Antithrombin Morioka (Cys 95-Arg): a novel missense mutation causing type I antithrombin deficiency.
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Adult↗
Biomedical subjects
K Niiya
Publications and source records attributed to K Niiya.
[The application of molecular biology to thrombosis and hemostasis].
Current biological science has been revolutionized by a series of new investigative molecular techniques developed within the last 15 years. These techniques allow the detection of gene mutations responsible for congenital diseases including thrombophilia and hemorrhagic disorders. Here, we provide a short introduction to polymerase chain reaction (PCR), single-strand conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and northern blot techniques and present the results of DNA sequence analyses of 4 different types of antithrombin III abnormalities and one IIB-type von Willebrand disease examined in our laboratory. In addition, we briefly summarize the gene mutations reported in patients with activated protein C-resistance, hemophilia A and hemophilia B.
Antithrombin III↗
Factor VII Toyama (Thr 359 Met): a homozygous missense mutation causing severe type I deficiency.
We performed a DNA analysis on a patient with severe type I factor VII deficiency by the polymerase chain reaction amplification and a direct DNA sequencing method. The proband was a 66-year-old Japanese woman who had recurrent episodes of excessive bleeding after dental extraction. The functional and antigenic levels of plasma factor VII markedly reduced to 1.6% and 2% of normal, respectively. However, she had no serious symptoms such as intracranial or intraarticular hemorrhage. The analysis revealed that the patient was homozygous for a missense mutation, Thr (ACG) to Met (ATG) at codon 359 in the catalytic domain. Her deceased parents were first cousins, and their consanguineous marriage presumably resulted in the homozygosity in her. This patient was the first case of homozygote for the Thr359Met mutation, though heterozygotes for the mutation were previously found in an Italian family.
Adult↗
A novel polymorphism in exon 10 of the factor V gene inducing an amino acid replacement of arginine to lysine at codon 485.
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Amino Acid Substitution↗
Absence of factor V Leiden in the Japanese.
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Factor V↗
Isolated recombinant domain of von Willebrand factor displaying increased sensitivity to ristocetin.
Type 2B von Willebrand disease is characterized by an abnormal von Willebrand factor molecule with increased affinity for the platelet glycoprotein (GP) Ib-IX receptor. A diagnostic feature of type 2B von Willebrand disease is a characteristic loss of von Willebrand factor high molecular weight multimers. In vitro, the soluble interaction of normal von Willebrand factor with platelets can be initiated with exogenous modulators, the most common being the antibiotic ristocetin. The variant molecules resulting in type 2B von Willebrand disease can sustain binding to platelets at subnormal levels of ristocetin. We characterized the von Willebrand factor gene of an individual with type 2B von Willebrand disease and identified a nucleotide transition resulting in an Arg543-->Trp amino-acid substitution within the GP Ib-IX binding domain of von Willebrand factor. In this study we demonstrate that a recombinant plasmid capable of expressing the isolated GP Ib-IX binding domain of von Willebrand factor, and containing the Arg543-->Trp amino-acid substitution, secretes a dimeric molecule that supports platelet agglutination using subnormal levels of ristocetin. These results demonstrate that the mutation at position 543 increases the affinity between the variant molecule and platelet GP Ib-IX as an intrinsic feature of the isolated von Willebrand factor domain. Thus, structural perturbations within the GP Ib-IX binding domain that are independent of the von Willebrand factor multimeric structure can sufficiently increase the affinity of von Willebrand factor to sustain platelet aggregation, using subnormal levels of ristocetin.
Adult↗
Induction of c-fos protooncogene transcription and apoptosis by delta 12-prostaglandin J2 in human Pl-21 myeloid leukemia and RC-K8 pre-B lymphoma cells.
delta 12-prostaglandin J2 (PGJ2) is a dehydration product of PGD2 and thought to be the most potent antitumor agent among prostaglandin compounds. We examine the cytotoxic effects of PGJ2 on the cell growth of leukemia/lymphoma cells. PGJ2 inhibited the growth of both human PL-21 myeloid leukemia and RC-K8 pre-B lymphoma cells in culture in a dose-dependent manner with fragmentation of nucleus and formation of apoptotic body. Agarose gel electrophoresis revealed DNA ladder formation in the cells treated with PGJ2. Furthermore, PGJ2 induced a rapid and transient expression of apoptosis-related protooncogene, c-fos, in both cells. The gene transcriptional rate was remarkably increased approximately 3.3-fold in PGJ2 treated cells, but the stability of c-fos mRNA was not significantly changed. Inhibition of de novo protein synthesis with cycloheximide increased c-fos mRNA stability but not abrogated PGJ2-induced c-fos transcription. These data suggest that PGJ2 can induce apoptosis of human leukemia/lymphoma cells and the rapid activation of c-fos protooncogene transcription in which de novo protein synthesis is not required.
Antineoplastic Agents↗
Heparin cofactor II-dependent antithrombin activity of calcium spirulan.
Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, enhanced the antithrombin activity of heparin cofactor II (HC II) more than 10000-fold. The apparent second-order rate constant of thrombin inhibition by HC II was calculated to be 4.2 x 10(4) M-1 min-1 in the absence of Ca-SP, and it increased in the presence of 50 micrograms/ml Ca-SP to 4.5 x 10(8) M-1 min-1. Ca-SP effectively induced the formation of a thrombin-HC II complex in plasma. In the presence of Ca-SP, both the recombinant HC II variants Lys173-->Leu and Arg 189-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHC II. This result indicates that the binding site of HC II for Ca-SP is different from the heparin- or dermatan sulfate-binding site. When we removed the calcium from the Ca-SP, the compound did not exert any antithrombin activity. Furthermore, Na-SP, which was prepared by replacement of the calcium in Ca-SP with sodium, accelerated the antithrombin activity of HC II as Ca-SP did. We therefore suggest that the molecular conformation maintained by Ca or Na is indispensable to the antithrombin activity of Ca-SP. The HC II-dependent antithrombin activity of Ca-SP was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, but not by treatment with chondroitinase ABC and chondroitinase AC II, suggesting that a heparin- or dermatan sulfate-like structure is not responsible for the activation of HC II by Ca-SP. Ca-SP is therefore thought to be a unique sulfated polysaccharide which shows a strong antithrombin effect in an exclusively HC II-dependent manner.
Animals↗
The production of heparin cofactor II is not regulated by inflammatory cytokines in human hepatoma cells: comparison with plasminogen activator inhibitor type-1.
Using the Northern blot technique, we screened 6 human hepatoma cell lines to investigate the regulation mechanism of heparin cofactor II (HC II) biosynthesis. We found that HuH-7 and Hep G2 cells constitutively expressed the HC II gene. In conditioned medium, HuH-7 cells constantly produced HC II that was functionally active and formed a complex with thrombin in the presence of dermatan sulfate. HC II is thought be an acute phase reactant, and, therefore, we examined the effects of the major inflammatory cytokines, IL-6, IL-1 beta, and TNF-alpha, on the regulation of HC II production in HuH-7 and Hep G2 cells. In HuH-7 cells, the antigen and mRNA levels of plasminogen activator inhibitor type-1 (PAI-1), an acute phase protein produced by hepatocytes, were increased in response to stimulation with either IL-6 or IL-1 beta or both, but HC II antigen and mRNA levels were not changed by the same stimulation. Even when Hep G2 cells were treated with a combination of three cytokines, IL-6, IL-1 beta, and TNF-alpha, HC II antigen and mRNA levels were not changed; however, PAI-1 antigen and mRNA levels were clearly increased. These results suggest that the production of HC II in hepatoma cells is not regulated by the major inflammatory mediators, IL-6, IL-1 beta, and TNF-alpha.
Blotting, Northern↗
Inhibitory effect of a synthetic prostacyclin analogue, beraprost, on urokinase-type plasminogen activator expression in RC-K8 human lymphoma cells.
Plasminogen activation by urokinase-type plasminogen activator (uPA) is implicated in tumor invasion and metastasis by the breakdown of extracellular matrix. We have recently demonstrated the inhibitory effect of cAMP on uPA gene transcription in RC-K8 human lymphoma cells (Biochim Biophys Acta 1268: 293-9, 1995). Prostacyclin produced by endothelial cells is shown to increase cellular cAMP levels by activating adenylate cyclase. We, therefore, examined the effect of a stable analogue of prostacyclin, Beraprost, on uPA production in RC-K8 cells. uPA activity gradually increased in the conditioned medium with time. Beraprost (0.1 nM-1.0 microM) inhibited uPA accumulation in a dose-dependent manner without affecting cell viability. Fibrinzymography demonstrated that high and low molecular forms of uPA were present in the conditioned medium and that after Beraprost-treatment all forms of uPA decreased and no PA/PA inhibitor complex was present. Northern blot analysis revealed that after exposure to Beraprost, uPA mRNA levels increased transiently and then rapidly decreased to below control levels. Treatment with Beraprost resulted in a rapid activation of cellular cyclic AMP-dependent protein kinase (PKA). Beraprost completely negated uPA gene expression induced by phorbol myristate acetate, an activator of protein kinase C (PKC). These results suggest that Beraprost inhibits uPA production by suppressing uPA gene expression through the PKA pathway and that PKA-mediated signals are dominant in uPA gene expression as compared to those medicated by PKC. This inhibition of uPA expression by a prostacyclin analogue may be an important fact to explain the mechanism of anti-metastatic effects of prostacyclin.
Enzyme Inhibitors↗
Protein kinase activity-dependent inhibition of urokinase-type plasminogen activator gene transcription by cyclic AMP in human pre-B lymphoma cell line RC-K8.
We investigated the effects of cAMP on the urokinase-type plasminogen activator (uPA) production in human pre-B lymphoma cell line RC-K8 that is consistently secreting uPA in the conditioned medium. Both Bt2cAMP and PGE1 inhibited the uPA accumulation in a dose-dependent manner. Northern blot analysis and nuclear run-on assay revealed that uPA gene transcription was repressed by Bt2cAMP and the repression was negated by inhibition of de novo protein synthesis by cycloheximide. Pretreatment with H89 (N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), a specific cAMP-dependent protein kinase (PKA) inhibitor, strongly inhibited both the PKA activation and the supression of uPA mRNA accumulation induced by cAMP. H85 (N-[2-(N-formyl-p-chlorocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), which closely resembles H89 in its chemical structure but is not a selective inhibitor of PKA, showed little effect on the regulation of uPA gene regulation by Bt2cAMP. These results suggest that cAMP represses uPA gene transcription in human pre-B lymphoma cells through PKA pathway and in which de novo protein synthesis is required.
Alprostadil↗
Carbazochrome sodium sulphonate (AC-17) decreases the accumulation of tissue-type plasminogen activator in culture medium of human umbilical vein endothelial cells.
This study investigated the effect of carbazochrome sodium sulphonate (AC-17) on the accumulation of tissue-type plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) in culture medium of human umbilical vein endothelial cells (HUVEC). HUVEC were cultured in the presence of AC-17, and the concentration of t-PA:Ag and PAI-1:Ag in the medium was measured by an enzyme-linked immunoassay. AC-17, after 48 and 72 h incubation, significantly decreased the t-PA:Ag in the culture medium, while it had no significant effect on the PAI-1:Ag. The result of fibrin zymography was consistent with the result obtained by ELISA. It also revealed that all of the t-PA present in the culture medium formed complexes with PAI-1, indicating that AC-17 did not interfere with t-PA/PAI-1 complex formation. Furthermore, AC-17 did not inhibit the activities of t-PA and plasmin when measured by a fibrin plate method. These results suggest that AC-17 may modulate the fibrinolytic balance of blood through changing the function of endothelial cells, a new aspect of action of AC-17 as a haemostatic drug.
Adrenochrome↗
Selective activation of heparin cofactor II by a sulfated polysaccharide isolated from the leaves of Artemisia princeps.
While checking anticoagulant activities in crude fractions from Wakan-Yakus (traditional herbal drugs), we detected antithrombin activity in the polysaccharide fraction of the leaves of Artemisia princeps Pamp. A sulfated polysaccharide purified from the crude fractions by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B potentiated the heparin cofactor II (HC II)-dependent antithrombin activity but not the antithrombin activity of antithrombin III (AT III). The polysaccharide enhanced the HC II-thrombin reaction more than 6000-fold. The apparent second-order rate constant of thrombin inhibition by HC II increased from 3.8 x 10(4) (in the absence of the polysaccharide) to 2.5 x 10(8) M-1 min-1 in the presence of 25-125 micrograms/ml of the polysaccharide. In human plasma, the polysaccharide accelerated the formation of thrombin-HC II complex. The stimulating effect on HC II-dependent antithrombin activity was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, while chondroitinase ABC or chondroitinase AC II had little or no effect. These results suggest that the polysaccharide is a glycosaminoglycan-like material with properties that are quite distinct from heparin or dermatan sulfate.
Antithrombin III↗
Sequential changes in the distribution of type I and III collagens in the infarct zone: immunohistochemical study of experimental myocardial infarction in the rat.
AIM: Ventricular remodeling following acute myocardial infarction is an important factor in prognosis. The healing process, involving changes in type I and III collagens, is one of the major factors in remodelling. We therefore examined sequential changes in type I and III collagens after experimental myocardial infarction. MATERIALS AND METHODS: Hearts were excised from 1 day to 10 weeks after permanent left coronary ligation in rats. Immunohistochemical staining with a polyclonal antibody to each collagen was performed by the avidin-biotin-peroxidase method. RESULTS: Type I collagen initially appeared in the peripheral zone of the infarct from 3 days after ligation, the extent of staining gradually increasing until it reached a maximal level on days 21-28, after which the distribution remained unchanged. Type III collagen appeared in the peripheral zone of the infarct from 3 days after ligation; the extent of staining reached the maximal level after 11-28 days, after which a slight decrease in the distribution was observed, although the staining did not entirely disappear. CONCLUSIONS: Type I collagen was a major factor in collagen matrix formation, especially in the relatively late phase. Type III collagen, however, contributed particularly to collagen matrix formation in the relatively early phase. This study improves current understanding of the time-dependent alterations in type I and III collagens involved in the healing process after coronary artery occlusion.
Animals↗
Changes in extracellular matrix components in cardiomyopathic Syrian hamster, BIO 14.6.
We examined changes in the distribution of extracellular matrix components in the myocardium of cardiomyopathic Syrian hamsters (BIO 14.6). Fibronectin, laminin, and type IV collagen, and type I and III collagens were immunohistochemically stained by the avidin-biotin-peroxidase complex method, using a polyclonal antibody for each component. Hearts obtained from 4 stages of BIO 14.6 cardiomyopathy were examined. Peri- and endomysial fibrosis increased as the disease progressed. Replacement and meshwork (perimysial fibrosis penetrating the intercellular space) fibrotic lesions appeared beginning in the 2nd stage, ie, the fibrotic and healing stage. All of the components examined, ie, fibronectin, laminin and type IV collagen, and type I and III collagens, were present in various fibrotic lesions and played a significant role in fibrotic changes throughout all of the stages of the disease. No primary deficit of any of these components was seen. An increased distribution of fibronectin was observed in both the enlarged peri-and endomysial spaces beginning in the initial stage, ie, the necrotic stage, when myocyte hypertrophy was inconspicuous, and distribution throughout the myocardium increased further as the disease progressed. Laminin and type IV collagen in the fibrotic lesions were not restricted to the myocyte membrane. Type III collagen was distributed in replacement and meshwork fibrotic lesions, and the extent of its distribution increased in proportion to that of type I collagen. The continuous increases in the distribution of fibronectin, laminin and type III collagen indicate that fibrotic changes occurred continuously in this model.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Transcriptional regulation of tissue- and urokinase-type plasminogen activator genes by thrombin in human fetal lung fibroblasts.
The 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Modulation of urokinase-type plasminogen activator gene expression by inflammatory cytokines in human pre-B lymphoma cell line RC-K8.
We examined the effects of inflammatory cytokines, such as interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), transforming growth factor-beta (TGF beta) and lipopolysaccharide (LPS), on the urokinase-type plasminogen activator (uPA) gene expression in RC-K8 human pre-B lymphoma cells. Recombinant IL-1 alpha, recombinant IL-1 beta and LPS but not recombinant IL-6, recombinant TNF alpha and TGF beta dose-dependently increased uPA accumulation in the conditioned medium. Northern blot analysis revealed that uPA mRNA levels rapidly increased with a peak induction at 2 h after stimulation with IL-1 alpha and IL- 1 beta, but uPA mRNA increase by LPS began at 9 h after stimulation and the increase was maintained until the experiment ended at 24 h. These responses were independent of de novo synthesis, rather amplified in the presence of a protein synthesis inhibitor. The effects by IL-1 alpha and Il-1 beta were prevented by addition of anti-IL-1 alpha and anti-IL-1 beta neutralizing antibodies, respectively. In contrast, both antibodies did not prevent LPS-induced uPA gene expression. Therefore, it is unlikely that the effect by LPS is through induction of IL-1. Both IL-1 alpha and IL- 1 beta rapidly activated uPA gene transcription, but not increased stability of uPA mRNA. These results suggest that both IL-1 alpha and IL-1 beta cause a rapid activation of uPA gene transcription in which de novo protein synthesis is not required and that LPS induces uPA gene expression independently of the IL-1 pathway. These modulations of uPA production by inflammatory mediators may be implicated in tumor growth and metastasis.
Antigen-Antibody Reactions↗
Ellagic acid/phospholipid-induced coagulation and dextran sulfate-induced fibrinolytic activities in beta 2-glycoprotein I-depleted plasma.
Beta 2-glycoprotein I (beta 2-GPI) binds negatively charged substances and inhibits intrinsic blood coagulation in the presence of ellagic acid-phospholipid suspension. Beta 2-GPI is thought to be an important protein in the reaction between negatively charged phospholipids and anti-phospholipid antibodies which appear in patients with lupus anticoagulant/antiphospholipid antibody syndrome. We prepared a monoclonal antibody against beta 2-GPI purified from human plasma and obtained beta 2-GPI-depleted plasma using a monoclonal antibody-coupled column. Either partial thromboplastin time or the activation of prekallikrein induced by diluted ellagic acid-phospholipid suspension in beta 2-GPI-depleted plasma was not different from that in control plasma. Beta 2-GPI inhibited the intrinsic blood coagulation only when added to control or beta 2-GPI-depleted plasma in excess (more than physiological concentrations). The intrinsic fibrinolysis in beta 2-GPI-depleted plasma induced by dextran sulfate was not impaired and, again, beta 2-GPI inhibited the intrinsic fibrinolysis only when added to control or beta 2-GPI-depleted plasma in excess. These results indicate that both in vitro Actin-induced intrinsic coagulation and dextran sulfate-induced fibrinolytic activities are significantly inhibited by more than physiological concentrations of beta 2-GPI.
Amino Acid Sequence↗