DNMT3L inhibits hepatocellular carcinoma progression through DNA methylation of CDO1: insights from big data to basic research.

BACKGROUND: DNMT3L is a crucial DNA methylation regulatory factor, yet its function and mechanism in hepatocellular carcinoma (HCC) remain poorly understood. Bioinformatics-based big data analysis has increasingly gained significance in cancer research. Therefore, this study aims to elucidate the role of DNMT3L in HCC by integrating big data analysis with experimental validation. METHODS: Dozens of HCC datasets were collected to analyze the expression of DNMT3L and its relationship with prognostic indicators, and were used for molecular regulatory relationship evaluation. The effects of DNMT3L on the malignant phenotypes of hepatoma cells were confirmed in vitro and in vivo. The regulatory mechanisms of DNMT3L were explored through MSP, western blot, and dual-luciferase assays. RESULTS: DNMT3L was found to be downregulated in HCC tissues and associated with better prognosis. Overexpression of DNMT3L inhibits cell proliferation and metastasis. Additionally, CDO1 was identified as a target gene of DNMT3L and also exhibits anti-cancer effects. DNMT3L upregulates CDO1 expression by competitively inhibiting DNMT3A-mediated methylation of CDO1 promoter. CONCLUSIONS: Our study revealed the role and epi-transcriptomic regulatory mechanism of DNMT3L in HCC, and underscored the essential role and applicability of big data analysis in elucidating complex biological processes.

Epigenome-wide development and validation of a prognostic methylation score in intrahepatic cholangiocarcinoma based on machine learning strategies.

Background: Clinical parameter-based nomograms and staging systems provide limited information for the prediction of survival in intrahepatic cholangiocarcinoma (ICC) patients. In this study, we developed a methylation signature that precisely predicts overall survival (OS) after surgery. Methods: An epigenome-wide study of DNA methylation based on whole-genome bisulfite sequencing (WGBS) was conducted for two independent cohorts (discovery cohort, n=164; validation cohort, n=170) from three hepatobiliary centers in China. By referring to differentially methylated regions (DMRs), we proposed the concept of prognostically methylated regions (PMRs), which were composed of consecutive prognostically methylated CpGs (PMCs). Using machine learning strategies (Random Forest and the least absolute shrinkage and selector regression), a prognostic methylation score (PMS) was constructed based on 14 PMRs in the discovery cohort and confirmed in the validation cohort. Results: The C-indices of the PMS for predicting OS in the discovery and validation cohorts were 0.79 and 0.74, respectively. In the whole cohort, the PMS was an independent predictor of OS [hazard ratio (HR) =8.12; 95% confidence interval (CI): 5.48-12.04; P<0.001], and the C-index (0.78) of the PMS was significantly higher than that of the Johns Hopkins University School of Medicine (JHUSM) nomogram (0.69, P<0.001), the Eastern Hepatobiliary Surgery Hospital (EHBSH) nomogram (0.67, P<0.001), American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging system (0.61, P<0.001), and MEGNA prognostic score (0.60, P<0.001). The patients in quartile 4 of PMS could benefit from adjuvant therapy (AT) (HR =0.54; 95% CI: 0.32-0.91; log-rank P=0.043), whereas those in the quartiles 1-3 could not. However, other nomograms and staging system failed to do so. Further analyses of potential mechanisms showed that the PMS was associated with tumor biological behaviors, pathway activation, and immune microenvironment. Conclusions: The PMS could improve the prognostic accuracy and identify patients who would benefit from AT for ICC patients, and might facilitate decisions in treatment of ICC patients.

Identification of multi-omics biomarkers and construction of the novel prognostic model for hepatocellular carcinoma.

Genome changes play a crucial role in carcinogenesis, and many biomarkers can be used as effective prognostic indicators in various tumors. Although previous studies have constructed many predictive models for hepatocellular carcinoma (HCC) based on molecular signatures, the performance is unsatisfactory. Because multi-omics data can more comprehensively reflect the biological phenomenon of disease, we hope to build a more accurate predictive model by multi-omics analysis. We use the TCGA to identify crucial biomarkers and construct prognostic models through difference analysis, univariate Cox, and LASSO/stepwise Cox analysis. The performances of predictive models were evaluated and validated through survival analysis, Harrell's concordance index (C-index), receiver operating characteristic (ROC) curve, and decision curve analysis (DCA). Multiple mRNAs, lncRNAs, miRNAs, CNV genes, and SNPs were significantly associated with the prognosis of HCC. We constructed five single-omic models, and the mRNA and lncRNA models showed good performance with c-indexes over 0.70. The multi-omics model presented a robust predictive ability with a c-index over 0.77. This study identified many biomarkers that may help study underlying carcinogenesis mechanisms in HCC. In addition, we constructed multiple single-omic models and an integrated multi-omics model that may provide practical and reliable guides for prognosis assessment.

Raman spectroscopy: A prospective intraoperative visualization technique for gliomas.

The infiltrative growth and malignant biological behavior of glioma make it one of the most challenging malignant tumors in the brain, and how to maximize the extent of resection (EOR) while minimizing the impact on normal brain tissue is the pursuit of neurosurgeons. The current intraoperative visualization assistance techniques applied in clinical practice suffer from low specificity, slow detection speed and low accuracy, while Raman spectroscopy (RS) is a novel spectroscopy technique gradually developed and applied to clinical practice in recent years, which has the advantages of being non-destructive, rapid and accurate at the same time, allowing excellent intraoperative identification of gliomas. In the present work, the latest research on Raman spectroscopy in glioma is summarized to explore the prospect of Raman spectroscopy in glioma surgery.

Effect of unfolded protein response on the immune infiltration and prognosis of transitional cell bladder cancer.

Background: Bladder cancer (BC) is one of the most common human malignancies worldwide. Previous researches have shown that the unfolded protein response (UPR) pathway could contribute to the tumorigenesis of BC. However, the role of UPR in the immune infiltration, progression, and prognosis of BC is unclear.Methods: The GSVA and ssGSEA methods were used for assessing the UPR score and immune cells infiltration score in three BC public datasets, respectively. The relationship between the UPR pathway and clinicopathological characteristics was analyzed by the Kruskal-Wallis, Wilcox test, and log-rank test. The association of the UPR pathway with various tumor-infiltrating immune cells was evaluated with the correlation analysis. Univariate Cox regression analysis was performed to identify risk factors significantly associated with prognosis. The predictive models were built based on risk factors and visualized with nomograms. The performance of our models was evaluated with the calibration curve, Harrell's concordance index (c-index), and receiver operating characteristic (ROC) analysis.Results: We found that the UPR pathway and many UPR-related genes were significantly associated with the pathologic grade, tumor type, and invasive progression of transitional cell bladder cancer (TCBC), and a high UPR score predicted a poor prognosis in patients. The UPR score was positively correlated with the infiltration abundance of many tumor immune cells in TCBC. Besides, we constructed predictive models based on the UPR score, and good performance was observed, with c-indexes ranging from 0.74 to 0.87.Conclusions: Our study proved that the UPR pathway may have an important impact on the progression, prognosis, and tumor immune infiltration in TCBC, and the models we built may provide effective and reliable guides for prognosis assessment and treatment decision-making for TCBC patients.

Dose-effect relationship and molecular mechanism by which BMSC-derived exosomes promote peripheral nerve regeneration after crush injury.

BACKGROUND: The development of new treatment strategies to improve peripheral nerve repair after injury, especially those that accelerate axonal nerve regeneration, is very important. The aim of this study is to elucidate the molecular mechanisms of how bone marrow stromal cell (BMSC)-derived exosomes (EXOs) participate in peripheral nerve regeneration and whether the regenerative effect of EXOs is correlated with dose. METHOD: BMSCs were transfected with or without an siRNA targeting Ago2 (SiAgo2). EXOs extracted from the BMSCs were administered to dorsal root ganglion (DRG) neurons in vitro. After 48 h of culture, the neurite length was measured. Moreover, EXOs at four different doses were injected into the gastrocnemius muscles of rats with sciatic nerve crush injury. The sciatic nerve functional index (SFI) and latency of thermal pain (LTP) of the hind leg sciatic nerve were measured before the operation and at 7, 14, 21, and 28 days after the operation. Then, the number and diameter of the regenerated fibers in the injured distal sciatic nerve were quantified. Seven genes associated with nerve regeneration were investigated by qRT-PCR in DRG neurons extracted from rats 7 days after the sciatic nerve crush. RESULTS: We showed that after 48 h of culture, the mean number of neurites and the length of cultured DRG neurons in the SiAgo2-BMSC-EXO and SiAgo2-BMSC groups were smaller than that in the untreated and siRNA control groups. The average number and diameter of regenerated axons, LTP, and SFI in the group with 0.9 × 1010 particles/ml EXOs were better than those in other groups, while the group that received a minimum EXO dose (0.4 × 1010 particles/ml) was not significantly different from the PBS group. The expression of PMP22, VEGFA, NGFr, and S100b in DRGs from the EXO-treated group was significantly higher than that in the PBS control group. No significant difference was observed in the expression of HGF and Akt1 among the groups. CONCLUSIONS: These results showed that BMSC-derived EXOs can promote the regeneration of peripheral nerves and that the mechanism may involve miRNA-mediated regulation of regeneration-related genes, such as VEGFA. Finally, a dose-effect relationship between EXO treatment and nerve regeneration was shown.

KIAA1429 contributes to liver cancer progression through N6-methyladenosine-dependent post-transcriptional modification of GATA3.

BACKGROUND: N6-methyladenosine (m6A) modification, the most abundant internal methylation of eukaryotic RNA transcripts, is critically implicated in RNA processing. As the largest known component in the m6A methyltransferase complex, KIAA1429 plays a vital role in m6A methylation. However, its function and mechanism in hepatocellular carcinoma (HCC) remain poorly defined. METHODS: Quantitative PCR, western blot and immunohistochemistry were used to measure the expression of KIAA1429 in HCC. The effects of KIAA1429 on the malignant phenotypes of hepatoma cells were examined in vitro and in vivo. MeRIP-seq, RIP-seq and RNA-seq were performed to identify the target genes of KIAA1429. RESULTS: KIAA1429 was considerably upregulated in HCC tissues. High expression of KIAA1429 was associated with poor prognosis among HCC patients. Silencing KIAA1429 suppressed cell proliferation and metastasis in vitro and in vivo. GATA3 was identified as the direct downstream target of KIAA1429-mediated m6A modification. KIAA1429 induced m6A methylation on the 3' UTR of GATA3 pre-mRNA, leading to the separation of the RNA-binding protein HuR and the degradation of GATA3 pre-mRNA. Strikingly, a long noncoding RNA (lncRNA) GATA3-AS, transcribed from the antisense strand of the GATA3 gene, functioned as a cis-acting element for the preferential interaction of KIAA1429 with GATA3 pre-mRNA. Accordingly, we found that the tumor growth and metastasis driven by KIAA1429 or GATA3-AS were mediated by GATA3. CONCLUSION: Our study proposed a complex KIAA1429-GATA3 regulatory model based on m6A modification and provided insights into the epi-transcriptomic dysregulation in hepatocarcinogenesis and metastasis.

Chloroplast Genomes and Comparative Analyses among Thirteen Taxa within Myrsinaceae s.str. Clade (Myrsinoideae, Primulaceae).

The Myrsinaceae s.str. clade is a tropical woody representative in Myrsinoideae of Primulaceae and has ca. 1300 species. The generic limits and alignments of this clade are unclear due to the limited number of genetic markers and/or taxon samplings in previous studies. Here, the chloroplast (cp) genomes of 13 taxa within the Myrsinaceae s.str. clade are sequenced and characterized. These cp genomes are typical quadripartite circle molecules and are highly conserved in size and gene content. Three pseudogenes are identified, of which ycf15 is totally absent from five taxa. Noncoding and large single copy region (LSC) exhibit higher levels of nucleotide diversity (Pi) than other regions. A total of ten hotspot fragments and 796 chloroplast simple sequence repeats (SSR) loci are found across all cp genomes. The results of phylogenetic analysis support the notion that the monophyletic Myrsinaceae s.str. clade has two subclades. Non-synonymous substitution rates (dN) are higher in housekeeping (HK) genes than photosynthetic (PS) genes, but both groups have a nearly identical synonymous substitution rate (dS). The results indicate that the PS genes are under stronger functional constraints compared with the HK genes. Overall, the study provides hypervariable molecular markers for phylogenetic reconstruction and contributes to a better understanding of plastid gene evolution in Myrsinaceae s.str. clade.

LncRNA SNHG10 Facilitates Hepatocarcinogenesis and Metastasis by Modulating Its Homolog SCARNA13 via a Positive Feedback Loop.

Understanding the roles of noncoding RNAs (ncRNA) in tumorigenesis and metastasis would establish novel avenues to identify diagnostic and therapeutic targets. Here, we aimed to identify hepatocellular carcinoma (HCC)-specific ncRNA and to investigate their roles in hepatocarcinogenesis and metastasis. RNA-seq of xenografts generated by lung metastasis identified long noncoding RNA small nucleolar RNA host gene 10 (SNHG10) and its homolog SCARNA13 as novel drivers for the development and metastasis of HCC. SNHG10 expression positively correlated with SCARNA13 expression in 64 HCC cases, and high expression of SNHG10 or SCARNA13 was associated with poor overall survival. As SCARNA13 showed significant rise and decline after overexpression and knockdown of SNHG10, respectively, we hypothesized that SNHG10 might act as an upstream regulator of SCARNA13. SNHG10 and SCARNA13 coordinately contributed to the malignant phenotype of HCC cells, where SNHG10 served as a sponge for miR-150-5p and interacted with RPL4 mRNA to increase the expression and activity of c-Myb. Reciprocally, upregulated and hyperactivated c-Myb enhanced SNHG10 and SCARNA13 expression by regulating SNHG10 promoter activity, forming a positive feedback loop and continuously stimulating SCARNA13 expression. SCARNA13 mediated SNHG10-driven HCC cell proliferation, invasion, and migration and facilitated the cell cycle and epithelial-mesenchymal transition of HCC cells by regulating SOX9. Overall, we identified a complex circuitry underlying the concomitant upregulation of SNHG10 and its homolog SCARNA13 in HCC in the process of hepatocarcinogenesis and metastasis. SIGNIFICANCE: These findings unveil the role of a noncoding RNA in carcinogenesis and metastasis of hepatocellular carcinoma.

Importance of gene expression signatures in pancreatic cancer prognosis and the establishment of a prediction model.

BACKGROUND AND AIM: Pancreatic cancer (PC) is one of the most common tumors with a poor prognosis. The current American Joint Committee on Cancer (AJCC) staging system, based on the anatomical features of tumors, is insufficient to predict PC outcomes. The current study is endeavored to identify important prognosis-related genes and build an effective predictive model. METHODS: Multiple public datasets were used to identify differentially expressed genes (DEGs) and survival-related genes (SRGs). Bioinformatics analysis of DEGs was used to identify the main biological processes and pathways involved in PC. A risk score based on SRGs was computed through a univariate Cox regression analysis. The performance of the risk score in predicting PC prognosis was evaluated with survival analysis, Harrell's concordance index (C-index), area under the curve (AUC), and calibration plots. A predictive nomogram was built through integrating the risk score with clinicopathological information. RESULTS: A total of 945 DEGs were identified in five Gene Expression Omnibus datasets, and four SRGs (LYRM1, KNTC1, IGF2BP2, and CDC6) were significantly associated with PC progression and prognosis in four datasets. The risk score showed relatively good performance in predicting prognosis in multiple datasets. The predictive nomogram had greater C-index and AUC values, compared with those of the AJCC stage and risk score. CONCLUSION: This study identified four new biomarkers that are significantly associated with the carcinogenesis, progression, and prognosis of PC, which may be helpful in studying the underlying mechanism of PC carcinogenesis. The predictive nomogram showed robust performance in predicting PC prognosis. Therefore, the current model may provide an effective and reliable guide for prognosis assessment and treatment decision-making in the clinic.

Integrative analysis of h-prune as a potential therapeutic target for hepatocellular carcinoma.

BACKGROUND: Drosophila prune protein (h-prune) has been proved to play an essential role in regulating tumor metastasis. However, the clinical relevance of h-prune and its potential mechanism in regulating hepatocellular carcinoma (HCC) are still poorly understood. METHODS: In this study, we used tissue microarrays (TMA) containing 304 HCC tumor samples to evaluate the expression of h-prune and its correlation with prognosis. Data of RNAseq, mutation profiles, copy number variation (CNV), miRNAseq and methylation array from The Cancer Genome Atlas (TCGA) dataset were adopted to analyze the distinctive genomic patterns associated with h-prune expression. RESULTS: By using TMA, we found increased expression of h-prune in HCC tumor cells compared with adjacent normal tissues. Higher expression of h-prune was correlated with poorer OS and DFS outcomes. In addition, multivariate analysis showed that h-prune expression was an independent risk factor for both OS and DFS. Gene enrichment analysis showed that the gene signatures of cell proliferation, DNA methylation and canonical Wnt signaling pathway were enriched in h-prune-high patients. Notably, somatic mutation analysis demonstrated that higher mutation burden of RB1 and RPS6KA3 could be observed in h-prune-high patients. Moreover, integrative analysis revealed a strong correlation between h-prune expression and epigenetic changes. INTERPRETATION: This study has highlighted the clinical value of h-prune in predicting the prognosis of HCC patients and its essential role in promoting tumorigenesis of HCC.

The Prognostic Value and Regulatory Mechanisms of microRNA-145 in Various Tumors: A Systematic Review and Meta-analysis of 50 Studies.

Acting as an important tumor-related miRNA, the clinical significance and underlying mechanisms of miR-145 in various malignant tumors have been investigated by numerous studies. This study aimed to comprehensively estimate the prognostic value and systematically illustrate the regulatory mechanisms of miR-145 based on all eligible literature.Relevant studies were acquired from multiple online databases. Overall survival (OS) and progression-free survival (PFS) were used as primary endpoints. Detailed subgroup analyses were performed to decrease the heterogeneity among studies and recognize the prognostic value of miR-145. All statistical analyses were performed with RevMan software version 5.3 and STATA software version 14.1. A total of 48 articles containing 50 studies were included in the meta-analysis. For OS, the pooled results showed that low miR-145 expression in tumor tissues was significantly associated with worse OS in patients with various tumors [HR = 1.70; 95% confidence interval (CI), 1.46-1.99; P < 0.001). Subgroup analysis based on tumor type showed that the downregulation of miR-145 was associated with unfavorable OS in colorectal cancer (HR = 2.17; 95% CI, 1.52-3.08; P < 0.001), ovarian cancer (HR = 2.15; 95% CI, 1.29-3.59; P = 0.003), gastric cancer (HR = 1.78; 95% CI, 1.35-2.36; P < 0.001), glioma (HR = 1.65; 95% CI, 1.30-2.10; P < 0.001), and osteosarcoma (HR = 2.28; 95% CI, 1.50-3.47; P < 0.001). For PFS, the pooled results also showed that the downregulation of miR-145 was significantly associated with poor PFS in patients with multiple tumors (HR = 1.39; 95% CI, 1.16-1.67; P < 0.001), and the subgroup analyses further identified that the low miR-145 expression was associated with worse PFS in patients with lung cancer (HR = 1.97; 95% CI, 1.25-3.09; P = 0.003) and those of Asian descent (HR = 1.50; 95% CI, 1.23-1.82; P < 0.001). For the regulatory mechanisms, we observed that numerous tumor-related transcripts could be targeted by miR-145-5p or miR-145-3p, as well as the expression and function of miR-145-5p could be regulated by multiple molecules.This meta-analysis indicated that downregulated miR-145 in tumor tissues or peripheral blood predicted unfavorable prognostic outcomes for patients suffering from various malignant tumors. In addition, miR-145 was involved in multiple tumor-related pathways and the functioning of significant biological effects. miR-145 is a well-demonstrated tumor suppressor, and its expression level is significantly correlated with the prognosis of patients with multiple malignant tumors.

The complete chloroplast genome of Primula tsiangii W. W. Smith (Primulaceae): a karst endemic primrose in Southwest China.

Primula tsiangii W. W. Smith is a karst endemic species in Southwest China. Here, we report the complete chloroplast genome of P. tsiangii. The chloroplast (cp) genome was determined to be 153,281 bp and the GC contents was 37.2%. The sequence includes a large single copy (LSC) region of 84,524 bp, a small single copy (SSC) region of 17,357 bp, and two separated inverted regions of 25,700 bp each. It contains 127 unique genes, including 82 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. This is the first report of cp genomes for karst endemic primrose, and will be useful for the genetic diversity of karst plants.

Sofosbuvir in combination with daclatasvir in liver transplant recipients with HCV infection: A systematic review and meta-analysis.

BACKGROUND: Studies focusing on the efficacy of SOF+DCV regimen on liver transplantation recipients with HCV infection are still limited. In the current study, we aimed to perform a systematic review and meta-analysis to evaluate the efficacy and tolerability of SOF+DCV regimen, with or without ribavirin, on post-LT setting. METHODS: A systematic literature search of various databases as well as abstracts of major liver diseases conferences was performed. Studies with SVR data in HCV infected liver transplantation recipients treated with daclatasvir/sofosbuvir regimen were included. All statistical analyses were conducted by R version 3.3.1 (The R Foundation for Statistical Computing, Vienna, Austria). RESULTS: Seven studies with a total of 379 LT recipients were included in this study. Most of these LT recipients had genotype 1 HCV infection. The overall rate of SVR12 reached 93.3% (95% CI: 83.3% to 99.4%). After excluding the study of Fontana et al., the SVR12 reached 96.8% and heterogeneity was lowered down (P=0.17). In three studies, patients treated with SOF+DCV (n=146) had a higher SVR12 rate than that of patients treated with SOF+DCV+RBV (n=83) (OR 0.33, 95% CI: 0.12 to 0.87; P=0.02). There was no difference in SVR12 between patients infected with HCV genotype 1 and genotype 3 (P=0.57) and no difference was found in SVR12 rate between 12-week therapy and 24-week therapy (P=0.82). The most common adverse effects (AEs) were: anemia 32% (n=64/202), infections 26% (n=38/149), neutropenia 23% (n=35/149), thrombocytopenia 21% (n=32/149) and renal failure 8% (n=12/149). CONCLUSION: SOF+DCV±RBV regimen is of high efficacy and tolerability in LT recipients with HCV infection.

Beta2-glycoprotein I protects thrombin from inhibition by heparin cofactor II: potentiation of this effect in the presence of anti-beta2-glycoprotein I autoantibodies.

OBJECTIVE: Beta2-glycoprotein I (beta2GPI) is an important autoantigen in the antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiologic functions, since it displays both anticoagulant and procoagulant properties. We have previously reported that beta2GPI can directly bind thrombin, a key serine protease in the coagulation pathway. The present study was undertaken to examine the influence of beta2GPI on thrombin inactivation by the serpin heparin cofactor II (HCII). The effect of anti-beta2GPI antibodies was also examined. METHODS: HCII inactivation of thrombin was assessed using chromogenic and various platelet functional assays. The influence of intact and proteolytically cleaved beta2GPI and anti-beta2GPI antibodies was determined in these systems. RESULTS: beta2GPI protected thrombin against inactivation by HCII/heparin. Cleavage of beta2GPI at Lys317-Thr318 abrogated its protective effect. Patient polyclonal IgG and murine monoclonal anti-beta2GPI antibodies potentiated the procoagulant influence of beta2GPI in this system. CONCLUSION: These novel findings suggest that beta2GPI may regulate thrombin inactivation by HCII/heparin. The observation that anti-beta2GPI antibodies potentiate the protective effect of beta2GPI on thrombin in this system, thereby promoting a procoagulant response, may potentially delineate one of the pathophysiologic mechanisms contributing to the prothrombotic tendency in patients with APS.

Beta2-glycoprotein I binds thrombin via exosite I and exosite II: anti-beta2-glycoprotein I antibodies potentiate the inhibitory effect of beta2-glycoprotein I on thrombin-mediated factor XIa generation.

OBJECTIVE: Beta(2)-glycoprotein I (beta(2)GPI) is a dominant antigenic target in antiphospholipid syndrome (APS). Beta(2)-glycoprotein I may bind to factor XI and serve a physiologic function as a regulator of factor XI activation by thrombin. We undertook this study to investigate the possible interactions of beta(2)GPI with thrombin in beta(2)GPI-regulated factor XI activation by thrombin and to evaluate the effect of anti-beta(2)GPI antibodies on this system. METHODS: The beta(2)GPI interaction with thrombin was investigated in direct and competitive assays using beta(2)GPI domain mutants and thrombin-binding exosite oligonucleotides. Beta(2)-glycoprotein I inhibition of thrombin-mediated factor XI activation was assessed in the presence of 8 anti-beta(2)GPI monoclonal antibodies (mAb) directed against domain I. RESULTS: Domain V of beta(2)GPI was involved in direct binding to thrombin, and exosite I and exosite II on thrombin took part in this interaction. Anti-beta(2)GPI mAb produced a >70% inhibition of thrombin-mediated factor XI activation in the presence of beta(2)GPI. CONCLUSION: We demonstrate that beta(2)GPI interacts with thrombin exosites I and II. This novel finding necessitates a reinterpretation of previous studies from which the detection of anti-human thrombin antibodies in APS has been reported. We also show that anti-beta(2)GPI antibodies potentiate the inhibitory effect of beta(2)GPI on thrombin-mediated factor XIa generation.

Anti-beta2-glycoprotein I antibodies in complex with beta2-glycoprotein I can activate platelets in a dysregulated manner via glycoprotein Ib-IX-V.

OBJECTIVE: Results of previous studies suggest that anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in complex with beta2GPI activate platelets in a dysregulated manner, potentially contributing to the prothrombotic tendency associated with the antiphospholipid syndrome (APS). We undertook this study to investigate the possible contribution of the GPIb-IX-V receptor to platelet activation mediated by the anti-beta2GPI antibody-beta2GPI complex. METHODS: In vitro methods were used in the present study. The interaction between beta2GPI and the GPIbalpha subunit of the GPIb-IX-V receptor was delineated using direct binding and competitive inhibition assays. The interaction between the anti-beta2GPI antibody-beta2GPI complex and platelets was studied using a novel method in which the Fc portion of the antibody was immobilized using protein A coated onto a microtiter plate. Platelet activation was assessed by two methods; one involved measuring thromboxane B2 production and the other involved assessment of the activation of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta intracellular signaling pathway. The contribution of the GPIbalpha receptor to platelet activation induced by the anti-beta2GPI antibody-beta2GPI complex was assessed by observing the influence of 2 anti-GPIbalpha antibodies (AK2 and SZ2) directed against distinct epitopes. RESULTS: This study showed that beta(2)GPI could bind to the GPIbalpha receptor. The anti-beta2GPI antibody-beta2GPI complex was able to activate platelets, and this effect was inhibited by anti-GPIbalpha antibody directed against epitope Leu-36-Gln-59, but not by anti-GPIbalpha antibody directed against residues Tyr-276-Glu-282. CONCLUSION: Our findings show that inappropriate platelet activation by the anti-beta2GPI antibody-beta2GPI complex via the GPIbalpha receptor may contribute to the prothrombotic tendency associated with APS.