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This recommended practice enables the quantification of volatile compounds in flavourings to be made by gas chromatography with flame-ionization detection, without having authentic compounds available, and also in many cases it can avoid time-consuming calibration procedures. The relative-response factors (RRF) can be predicted from the molecular formula of the compound, and this approach can be applied to compounds containing the atoms C, H, O, N, S, F, Cl, Br, I, and Si, providing that the molecular formula and number of benzene rings in the analytes are known. The purity of chemically-defined flavouring substances or chromatographic standards can also be estimated using these predicted RRF, and this procedure can also be used to quantify (poly)hydroxylated compounds, after their derivatization into trimethylsilyl ethers or esters. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Many flavour regulations around the world contain a list of so-called 'restricted substances' (RS), i.e. substances that occur naturally in source materials for flavourings and food ingredients with flavouring properties, but whose presence in certain foods is restricted and/or for which maximum levels are set, for example, the European regulation 1334/2008. Only a few publications refer to the determination of RS in compound flavourings or their raw materials, and the latter only concern the analysis of one or two individual RS in single essential oils. The Working Group on Methods of Analysis of the International Organization of the Flavor Industry (IOFI) has developed a method for the rapid routine determination of β-asarone, coumarin, menthofuran, methylchavicol, methyleugenol, pulegone, safrole and α- and β-thujones in flavourings and their raw materials by gas chromatography-mass spectrometry (GC-MS), using selected-ion monitoring and internal standards. The method has been evaluated by nine flavour-industry laboratories using a complex surrogate flavouring containing all of the above analytes, at concentrations that would be likely to produce levels in finished foods of around typical maximum limits for these RS. Results were obtained from a total of 15 columns and sets of analytical conditions, using 11 GC/MS instruments, with in each case a determination of the analyte in two versions of the flavouring. With reproducibility relative standard deviations (RSDR) of less than about 20%, and recoveries of 80-120%, the method performance can be considered as satisfactory for rapid routine checks on the levels of restricted substances in compound flavourings. The method is intended for flavour-industry laboratories in order for them to fulfil their obligation to inform food-industry clients of the amounts of these substances in commercial flavourings, but is not intended for their analysis in finished foods. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

ABSTRACT Simultaneous distillation-extraction (SDE) combines, in a single step, hydro-distillation of volatiles from a sample with continuous solvent extraction of the solutes dissolved in the aqueous distillate. The solvent extract containing the isolated flavour volatiles can be subsequently analysed by gas chromatography. The technique is applicable to compounds possessing a wide range of volatility, but it is suitable neither for compounds of low volatility (e.g. vanillin), nor for highly volatile materials (e.g. methanethiol). The present guidelines concern the isolation of flavouring substances by simultaneous distillation-extraction (SDE), based on the experience of the authors in laboratories of the flavour industry. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

We performed a safety evaluation using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) of the following four flavouring substances that belong to the class of 'aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and esters containing additional oxygenated functional groups' and are uniquely used in Japan: butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate. Although no genotoxicity study data were found in the published literature, none of the four substances had chemical structural alerts predicting genotoxicity. All four substances were categorised as class I by using Cramer's classification. The estimated daily intake of each of the four substances was determined to be 0.007-2.9 μg/person/day by using the maximised survey-derived intake method and based on the annual production data in Japan in 2001, 2005 and 2010, and was determined to be 0.250-600.0 μg/person/day by using the single-portion exposure technique and based on average-use levels in standard portion sizes of flavoured foods. Both of these estimated daily intake ranges were below the threshold of toxicological concern for class I substances, which is 1800 μg/person/day. Although no information from in vitro and in vivo toxicity studies for the four substances was available, these substances were judged to raise no safety concerns at the current levels of intake.

Rheumatoid arthritis (RA) is a systemic inflammatory disease in which the predominant symptom is polyarthritis that follows a chronic and progressive clinical course characterized by destructive synovitis and various immune disorders. Striking progress in RA treatment was achieved with the emergence of monoclonal antibodies to target cytokines. However, drug choices are limited for many patients due to resistance to multidrug antirheumatic therapy, concomitant disease, and infection. We evaluated the efficacy of treatment in 85 patients with RA for whom leukocytapheresis (LCAP) was initiated at our hospital between 2006 and 2015. All patients continued drug therapy and were treated with LCAP once a week for up to 5 weeks. The clinical response was evaluated at the completion of LCAP series and 4 weeks later using the American College of Rheumatology (ACR) criteria and the 28-joint disease activity score (DAS28) of European League Against Rheumatism (EULAR). The tender joint counts, swollen joint counts, and C-reactive protein (CRP) levels decreased remarkably. DAS28-CRP was significantly improved by LCAP. And furthermore, the efficacy lasted at least 4 weeks after the completion of LCAP. These results suggest that LCAP is a beneficial and are consistent with several trials' reported effect of LCAP. This treatment can contribute to improvements in activities of daily living (ADLs) and long-term outcome by improving swollen and tender joint counts and CRP levels even in refractory patients for whom the use of conventional disease-modifying antirheumatic drugs (DMARDs) and biopharmaceuticals is problematic. LCAP might be a promise therapy to refractory RA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Ankylosing spondylitis (AS) is characterized by chronic inflammation of the axial and peripheral joints and ligamentous attachments. Gut immunity is thought to be involved in AS, because a prominent coexistence of gut and joint inflammation has been observed in patients with AS. Mucosal-associated invariant T (MAIT) cells are preferentially located in the gut lamina propria and produce inflammatory cytokines such as interleukin 17 (IL-17) and tumor necrosis factor-α (TNF-α), which are therapeutic targets for AS. This study aimed to investigate the involvement of MAIT cells in AS., Methods: The frequency of MAIT cells and their cytokine production were determined in patients with AS and healthy controls (HC). The expression of a MAIT cell activation marker (CD69) was analyzed in patients with AS by using flow cytometry., Results: The frequency of MAIT cells in the peripheral blood was lower in patients with AS compared with HC. The levels of IL-17 produced by MAIT cells after activation were higher in patients with AS than in the HC. CD69 expression on MAIT cells correlated with the Ankylosing Spondylitis Disease Activity Score in patients with AS., Conclusion: These results suggest the involvement of MAIT cells in the pathogenesis of AS.

MicroRNAs (miRNAs), which are important inhibitors of mRNA translation, participate in differentiation, migration, cell proliferation, and cell death. The pathology of miRNAs results in alterations in protein expression. Recently, miRNAs circulating in peripheral blood have been shown to control the synthesis and translation of proteins at distal sites after intake into local cells. A number of studies are currently being conducted to investigate how to use miRNAs in disease treatment, but no studies have attempted to alleviate disease by directly eliminating miRNAs from blood. Therefore, we examined whether the removal or reduction of circulating miRNAs with apheresis improved pathologies caused by miRNAs. After approval of the study by our medical school's ethics committee, we collected blood and separated plasma samples from three patients with systemic lupus erythematosus who were undergoing plasmapheresis at our hospital. Peripheral blood was collected before and after it was passed through a primary membrane, centrifuged, and used to extract circulating miRNAs. A comprehensive expression analysis was then performed with a miRNA array chip. The levels of expression of a large number of circulating miRNAs were measured in the plasma samples separated by the primary membranes from all 3 patients with systemic lupus erythematosus. We present the first report that circulating miRNAs in peripheral blood can be separated and possibly directly removed using membrane separation apheresis., (© 2016 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.)

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on five acetal flavouring substances uniquely used in Japan: acetaldehyde 2,3-butanediol acetal, acetoin dimethyl acetal, hexanal dibutyl acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal. As no genotoxicity study data were available in the literature, all five substances had no chemical structural alerts predicting genotoxicity. Using Cramer's classification, acetoin dimethyl acetal and hexanal dibutyl acetal were categorised as class I, and acetaldehyde 2,3-butanediol acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal as class III. The estimated daily intakes for all five substances were within the range of 1.45-6.53 µg/person/day using the method of maximised survey-derived intake based on the annual production data in Japan from 2001, 2005, 2008 and 2010, and 156-720 µg/person/day using the single-portion exposure technique (SPET), based on the average use levels in standard portion sizes of flavoured foods. The daily intakes of the two class I substances were below the threshold of toxicological concern (TTC) - 1800 μg/person/day. The daily intakes of the three class III substances exceeded the TTC (90 μg/person/day). Two of these, acetaldehyde 2,3-butanediol acetal and hexanal glyceryl acetal, were expected to be metabolised into endogenous products after ingestion. For 4-methyl-2-pentanone propyleneglycol acetal, one of its metabolites was not expected to be metabolised into endogenous products. However, its daily intake level, based on the estimated intake calculated by the SPET method, was about 1/15 000th of the no observed effect level. It was thus concluded that all five substances raised no safety concerns when used for flavouring foods at the currently estimated intake levels. While no information on in vitro and in vivo toxicity for all five substances was available, their metabolites were judged as raising no safety concerns at the current levels of intake.

We evaluated the bradykinin generation level during leukocytapheresis (LCAP) using novel Cellsorba(TM) CS-180S, which has sodium pyrosulfite and sodium carbonate as a filling solution. Subjects of this study were 14 rheumatoid arthritis patients. Regardless of the type of anticoagulant used, bradykinin levels were lower with the novel CS-180S than with the conventional CS-180S (28.7 ± 53.3 vs. 8.0 ± 2.7 as the mean ± standard deviation). When anticoagulants other than nafamostat mesilate were used with the conventional CS-180S, bradykinin levels increased at the column outlet compared with the column inlet, and adverse effects of bradykinin were seen in several cases. In contrast, bradykinin levels remained low and no bradykinin-associated adverse events were observed with the novel CS-180S. We recommend using the novel column instead of the conventional column in the treatment of LCAP., (Copyright © 2013 Wiley Periodicals, Inc.)

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on four flavoring substances structurally related to menthol (L-menthyl 2-methylbutyrate, DL-menthyl octanoate, DL-menthyl palmitate, and DL-menthyl stearate) uniquely used in Japan. While no genotoxicity study data were available in the literature, all four substances had no chemical structural alerts predictive of genotoxicity. Moreover, they all four are esters consisting of menthol and simple carboxylic acids that were assumed to be immediately hydrolyzed after ingestion and metabolized into innocuous substances for excretion. As menthol and carboxylic acids have no known genotoxicity, it was judged that the JECFA procedure could be applied to these four substances. According to Cramer's classification, these substances were categorized as class I based on their chemical structures. The estimated daily intakes for all four substances were within the range of 1.54-4.71 μg/person/day and 60-1250 μg/person/day, using the methods of Maximized Survey-Derived Intake and Single Portion Exposure Technique, respectively, based on the annual usage data of 2001, 2005, and 2010 in Japan. As the daily intakes of these substances were below the threshold of concern applied to class I substances viz., 1800 μg/person/day, it was concluded that all four substances raise no safety concerns when used for flavoring foods under the currently estimated intake levels., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: We aim to examine changes in usage of nonbiologic, disease-modifying antirheumatic drugs (DMARDs) and evaluate their continuation rates in Japan., Methods: We analyzed DMARD treatment data for 3,734 patients with rheumatoid arthritis (RA) from 1998 to 2009 at Juntendo Hospital in Tokyo, Japan. The DMARD usage rate per month was determined to evaluate RA treatment history in the last decade. We also evaluated continuation rates of nonbiologic DMARDs in single and combination therapies and number of nonbiologic DMARD combination therapies used in each patient., Results: We found that nonbiologic DMARD usage has dramatically changed in the last decade, with the most commonly used DMARD shifting from bucillamine to methotrexate (MTX). MTX showed the highest continuation rate; however, much lower continuation rate was observed when used alone rather than in combination treatments. Further, MTX was also used in the highest number of different combination therapies for a particular patient., Conclusions: These findings indicate that single MTX treatment may be unable to keep patients in clinical remission or lower disease activity compared with several combination therapies. Recent change in permitted maximum dosage of MTX from 8 to 16 mg/week may improve its efficacy and continuation rate in treating Japanese RA patients.

Leukocytapheresis (LCAP) is a safe, unique therapy pertaining to intractable rheumatoid arthritis (RA) even in cases of drug allergy or infectious states. To investigate how to represent LCAP efficacy, we have conducted gene expression analyses from the peripheral blood of RA patients treated with non-woven polyethylene terephthalate filters. Peripheral blood samples were collected immediately before and after treatment from eight RA patients who received LCAP. Among these patients, all of them achieved 20% improvement in the core set of the American College of Rheumatology (ACR20), and thus, they were confirmed as LCAP responders. Gene expression analysis was done with a high-resolution DNA microarray. The results of each of the two groups' gene expression values (immediately before and after LCAP) were calculated using Welch's t-test. Calculations were performed with a statistical software R.basic package: if the P-value was less than 0.05, this was seen as a significant change. In a comparison of 25,370 gene expressions, the number of genes showing a P-value < 0.05 in the upregulating group was 2110, and in the downregulating group it was 1864. The results of pathway analysis using the MetaCore program indicate that gene groups work for cytoskeletal remodeling are upregulated, and genes related to immune responses, such as antigens presenting via major histocompatibility complex class I and II, are downregulated just after LCAP. These findings may relate to LCAP efficacy for RA patients, but this needs further investigation., (© 2012 The Authors. Therapeutic Apheresis and Dialysis © 2012 International Society for Apheresis.)

Objective: Joint swelling, an important factor in the classification criteria and disease activity assessment in rheumatoid arthritis (RA), renders joint palpation a necessary skill for physicians. Ultrasound (US) examination that visualizes soft tissue abnormalities is now used to assess musculoskeletal disease. We assessed the usefulness of US assessments in enhancing physical joint examination skills., Methods: We examined 1944 joints (bilateral shoulder, elbow, wrist, metacarpophalangeal joints 1-5, and knee joints) in 108 patients with RA during April-July 2011. We first physically examined and confirmed joint swelling; subsequently, the same rheumatologist conducted US examinations and multiple assessors graded the joint swelling. When the 2 results differed, we received autofeedback from the US results to improve the physical examination skills., Results: The sensitivities and specificities of physical examination for US-detected swollen joint, the correlation coefficient (CC) of the swollen joint counts, and the concordance rate in each patient for joint swelling sites and power Doppler (PD)-positive sites with the κ coefficients between the physical and US examinations were compared over time. We found that the sensitivity of physical examination increased by 42 percentage points (pp), while the specificity decreased by 18 pp. The average CC in June-July was greater than that in April-May. The percentage of κ coefficients > 0.8 increased from 8.8% to 17% for joint swelling and from 8.3% to 14% for PD-positive sites., Conclusion: Our results suggest that autofeedback from US assessment provides quick improvement in palpation skills for identifying joint swelling in patients with RA.

To retrospectively evaluate the efficacy and safety of combination therapy with tacrolimus (TAC) and other disease-modifying antirheumatic drugs (DMARDs). One hundred fifteen rheumatoid arthritis (RA) patients treated with tacrolimus were enrolled in this retrospective analysis. We collected clinical information, including patient background, treatment efficacy (evaluated using the DAS score), and adverse events observed. Multiple logistic regression analysis was conducted to analyze factors contributing to clinical response and adverse effects. The disease activity score of 28 joints (DAS28) improved significantly at 24 weeks, and continuation rate at 1 year was 57.9%. There was no difference in continuation rate between different DMARD combinations, and not only methotrexate (MTX) but also bucillamine (BUC) and salazosulfapyridine (SSZ) were effective combination partners with TAC. No serious adverse events were observed, and no different inefficacy or safety was observed between non-elderly (<65 years old) and elderly (≥65 years old) RA patients. By conducting multiple logistic regression analysis, combination therapy with MTX and TAC, the number of baseline DMARDs (specifically, ≥3), and old age were identified as risk factors for adverse events. Our findings indicate that TAC is a valuable DMARD for second-line combination therapy in RA.

Objective: To study the significance of anti-Ro/SSA antibodies (anti-Ro) in the clinical response to tumor necrosis factor (TNF) inhibitors in patients with rheumatoid arthritis (RA)., Methods: The clinical responses of a cohort of 190 patients with RA who were treated with infliximab, etanercept, or adalimumab (n = 112, 64, and 14, respectively) as the first biologics were examined using the Disease Activity Score in 28 joints (DAS28) at 24 weeks and the discontinuation rate at 56 weeks. The baseline characteristics of responders and the nonresponders were compared. The clinical response was compared between anti-Ro-negative and -positive patients. The factors associated with the inefficiency of TNF inhibitors were estimated with a multivariable logistic regression analysis., Results: The positive rate of anti-Ro was significantly higher in patients with no European League Against Rheumatism (EULAR) response at 24 weeks (OR 3.64, 95% CI 1.45-9.01, p = 0.003). In anti-Ro-positive patients, a moderate or good EULAR response rate was significantly lower with a sustaining higher median DAS28 (p = 0.006), and this difference was greater among infliximab-treated patients. The discontinuation rate for TNF inhibitors due to inefficacy at 56 weeks was also higher in anti-Ro-positive patients (OR 4.68, 95% CI 1.82-11.99, p = 0.0005), and 75% of these patients received infliximab. The presence of anti-Ro was strongly associated with no EULAR response at 24 weeks and a higher discontinuation rate of TNF inhibitors by 56 weeks (OR 5.22, 95% CI 1.75-15.57, p = 0.003 and OR 10.18, 95% CI 2.18-49.56, p = 0.003)., Conclusion: The presence of anti-Ro might be related to the lesser clinical response to infliximab compared to other TNF inhibitors, suggesting that the presence of anti-Ro should be considered when choosing the appropriate biologics for patients with RA.

We conducted a study to evaluate the effect of various anticoagulant agents on large-volume leukocytapheresis using the new Cellsorba CS-180S Filter filled with a changed solution of sodium pyrosulfite and sodium carbonate. We conducted the study on a total of 12 cases of rheumatoid arthritis. As the anticoagulant agents we used sodium citrate, nafamostat mesilate and low molecular weight heparin. The new Cellsorba CS-180S was safely used with the various blood anticoagulant agents. Also, through adjustment of the sodium citrate percentage to the blood flow volume, it is hypothesized that it is possible to increase the neutrophil removal rate., (© 2011 The Authors. Therapeutic Apheresis and Dialysis © 2011 International Society for Apheresis.)

Background: Elderly patients with rheumatoid arthritis (RA) have more aging-related complications than nonelderly patients with RA., Objectives: The objective of the study was to investigate the treatment status of elderly patients with RA., Methods: Between January and March 2008, 969 patients with RA were enrolled in this observational cross-sectional study. Prescription of disease-modifying antirheumatic drugs (DMARDs) and corticosteroids and laboratory data related to RA, including matrix metalloproteinase 3, rheumatoid factor, and anti-cyclic citrullinated peptide antibody levels, were compared between the elderly and the nonelderly patients., Results: Fewer DMARDs were prescribed to the elderly patients (1.40 [SD, 0.57] vs. 1.51 [SD, 0.61]; P = 0.029). Furthermore, a lower percentage of patients received methotrexate (MTX) (47.2% vs. 56.9%; P = 0.0001), a lower average dosage of MTX was administered (5.46 [SD, 1.66] mg/wk vs. 5.96 [SD, 1.77] mg/wk; P = 0.0001), and fewer biologic DMARDs were used (1.46% vs. 5.59% for infliximab, P = 0.0008; 0.58% vs. 3.19% for etanercept, P = 0.0038) in the elderly group. The laboratory data suggested that the disease status was uncontrolled to a greater extent, and complications were more common in the elderly group., Conclusion: Elderly patients with RA receive less aggressive treatment than nonelderly patients with RA, despite laboratory evidence for poorly controlled disease status among the elderly. The use of a less aggressive regimen could be attributed to the higher prevalence of complications and problems. Therefore, the elderly with RA should be considered a different patient population from the viewpoint of treatment and be administered specialized medical care.

Osteonecrosis (ON) of the femoral head is known to occur commonly in cases with systemic lupus erythematosus (SLE) that received corticosteroid (CS) treatment. However, there have been no detailed reports about the onset of ON in cases with recurrence of SLE. Using MRI, we followed up 17 patients who experienced recurrence of SLE for at least 1 year at our hospital and in whom the CS dose was increased from a maintenance dose to middle to high dose to see if ON would occur. We then compared the group that developed ON and the group that did not with respect to patient characteristics, blood test results, changes in serum lipid levels, and CS dose. ON occurred in five subjects (29.4%), revealing that osteonecrosis occurs not only when CS are first administered but also in cases which the CS dose is increased for recurrence of SLE. Especially, serum cholesterol levels and its rate of increase soared rapidly soon after increasing the CS dose in the ON group as compared with the non-ON group (P < 0.05). This suggests that increased serum lipid levels might be a contributing factor to onset of ON. Moreover, SLE disease activity index 2000 (SLEDAI-2K) scores when the CS dose was increased were significantly (P < 0.05) higher in the ON group, suggesting that SLE disease activity itself is a risk factor for onset of ON.

Phospholipase C-gamma1 (PLC-gamma1) contains two tandem Src homology 2 (SH2) domains. The NH(2)-terminal SH2 domain has been known to mediate the binding of PLC-gamma1 to receptor protein tyrosine kinases, which then activate PLC-gamma1 via phosphorylation at Y783. We now show that the phosphorylated Y783 residue (pY783) associates with the COOH-terminal SH2 domain [SH2(C)] within the same molecule of PLC-gamma1. The specificity of this intramolecular interaction is demonstrated in several ways. The mutation of SH2(C), but not of the NH(2)-terminal SH2 domain, exposes pY783 and makes it available for binding by anti-pY783 antibodies, for intermolecular association with a GST fusion protein containing the tandem SH2 domains of PLC-gamma1 and for dephosphorylation by phosphatases. The intramolecular interaction between pY783 and SH2(C) induces a rearrangement of surface charge such that PLC-gamma1 molecules phosphorylated at Y783 are retained more strongly by heparin resins than are unphosphorylated molecules. Finally, the intramolecular interaction of pY783 with SH2(C) results in activation of phospholipase activity. Our results thus clarify the molecular mechanism of PLC-gamma1 activation, revealing the specific function of pY783 and the distinct roles of the two SH2 domains in this process.

Phospholipase C-gamma2 (PLC-gamma2) plays an important role in B-cell signaling. Phosphorylation of various tyrosine residues of PLC-gamma2 has been implicated in regulation of its lipase activity. With the use of antibodies specific for each of the putative phosphorylation sites, we have now shown that PLC-gamma2 is phosphorylated on Y753, Y759, and Y1217 in response to engagement of the B-cell receptor in Ramos cells, as well as in murine splenic B cells. In cells stimulated maximally via this receptor, the extent of phosphorylation of Y1217 was three times that of Y753 or of Y759. Stimulation of Jurkat T cells or platelets via their immunoreceptors also elicited phosphorylation of Y753 and Y759 but not that of Y1217. A basal level of phosphorylation of Y753 was apparent in unstimulated lymphocytes. The extent of phosphorylation of Y753 and Y759, but not that of Y1217, correlated with the lipase activity of PLC-gamma2. Examination of the effects of various pharmacological inhibitors and of RNA interference in Ramos cells suggested that Btk is largely, but not completely, responsible for phosphorylation of Y753 and Y759, whereas phosphorylation of Y1217 is independent of Btk. Finally, phosphorylation of Y1217 and that of Y753 and Y759 occurred on different PLC-gamma2 molecules.

The discovery of sequence motifs that mediate protein-protein interactions, coupled with the availability of protein amino acid sequence data, allows for the identification of putative protein binding pairs. The present studies were based on our identification of an amino acid sequence in phosphatidylinositol-specific phospholipase C-gamma1 (PLC-gamma1) that fits the consensus sequence for a mitogen-activated protein kinase (MAPK) binding site, termed the D-domain. Extracellular signal-regulated kinase 2 (ERK2), an MAPK, and phospho-ERK2 were bound by an immobilized peptide sequence containing the identified PLC-gamma1 D-domain. Furthermore, a peptide containing the PLC-gamma1 D-domain was able to competitively inhibit the in vitro phosphorylation of recombinant PLC-gamma1 by recombinant phospho-ERK2, whereas a control peptide derived from a distant region of PLC-gamma1 was ineffective. Similarly, the peptide containing the PLC-gamma1 D-domain, but not the control peptide, competitively inhibited the in vitro phosphorylation of Elk-1 and c-Jun catalyzed by recombinant phospho-ERK2 and phospho-c-Jun N-terminal kinase 3 (phospho-JNK3), another type of MAPK, respectively. Incubation of anti-PLC-gamma1 immunocomplexes isolated from rat brain with recombinant phospho-ERK2 opposed the increase in PLC-gamma1-catalyzed hydrolysis of phosphatidylinositol 4,5-P(2) (PtdIns(4,5)P(2)), which was produced by a tyrosine kinase associated with the immunocomplexes, whereas in vitro phosphorylation of recombinant PLC-gamma1 by recombinant phospho-ERK2 did not alter PLC-gamma1-catalyzed PtdIns(4,5)P(2) hydrolysis. These studies have uncovered a previously unidentified mechanism for the integration of PLC-gamma1- and ERK2-dependent signaling.

Phospholipase C-gamma 1 (PLC-gamma 1) is phosphorylated on three tyrosine residues: Tyr-771, Tyr-783, and Tyr-1253. With the use of antibodies specific for each of these phosphorylation sites, we have now determined the kinetics and magnitude of phosphorylation at each site. Phosphorylation of Tyr-783, which is essential for lipase activation, was observed in all stimulated cell types examined. The extent of phosphorylation of Tyr-1253 was approximately 50 to 70% of that of Tyr-783 in cells stimulated with platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), but Tyr-1253 phosphorylation was not detected in B or T cell lines stimulated through B- and T-cell antigen receptors, respectively. Tyr-771 was phosphorylated only at a low level in all cells studied. In cells stimulated with PDGF, phosphorylation and dephosphorylation of Tyr-783 and of Tyr-1253 occurred with similar kinetics; the receptor kinase appeared to phosphorylate both sites, albeit with Tyr-783 favored over Tyr-1253, before the bound PLC-gamma 1 was released, and phosphorylation at the two sites occurred independently. PDGF and EGF induced similar levels of phosphorylation of Tyr-783 and of Tyr-1253 in a cell line that expressed receptors for both growth factors. However, only PDGF, not EGF, elicited substantial PLC activity, suggesting that Tyr-783 phosphorylation was not sufficient for enzyme activation. Finally, concurrent production of phosphatidylinositol 3,4,5-trisphosphate was found to contribute to the activation of phosphorylated PLC-gamma 1.

Glycoproteins GPVI and GPIb-IX-V stimulate robust tyrosine phosphorylation of Syk and PLCg2 (phospholipase Cg2) in washed platelets, but only the former stimulates pronounced activation of phospholipase. Using phospho-specific antibodies, we demonstrate that GPVI, but not GPIb-IX-V, stimulates significant tyrosine phosphorylation of Syk at the autophosphorylation site pY525/526, a marker of Syk activity. In addition, GPVI stimulates tyrosine phosphorylation of PLCg2 at Tyr753 and Tyr759, whereas GPIb-IX-V only induces significant phosphorylation at Tyr753. Both receptors stimulate tyrosine phosphorylation of Btk at the regulatory Tyr223 and Tyr551. Syk and Btk phosphorylate peptides from PLCg2 containing Tyr753 and Tyr759 respectively, suggesting that they may stimulate phosphorylation at these sites in phospholipase. Studies using PLCg2-deficient platelets demonstrated that phospholipase is not required for the activation of integrin aIIbb3 by GPIb-IX-V. Our results demonstrate fundamental differences between GPVI and GPIb-IX-V in the regulation of tyrosine phosphorylation of Syk and PLCg2 consistent with the functional impairment of phospholipase in signalling by GPIb-IX-V.

Species in the genus Echis have been classified mainly based on their morphological appearance and the analytical patterns of their serum. However, re-classification of the genus Echis has recently been suggested by taxonomists, toxicologists, and clinicians, since there have been problems with the current classification, such as the efficacy of antivenoms used for treating bites and the broad geographical distribution of Echis snakes. In this study, we purified five novel disintegrins, the platelet aggregation inhibitors pyramidin A and B from the venom of Echis pyramidum, ocellatin from the venom of Echis ocellatus, and leucogastin A and B from the venom of Echis leucogaster, to compare their sequences and allow us to re-evaluate the classification of various species in the genus Echis. Comparison of the amino acid sequences of five new and four known isolated disintegrins from snake venoms of six Echis species and their distribution strongly support the recent re-classification of the genus Echis.

Upon stimulation of cells with platelet-derived growth factor (PDGF), phospholipase C-gamma1 (PLC-gamma1) binds to the tyrosine-phosphorylated PDGF receptor through one or both of its Src homology 2 (SH2) domains, is phosphorylated by the receptor kinase, and is thereby activated to hydrolyze phosphatidylinositol 4, 5-bisphosphate. Association of PLC-gamma1 with the insoluble subcellular fraction is also enhanced in PDGF-stimulated cells. The individual roles of the two SH2 domains of PLC-gamma1 in mediating the interaction between the enzyme and the PDGF receptor have now been investigated by functionally disabling each domain. A critical Arg residue in each SH2 domain was mutated to Ala. Both wild-type and mutant PLC-gamma1 proteins were transiently expressed in a PLC-gamma1-deficient fibroblast cell line, and these transfected cells were stimulated with PDGF. The mutant protein in which the COOH-terminal SH2 domain was disabled bound to the PDGF receptor. Accordingly, it was phosphorylated by the receptor, catalyzed the production of inositol phosphates, and mobilized intracellular calcium to extents similar to (but slightly less than) those observed with the wild-type enzyme. In contrast, the mutant in which the NH(2)-terminal SH2 domain was impaired did not bind to the PDGF receptor and consequently was neither phosphorylated nor activated. These results suggest that the NH(2)-terminal SH2 domain, but not the COOH-terminal SH2 domain, of PLC-gamma1 is required for PDGF-induced activation of PLC-gamma1. Functional impairment of the SH2 domains did not affect the PDGF-induced redistribution of PLC-gamma1, suggesting that recruitment of PLC-gamma1 to the particulate fraction does not involve the SH2 domains.

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-gamma in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70-130 kDa. They were identified as fragments derived from an unusually large protein (approximately 700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins activated PLC-gamma1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-gamma activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-gamma1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent Km toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2 can act as an additional trigger for PLC-gamma activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation.

Activation of PLC-gamma isozymes in response to various agonists involves tyrosine phosphorylation of the effector enzymes. Recent evidence indicates that PLC-gamma isozymes are additionally activated by phosphatidic acid, phosphatidylinositol 3,4,5-trisphosphate and arachidonic acid in the absence of PLC-gamma tyrosine phosphorylation. These lipid-derived messengers are the immediate products of phospholipase D, phosphatidylinositol 3-kinase, and phospholipase A2, enzymes which are often stimulated along with PLC-gamma in response to an agonist. Furthermore, phosphatidylinositol 4,5-bisphosphate acts as a substrate for both PLC-gamma and phosphatidylinositol 3-kinase and as an activator for phospholipase D and phospholipase A2. These results reveal an elaborate mechanism of cross-talk and mutual regulation between four effector enzymes that participate in receptor signaling by acting on phospholipids.

A Ca(2+)-dependent prothrombin activator, carinactivase-1 (CA-1), was previously found in the venom of Echis carinatus leucogaster. In the present study, the activities of CA-1-like enzymes were screened in the venoms of various Viperidae snakes. The addition of 1 mM Ca2+ ions to the venoms of only Echis snakes in Viperidae produced considerably high prothrombin activator activity, indicating that only the Echis snake venoms contain not only the Ca(2+)-independent prothrombin activator, ecarin, but also Ca(2+)-dependent activator(s). CA-1-like activators and ecarin in the venom of each Echis snake were efficiently separated by Blue Sepharose column chromatography. The venoms of the various Viperidae snakes were also examined for factor X activator activity. The venoms of genera Daboia, Vipera, Cerastes, Echis, Calloselasma and Bothrops contained factor X activator activity in the presence of Ca2+ ions. Cerastes cerastes and Calloselasma rhodostoma venoms also had Ca(2+)-independent factor X activator activity.

In the accompanying paper (Chung, J.-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y. S., Morris, A. J., and Rhee, S. G. (1997) J. Biol. Chem. 272, 15980-15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P2 with low affinity. But the PI(4,5)P2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P2. A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.

A 150-kDa protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) was previously purified from rat brain. The sequences of peptides derived from the purified PLD inhibitor now identify it as synaptojanin, a nerve terminal protein that has been implicated in the endocytosis of fused synaptic vesicles and shown to be a member of the inositol polyphosphate 5-phosphatase family. Further characterization of the enzymatic properties of synaptojanin now shows that it hydrolyzes only the 5-phosphate from inositol 1,4,5-trisphosphate (I(1,4,5)P3) and that it does not catalyze the dephosphorylation of either I(1,3,4)P3 or inositol 1, 4-bisphosphate. However, synaptojanin hydrolyzes both the 4- and 5-phosphates of PI(4,5)P2 and the 4-phosphate of phosphatidylinositol 4-phosphate, converting both compounds to phosphatidylinositol. Magnesium is required for the hydrolysis of I(1,4,5)P3, but not for that of phosphoinositides, by synaptojanin. The inhibition of PLD by synaptojanin is attributable to its ability to hydrolyze PI(4,5)P2. Synaptojanin did not inhibit PLD in the absence of PI(4,5)P2, and the extent of PLD inhibition was related to the extent of PI(4,5)P2 hydrolysis in substrate vesicles. It has been proposed that the biosynthesis of PI(4,5)P2 and the activation of PLD by ADP-ribosylation factor constitute a positive loop to increase rapidly the concentrations of PI(4,5)P2 and phosphatidic acid (PA) during membrane vesiculation. The PA thus produced, probably together with PI(4,5)P2, facilitates vesicle coat assembly. The hydrolysis of PI(4,5)P2, and consequent inhibition of PLD, by synaptojanin might therefore constitute a mechanism to halt the positive loop connecting PI(4,5)P2 and PA during the endocytotic cycle of synaptic vesicles and serve as a signal for uncoating.

We demonstrated recently that coagulation factor IX has a specific binding site(s) for Mg2+ ions, independent of the (Ca2+)-binding sites, and that binding of Mg2+ ions is very important for expression of the functional conformation of this protein. We report here the localization of this Mg2+-specific binding site. We prepared three Gla-containing fragments of bovine factor IX, namely GlaEGF(NC) (residues 1-144+286-296), GlaEGF(N) (1-83) and the Gla domain peptide (1-46). Fragments GlaEGF(NC) and GlaEGF(N) retained the ability to undergo a conformational change upon binding of Mg2+ ions in the presence of excess Ca2+ ions. This change could be detected by a conformation-specific antibody. Furthermore, the Gla domain peptide was capable of binding Mg2+ ions, as determined by the metal ion-induced quenching of the intrinsic fluorescence. It appears that the (Mg2+)-specific binding site of factor IX is located in the N-terminal Gla domain.

We recently showed that not only Ca2+ ions but also Mg2+ ions play a crucial role in stabilizing the native conformation of coagulation factor IX. We here report that Mg2+ ions at physiological concentrations greatly augment the biological activities of factor IX. In clotting assays with dialyzed plasma, addition of Mg2+ ions enhanced the apparent coagulant activity of factor IXa, while that of factor Xa was scarcely affected. Activation of factor X by factor IXa in the presence of factor VIIIa, phospholipids, and Ca2+ ions was accelerated by Mg2+ ions. It appeared that the cation increased the affinity between factor IXa and factor VIIIa, thereby increasing the apparent catalytic efficacy of the enzyme. We also evaluated the effect of Mg2+ ions in the coagulation pathway initiated by tissue factor and found that activation of factor IX by factor VIIa*tissue factor was accelerated by the cation. Consequently, clotting of normal plasma induced by factor VIIa*tissue factor was shortened by the cation, while no such effect was observed in plasma deficient in factor IX or VIII. These results indicate that the previously unrecognized plasma component, Mg2+ ions, plays crucial roles in blood coagulation and, moreover, that contributions of factors IX and VIII in the coagulation cascade have been seriously underestimated in previous investigations.

The venom of the viper Echis carinatus contains a metalloprotease, ecarin, that is a potent prothrombin activator. We here show that the venom is also rich in another prothrombin activator, which does not belong to any known category of prothrombin activators. The novel enzyme, designated carinactivase-1 (CA-1), consists of two subunits held together non-covalently but very tightly. One subunit is a 62-kDa polypeptide that has metalloprotease activity and is homologous to the single-chain enzyme ecarin; the other subunit of 25 kDa consists of two disulfide-linked polypeptides of 17 and 14 kDa, and this subunit resembles the anticoagulant in the habu snake venom, IX/X-bp, that specifically binds the Gla domains of coagulation factors IX and X in a Ca2+-dependent fashion. The activation of prothrombin by CA-1 requires Ca2+ ions at millimolar concentrations and in the absence of Ca2+ ions this enzyme is virtually inactive. By contrast, activation by ecarin is completely independent of Ca2+ ions. CA-1, unlike ecarin, does not activate prothrombin derivatives, in which binding of Ca2+ ions has been perturbed, namely prethrombin-1 and acarboxyprothrombin. Furthermore, the isolated catalytic subunit, although its activity is greatly reduced as compared to that of the holoenzyme, no longer requires Ca2+ ions for the activation of prothrombin. Reconstitution with the non-catalytic 25-kDa subunit restores high level activity and the dependence on Ca2+ ions. Finally, prothrombin activation by CA-1 is inhibited by prothrombin fragment 1, and the isolated non-catalytic subunit is capable of binding fragment 1 in the presence of Ca2+ ions. From these observations, we postulate the following unique mechanism for the activation of prothrombin by CA-1. The enzyme primarily recognizes the Ca2+-bound conformation of the Gla domain in prothrombin via the 25-kDa regulatory subunit, and the subsequent conversion of prothrombin to active thrombin is catalyzed by the 62-kDa catalytic subunit.

The coagulation factor IX/factor X-binding protein (IX/X-bp) from the venom of Trimeresurus flavoviridis is a heterogeneous two-chain protein, and the structure of each chain is similar to that of the carbohydrate-recognition domain of C-type lectins, such as asialoglycoprotein receptors, pancreatic stone protein, and the Fc epsilon receptor for immunoglobulin E. Analysis of the binding properties of IX/X-bp revealed that it binds to the gamma-carboxyglutamic acid (Gla)-containing domains of factors IX and X [Atoda, H. et al. (1994) Eur. J. Biochem. 224, 703-708]. In the present study, we isolated another anticoagulant protein that binds to factor IX but is not to factor X. This protein, designated IX-bp, inhibited factor IXa-induced clotting but not factor Xa-induced clotting, whereas IX/X-bp inhibits both. The concentration of IX-bp for half-maximal binding to solid-phase bovine factor IX was 0.4 nM whereas IX-bp did not bind to factor X even at 40 nM. The binding of IX-bp to solid-phase factor IX was inhibited by the addition of Gla-domain peptide of factor IX, indicating that IX-bp binds to the Gla-domain region of factor IX. IX-bp had two Ca(2+)-binding sites with different affinities for Ca2+ ions. At pH 7.5, the apparent Kd values for these sites were 14 and 130 microM, respectively. IX-bp was a two-chain protein (27.5-kDa band before reduction and 16.8- and 15.7-kDa bands after reduction on SDS-PAGE) and it reacted with immunoglobulin G against IX/X-bp. The complete amino acid sequence of IX-bp was determined. The 16.8-kDa chain (A chain) of IX-bp consisted of 129 residues, of which 19 were different from those in the A chain of IX/X-bp (129 residues). The sequence of the 15.7-kDa chain (B chain) was identical to that of the B chain of IX/X-bp (123 residues). We conclude that IX-bp is a protein that is structurally similar to but functionally different from IX/X-bp. The difference of binding specificity between IX-bp and IX/X-bp presumably arises from the sequence differences in the A chains.

IX/X-bp, an anticoagulant protein isolated from the venom of the habu snake Trimeresurus flavoviridis, has a structure homologous to the carbohydrate-recognition domains of C-type (Ca(2+)-dependent) animal lectins, and it binds to the gamma-carboxyglutamic acid (Gla) domains of coagulation factors IX and X in a Ca(2+)-dependent fashion. In the present study, we elucidated the role of Ca2+ ions in this binding. The binding of 125I-labeled IX/X-bp to both coagulation factors required about 1 mM Ca2+ ions in this at pH 7.5. A decrease in the pH to 6.5 had a striking negative effect on the binding, and the Ca(2+)-requirement curve was shifted rightward. We investigated the binding of Ca2+ ions to IX/X-bp directly by equilibrium dialysis and identified two independent binding sites with different affinities. At pH 7.5, the apparent Kd values for these sites were 25 and 200 microM, respectively. When the pH was decreased to 6.5, the affinity of the high-affinity binding site was reduced only slightly but that of the low-affinity site was reduced considerably. Moreover, it was evident from observations of Ca(2+)-induced changes in the intrinsic fluorescence that IX/X-bp underwent a conformational change upon binding of Ca2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)

The indispensable role of Ca2+ ions in the maintenance of the functional tertiary structures of vitamin K-dependent coagulation factors has been definitively established but the participation of Mg2+ ions, another alkaline-earth metal that is present abundantly in blood plasma, in such a process is not yet understood. We show here that the Ca(2+)-stabilized conformation of coagulation factor IX undergoes a further conformational change upon binding of Mg2+ ions using three independent structural probes. The probes we used were (i) IX/X-bp, a snake venom anticoagulant that recognizes the Gla domains in coagulation factors IX and X, (ii) conformation-specific polyclonal antibodies against bovine factor IX, and (iii) monoclonal antibodies against the Gla domain of human factor IX. The binding of all these probes had an absolute requirement for Ca2+ ions, and Mg2+ ions alone were ineffective. However, when added together with Ca2+ ions, Mg2+ ions at physiological concentrations greatly augmented the binding of these probes to factor IX; the required concentration of Ca2+ ions was much reduced, and the affinity of each probe for factor IX was increased even in the presence of an excess of Ca2+ ions. These results suggest the presence of a Mg(2+)-specific binding site that does not interact with Ca2+ ions in factor IX. Furthermore, Mg2+ ions potentiated the susceptibility of factor IX to activation by factor XIa, concomitant with their effect on the conformation. Similarly, the required Ca2+ concentration was reduced by Mg2+ ions, and the rate of conversion to factor IXa was increased by Mg2+ ions in the presence of an excess of Ca2+ ions. At a saturating concentration of Ca2+ ions (5 mM), addition of 1 mM Mg2+ reduced the apparent Km value for factor IX from 0.31 to 0.18 microM, and in the presence of a physiological concentration of Ca2+ ions (1 mM), the reduction in Km by Mg2+ ions was far more striking (from 0.91 to 0.24 microM). The apparent Vmax values were hardly affected by Mg2+ ions. Our present data reveal a hitherto novel physiological role of the Mg2+ ions in plasma. Not only Ca2+ ions but also Mg2+ ions are important regulators of the stabilization of the native conformation of factor IX as well as of its efficient activation.

We herein report that a novel membrane-associated protease capable of activating prothrombin is present in several mammalian cells. This protease can directly convert prothrombin to active thrombin and induces blood clotting both in vivo and in vitro but is apparently different from coagulation factor Xa, which has been thought to be the only physiological activator of prothrombin. This protease activity was initially found and was very high in 8C feline kidney fibroblast cells, and we characterized its enzymological features using this cell line. Activity was detected in neither the cytosolic fraction nor the culture medium but found in the membranes and identified on the surface of intact cells. The activation of prothrombin required Ca2+ ions, and the apparent Km value for prothrombin was 0.2 microM. The activity was irreversibly inhibited by exposure to EDTA, but various inhibitors for serine proteases including antithrombin III were without effect. Based on these results, we propose that this novel enzyme, membrane-associated prothrombin activator, catalyzes an alternative pathway for generation of thrombin, which is independent of the blood coagulation cascade, and that the thrombin generated is involved in certain pathological states and/or in activation of cells that are spatially separated from the bloodstream.

We previously isolated a unique anticoagulant protein named IX/X-bp (factor IX/factor X-binding protein) from the venom of the habu snake Trimeresurus flavoviridis. We recently determined its primary structure and found that this protein had a structure homologous to the carbohydrate-recognition domains of C-type lectins. Most interestingly, a high homology was found between this protein and botrocetin, an inducer of platelet agglutination found in the venom of the jararaca snake Bothrops jararaca. To examine the possible identity of these proteins, we searched for IX/X-bp-like protein(s) in the venom of B. jararaca. When the venom was subjected to DEAE anion-exchange chromatography, such an activity was eluted separately from that of botrocetin. This activity was purified to homogeneity and designated jararaca IX/X-bp. Jararaca IX/X-bp was a disulfide-linked heterodimer consisting of 16- and 15-kDa subunits, being structurally similar to botrocetin. The respective NH2-terminal amino acid sequences were also very similar. Jararaca IX/X-bp had no botrocetin-like activity. However, this protein did have an activity to bind to factors IX and X and protein S in a Ca(2+)-dependent fashion, that resulted in interference with coagulation, while botrocetin did not. The binding to coagulation factors appeared not to be mediated by the lectin-like activity of jararaca IX/X-bp, because a derivative of factor X free of carbohydrates retained the ability to bind. It is concluded, therefore, that the two proteins isolated from the same venom have different biological activities despite the high degree of structural similarity between them.

In a previous paper we demonstrated using immunochemical techniques that propolypeptide of von Willebrand factor was present on the surface of resting platelets. In the present paper we show that polyclonal antibodies against propolypeptide of von Willebrand factor induce activation of phospholipase(s) in platelets and lead to platelet aggregation. The antibody-stimulation of platelets induced the synthesis of thromboxane A2 (TXA2). Furthermore, the aggregation was inhibited by aspirin and an antagonist of TXA2. Aspirin inhibited not only the aggregation but also the activation of arachidonic acid liberation from phospholipids, but the effect of aspirin on arachidonic acid liberation was cancelled by the combined effect of the antibodies and a TXA2 mimetic agonist, which itself did not activate arachidonic acid liberation. The antibody-induced activation of arachidonic acid liberation and the aggregation were blocked by cytochalasin B. All these results obtained with antibodies were quite similar to the results obtained with collagen.

When platelets are activated by the recognition of exposed collagen fibers, they start synthesizing two major arachidonic acid metabolites, i.e. thromboxane A2 and 12S-hydroxyeicosatetraenoic acid (12-HETE) via cyclooxygenase and 12-lipoxygenase pathways, respectively. Although the physiological role of the former is well established, that of the latter has not been fully elucidated. Recently, we have revealed that 12-HETE interferes with collagen-induced platelet aggregation [Sekiya, F. et al. (1990) Biochim. Biophys. Acta 1044, 165-168]. In the present paper, we show that this substance enhances thrombin-induced aggregation of bovine platelets, in sharp contrast with the case of collagen. Additionally, 12-HETE is able to prevent the prostaglandin E1-induced elevation of platelet cAMP level and counteracts its inhibitory effect on platelet aggregations. With these observations, we propose a novel self-regulatory mechanism of platelets where 12-HETE plays a key role; it switches sensitivity of platelets from the primary agonist (collagen) to the secondary one (thrombin), and cancels the inhibitory effect of cAMP elevators.

Recently we have found that propolypeptide of von Willebrand factor (pp-vWF) obtained from platelets binds to type I collagen. It is known that pp-vWF is present in platelet alpha-granules and is secreted upon activation. In this paper, we demonstrate the two following evidences to show that it is also present on the surface of resting platelets. [1] The antibody against pp-vWF bound to the surface of platelets. [2] The antibody induced aggregation of platelets. The binding of the antibody and the antibody-induced aggregation of platelets were inhibited in a dose-dependent manner by Fab fragment of the antibody. Platelets from von Willebrand disease patients bound less of the antibody and responded weakly to the antibody.

We reported previously that bovine propolypeptide of von Willebrand factor (pp-vWF) binds to type I collagen. To determine the collagen-binding sites of pp-vWF we generated monoclonal antibodies (mAbs) against bovine pp-vWF. One mAb, designated TC8, very strongly inhibited the binding of pp-vWF to type I collagen; three other mAbs, designated TC2, TC6 and TC7, exhibited moderate inhibition. Competition between the mAbs for binding to intact pp-vWF revealed that the epitope for TC8 was structurally independent of that for TC6 and TC7. To determine directly the location of the epitope for each mAb on the bovine pp-vWF molecule, we tested the reactivity of mAbs by immunoblotting toward peptide fragments obtained by digestion with lysylendopeptidase. TC2 and TC8 recognized a fragment of mass 21 kDa, while TC6 and TC7 recognized a distinct fragment of 18 kDa. These two fragments were purified to homogeneity and their N-terminal amino acid sequences were determined. Comparing these sequences with the sequence of human pp-vWF, the locations of these fragments in the primary structure were estimated to be Phe570--Lys682 for the 21-kDa fragment and Glu281--Lys375 for the 18-kDa fragment. These data suggest that pp-vWF contains at least two collagen-binding sites which lie within or close to the regions between Phe570--Lys682 and Glu281--Lys375.

Two reduced/alkylated fragments of bovine propolypeptide of von Willebrand factor (pp-vWF) that inhibit pp-vWF binding to collagen were isolated. One is a tryptic fragment of molecular mass of about 30 kDa and inhibits the binding at a molar concentration about 20 times higher than the intact pp-vWF. Amino acid sequence of this fragment was determined almost completely, and it was revealed that this fragment corresponded to the carboxyl-terminal region of pp-vWF molecule beginning with Phe557. The other active fragment was obtained by lysyl endopeptidase digestion. This migrated as a 21.5/21-kDa doublet in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but deglycosylation of this doublet resulted in production of single species of 19 kDa. The difference between the doublet constituents, therefore, was of carbohydrate composition. The extent of inhibition of collagen-binding by this 21.5/21-kDa fragment was comparable to that of the 30-kDa fragment, and furthermore, location of this fragment in the molecule was established to be between Phe570 and Lys682. These were the only fragments among those obtained by proteolytic digestions that had significant competitive effect on the binding of intact pp-vWF to collagen. These results strongly suggest that at least one collagen-binding site should be present in the carboxyl-terminal region of bovine pp-vWF extending from residue 570 to 682.

The Amadori rearrangement compound, the product in the early step of the Maillard reaction of proteins with glucose, is known to be degraded into 3-deoxyglucosone (3DG), a 2-oxoaldehyde. In order to elucidate the metabolic pathway of 3DG, [14C]3DG was synthesized from [14C]-glucose and administered to rats orally and intravenously. 2 h after oral administration of [14C]3DG, the percentages of radioactivity (RaI%) in stomach, small intestine and urine were 3.9, 60 and 6.4%, respectively, while RaI% in liver, kidney, spleen, blood and CO2 were less than 0.5%. The absorption rate of 3DG was obviously lower in comparison with that of glucose. 3 h after intravenous administration of [14C]3DG, the RaI% in urine was 72% and those in liver, kidney, spleen, blood and CO2 were less than 1%. It therefore appeared that the absorbed 3DG was not biologically utilized by the rats, but was rapidly excreted in the urine. Some metabolites of [14C]3DG were detected in urine by TLC-autoradiography. The main metabolite was purified and identified as 3-deoxyfructose by FD-MS and 13C-NMR spectroscopy, indicating that the aldehyde group of 3DG was reduced to an alcohol.

In cooperative aggregation of bovine platelets induced by simultaneous addition of PAF and collagen at subthreshold concentrations the following observations were made. (i) Formation of phosphatidic acid and arachidonic acid metabolites, which characterize PAF and collagen alone-induced aggregation, respectively, was observed very obviously. (ii) A thromboxane antagonist did not completely block the cooperative aggregation. The extent of residual aggregation activity was dependent on concentration of collagen used in the simultaneous administration with PAF. These results suggest that both signal transduction pathways activated by PAF and collagen alone at high concentrations are attained by simultaneous addition of both agonists at subthreshold concentrations through unknown mechanisms.

We have proposed a mechanism that platelet aggregation is regulated by its 12-lipoxygenase product, 12S-hydroxyeicosatetraenoic acid (12-HETE) (Sekiya, F., Takagi, J. and Saito, Y. (1989) Thrombos. Res. 56, 407-415). Inhibition of endogenous 12-HETE production by 15-HETE, a specific inhibitor of 12-lipoxygenase, accelerated aggregation of bovine platelets in response to collagen and arachidonic acid liberation from phospholipids was enhanced. Exogenously added 12-HETE suppressed collagen-induced liberation of arachidonic acid and the aggregation was also inhibited. On the other hand, 12-HETE did not interfere with thromboxane synthesis from free arachidonic acid in a cell-free system. These observations suggest that 12-HETE exerts a negative feedback to prevent excess aggregation through interference with arachidonic acid liberation from membrane phospholipids.

Distribution of B subunit of coagulation factor XIII(FXIII B) in bovine platelets was determined. Intracellular location of FXIII B in platelets was confirmed by fluorescent antibody technique. Positive staining of FXIII B was observed with Triton-permeabilized platelets but not with non-permeabilized platelets. Immunoblots of plasma and platelet lysate revealed that the antibody was specific for FXIII B and this antigen existed in the supernatant of sonication-solubilized platelets. The amount of FXIII B in platelets was determined to be 17.3 +/- 6.7 ng/10(8) platelets by radioimmunoassay. This is the first visual and quantitative demonstration of intracellular FXIII B.

Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose. Among these proteins, a protein of about 100,000 daltons was purified to homogeneity by two additional affinity chromatographies, an organomercurial-agarose and a lentil lectin-agarose. This protein consisted of a single polypeptide chain which contains carbohydrate moiety and many intrapolypeptide disulfide bridges. In addition to platelets, this protein was present in plasma and cultured endothelial cells but not in red blood cells, leukocytes, and smooth muscle cells. Furthermore, it was released from platelets upon stimulation by various agonists. The purified 100-kDa protein was labeled with 125I to quantitate its binding to fibrillar type I collagen. The protein specifically bound to fibrillar collagen with the apparent dissociation constant of 5.6 x 10(-8) M for the high affinity site and 5.5 x 10(-7) M for the low affinity site. Analyses of amino acid sequences of both intact and tryptic fragments of this protein revealed that it had strong homology to the propolypeptide of human von Willebrand factor, which is also known as von Willebrand antigen II. Various properties of this protein listed above also strongly suggest that it was indeed the propolypeptide of bovine von Willebrand factor.