library. The level of PAI-2 mRNA was markedly increased in response to PMA and reached a maximum 5-9 h after stimulation. Nuclear run-on assay revealed an increase in PAI-2 gene transcription in PMA-treated cells. The induction was inhibited by inhibiting de novo protein synthesis with cycloheximide (CHX). cAMP also increased PAI-2 mRNA level in a dose-dependent manner. The increase began within 2 hours and, contrary to the case of PMA, the mRNA levels were maintained. Moreover, cAMP-induced increase in PAI-2 mRNA was not inhibited by CHX, rather enhanced. PMA and cAMP synergistically induced PAI-2 gene expression, which was completely inhibited by CHX. The cells pretreated with PMA for 24 h did not any more respond to stimulation with PMA but responded to cAMP and PAI-2 mRNA level was increased. The apparent half-life of constitutive level PAI-2 mRNA in PL-21 cells, determined by actinomycin-D-decay experiments, was approximately 2 h. Those induced by PMA and cAMP were approximately 5 h and 2 h, respectively. These data suggest that PAI-2 mRNA induced by PMA is relatively stable and the expression requires de novo protein synthesis, whereas cAMP increases PAI-2 mRNA level without affecting the stability and the induction does not require de novo protein synthesis. Judging from these data, PAI-2 gene expression appears to be differently regulated by the PKC and cAMP signalling pathways.
SummaryThe mechanism of thrombin induction of tissue- and urokinase-type plasminogen activator (t-PA and u-PA) biosynthesis was investigated in cultured human fetal lung fibroblast cells, IMR-90. Northern blot analysis of total RNA from thrombin-treated cells showed marked accumulations of both t-PA and u-PA mRNA during 24 h. Nuclear run-on experiments showed that the transcription rates of both genes were increased in the thrombin-treated cells.These thrombin effects were inhibited by cycloheximide (CHX), an inhibitor of protein biosynthesis. Treatment of IMR-90 cells with CHX alone caused an increase in u-PA mRNA but not in t-PA mRNA. CHX, however, did not affect the transcription rates of both genes in the cells. Thus, on-going protein synthesis is required for increased accumulations of both t-PA and u-PA mRNA by thrombin but not for the constitutive expression of u-PA gene in IMR-90 cells. Therefore, we conclude that the accumulations of t-PA and u-PA mRNA due to thrombin result mainly from increased rates of their gene transcriptions, and that this influence is exerted in part by proteins synthesized by thrombin stimulation.Thrombin also increased plasminogen activator inhibitor type-1 (PAI-1) in the levels of both antigen and mRNA more rapidly than it increased t-PA in IMR-90 cells. In conditioned medium, most of the secreted PAI-1 seemed to form a complex with t-PA. Northern blot analysis using a PAI-2 cDNA probe showed that the levels of PAI-2 mRNA were markedly increased in response to thrombin. PAI-2 appeared not to have been secreted by the cells because the antigen was rarely detectable in the conditioned medium. Thus, thrombin increases not only plasminogen activators, but their inhibitors as well.
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