Tuberculosis and Nontuberculous Mycobacterial Infections in Patients with Spondyloarthritis: A Population-Based Study.

Background and Objectives: Tuberculosis is caused by Mycobacterium tuberculosis (MTB), while nontuberculous mycobacteria (NTM) encompass a group of mycobacterial species that are distinct from the MTB complex and leprae. Spondyloarthritis (SpA) is a group of chronic inflammatory diseases with shared clinical characteristics and is treated with biological agents; however, their use may elevate the risk of MTB and NTM infections. This study aimed to compare the incidence and risk of MTB and NTM infections in patients with SpA, including ankylosing spondylitis (AS) and psoriatic arthritis (PsA), using a population-based approach. Materials and Methods: This study included 2333 patients with SpA and 9332 age- and sex-matched controls from the Korea National Health Insurance Service-National Sample Cohort database from 2002 to 2019. The patients were identified using the International Classification of Diseases-10 codes for AS, PsA, MTB, and NTM. Results: The results showed that a negligible percentage of patients with SpA developed NTM (0.002%) and MTB (0.016%), with no significant difference in the incidence rate ratio (IRR) compared to controls. Among patients with SpA treated with biologics, the IRRs for NTM and MTB were 5.66 and 3.069, respectively; however, these were not statistically significant. No cases of NTM or MTB infection were reported in female patients with SpA treated with biologics. In both the SpA patient group and the control group, the incidence of MTB was higher in individuals over 60 years old compared to those under 60 years old. Cox proportional hazard analysis revealed a significant adjusted hazard ratio of 1.479 for MTB in patients with SpA after adjusting for age, sex, smoking history, insurance level, and comorbidities. However, this significance was not maintained when biological therapy was further adjusted. Conclusions: Our study indicated that the risks of NTM and MTB infection are not elevated in patients with SpA. Although biological use may potentially increase the risk of MTB infection, it does not lead to a significant increase in incidence rates. Proactive screening for latent tuberculosis and adequate prophylaxis using biologics can effectively manage the risk of NTM and MTB infections.

Target and non-target analytical method for potential hazardous substances in livestock and pet hair using liquid- and gas chromatography quadrupole time-of-flight mass spectrometry.

Extraction using acetonitrile and water and quadrupole time-of-flight mass spectrometry (LC and GC-QTOF/MS) techniques were used to screen for potential hazardous substances in livestock and pet hair. In addition, LC-MS/MS and GC-MS/MS techniques were used for verification of the analytical method and quantitative analysis of pesticides, veterinary drugs, mycotoxins and antioxidants in hair. Optimized sample preparation involves extracting 0.05 g of sample with 0.6 mL of ACN and 0.4 mL of distilled water. In addition, the two layers were separated by adding 0.1 g of NaCl. Then, both the ACN and water layers were analyzed by LC-TOF/MS, and the ACN layer was analyzed by GC-TOF/MS. Most of the matrix effects of livestock and pet hair were less than 50%, but some matrices and components showed high results, so matrix matching correction was applied for more precise quantification. Method validation was performed for 394 constituents (293 pesticides, 93 veterinary drugs, 6 mycotoxins and 2 preservatives) in dog, cat, cow and pig hair and chicken and duck feathers. All components showed good linearity (r2 ≥0.98) in the developed assay. The quantification limit of all compounds was set at 0.02 mg/kg, which is the lowest level that satisfies the recovery rate standard. The recovery experiment was repeated 8 times at 3 concentrations. Most of the components were extracted with the ACN layer, and the recovery rate was 63.35-119.98%. In order to confirm the efficiency of extracting harmful substances from actual samples, 30 hairs of livestock and pets were screened.

Development of a Quantitative Method for Detection of Multiclass Veterinary Drugs in Feed Using Modified QuPPe Extraction and LC-MS/MS.

A method was developed for the rapid and quantitative analysis of 30 veterinary drugs belonging to 17 classes (amphenicols (1), anthelmintics (1), cephalosporins (4), coccidiostats (1), lincosamides (1), macrolide (1), nitroimidazole (1), penicillins (3), phenylhydrazines (1), polypeptides (1), pyrethrins (1), quinolones (5), sulfonamides (3), tetracycline (3), neuroleptic agents (1), triazene trypanocidal agents (1), other. (1)) in feeds. The proposed method with a modified Quick Polar Pesticides (QuPPe) sample preparation was validated for the determination of 30 veterinary drugs in feed samples by liquid chromatography triple-quadrupole mass spectrometry (LC-MS/MS). The sample was extracted with methanol containing 1% acetic acid and purified by dispersive solid-phase extraction (d-SPE) with C18. Good linearity (r2 ≥ 0.98) was observed, and the LOQ values ranged from 10 to 200 µg/kg. Average recoveries ranged from 70.8 to 118.4%, and the relative standard deviation was ≤ 18.7%. This validated method was used in the determination of 30 veterinary drugs in 142 feed samples obtained from South Korea. The results show that lincomycin was present in only one of the tested feed samples, although it was detected at a value lower than the LOQ. In conclusion, this multi-residue method can be used for screening through the detection and quantitation of residual multiclass veterinary drugs in feed samples.

Multi-residue analytical method for detecting pesticides, veterinary drugs, and mycotoxins in feed using liquid- and gas chromatography coupled with mass spectrometry.

Modified QuEChERS and triple quadrupole mass spectrometry (LC and GC-MS/MS) technology were used to sequentially analyze pesticides, veterinary drugs, and mycotoxins in feed. In order to analyze the harmful substances that may remain or occur in the feed, we performed optimization experiments for sample preparation and LC-MS/MS and GC-MS/MS conditions. Optimized sample preparation involves extracting 5 g of sample with 15 mL of 0.25 M EDTA and 10 mL of acetonitrile. And some extracts were diluted 10-fold with 100 mM ammonium formate aqueous solution and analyzed by LC-MS/MS, and some extracts were purified through 25 mg PSA and analyzed by GC-MS/MS by adding an analyte protectant. We confirmed the matrix effect of feed ingredients and compound feeds, and added a dilution process after extraction to increase on-site efficiency. Matrix-matched calibration was applied for quantification. Method validation was performed for 197 pesticides, 56 components for veterinary drugs, and 5 components for toxins. All the components showed good linearity (r2 ≥ 0.98) in the developed analytical method. For most compounds, the limit of quantitation was 0.05 mg/kg. The recovery rate experiment was repeated three times at three concentrations including LOQ in feed ingredient, compound feed for livestock, and compound feed for pets. The recovery rate was 70.09-119.76% and relative standard deviations were ≤ 18.91%. And the accuracy and precision were further verified through cross-validation between laboratories. The developed analytical method was used to monitor 414 domestically distributed and imported feeds.

Effect of the similarity of gut microbiota composition between donor and recipient on graft function after living donor kidney transplantation.

Graft outcomes of unrelated donor kidney transplant are comparable with those of related donor kidney transplant despite their genetic distance. This study aimed to identify whether the similarity of donor-recipient gut microbiota composition affects early transplant outcomes. Stool samples from 67 pairs of kidney transplant recipients and donors were collected. Gut microbiota differences between donors and recipients were determined using weighted UniFrac distance. Among the donor-recipient pairs, 30 (44.8%) pairs were related, while 37 (55.2%) were unrelated. The unrelated pairs, especially spousal pairs, had similar microbial composition, and they more frequently shared their meals than related pairs did. The weighted UniFrac distance showed an inverse correlation with the 6-month allograft function (p = 0.034); the correlation was significant in the unrelated pairs (p = 0.003). In the unrelated pairs, the microbial distance showed an excellent accuracy in predicting the estimated glomerular filtration rate of < 60 mL/min/1.73 m2 at 6-months post-transplantation and was better than human leukocyte antigen incompatibility and rejection. The incidence of infection within 6 months post-transplantation increased in the recipients having dissimilar microbiota with donors compared to the other recipients. Thus, pre-transplantation microbial similarity in unrelated donors and recipients may be associated with 6-month allograft function.

Regulation of Anti-Oxidative, Anti-Inflammatory, and Anti-Apoptotic Activity of Advanced Cooling Composition (ACC) in UVB-Irradiated Human HaCaT Keratinocytes.

We recently demonstrated that advanced cooling composition (ACC) has effective ingredients that exhibit anti-inflammatory effects in RAW 264.7 cells stimulated with lipopolysaccharide (LPS) and exhibit strong antimicrobial effects on Pseudomonas aeruginosa, Staphylococcus aureus, MRSA (methicillin-resistant Staphylococcus aureus), Candida albicans, and Streptococcus mutans. To further investigate whether ACC has beneficial effects in ultraviolet B (UVB)-irradiated human keratinocytes (HaCaT cells), HaCaT cells were pretreated with ACC prior to UVB irradiation. Our data showed that ACC, which is effective at 100 µg/mL, is nontoxic and has an antioxidative effect against UVB-induced intracellular reactive oxygen species (ROS) in HaCaT cells. In addition, ACC exerts cytoprotective effects against UVB-induced cytotoxicity in HaCaT cells by inhibiting abnormal inflammation and apoptosis through the regulation of mitogen-activated protein kinase (MAPK) signals, such as jun-amino-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). Therefore, these results indicate that ACC is a potentially beneficial raw material that possesses antioxidative, anti-inflammatory, and antiapoptotic effects against UVB-induced keratinocytes and may have applications in skin health.

Healthcare resource use and costs of diabetic macular oedema for patients with antivascular endothelial growth factor versus a dexamethasone intravitreal implant in Korea: a population-based study.

OBJECTIVES: To estimate the costs and healthcare resources of patients with diabetic macular oedema (DME) who received intravitreal antivascular endothelial growth factor (anti-VEGF) agents or a dexamethasone intravitreal implant (DEX-implant) in Korea. DESIGN: Retrospective cohort study. SETTING: The Korean National Health Insurance claim data from 1 January 2015 to 30 June 2017 were retrieved from the Health Insurance Review and Assessment Service. PARTICIPANTS: Adult patients with DME who were diagnosed with diabetic retinopathy or DME and received ranibizumab, aflibercept or a DEX-implant in conjunction with intravitreal injection were included. Patients whose primary diagnoses were age-related macular degeneration or retinal vein occlusion were excluded. MAIN OUTCOME MEASURES: Healthcare resource utilisation and costs related to DME in the 12-month postindex period. RESULTS: During the study period, 182 patients and 414 patients were identified in the anti-VEGF and DEX-implant groups, respectively, and there was no significant difference in the demographic characteristics between the two groups. The outpatient eye care-related medical costs were US$3002.33 for the anti-VEGF group vs US$2250.35 for the DEX-implant group (p<0.0001). After adjusting the relevant covariates based on the generalised linear model, the estimated outpatient eye care-related medical costs were 33% higher in the anti-VEGF group than in the DEX-implant group (p<0.0001, 95% CI 22% to 45%). The utilisation pattern of the two groups showed no significant difference except for the number of intravitreal injections, which was higher in the anti-VEGF group (2.69±2.29) than in the DEX-implant group (2.09±1.37, p<0.001). CONCLUSION: The average annual eye-related medical cost of the DEX-implant group was significantly lower than that of the anti-VEGF group during the study period, which was mainly due to decreased utilisation of eye care-related injections. Further long-term studies are needed.

Glomerular crescents are associated with worse graft outcome in allograft IgA nephropathy.

The prognosis of patients with allograft IgA nephropathy (IgAN) requires further investigation. We performed a bicenter retrospective cohort study on kidney transplant recipients diagnosed with IgAN in allograft biopsy. Recipients without allograft IgAN but with known IgAN before transplantation were included as the control group. We investigated the associations between clinicopathological characteristics, including allograft crescents, and the risk of death-censored graft failure. In total, 1256 IgAN patients in both pre- and posttransplant stages were included. Among them, 559 were diagnosed with allograft IgAN, which was a time-dependent risk factor for worse prognosis (adjusted hazard ratio = 5.009 [3.610-6.951]; P < .001) during a median of 8.1 years of follow-up. Of the patients with allograft IgAN, 88 (15.9%) had glomerular crescents, including 40 patients (7.2%) with >10% crescent formation in the total biopsied glomeruli. The presence of glomerular crescents in IgAN was associated with a worse graft prognosis, and the association was still valid with the C scores of the current Oxford classification. In conclusion, allograft IgAN is a time-dependent event and is associated with worse graft outcomes. The pathological characteristics of allograft, particularly the degree of glomerular crescent formation, may represent important risk factors for a poor prognosis.

Simple Postoperative AKI Risk (SPARK) Classification before Noncardiac Surgery: A Prediction Index Development Study with External Validation.

BACKGROUND: Researchers have suggested models to predict the risk of postoperative AKI (PO-AKI), but an externally validated risk index that can be practically implemented before patients undergo noncardiac surgery is needed. METHODS: We performed a retrospective observational study of patients without preexisting renal failure who underwent a noncardiac operation (≥1 hour) at two tertiary hospitals in Korea. We fitted a proportional odds model for an ordinal outcome consisting of three categories: critical AKI (defined as Kidney Disease Improving Global Outcomes AKI stage ≥2, post-AKI death, or dialysis within 90 days after surgery), low-stage AKI (defined as PO-AKI events not fulfilling the definition of critical AKI), and no PO-AKI. RESULTS: The study included 51,041 patients in a discovery cohort and 39,764 patients in a validation cohort. The Simple Postoperative AKI Risk (SPARK) index included a summation of the integer scores of the following variables: age, sex, expected surgery duration, emergency operation, diabetes mellitus, use of renin-angiotensin-aldosterone inhibitors, baseline eGFR, dipstick albuminuria hypoalbuminemia, anemia, and hyponatremia. The model calibration plot showed tolerable distribution of observed and predicted probabilities in both cohorts. The discrimination power of the SPARK index was acceptable in both the discovery (c-statistic 0.80) and validation (c-statistic 0.72) cohorts. When four SPARK classes were defined on the basis of the sum of the risk scores, the SPARK index and classes fairly reflected the risks of PO-AKI and critical AKI. CONCLUSIONS: Clinicians may consider implementing the SPARK index and classifications to stratify patients' PO-AKI risks before performing noncardiac surgery.

Interstitial Nephritis Caused by Anorexia Nervosa in Young Male; A Case Report and Literature Review.

Severe eating disorders characterized by repetitive episodes of purging and vomiting can occasionally trigger acute kidney injury. However, interstitial nephritis induced by episodes of repeated vomiting has rarely been reported, and the pathophysiology of this entity remains unknown. A 26-year-old man was admitted to our hospital because of known hypokalemia. His serum electrolyte profile showed: sodium 133 mEq/L, potassium 2.6 mEq/L, chloride 72 mEq/L, total carbon dioxide 50 mEq/L, blood urea nitrogen/creatinine ratio (BUN/Cr) 21.9/1.98 mg/dL, and magnesium 2.0 mg/dL. Arterial blood gas analysis showed: pH 7.557, partial pressure of carbon dioxide 65.8 mmHg, and bicarbonate 58.5 mEq/L. His urinary potassium concentration was 73.2 mEq/L, and Cr was 111 mg/dL. Renal biopsy revealed acute tubular necrosis and tubulointerstitial nephritis with a few shrunken glomeruli. Repeated psychogenic vomiting may precipitate acute kidney injury and interstitial nephritis secondary to volume depletion and hypokalemia. Serum electrolyte levels and renal function should be carefully monitored in patients diagnosed with eating disorders to prevent tubular ischemia and interstitial nephritis.

Prevalence and evaluation strategies for viral contamination in food products: Risk to human health-a review.

Nowadays, viruses of foodborne origin such as norovirus and hepatitis A are considered major causes of foodborne gastrointestinal illness with widespread distribution worldwide. A number of foodborne outbreaks associated with food products of animal and non-animal origins, which often involve multiple cases of variety of food streams, have been reported. Although several viruses, including rotavirus, adenovirus, astrovirus, parvovirus, and other enteroviruses, significantly contribute to incidence of gastrointestinal diseases, systematic information on the role of food in transmitting such viruses is limited. Most of the outbreak cases caused by infected food handlers were the source of 53% of total outbreaks. Therefore, prevention and hygiene measures to reduce the frequency of foodborne virus outbreaks should focus on food workers and production site of food products. Pivotal strategies, such as proper investigation, surveillance, and reports on foodborne viral illnesses, are needed in order to develop more accurate measures to detect the presence and pathogenesis of viral infection with detailed descriptions. Moreover, molecular epidemiology and surveillance of food samples may help analysis of public health hazards associated with exposure to foodborne viruses. In this present review, we discuss different aspects of foodborne viral contamination and its impact on human health. This review also aims to improve understanding of foodborne viral infections as major causes of human illness as well as provide descriptions of their control and prevention strategies and rapid detection by advanced molecular techniques. Further, a brief description of methods available for the detection of viruses in food and related matrices is provided.

Effects of renal impairment on the pharmacokinetics and pharmacodynamics of a novel dipeptidyl peptidase-4 inhibitor, evogliptin (DA-1229).

Evogliptin is a novel potent and selective dipeptidyl peptidase-4 (DPP-4) inhibitor. The aim of the present study was to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of evogliptin in participants with renal impairment (RI). An open-label, parallel-group clinical study was conducted in participants with mild, moderate and severe RI and in matched participants with normal renal function (NRF). A single oral 5-mg dose of evogliptin was administered and serial blood and urine samples were obtained to assess the PK and PD characteristics of evogliptin. Baseline urine samples were collected to evaluate endogenous CYP3A metabolic markers. The plasma exposure to evogliptin and degree of DPP-4 activity inhibition increased with decreasing renal function. The mean areas under the concentration-time curves from 0 to 120 hours were increased 1.2-, 1.8- and 1.98-fold in the mild, moderate and severe RI groups, respectively, compared with the NRF group. The levels of CYP3A metabolic markers were lower in the RI group than in the NRF group. The increase in the plasma concentration of evogliptin is unlikely to result in changes in its efficacy or safety, considering the results of previous clinical studies.

Vasodilator-stimulated phosphoprotein-phosphorylation by ginsenoside Ro inhibits fibrinogen binding to αIIb/ß3 in thrombin-induced human platelets.

BACKGROUND: Glycoprotein IIb/IIIa (αIIb/ß3) is involved in platelet adhesion, and triggers a series of intracellular signaling cascades, leading to platelet shape change, granule secretion, and clot retraction. In this study, we evaluated the effect of ginsenoside Ro (G-Ro) on the binding of fibrinogen to αIIb/ß3. METHODS: We investigated the effect of G-Ro on regulation of signaling molecules affecting the binding of fibrinogen to αIIb/ß3, and its final reaction, clot retraction. RESULTS: We found that G-Ro dose-dependently inhibited thrombin-induced platelet aggregation and attenuated the binding of fibrinogen to αIIb/ß3 by phosphorylating cyclic adenosine monophosphate (cAMP)-dependently vasodilator-stimulated phosphoprotein (VASP; Ser157). In addition, G-Ro strongly abrogated the clot retraction reflecting the intensification of thrombus. CONCLUSION: We demonstrate that G-Ro is a beneficial novel compound inhibiting αIIb/ß3-mediated fibrinogen binding, and may prevent platelet aggregation-mediated thrombotic disease.

Total saponin from Korean Red Ginseng inhibits binding of adhesive proteins to glycoprotein IIb/IIIa via phosphorylation of VASP (Ser(157)) and dephosphorylation of PI3K and Akt.

BACKGROUND: Binding of adhesive proteins (i.e., fibrinogen, fibronectin, vitronectin) to platelet integrin glycoprotein IIb/IIIa (αIIb/ß3) by various agonists (thrombin, collagen, adenosine diphosphate) involve in strength of thrombus. This study was carried out to evaluate the antiplatelet effect of total saponin from Korean Red Ginseng (KRG-TS) by investigating whether KRG-TS inhibits thrombin-induced binding of fibrinogen and fibronectin to αIIb/ß3. METHODS: We investigated the effect of KRG-TS on phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and dephosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, affecting binding of fibrinogen and fibronectin to αIIb/ß3, and clot retraction. RESULTS: KRG-TS had an antiplatelet effect by inhibiting the binding of fibrinogen and fibronectin to αIIb/ß3 via phosphorylation of VASP (Ser(157)), and dephosphorylation of PI3K and Akt on thrombin-induced platelet aggregation. Moreover, A-kinase inhibitor Rp-8-Br-cyclic adenosine monophosphates (cAMPs) reduced KRG-TS-increased VASP (Ser(157)) phosphorylation, and increased KRG-TS-inhibited fibrinogen-, and fibronectin-binding to αIIb/ß3. These findings indicate that KRG-TS interferes with the binding of fibrinogen and fibronectin to αIIb/ß3 via cAMP-dependent phosphorylation of VASP (Ser(157)). In addition, KRG-TS decreased the rate of clot retraction, reflecting inhibition of αIIb/ß3 activation. In this study, we clarified ginsenoside Ro (G-Ro) in KRG-TS inhibited thrombin-induced platelet aggregation via both inhibition of [Ca(2+)]i mobilization and increase of cAMP production. CONCLUSION: These results strongly indicate that KRG-TS is a beneficial herbal substance inhibiting fibrinogen-, and fibronectin-binding to αIIb/ß3, and clot retraction, and may prevent platelet αIIb/ß3-mediated thrombotic disease. In addition, we demonstrate that G-Ro is a novel compound with antiplatelet characteristics of KRG-TS.

Onion (Allium cepa L.) peel extract has anti-platelet effects in rat platelets.

The effects of onion peel extract (OPE) in collagen (5 µg/mL)-stimulated washed rat platelet aggregation were investigated. OPE inhibited platelet aggregation via inhibition of aggregation-inducing molecules, intracellular Ca(2+) and thromboxane A2 (TXA2) by blocking cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS) activities in a dose-dependent manner. In addition, OPE elevated the formation of cyclic adenosine monophosphate (cAMP), aggregation-inhibiting molecule, but not cyclic guanosine monophosphate (cGMP). High performance liquid chromatography (HPLC) analysis of OPE revealed that OPE contains quercetin, one of the major flavonoids, which has anti-platelet effect. In conclusion, we suggest that OPE is an effective inhibitor of collagen-stimulated platelet aggregation in vitro. Therefore, it can be a promising and safe strategy for anti-cardiovascular diseases.

Inhibitory effects of total saponin from Korean Red Ginseng on [Ca(2+)]i mobilization through phosphorylation of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and inositol 1,4,5-trisphosphate receptor type I in human platelets.

BACKGROUND: Intracellular Ca(2+)([Ca(2+)]i) is a platelet aggregation-inducing molecule. Therefore, understanding the inhibitory mechanism of [Ca(2+)]i mobilization is very important to evaluate the antiplatelet effect of a substance. This study was carried out to understand the Ca(2+)-antagonistic effect of total saponin from Korean Red Ginseng (KRG-TS). METHODS: We investigated the Ca(2+)-antagonistic effect of KRG-TS on cyclic nucleotides-associated phosphorylation of inositol 1,4,5-trisphosphate receptor type I (IP3RI) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation. RESULTS: The inhibition of [Ca(2+)]i mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-Br-cAMPS), which was more stronger than the inhibition by a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) (Thr(197)) by KRG-TS. The phosphorylation of IP3RI (Ser(1756)) by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS compared with that by Rp-8-Br-cGMPS. These results suggest that the inhibitory effect of [Ca(2+)]i mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. KRG-TS also inhibited the release of adenosine triphosphate and serotonin. In addition, only G-Rg3 of protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation. CONCLUSION: These results strongly indicate that KRG-TS is a potent beneficial compound that inhibits [Ca(2+)]i mobilization in thrombin-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease.

Antiplatelet effects of caffeic acid due to Ca(2＋) mobilizationinhibition via cAMP-dependent inositol-1, 4, 5-trisphosphate receptor phosphorylation.

AIM: In this study, we investigated the effects of caffeic acid (CAFA), a phenolic acid, on Ca(2＋)-antagonistic cyclic nucleotides associated with the phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and vasodilator-stimulated phosphoprotein (VASP) and the thromboxane A2 (TXA2)-associated microsomal cyclooxygenase-1 (COX-1) activity in collagen (10 µg/mL)-stimulated platelet aggregation. METHODS: Washed platelets (10(8)/mL) obtained from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 minutes at 37℃ in the presence of 2 mM exogenous CaCl2 with or without CAFA or other materials, stimulated with collagen (10 µg/mL) for 5 minutes, then used to determine the levels of intracellular cytosolic Ca(2＋) ([Ca(2＋)]i), TXA2, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), COX-1 activity, VASP and IP3R phosphorylation. RESULTS: CAFA dose-dependently inhibited collagen-induced platelet aggregation and suppressed the production of TXA2, an aggregation-inducing autacoid associated with the strong inhibition of COX-1 in platelet microsomes exhibiting cytochrome C reductase activity. CAFA dose-dependently inhibited collagen-elevated [Ca(2＋)]i mobilization, which was increased by a cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS, but not a cGMP-dependent protein kinase (G-kinase) inhibitor, Rp-8-Br-cGMPS. In addition, CAFA significantly increased the formation of cAMP and cGMP, intracellular Ca(2＋)-antagonists that function as aggregation-inhibiting molecules. CAFA increased IP3R (320 kDa) phosphorylation, indicating the inhibition of IP3-mediated Ca(2＋) release from internal stores (i.e. the dense tubular system) via the IP3R on collagen-activated platelets. Furthermore, CAFA-induced IP3R phosphorylation was strongly inhibited by an A-kinase inhibitor, Rp-8-Br-cAMPS, but only mildly inhibited by a G-kinase inhibitor, Rp-8-Br-cGMPS. These results suggest that the inhibition of [Ca(2＋)]i mobilization by CAFA is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CAFA elevated the phosphorylation of VASP-Ser(157), an A-kinase substrate, but not the phosphorylation of VASP-Ser(239), a G-kinase substrate. We demonstrate that CAFA increases cAMP and subsequently phosphorylates both IP3R and VASP-Ser(157) through A-kinase activation to inhibit [Ca(2＋)]i mobilization and TXA2 production via the inhibition of the COX-1 activity. CONCLUSIONS: These results strongly indicate that CAFA is a potent beneficial compound that elevates the level of cAMP-dependent protein phosphorylation in collagen-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.

Inhibitory effects of total saponin from Korean red ginseng via vasodilator-stimulated phosphoprotein-Ser(157) phosphorylation on thrombin-induced platelet aggregation.

In this study, we have investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-induced platelet aggregation. TSKRG dose-dependently inhibited thrombin-induced platelet aggregation with IC50 value of about 81.1 µg/mL. In addition, TSKRG dose-dependently decreased thrombin-elevated the level of cytosolic-free Ca(2+) ([Ca(2+)]i), one of aggregation-inducing molecules. Of two Ca(2+)-antagonistic cyclic nucleotides as aggregation-inhibiting molecules, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), TSKRG significantly dose-dependently elevated intracellular level of cAMP, but not cGMP. In addition, TSKRG dose-dependently inhibited thrombin-elevated adenosine triphosphate (ATP) release from platelets. These results suggest that the suppression of [Ca(2+)]i elevation, and of ATP release by TSKRG are associated with upregulation of cAMP. TSKRG elevated the phosphorylation of vasodilator-stimulated phosphoprotein (VASP)-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMPdependent protein kinase substrate, in thrombin-activated platelets. We demonstrate that TSKRG involves in increase of cAMP level and subsequent elevation of VASP-Ser(157) phosphorylation through A-kinase activation to inhibit [Ca(2+)]i mobilization and ATP release in thrombin-induced platelet aggregation. These results strongly indicate that TSKRG is a beneficial herbal substance elevating cAMP level in thrombin-platelet interaction, which may result in preventing of platelet aggregation-mediated thrombotic diseases.

Inhibition of platelet aggregation by chlorogenic acid via cAMP and cGMP-dependent manner.

In this study, we investigated the effect of chlorogenic acid, a phenolic acid, on collagen (10 µg/ml)-stimulated platelet aggregation. Chlorogenic acid dose-dependently inhibited collagen-induced platelet aggregation, and suppressed the production of thromboxane A2 (TXA2), an intracellular Ca-agonist as an aggregation-inducing autacoidal molecule, which was associated with the strong inhibition of cyclooxygenase (COX)-1 in platelet microsomes having cytochrome c reductase activity. In addition, chlorogenic acid increased significantly the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), intracellular Ca-antagonists as aggregation-inhibiting molecules. These results suggest that chlorogenic acid has antiplatelet activity through the reduction of TXA2 and the increase of cAMP and cGMP levels. Therefore, our data demonstrate that chlorogenic acid is a potent inhibitor of collagen-stimulated platelet aggregation, and may be a crucial tool for a negative regulator during platelet activation in thrombotic diseases.

Epigallocatechin-3-gallate has an anti-platelet effect in a cyclic AMP-dependent manner.

AIM: In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cyclic nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in collagen (10 µg/mL)-stimulated platelet aggregation. METHODS: Washed platelets (10(8)/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 min at 37°C in the presence of 2 mM exogenous CaCl(2) with or without EGCG or other materials, stimulated with collagen (10 µg/mL) for 5 min, and then used for the determination of intracellular cytosolic Ca(2+) ([Ca(2+)](i)), thromboxane A(2) (TXA(2)), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and VASP phosphorylation. RESULTS: EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both [Ca(2+)](i) mobilization and TXA(2) production. Of two aggregation-inhibiting molecules, cAMP and cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMPspecific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASP-Ser(157) phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenylate cyclase activation and subsequently phosphorylates VASP-Ser(157) through A-kinase activation to inhibit [Ca(2+)](i) mobilization and TXA(2) production on collagen-induced platelet aggregation. CONCLUSIONS: These results strongly indicate that EGCG is a beneficial compound elevating cAMP level in collagen-platelet interaction, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.