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Biomedical subjects

H Nagase

Publications and source records attributed to H Nagase.

At least 199 records · Page 11Linked to original sources
PubMed1997

Involvement of central neurotensin in thermoregulatory and neuroimmune function in pregnant rats exposed to heat.

To examine a functional relationship among pregnancy and central neurotensin and thermoregulatory and neuroimmune systems during heat stress, we monitored colonic temperature in six virgin female rats and six pregnant rats (9 to 11 days gestation) exposed to a microwave source. We also assayed splenic natural killer cell activity (NKCA), blood corticosterone (CS), and ACTH as indicators of the hypothalamic-pituitary-adrenal axis, beta-endorphin (beta-EP), and neurotensin (NT) in discrete brain regions. Additionally, we clarified the effects of intracerebroventricular (icv) administration of NT antiserum on these same responses in pregnant rats exposed to heat stress. Repeated-measures analysis of variance showed significant main effects of heat and pregnancy and a significant interactive effect on colonic temperature. Significant elevation in blood CS, ACTH, beta-EP, and NT in the hypothalamus and significant reductions in splenic NKCA and NT in the nucleus accumbens were produced by heat. In the experiment examining the effect of icv administration of NT antiserum, significant main effects of heat and administration and a significant interactive effect on colonic temperature were observed. Icv administration of NT antiserum increased splenic NKCA and decreased blood beta-EP. These results show that pregnancy enhances thermal homeostasis, suggesting central thermoregulatory mechanisms through NT in nucleus accumbens and hypothalamus in which placental or pituitary beta-EP may be involved. NT and beta-EP seem to play central roles simultaneously in heat-induced immunosuppression during pregnancy. Clarification for the effects of NT antiserum on beta-EP in virgin rats or manipulation of agents related to opioid system should be the focus of future work.

Adrenocorticotropic Hormone↗
PubMed1997

Insulin-like growth factor binding protein (IGFBP) substrate zymography. A new tool to identify and characterize IGFBP-degrading proteinases.

Insulin-like growth factor binding protein (IGFBP) degrading proteinase activities have been described in biological fluids and conditioned media from numerous cell lines. To identify and characterize IGFBP-degrading proteinases, our laboratory has developed IGFBP substrate zymography. Herein, we illustrate how IGFBP substrate zymography can be used both to identify candidate IGFBP-degrading proteinases and characterize their degradative capabilities. For this purpose, human matrix metalloproteinase-3 (MMP-3), a proteinase that degrades IGFBP-3 in human fibroblast cultures, was first electrophoresed through a polyacrylamide gel containing IGFBP-3 as substrate and then analyzed for its ability to degrade the substrate into immunoreactive fragments that were absorbed onto a polyvinylidene difluoride membrane. IGFBP-3 substrate zymography was capable of detecting as little as 20 ng of human MMP-3, demonstrating a sensitivity similar to casein substrate zymography. Using the zymogram as a template, MMP-3 was identified in a standard SDS-polyacrylamide gel run in parallel with the zymogram, and the corresponding area of the gel was excised. Electroelution of the gel slice yielded active MMP-3 when examined by casein substrate zymography. Furthermore, digestion of IGFBP-3 in solution by the electroeluted MMP-3 revealed the same fragmentation pattern of the binding protein as that produced by MMP-3, which had not been electroeluted. Together, these studies demonstrate that IGFBP substrate zymography can be a useful tool for both the identification and the characterization of IGFBP-degrading proteinases.

Caseins↗
PubMed1997

Use of antisense oligodeoxynucleotide to delta-opioid receptor mRNA in the study of turnover of delta-opioid receptors in the spinal cord of the mouse.

Pretreatment of male ICR mice with an antisense oligodeoxynucleotide to delta-opioid receptor mRNA (DOR AS oligo, 163 pmol) given intrathecally (i.t.) once a day for 1-3 days produced a time-dependent attenuation of antinociception produced by i.t.-challenged [D-Ala2] deltorphin II (6.4 nmol), a delta-opioid receptor agonist. The attenuation of the [D-Ala2]deltorphin II-induced antinociception caused by pretreatment with DOR AS oligo given i.t. daily was blocked by co-pretreatment with naltriben (14.5 nmol), a delta-opioid receptor antagonist, but was markedly enhanced by concomitant pretreatment with thiorphan (19.7 nmol) or bestatin (14.5 nmol), which inhibits the degradation of endogenously released Met-enkephalin. Concomitant pretreatment with antiserum to Met-enkephalin, but not with antiserum to Leu-enkephalin, beta-endorphin or dynorphin A (1-17), and DOR AS oligo given i.t. daily for 3 days prevented the attenuation of i.t.-challenged [D-Ala2]deltorphin II-induced antinociception caused by the DOR AS oligo pretreatment. Our results support the existence of a turnover of delta-opioid receptors in the mouse spinal cord caused by the release of Met-enkephalin.

Animals↗
PubMed1997

Antinociceptive effect of buprenorphine in mu1-opioid receptor deficient CXBK mice.

The antinociceptive effect of buprenorphine was examined in mu1-opioid receptor-deficient CXBK mice. I.p. administration of buprenorphine at a dose of 3 mg/kg produced marked antinociception in the tail-flick test in C57BL/6 mice, a progenitor strain of CXBK mice. The antinociceptive effect of buprenorphine in C57BL/6 mice was antagonized by pretreatment with either beta-funaltrexamine (beta-FNA), a mu-opioid receptor antagonist, or naloxonazine (NXZ), a selective mu1-opioid receptor antagonist. The antinociceptive effect of buprenorphine (3 mg/kg, i.p.) in CXBK mice was significantly less than that in C57BL/6 mice. Neither beta-FNA nor NXZ reduced the antinociceptive effect of buprenorphine in CXBK mice. There was no significant difference between the buprenorphine-induced antinociceptive effect in CXBK mice and NXZ-treated C57BL/6 mice. Furthermore, neither naltrindole, a selective delta-opioid receptor antagonist, nor norbinaltorphimine, a selective kappa-opioid receptor antagonist, had a significant effect on the antinociceptive effects of buprenorphine in both CXBK and C57BL/6 mice. These results support our previous hypothesis that mu1- rather than mu2-, delta- or kappa-opioid receptors are involved in the antinociceptive effects of buprenorphine.

Animals↗
PubMed1997

Inhibitory effect of pregnancy on stress-induced immunosuppression through corticotropin releasing hormone (CRH) and dopaminergic systems.

To clarify the involvement of pregnancy in the response of the neuroendocrine-immune system to stress, we examined splenic natural killer-cell-activity-(NKCA) and its relevant central and blood parameters in female virgin and pregnant rats (10 to 11 days gestation) exposed to forced water-immersion stress with durations of 90 min and 180 min. Decreases in splenic NKCA, corticotropin releasing hormone (CRH) in the hypothalamus, and increases in progesterone (P), beta-endorphin (beta EP), and dopamine (DA) metabolic ratios in the frontal cortex and nucleus accumbens produced by stress were recognized in the virgin rats, but not in the pregnant rats. Pregnancy reduced splenic NKCA in rats without stress, but elevated it in the rats exposed to stress with a duration of 180 min. These findings suggest inhibitory effects of pregnancy on stress-induced immunosuppression and neuroendocrine changes, thereby promoting homeostasis in the neuroendocrine-immune system against stress. Such enhanced homeostasis associated with pregnancy seemed to be mediated by the activation of placental P and placental or pituitary beta EP in cooperation with mesocortical and mesolimbic DA systems and hypothalamic CRH.

Animals↗
PubMed1997

Nonenzymatic glycation of type IV collagen and matrix metalloproteinase susceptibility.

The glomerular basement membrane (GBM) is damaged in diabetes through complex mechanisms that are not fully understood. Prominent among them is nonenzymatic protein glycation leading to the formation of so-called advanced glycation end products (AGEs). We examined the effects of in vitro glycation of intact collagen type IV in bovine lens capsule (LBM) and kidney glomerular (GBM) basement membranes on their susceptibility to matrix metalloproteinases, using stromelysin 1 (MMP-3) and gelatinase B (MMP-9). Sites of cleavage of unmodified LBM collagen were located in the triple helical region. In vitro glycation by glucose severely inhibited the release of soluble collagen cleavage peptides by MMP-3 and MMP-9. The distribution of AGEs within the three domains of collagen IV (7S, triple helical, and noncollagenous NC1) were compared for LBM glycation using AGE fluorescence, pentosidine quantitation, and immunoreactivity towards anti-AGE antibodies that recognize the AGE carboxymethyllysine (CML). Marked asymmetry was observed, with the flexible triple helical domain having the most pentosidine and fluorescent AGEs but the least CML. The in vivo relevance of these findings is supported by preliminary studies of AGE distribution in renal basement membrane (RBM) collagen IV domains from human kidneys of two insulin-dependent diabetics and one normal subject. Pentosidine and fluorescent AGE distributions of diabetic RBM were similar to LBM, but the CML AGE in diabetic kidney was less in the triple helical domain than in NC1. Our results support the hypothesis that nonenzymatic glycation of collagen IV contributes to the thickening of basement membranes, a hallmark of diabetic nephropathy.

Adult↗
PubMed1997

Alpha-fetoprotein (AFP) levels in normal children.

Alpha-fetoprotein (AFP) is an important tumor marker for yolk sac tumor and hepatoblastoma in childhood. We have been using the graph of the normal range of serum AFP made by Tsuchida et al, when we evaluate the serum AFP levels in early infancy. We measured the serum AFP levels by an immunoradiometric assay in 163 normal infants under 2 years of age, in order to make a more precise graph. Our normal range was a little wider than that of Tsuchida et al. According to our graph, false-positive cases would be fewer. Referring to the half-lives of serum AFP levels in normal infancy is also useful, when it is difficult to evaluate the AFP level.

Age Factors↗
PubMed1997

Hormonal regulation of matrix metalloproteinase 9/gelatinase B gene expression in rabbit uterine cervical fibroblasts.

Since the degradation of uterine cervical extracellular matrix is an essential process in cervical ripening and dilatation at term, we examined the effect of pregnancy on the level of pro-matrix metalloproteinase 9 (proMMP-9)/progelatinase B in the rabbit uterine cervix and its regulation in uterine cervical fibroblasts. Uterine cervices shortly before term parturition contained high levels of proMMP-9 compared with those of nonpregnant rabbits. Uterine cervical fibroblasts prepared from rabbits at 23 days of gestation did not produce proMMP-9; in contrast, interleukin (IL)-1 alpha, IL-1 beta, or phorbol 12-myristate 13-acetate (PMA) induced proMMP-9 production along with an increase of proMMP-9 mRNA, and the IL-1-mediated induction of proMMP-9 was synergistically enhanced by PMA. On the other hand, physiological concentrations of progesterone suppressed IL-1-and/or PMA-mediated production of proMMP-9 in a dose-dependent manner. This suppressive effect of progesterone on proMMP-9 production was accompanied by a decrease in matrix metalloproteinase 9 (MMP-9) mRNA, indicating that progesterone down-regulates the production of proMMP-9 at the transcriptional level. These results indicate that in the rabbit uterine cervix, IL-1 alpha and IL-1 beta are effective inducers of the proMMP-9 production, and progesterone is a physiological suppressor. Thus, MMP-9 and progesterone are very likely to play essential roles in cervical ripening and dilatation in the rabbit, and the production of MMP-9 in the cervix is finely regulated during pregnancy.

Animals↗
PubMed1997

Effects of exposure to microwaves on cellular immunity and placental steroids in pregnant rats.

OBJECTIVES: Microwaves produce various detrimental changes based on actions of heat or non-specific stress, although the effects of microwaves on pregnant organisms has not been uniform. This study was designed to clarify the effect of exposure to microwaves during pregnancy on endocrine and immune functions. METHODS: Natural killer cell activity and natural killer cell subsets in the spleen were measured, as well as some endocrine indicators in blood--corticosterone and adrenocorticotrophic hormone (ACTH) as indices of the hypothalamic-pituitary-adrenal axis--beta-endorphin, oestradiol, and progesterone in six female virgin rats and six pregnant rats (nine to 11 days gestation) exposed to microwaves at 10 mW/cm2 incident power density at 2450 MHz for 90 minutes. The same measurements were performed in control rats (six virgin and six pregnant rats). RESULTS: Skin temperature in virgin and pregnant rats increased immediately after exposure to microwaves. Although splenic activity of natural killer cells and any of the subset populations identified by the monoclonal antibodies CD16 and CD57 did not differ in virgin rats with or without exposure to microwaves, pregnant rats exposed to microwaves showed a significant reduction of splenic activity of natural killer cells and CD16+CD57-. Although corticosterone and ACTH increased, and oestradiol decreased in exposed virgin and pregnant rats, microwaves produced significant increases in beta-endorphin and progesterone only in pregnant rats. CONCLUSIONS: Microwaves at the power of 10 mW/cm2 produced activation of the hypothalamic-pituitary-adrenal axis and increased oestradiol in both virgin and pregnant rats, suggesting that microwaves greatly stress pregnant organisms. These findings in pregnant rats suggest that--with exposure to microwaves--pregnancy induces immunosuppression, which could result in successful maintainance of pregnancy. This enhancement of adaptability to heat stress with pregnancy may be mediated by activation of placental progesterone and placental or pituitary beta-endorphin.

Adrenocorticotropic Hormone↗
PubMed1997

Altered kidney matrix gene expression in early stages of experimental diabetes.

The expression of mRNA and distribution of alpha 1(IV), alpha 3(IV) chains of type IV collagen, matrix metalloproteinase 2 (MMP-2), and tissue inhibitor of metalloproteinase 1 (TIMP-1) were examined in kidneys from streptozotocin-diabetic rats, 2.5 months after administration of the drug, an early time point when specific diabetic glomerular changes were still minimal. Ten age-matched Sprague-Dawley rats were assigned to control and diabetic groups. Compared to the controls, the diabetic rats had a significantly lower body weight, higher kidney weight and serum glucose levels, but no significant changes of glomerular surface area and urine albumin were observed. Northern blot analysis, using whole kidney mRNA, revealed that diabetic rat kidneys expressed 113.5% more alpha 1(IV), 46.5% more alpha 3(IV), 54.8% less MMP-2 and 246% more TIMP-1 (in all instances: p < 0.05). These results were corroborated by in situ hybridization for RNA expression. A quantitative analysis of the data indicated the following changes in glomeruli: (1) 74.6% more alpha 1(IV), (2) 103.8% more alpha 3(IV), (3) 40.7% less MMP-2 and (4) 80.9% more TIMP-1. Similar changes were observed in tubular (proximal and distal) cells. We conclude that an increased synthesis and decreased degradation of renal extracellular matrix components occur early after induction of experimental diabetes, before the onset of typical structural changes in the kidneys, and represent changes of specific gene expression at the transcriptional level. All the cell types in the glomerulus as well as the proximal and distal tubules appear to be involved in this alteration of expression, and this is a novel finding.

Animals↗
PubMed1997

[Research and development of beraprost sodium, a new stable PGI2 analogue].

A novel prostaglandin I2 (PGI2) analogue, beraprost sodium, is the first launched drug as an orally active PGI2. PGI2 was discovered in 1976, and has attracted much attention as a medicine for cardiovascular diseases such as strokes and heart attacks because of its potent antiplatelet and vasodilating effect. However, PGI2 is extremely unstable for the use as practical medicines. Thus, stable PGI2 analogues have been explored by a large number of researchers in the world. Just after the discovery of PGI2, we started a research on chemically and metabolically stable PGI2 derivatives with longer duration of action and less adverse reaction. We invented a novel class of stable PGI2, 5,6,7-trinor-4,8-inter-m-phenylenePGI2 analogues that have the phenol moiety instead of the enolether moiety of PGI2. Further efforts were devoted to enhance the efficacy of the PGI2 analogues and to eliminate their side effects, and an orally active analogue, beraprost sodium, was obtained. In order to establish the synthetic route of beraprost sodium, various novel processes were invented, including ortho-selective metalation of bromoanisoles by means of Grignard reagents, copper-catalyzed SN2' cyclization to prepare cyclopenta[b]benzofuran, and stereo-selective elongation of the omega-side chain by Prins reaction. Beraprost sodium inhibit platelet aggregation induced by adenosine 5'-diphosphate (ADP), collagen and arachidonic acid. It was shown that the drug has a potent antiplatelet effect both in vitro and ex vivo in human and several animal species. In clinical studies, beraprost sodium exerted a marked effect to improve arteriosclerosis obliterans. No serious adverse effects related with the drug have been reported. It was highly evaluated as an orally active PGI2 by pharmaceutical companies overseas as well, and now clinical trials are under way in U.S.A. and Europe.

Animals↗
PubMed1997

Non-peptidic cysteine derivatives as inhibitors of matrix metalloproteinases.

By exploiting the thiol function of L-cysteine as a chelating group of the active-site zinc atom of matrix metalloproteinases (MMPs), N- and C-terminal derivatization of this amino acid with aliphatic and aromatic groups allowed us to explore the selectivity of the S and/or S' binding subsites of human neutrophil collagenase (MMP8) and stromelysin (MMP3). With N-benzyloxycarbonyl-L-cysteine-(2-phenyl)ethylamide a submicromolar inhibitor of MMP8 was discovered.

Cysteine↗
PubMed1997

Delta-1 opioid receptor-mediated antinociceptive properties of a nonpeptidic delta opioid receptor agonist, (-)TAN-67, in the mouse spinal cord.

The effects of enantiomorphs of TAN-67 (2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino[2,3,3-g]isoquinoline), (-)TAN-67 and (+)TAN-67, given intrathecally (i.t.) on antinociceptive response with the tail-flick test were studied in male ICR mice. (-)TAN-67 at doses from 17.9 to 89.4 nmol given i.t. produced a dose- and time-dependent inhibition of the tail-flick response, whereas its enantiomer (+)TAN-67 even at smaller doses (1.8, 4.5 and 8.9 nmol) given i.t. decreased the latencies of the tail-flick response. In addition, (+)TAN-67 at higher doses (17.9-89.4 nmol) given i.t. produced scratching and biting pain-like responses. The antinociceptive response induced by i.t.-administered (-)TAN-67 was mediated by the stimulation of delta-1 but not by delta-2, mu or kappa opioid receptors, because the effect was blocked by the i.t. pretreatment with BNTX, but not by naltriben, [D-Phe-Cys-Tyr-[D-Try-Orn-Thr-Pen-Thr-NH2 or nor-binaltorphimine dihydrochloride. Pretreatment with (-)TAN-67 given i.t. 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of (-)TAN-67 and by [D-Pen2,5]enkephalin (DPDPE). However, the tail-flick inhibition induced by [D-Ala2]deltorphin II, [D-Ala2,NMePhe4,Gly5-ol]enkephalin and U50,488H were not affected by (-)TAN-67 pretreatment. Conversely, pretreatment with DPDPE given i.t. 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of (-)TAN-67 and by DPDPE. However, the tail-flick inhibition induced by [D-Ala2]deltorphin II was not affected by i.t. DPDPE pretreatment. It is concluded that (-)TAN-67 given i.t. produces delta-1 opioid receptor-mediated antinociception; on the other hand, its enantiomer (+)TAN-67 produces hyperalgesia. Present studies provide other evidence that delta-1 opioid receptors exist separated from delta-2 opioid receptor.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1997

Antithrombin III-independent effect of depolymerized holothurian glycosaminoglycan (DHG) on acute thromboembolism in mice.

A previous study in this laboratory showed that depolymerized holothurian glycosaminoglycan (DHG) has two different antithrombin III (ATIII)-independent inhibitory effects on the in vitro blood coagulation system: heparin cofactor II (HCII)-dependent inhibition of thrombin, and ATIII- and HCII-independent inhibition of factor X activation by factor IXa-factor VIIIa complex (Nagase et al. Blood 85, 1527-1534, 1995). In the present study, we compared the antithrombotic effects of DHG in normal and in ATIII-deficient mice with those of unfractionated heparin (UFH) and low molecular weight heparin (LMWH). DHG, unlike UFH and LMWH, exerted an in vivo antithrombotic effect even in mice with decreased plasma ATIII activity (about 30% of normal). We then compared the anticoagulant and antithrombotic effects of DHG in mice with those of high molecular weight (HMW)-DHG, low molecular weight (LMW)-DHG, and dermatan sulfate (DS). In terms of in vitro anticoagulant activity assessed by use of purified human components, DHGs (DHG, HMW-DHG, and LMW-DHG) had different anti-thrombin activity in the presence of HCII and anti-factor Xase activities, which differences were dependent on the molecular weight. With respect to in vivo antithrombotic activity, DHG, HMW-DHG, and LMW-DHG showed almost the same inhibitory effect on acute thromboembolism in mice (minimum effective dose [MED]: > 0.3 mg/kg). Since the antithrombotic activities of DHGs were not correlated with the anticoagulant-specific activities, the contribution of the two anticoagulant activities to the in vivo antithrombotic effect of DHGs remains unknown. However, DHG was more effective against acute thromboembolism in mice than DS (MED > 1 or > 3 mg/kg), which showed no inhibitory activity toward factor Xase. Therefore, it seems that factor Xase inhibition contributes greatly to the antithrombotic effect of DHG and that DHG exerts this effect in mice mainly by inhibiting factor Xase.

Animals↗
PubMed1997

Invasive properties of cadmium-resistant human fibrosarcoma HT-1080 cells.

Invasive properties of tumor cells having acquired heavy metal resistance were investigated. We selected the cadmium-resistant (Cd-R) cells from human fibrosarcoma HT-1080 cells. Total metallothionein levels in cytosol of HT-1080 Cd-R cells were significantly higher than original lines, and were of a highly resistant potency to cytotoxicity of cisplatin, as well as heavy metals. The HT-1080 Cd-R cells showed higher invasiveness into recombinant basement membrane Matrigel. However, HT-1080 Cd-R cells were inferior in locomotion ability. Significant differences in adhesive ability to extracellular matrix proteins were not observed between HT-1080 and HT-1080 Cd-R cells. High invasiveness of HT-1080 Cd-R cells was caused by their extremely strong enzymatic activities. High level of 92kDa matrix metalloproteinase-9 (MMP-9) was recognized from the conditioned medium of HT-1080 Cd-R cells, whereas 72kDa MMP-2 was secreted equally from both cell lines. Our investigation suggests that drug resistance acquired through the mechanisms of cellular metal-tolerance may promote malignancy and tumor metastasis during cancer chemotherapy.

Cadmium↗
PubMed1997

The anticoagulant and hemorrhagic effects of DHG, a new depolymerized holothurian glycosaminoglycan, on experimental hemodialysis in dogs.

We studied the use of depolymerized holothurian glycosaminoglycan (DHG) as an anticoagulant in experimental beagle-dog hemodialysis using a hollow-fiber dialyzer compared to that using unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and nafamostat mesilate (FUT). Effectiveness was based on 5 h hemodialysis and no marked clot deposition in the extracorporeal circuit. At effective doses, UFH and LMWH significantly prolonged template bleeding time, in sharp contrast to FUT and DHG, which scarcely prolonged bleeding time during hemodialysis. DHG prolonged activated partial thromboplastin time (APTT) about 6 times that of normal plasma and prolonged thrombin clotting time (TCT) markedly; FUT showed marked APTT prolongation but hardly prolonged TCT in the hemodialysis circuit at the effective dose. The anticoagulant profile of DHG thus differs completely from that of FUT. These results suggest that DHG may be useful as anticoagulant for hemodialysis with low hemorrhagic risk.

Animals↗
PubMed1997

Effect of depolymerized holothurian glycosaminoglycan (DHG) on tissue factor pathway inhibitor: in vitro and in vivo studies.

Depolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicus Selenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor VIIa by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor VIIa inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitro activity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

Animals↗