Falsely elevated thyroid hormone levels associated with fibrin interference in patients receiving oral anticoagulant therapy.

OBJECTIVE: Unique clinical courses were observed in two asymptomatic patients receiving warfarin who referred to our hospital because of suspected central hyperthyroidism. We eventually diagnosed these patients with falsely elevated thyroid hormone levels caused by macroscopically invisible fibrin. Although false results caused by fibrin interference in vitro have been identified in various immunoassays, especially in blood samples from patients receiving anticoagulant therapy, no studies on thyroid function testing have been reported. The experience in evaluating these cases prompted us to investigate the independent influence of oral anticoagulants via putative fibrin interference on thyroid function testing. METHODS: We retrospectively reviewed known contributing factors that affect thyroid function testing including age, gender, medication history, body mass index, estimated glomerular filtration rate, smoking status, alcohol consumption, and the seasons of hospital visits from participants who presented the Department of Health Checkup between April 2010 and December 2020. RESULTS: A propensity-matched analysis revealed that the median serum free thyroxine levels under oral anticoagulant were significantly higher (17.9 pmol/L, n = 60) than those without anticoagulants (16.0 pmol/L, n = 60; p < 0.001). It was noted that this difference was the largest among contributing factors we analyzed. No significant differences were noted in serum thyroid-stimulating hormone levels. CONCLUSIONS: We report two patients receiving warfarin with falsely elevated thyroid hormone levels caused by fibrin interference resembling central hyperthyroidism for the first time. Our retrospective study suggests that the medication status of oral anticoagulants should be considered when evaluating thyroid function tests.

Role of thyroid hormone in an experimental model of atherosclerosis: the potential mediating role of immune response and autophagy.

Accumulating evidence has revealed that several conditions related to abnormal thyroid hormone status, such as dyslipidemia, hypertension, or hypercoagulable state, can exacerbate atherosclerotic vascular disease. Thyroid hormone effects on vascular smooth muscle cells and endothelial cells have also been studied extensively. However, only limited information is available on thyroid hormone-mediated immune response in current review articles on the pathophysiology of atherosclerosis. This report thus presents an overview of the recent advances in the understanding of the dynamic interactions taking place between thyroid hormone status and immune response in the pathogenesis of atherosclerosis. In particular, we focus on macrophages and T-lymphocytes, which have been recognized as important determinants for the initiation and development of atherosclerosis. Numerous studies have revealed the role of autophagy in immune cells produced in atherosclerosis. In addition, thyroid hormones induce autophagy in several cells and tissues, such as liver, skeletal muscles, lungs, and brown adipose tissue. Our research group, among others, have reported different targets of thyroid hormone-mediated autophagy, including lipid droplets (lipophagy), mitochondria (mitophagy), and aggregated proteins (aggrephagy). Based on these findings, thyroid hormone-mediated autophagy could serve as a novel therapeutic approach for atherosclerosis. We also consider the limitations of the current murine models for studies on atherosclerosis, especially in relation to low-density lipoprotein-cholesterol driven atherosclerotic plaque.

Dasatinib suppresses atherosclerotic lesions by suppressing cholesterol uptake in a mouse model of hypercholesterolemia.

Although the use of BCR-ABL1 tyrosine kinase inhibitors (TKIs) for chronic myeloid leukemia is known to cause vascular adverse events (VAEs), the frequency of VAEs during dasatinib administration is not high, and the same holds for atherosclerosis-related VAEs. However, its effect on atherosclerosis remains controversial. In this study, our primary objective was to investigate how dasatinib affects atherosclerosis. Ldlr-/-/Apobec1-/- mice, which are highly prone to develop atherosclerosis, were administered dasatinib. After 16 weeks, we evaluated their atherosclerotic lesions. We used bone-marrow-derived macrophages to investigate the uptake of oxidized low-density lipoprotein (LDL) complexed with DiI dye (DiI-oxLDL). RNA sequencing and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were performed to explore the potential effects of dasatinib on cholesterol metabolism. Dasatinib administration significantly reduced atherosclerotic lesions (P < 0.001 and P = 0.013) and DiI-oxLDL uptake (P < 0.001) unlike other TKIs. RNA sequencing and RT-qPCR suggested that Sort1, which encodes sortilin, a known regulator of LDL uptake, and Cd36 were potential targets of dasatinib. In conclusion, dasatinib induced elevated LDL-C levels, but oxLDL uptake in macrophages were suppressed, resulting in reducing atherosclerotic lesions. These results further our understanding of the differences in VAEs between dasatinib and other TKIs.

Successful lactation in Plgrkt-deficient female mice caused by a 1-bp deletion of exon4.

Plasminogen (Pg) activation on the cell surface is important for various (patho)physiologic conditions, and Plg-RKT is a cell membrane protein that binds to Pg and promotes its activation. To evaluate the role of Plg-RKT in atherosclerosis, Plgrkt gene in Ldlr-/-/Apobec1-/- was modified using in vivo CRISPR/Cas9. Synthetic RNA for Plgrkt and Cas9 complex was electroporated into the fertilized eggs in the oviducts. Plgrkt deficient mice were established through a 1-bp deletion, and in this research communication we report their lactational ability. In contrast to Plgrkt-/- mice developed by a conventional method, these newly developed mice did not suffer lactation failure and could maintain their pups until weaning. The major obvious difference between these lines is the area of gene modification. The conventionally developed mouse possesses about 10 kb deletion of Plgrkt, which might relate to the lactation failure. Lactation failure is a lethal phenotype in mammals, and analyses of causative genes are especially important for dairy industries. Further genome-wide analyses with both Plgrkt-/- mice may help to establish causative genes for lactation failure.

Inhibition of plasminogen suppresses fibrosis and macrophage foaming in a nonalcoholic steatohepatitis mouse model.

Nonalcoholic steatohepatitis is a clinically important liver disease. Its symptoms are exacerbated by macrophage foaming, which is promoted by plasminogen in vitro. However, the influence of plasminogen on nonalcoholic steatohepatitis has not been reported. In this study, we evaluated the influence of plasminogen in a mouse model of nonalcoholic steatohepatitis with macrophage foaming. L-/- /A-/- mice, characterized by hypercholesterolemia, were injected with streptozotocin and fed a high-fat diet to develop nonalcoholic steatohepatitis with macrophage foaming. To confirm the influence of plasminogen, we used the well-known plasminogen inhibitor tranexamic acid and L-/- /A-/- /Plg-/- mice, which are deficient in plasminogen and investigated the influence on nonalcoholic steatohepatitis. The influence of plasminogen on the expression levels of proinflammatory cytokines involved in foaming in macrophages was also assessed. The formation of nonalcoholic steatohepatitis lesions with macrophage foaming was confirmed in the L-/- /A-/- mouse model. Tranexamic acid attenuated foaming and fibrosis in the L-/- /A-/- mice. Similarly, foaming and liver fibrosis were also attenuated in the L-/- /A-/- /Plg-/- mice. The mRNA expression levels of TGF-ß1 and IL-1ß in liver and peritoneal macrophages were reduced upon plasminogen inhibition. We show that inhibition of plasminogen suppressed macrophage foaming, cytokine expression, and consequently fibrosis in nonalcoholic steatohepatitis. Our results provide a clue toward various processes leading to fibrosis and may contribute to new therapeutic strategies for nonalcoholic steatohepatitis.

Plasminogen Deficiency Significantly Reduces Vascular Wall Disease in a Murine Model of Type IIa Hypercholesterolemia.

The fibrinolytic system has been implicated in the genesis and progression of atherosclerosis. It has been reported that a plasminogen (Pg) deficiency (Plg-/-) exacerbates the progression of atherosclerosis in Apoe-/- mice. However, the manner in which Plg functions in a low-density lipoprotein-cholesterol (LDL-C)-driven model has not been evaluated. To characterize the effect of Pg in an LDL-C-driven model, mice with a triple deficiency of the LDL-receptor (LDLr), along with the active component (apobec1) of the apolipoprotein B editosome complex, and Pg (L-/-/A-/-/Plg-/-), were generated. Atherosclerotic plaque formation was severely retarded in the absence of Pg. In vitro studies demonstrated that LDL uptake by macrophages was enhanced by plasmin (Pm), whereas circulating levels of LDL were enhanced, relative to L-/-/A-/- mice, and VLDL synthesis was suppressed. These results indicated that clearance of lipoproteins in the absence of LDLr may be regulated by Pg/Pm. Conclusions: The results from this study indicate that Pg exacerbates atherosclerosis in an LDL-C model of atherosclerosis and also plays a role in lipoprotein modification and clearance. Therefore, controlling the Pg system on macrophages to prevent foam cell formation would be a novel therapeutic approach.

Fibrin-mediated growth restriction of early-stage human trophoblasts is switched to growth promotion through fibrinolysis.

STUDY QUESTION: Does fibrin promote trophoblast growth in human and mouse blastocysts during early embryo implantation? SUMMARY ANSWER: Mouse blastocysts were unaffected by fibrin; however, human blastocysts were significantly suppressed by fibrin in trophoblast growth and then switched to growth promotion through increased fibrinolysis with urokinase-type plasminogen activator (uPA) activity. WHAT IS KNOWN ALREADY: Fibrin(ogen) plays an important role in various physiological processes and is also critical for maintaining feto-maternal attachment during pregnancy. The addition of fibrin to embryo transfer media has been used to increase implantation rates in human ART; however, its mechanism of action' in vitro has not yet been characterized. STUDY DESIGN, SIZE, DURATION: Vitrified mouse and human blastocysts were warmed and individually cultured in vitro for up to 120 and 168 h, respectively, on a fibrin substrate. Blastocysts were cultured at 37°C in 6% CO2, 5% O2 and 89% N2. Blastocyst development and related fibrinolytic factors were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICR strain mouse embryos were purchased from a commercial supplier. Human blastocysts were donated with informed consent from two fertility centers. Mouse and human blastocysts cultured on fibrin-coated plates were compared to those on non-coated and collagen-coated plates in vitro. Trophoblast growth and fibrin degradation were assessed based on the cell area and fibrin-free area, respectively. Fibrinolytic factors were detected in supernatants using plasminogen-casein zymography. The fibrinolytic activity of blastocysts was investigated using a selective uPA inhibitor, exogenous uPA, plasminogen activator inhibitor-1 (PAI-1) inhibitor and fibrin degradation products (FDPs). Fibrinolysis-related mRNA expression level was detected using quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Fibrin did not affect the developmental speed or morphology of mouse blastocysts, and a large fibrin-degrading region was observed in the attachment stage. In contrast, fibrin significantly suppressed the outgrowth of trophoblasts in human blastocysts, and trophoblasts grew after the appearance of small fibrin-degrading regions. uPA was identified as a fibrinolytic factor in the conditioned medium, and uPA activity was significantly weaker in human blastocysts than in mouse blastocysts. The inhibition of uPA significantly reduced the outgrowth of trophoblasts in mouse and human blastocysts. Human blastocysts expressed PLAU (uPA), PLAUR (uPA receptor), SERPINE1 (PAI-1) and SERPINB2 (PAI-2), whereas mouse blastocysts were limited to Plau, Plaur and Serpine1. In a subsequent experiment on human blastocysts, the addition of exogenous uPA and the PAI-1 inhibitor promoted trophoblast growth in the presence of fibrin, as did the addition of FDPs. LIMITATIONS, REASONS FOR CAUTION: This model excludes maternal factors and may not be fully reproduced in vivo. Donated human embryos are surplus embryos that may inherently exhibit reduced embryonic development. In addition, donated ART-derived embryos may exhibit weak uPA activity, because women with sufficient uPA-active embryos may not originally require ART. The present study used orthodox culture methods, and results may change with the application of recently developed protocols for culture blastocysts beyond the implantation stage. WIDER IMPLICATIONS OF THE FINDINGS: The present results suggest that the distinct features of trophoblast outgrowth in human blastocysts observed in the presence of fibrin are regulated by a phenotypic conversion induced by contact with fibrin and FDPs. Mouse embryos did not exhibit the human phenomenon, indicating that the present results may be limited to humans. STUDY FUNDING/COMPETING INTEREST(S): The present study was supported by the Department of Obstetrics and Gynecology at the Hamamatsu University School of Medicine and Kishokai Medical Corporation. None of the authors have any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Hyperfunctioning Papillary Thyroid Carcinoma with a BRAF Mutation: The First Case Report and a Literature Review.

INTRODUCTION: Hyperfunctioning papillary thyroid carcinoma (PTC) is rare and consequently, little information on its molecular etiology is available. Although BRAF V600E (BRAF c.1799T>A, p.V600E) is a prominent oncogene in PTC, its mutation has not yet been reported in hyperfunctioning PTC. CASE PRESENTATION: Ultrasonography detected a 26-mm nodule in the right lobe of the thyroid gland of a 48-year-old man. Thyroid function tests indicated that he was hyperthyroid with a TSH level of 0.01 mIU/L (reference range: 0.05-5.00) and a free thyroxine level of 23.2 pmol/L (reference range: 11.6-21.9). TSHR autoantibodies were <0.8 IU/L (reference value: <2.0 IU/L). The 99mTc thyroid scintigram revealed a round, right-sided focus of tracer uptake by the nodule with a decreased uptake in the remainder of the gland. The patient underwent total thyroidectomy because fine-needle aspiration cytology revealed a malignancy. The histopathological diagnosis was conventional PTC. Subsequent mutational analysis of BRAF (exon 15), TSHR (exons 1-10), GNAS (exons 7-10), EZH1 (exon 16), KRAS, NRAS, HRAS (codons 12, 13, and 61), and TERT promoter (C250T and C228T) identified a heterozygous point mutation in BRAF V600E in a tumor tissue sample. In addition, we identified a TSHR D727E polymorphism (TSHR c.2181C>G, p.D727E) in both the tumor and the surrounding normal thyroid tissue. DISCUSSION AND CONCLUSIONS: We report a case of hyperfunctioning PTC with a BRAF V600E mutation for the first time. Our literature search yielded 16 cases of hyperfunctioning thyroid carcinoma in which a mutational analysis was conducted. We identified TSHR mutations in 13 of these cases. One case revealed a combination of TSHR and KRAS mutations; the other case revealed a TSHR mutation with a PAX8/PPARG rearrangement. These findings suggest that the concomitant activation of oncogenes (in addition to constitutive activation of the TSHR-cyclic AMP cascade) are associated with the malignant phenotype in hyperfunctioning thyroid nodules.

Nanobody production can be simplified by direct secretion from Escherichia coli.

It is well known that camelids (camels and llamas) have fully functional antibodies with only a heavy chain consisting of a single variable domain and two constant domains. This single variable domain is called a "nanobody" and many nanobodies are synthesized in the cytosol of Escherichia coli, however, most of the nanobodies become inclusion bodies without tags to enhance their solubility. We generated a vector system to enable the secretary expression of nanobodies in Escherichia coli. In this system, several NBs were secreted into the culture supernatant. Since the vector contained 6xHis tag and AviTAG, biotinylation (even fluorescent-labeled) of AviTAG was achieved during cell culture, and purification of the supernatant was a step by immobilized metal ion adsorption chromatography. The procedure described in this study is believed to be as simple as regular plasmid minipreps. Therefore, many laboratories can use this method.

Recognition of Plasminogen Activator Inhibitor Type 1 as the Primary Regulator of Fibrinolysis.

The fibrinolytic system consists of a balance between rates of plasminogen activation and fibrin degradation, both of which are finely regulated by spatio-temporal mechanisms. Three distinct inhibitors of the fibrinolytic system that differently regulate these two steps are plasminogen activator inhibitor type-1 (PAI-1), α2-antiplasmin, and thrombin activatable fibrinolysis inhibitor (TAFI). In this review, we focus on the mechanisms by which PAI-1 governs total fibrinolytic activity to provide its essential role in many hemostatic disorders, including fibrinolytic shutdown after trauma. PAI-1 is a member of the serine protease inhibitor (SERPIN) superfamily and inhibits the protease activities of plasminogen activators (PAs) by forming complexes with PAs, thereby regulating fibrinolysis. The major PA in the vasculature is tissue-type PA (tPA) which is secreted from vascular endothelial cells (VECs) as an active enzyme and is retained on the surface of VECs. PAI-1, existing in molar excess to tPA in plasma, regulates the amount of free active tPA in plasma and on the surface of VECs by forming a tPA-PAI-1 complex. Thus, high plasma levels of PAI-1 are directly related to attenuated fibrinolysis and increased risk for thrombosis. Since plasma PAI-1 levels are highly elevated under a variety of pathological conditions, including infection and inflammation, the fibrinolytic potential in plasma and on VECs is readily suppressed to induce fibrinolytic shutdown. A congenital deficiency of PAI-1 in humans, in turn, leads to life-threatening bleeding. These considerations support the contention that PAI-1 is the primary regulator of the initial step of fibrinolysis and governs total fibrinolytic activity.

Characterization of Atherosclerosis Formation in a Murine Model of Type IIa Human Familial Hypercholesterolemia.

A murine genetic model of LDL-cholesterol- (LDL-C-) driven atherosclerosis, based on complete deficiencies of both the LDL-receptor (Ldlr-/-) and key catalytic component of an apolipoprotein B-edisome complex (Apobec1-/-), which converts apoB-100 to apoB-48, has been extensively characterized. These gene deficiencies allow high levels of apoB-100 to be present and inefficiently cleared, thus leading to very high levels of LDL-C in mice on a normal diet. Many key features of atherosclerotic plaques observed in human familial hypercholesterolemia are found in these mice as they are allowed to age through 72 weeks. The general characteristics include the presence of high levels of LDL-C in plasma and macrophage-related fatty streak formation in the aortic tree, which progressively worsens with age. More specifically, plaque found in the aortic sinuses contains a lipid core with relatively high numbers of macrophages and a smooth muscle cell α-actin- and collagen-containing cap, which thins with age. These critical features of plaque progression suggest that the Ldlr-/-/Apobec1-/- mouse line presents a superior model of LDL-C-driven atherosclerosis.

Elevated Plasma Levels of LDL Cholesterol Promote Dissecting Thoracic Aortic Aneurysms in Angiotensin II-Induced Mice.

BACKGROUND: Plasma low-density lipoprotein (LDL) cholesterol is implicated in abdominal aorta (AA) and aortic dissection (AD); however, its role in the pathogenesis of AA and AD, a disease with a high mortality rate, is unknown. The existing animal models such as apolipoprotein E-deficient (Apoe-/-) mice cannot reproduce all the conditions of AA/AD, including elevated LDL-cholesterol levels and spontaneous atheroma formation; therefore, a more reliable in vivo model is required. Here, we analyzed angiotensin II (Ang II)-induced mice with combined deficiency of the LDL receptor and the catalytic component of the apolipoprotein B-edisome complex (Ldlr-/-/Apobec1-/- [WKO]) to understand AA formation and AD occurrence in relation to plasma lipid composition. METHODS: AAs and ADs were created in 18- to 22- week-old male Apoe-/- and Ldlr-/-/Apobec1-/- mice by Ang II infusion. Immunostaining allowed assessment of smooth muscle cells and mural monocytes/macrophages. RESULTS: Ldlr-/-/Apobec1-/- mice had elevated LDL-cholesterol levels characteristic for human type IIa hyperlipidemia, resulting in atherogenesis, which promoted mortality, AA formation, and AD development. Interestingly, variations in the distribution of atheromas and inflammatory sites between Apoe-/- and Ldlr-/-/Apobec1-/- mice depending on lipid profiles resulted in differences in AA formation and AD occurrence in the thoracic aorta. CONCLUSIONS: Our results indicate the presence of a pathogenic pathway involving serum lipid composition that plays a key role in AA formation and AD occurrence in Ang II-induced mice.

Lysophosphatidylcholine Acyltransferase-3 Expression Is Associated with Atherosclerosis Progression.

Free arachidonic acid (AA) is an important precursor of lipid mediators such as leukotrienes and prostaglandins that induces inflammation and is associated with atherosclerosis progression. Recent studies have shown that lysophosphatidylcholine acyltransferase-3 (LPCAT3) converts lysophosphatidylcholine (LPC) and free AA into phosphatidylcholine (PC)-containing AA (arachidonyl-PC) and thereby can regulate intracellular free-AA levels. However, the association between LPCAT3 and atherosclerosis remains to be established. In this study, we analyzed human and mouse atherosclerotic tissues to gain insight into the arachidonyl-PC metabolism involving LPCAT3 using imaging mass spectrometry. The data revealed a complementary distribution of arachidonyl-PC and LPC in human atherosclerotic tissues with arachidonyl-PC decreasing and LPC increasing as atherosclerosis progressed. Furthermore, we found a homologous distribution of LPCAT3 expression and arachidonyl-PC based on atherosclerotic progression. In contrast, in ApoE-deficient mice, atherosclerosis increased both arachidonyl-PC accumulation and LPCAT3 expression. Taken together, these findings suggest that the regulation of LPCAT3 expression might be associated with atherosclerotic progression in humans.

Mutation in a highly conserved glycine residue in strand 5B of plasminogen activator inhibitor 1 causes polymerisation.

Serpinopathy is characterised as abnormal accumulation of serine protease inhibitors (SERPINs) in cells and results in clinical symptoms owing to lack of SERPIN function or excessive accumulation of abnormal SERPIN. We recently identified a patient with functional deficiency of plasminogen activator inhibitor-1 (PAI-1), a member of the SERPIN superfamily. The patient exhibited life-threatening bleeding tendencies, which have also been observed in patients with a complete deficiency in PAI-1. Sequence analysis revealed a homozygous single-nucleotide substitution from guanine to cytosine at exon 9, which changed amino acid residue 397 from glycine to arginine (c.1189G>C; p.Gly397Arg). This glycine was located in strand 5B and was well conserved in other serpins. The mutant PAI-1 was polymerised in the cells, interfering with PAI-1 secretion. The corresponding mutations in SERPINC1 (anti-thrombin III) at position 456 (Gly456Arg) and SERPINI1 (neuroserpin) at position 392 (Gly392Glu) caused an anti-thrombin deficiency and severe dementia due to intracellular retention of the polymers. Glycine is the smallest amino acid, and these mutated amino acids were larger and charged. To determine which factors were important, further mutagenesis of PAI-1 was performed. Although the G397A, C, I, L, S, T, and V were secreted, the G397D, E, F, H, K, M, N, P, Q, W, and Y were not secreted. The results revealed that the size was likely triggered by the polymerisation of SEPRINs at this position. Structural analyses of this mutated PAI-1 would be useful to develop a novel PAI-1 inhibitor, which may be applicable in the context of several pathological states.

Successful synthesis of active human coagulation factor VII by co-expression of mammalian gamma-glutamyl carboxylase and modification of vit.K cycle in Drosophila Schneider S2 cells.

Mammalian gamma-glutamyl carboxylase and reduced vitamin K are indispensable for synthesis of mature mammalian vitamin K dependent proteins including some of blood coagulation factors (factors II, VII, IX, and X). It was well known that Drosophila melanogaster expressed gamma-glutamyl carboxylase and possessed a vit.K cycle although native substrates for them have not been identified yet. Despite the potential capability of gamma carboxylation in D. melanogaster derived cells such as S2 cells, Drosophila gamma-glutamyl carboxylase failed to gamma carboxylate a peptide fused to the human coagulation factor IX propeptide. Thus, it had been believed that the Drosophila system was not adequate to synthesize mammalian vit.K dependent proteins. Indeed, we previously attempted to synthesize biologically active factor VII in S2 cells although we were not able to obtain it. However, recently, a successful transient expression of biologically active human factor IX from S2 cells was reported. In the present study, several expression vectors which enable expressing mammalian GGCX, VKORC1, and/or PDIA2 along with F7 were developed. S2 cells transfected with pMKA85, pMAK86, and pMAK219 successfully synthesized active FVII. Thus, mammalian GGCX was indispensable to synthesize active FVII while mammalian VKORC1 and PDIA2 were not critical but supportive factors for S2 cells.

Proper cytoskeletal architecture beneath the plasma membrane of red blood cells requires Ttll4.

Mammalian red blood cells (RBCs) circulate through blood vessels, including capillaries, for tens of days under high mechanical stress. RBCs tolerate this mechanical stress while maintaining their shape because of their elastic membrane skeleton. This membrane skeleton consists of spectrin-actin lattices arranged as quasi-hexagonal units beneath the plasma membrane. In this study, we found that the organization of the RBC cytoskeleton requires tubulin tyrosine ligase-like 4 (Ttll4). RBCs from Ttll4-knockout mice showed larger average diameters in smear test. Based on the rate of hemolysis, Ttll4-knockout RBCs showed greater vulnerability to phenylhydrazine-induced oxidative stress than did wild-type RBCs. Ultrastructural analyses revealed the macromolecular aggregation of cytoskeletal components in RBCs of Ttll4-knockout mice. Immunoprecipitation using the anti-glutamylation antibody GT335 revealed nucleosome assembly protein 1 (NAP1) to be the sole target of TTLL4 in the RBCs, and NAP1 glutamylation was completely lost in Ttll4-knockout RBCs. In wild-type RBCs, the amount of glutamylated NAP1 in the membrane was nearly double that in the cytosol. Furthermore, the absence of TTLL4-dependent glutamylation of NAP1 weakened the binding of NAP1 to the RBC membrane. Taken together, these data demonstrate that Ttll4 is required for proper cytoskeletal organization in RBCs.

The Pharmacokinetics and Pharmacodynamics of Prasugrel and Clopidogrel in Healthy Japanese Volunteers.

The dosing regimen of prasugrel adjusted for Japanese patients was compared with that of clopidogrel by analyzing the pharmacokinetics and pharmacodynamics in 40 healthy Japanese subjects in a randomized, single-blind crossover study. In period 1, the subjects received either 300 mg clopidogrel or 20 mg prasugrel; after a >2-week interval (period 2), the drug was switched. Blood samples of 36 of the 40 subjects were collected for analysis of pharmacokinetics, pharmacodynamics, and CYP2C19 genotypes. The plasma concentration of the active metabolite of prasugrel increased rapidly and reached its peak 30 minutes postadministration, whereas that of the active metabolite of clopidogrel reached its peak 1 hour postadministration. The mean AUC and Cmax of the active metabolite of clopidogrel, but not those of prasugrel, were CYP2C19 genotype dependent. Prasugrel rapidly inhibited platelet aggregation, reaching its maximum effect 1 hour postadministration. Clopidogrel, on the other hand, showed maximum inhibition 2 hours postadministration. Platelet aggregation inhibition by clopidogrel was significantly lower in the poor-metabolizer subjects than in the extensive-metabolizer subjects. Overall, prasugrel inhibited platelet aggregation more rapidly and more effectively in healthy Japanese subjects than was observed for clopidogrel.

[18F]FDG Uptake in the Aortic Wall Smooth Muscle of Atherosclerotic Plaques in the Simian Atherosclerosis Model.

Atherosclerosis is a self-sustaining inflammatory fibroproliferative disease that progresses in discrete stages and involves a number of cell types and effector molecules. Recently, [18F]fluoro-2-deoxy-D-glucose- ([18F]FDG-) positron emission tomography (PET) has been suggested as a tool to evaluate atherosclerotic plaques by detecting accumulated macrophages associated with inflammation progress. However, at the cellular level, it remains unknown whether only macrophages exhibit high uptake of [18F]FDG. To identify the cellular origin of [18F]FDG uptake in atherosclerotic plaques, we developed a simian atherosclerosis model and performed PET and ex vivo macro- and micro-autoradiography (ARG). Increased [18F]FDG uptake in the aortic wall was observed in high-cholesterol diet-treated monkeys and WHHL rabbits. Macro-ARG of [18F]FDG in aortic sections showed that [18F]FDG was accumulated in the media and intima in the simian model as similar to that in WHHL rabbits. Combined analysis of micro-ARG with immunohistochemistry in the simian atherosclerosis model revealed that most cellular [18F]FDG uptake observed in the media was derived not only from the infiltrated macrophages in atherosclerotic plaques but also from the smooth muscle cells (SMCs) of the aortic wall in atherosclerotic lesions.