Integrated amplification microarray system in a lateral flow cell for warfarin genotyping from saliva.

BACKGROUND: Genetic polymorphisms in the CYP2C9 and VKORC1 genes have been linked to sensitivity of the anticoagulant drug warfarin. The aim of this study is to demonstrate a method for warfarin sensitivity genotyping using gel element microarray technology in a simplified workflow from sample collection to analysis and detection. METHODS: We developed an integrated amplification microarray system combining PCR amplification, target labeling, and microarray hybridization within a single, closed-amplicon "lateral flow cell" for genotyping three single nucleotide polymorphisms (SNPs) that influence warfarin response. We combined nucleic acid extraction of saliva using the TruTip technology together with the lateral flow cell assay and with fully automated array detection and analysis. RESULTS: The analytical performance of the assay was tested using 20 genotyped human genomic DNA samples and found to be sensitive down to 330 input genomic copies (1 ng). A follow-up pre-clinical evaluation was performed with 65 blinded saliva samples and the genotyping results were in agreement with those determined by bidirectional sequencing. CONCLUSIONS: Combined with the use of non-invasive saliva samples, rapid DNA extraction, the lateral flow cell, automatic imaging and data analysis provides a simple and fast sample-to-answer microarray test for warfarin sensitivity genotyping.

Binding of anti-GRP78 autoantibodies to cell surface GRP78 increases tissue factor procoagulant activity via the release of calcium from endoplasmic reticulum stores.

The increased risk of venous thromboembolism in cancer patients has been attributed to enhanced tissue factor (TF) procoagulant activity (PCA) on the surface of cancer cells. Recent studies have shown that TF PCA can be modulated by GRP78, an endoplasmic reticulum (ER)-resident molecular chaperone. In this study, we investigated the role of cell surface GRP78 in modulating TF PCA in several human cancer cell lines. Although both GRP78 and TF are present on the cell surface of cancer cells, there was no evidence of a stable interaction between recombinant human GRP78 and TF, nor was there any effect of exogenously added recombinant GRP78 on cell surface TF PCA. Treatment of cells with the ER stress-inducing agent thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca(2+) pump that causes Ca(2+) efflux from ER stores, increased cytosolic [Ca(2+)] and induced TF PCA. Consistent with these findings, anti-GRP78 autoantibodies that were isolated from the serum of patients with prostate cancer and bind to a specific N-terminal epitope (Leu(98)-Leu(115)) on cell surface GRP78, caused a dose-dependent increase in cytosolic [Ca(2+)] and enhanced TF PCA. The ability to interfere with cell surface GRP78 binding, block phospholipase C activity, sequester ER Ca(2+), or prevent plasma membrane phosphatidylserine exposure resulted in a significant decrease in the TF PCA induced by anti-GRP78 autoantibodies. Taken together, these findings provide evidence that engagement of the anti-GRP78 autoantibodies with cell surface GRP78 increases TF PCA through a mechanism that involves the release of Ca(2+) from ER stores. Furthermore, blocking GRP78 signaling on the surface of cancer cells attenuates TF PCA and has the potential to reduce the risk of cancer-related venous thromboembolism.

Peroxynitrite causes endoplasmic reticulum stress and apoptosis in human vascular endothelium: implications in atherogenesis.

OBJECTIVE: Peroxynitrite, a potent oxidant generated by the reaction of NO with superoxide, has been implicated in the promotion of atherosclerosis. We designed this study to determine whether peroxynitrite induces its proatherogenic effects through induction of endoplasmic reticulum (ER) stress. METHODS AND RESULTS: Human vascular endothelial cells treated with Sin-1, a peroxynitrite generator, induced the expression of the ER chaperones GRP78 and GRP94 and increased eIF2alpha phosphorylation. These effects were inhibited by the peroxynitrite scavenger uric acid. Sin-1 caused the depletion of ER-Ca2+, an effect known to induce ER stress, resulting in the elevation of cytosolic Ca2+ and programmed cell death (PCD). Sin-1 treatment was also found, via 3-nitrotyrosine and GRP78 colocalization, to act directly on the ER. Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented Sin-1-induced PCD. Consistent with these in vitro findings, 3-nitrotyrosine was observed and colocalized with GRP78 in endothelial cells of early atherosclerotic lesions from apolipoprotein E-deficient mice. CONCLUSIONS: Peroxynitrite is an ER stress-inducing agent. Its effects include the depletion of ER-Ca2+, a known mechanism of ER stress induction. The observation that 3-nitrotyrosine-containing proteins colocalize with markers of ER stress within early atherosclerotic lesions suggests that peroxynitrite contributes to atherogenesis through a mechanism involving ER stress.