We have identified structural attributes required for signal transduction through a seven-transmembrane-domain receptor. Platelets from a patient (AC) with a congenital bleeding disorder had normal shape change but reduced and reversible aggregation in response to 4 μM ADP, similar to normal platelets with blocked P2Y ₁₂ receptor. The response to 20 μM ADP, albeit still decreased, was more pronounced and was reduced by a P2Y ₁₂ antagonist, indicating some residual receptor function. ADP failed to lower the adenylyl cyclase activity stimulated by prostaglandin E ₁ in the patient's platelets, even though the number and affinity of 2-methylthioadenosine 5′-[ ³³ P]diphosphate-binding sites was normal. Analysis of the patient's P2Y ₁₂ gene revealed a G-to-A transition in one allele, changing the codon for Arg-256 in the sixth transmembrane domain to Gln, and a C-to-T transition in the other allele, changing the codon for Arg-265 in the third extracellular loop to Trp. Neither mutation interfered with receptor surface expression but both altered function, since ADP inhibited the forskolin-induced increase of cAMP markedly less in cells transfected with either mutant P2Y ₁₂ as compared with wild-type receptor. These studies delineate a region of P2Y ₁₂ required for normal function after ADP binding.

Introduction: The advent of anti-tumor necrosis factor alpha (anti-TNFα) drugs has considerably improved medical management in rheumatoid arthritis (RA) patients, although it has been reported to be ineffective in a fraction of them. MicroRNAs (miRNAs) are small, non-coding RNAs that act as fine-tuning regulators of gene expression. Targeting miRNAs by gain or loss of function approaches have brought therapeutic effects in various disease models. The aim of this study was to investigate serum miRNA levels as predictive biomarkers of response to anti-TNFα therapy in RA patients. Methods: In total, 95 RA patients undergoing anti-TNFα/disease-modifying antirheumatic drugs (anti-TNFα/DMARDs) combined treatments were enrolled. Serum samples were obtained at 0 and 6 months and therapeutic efficacy was assessed. miRNAs were isolated from the serum of 10 patients before and after anti-TNFα/DMARDs combination therapy, cDNA transcribed and pooled, and human serum miRNA polymerase chain reaction (PCR) arrays were performed. Subsequently, selected miRNAs were analyzed in a validation cohort consisting of 85 RA patients. Correlation studies with clinical and serological variables were also performed. Results: Ninety percent of RA patients responded to anti-TNFα/DMARDs combination therapy according to European League Against Rheumatism (EULAR) criteria. Array analysis showed that 91% of miRNAS were overexpressed and 9% downregulated after therapy. Functional classification revealed a preponderance of target mRNAs involved in reduction of cells maturation - especially on chondrocytes - as well as in immune and inflammatory response, cardiovascular disease, connective tissue and musculoskeletal system. Six out of ten miRNAs selected for validation were found significantly upregulated by anti-TNFα/DMARDs combination therapy (miR-16-5p, miR-23-3p, miR125b-5p, miR-126-3p, miRN-146a-5p, miR-223-3p). Only responder patients showed an increase in those miRNAs after therapy, and paralleled the reduction of TNFα, interleukin (IL)-6, IL-17, rheumatoid factor (RF), and C-reactive protein (CRP). Correlation studies demonstrated associations between validated miRNAs and clinical and inflammatory parameters. Further, we identified a specific plasma miRNA signature (miR-23 and miR-223) that may serve both as predictor and biomarker of response to anti-TNFα/DMARDs combination therapy. Conclusions: miRNA levels in the serum of RA patients before and after anti-TNFα/DMARDs combination therapy are potential novel biomarkers for predicting and monitoring therapy outcome.

Summary. Background: Tissue factor (TF) is the main initiator of the coagulation cascade and elements that may upregulate its expression might provoke thrombotic events. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are autoimmune diseases characterized by a high TF expression in monocytes. Objectives: To examine the role of microRNAs (miRNAs) in TF expression and to evaluate their levels in SLE and APS patients. Methods: An in silico search was performed to find potential putative binding sites of miRNAs in TF mRNA. In vitro validation was performed transfecting cells expressing TF (THP‐1 and MDA‐MB‐231) with oligonucleotide miRNA precursors and inhibitors. Additionally, reporter assays were performed to test for the binding of miR‐20a to TF mRNA. Levels of miRNAs and TF were measured by quantitative (qRT‐PCR) in patients with APS and SLE. Results: Overexpression of miRNA precursors, but not inhibitors, of two of the members of cluster miR‐17∼92, for example miR‐19b and miR‐20a, in cells expressing TF decreased TF mRNA, protein levels, and procoagulant activity between 30% and 60%. Reporter assays showed that miR‐20a binds to TF mRNA. Finally, we measured levels of miR‐19b and miR‐20a in monocytes from patients with APS and SLE and observed significantly lower miRNAs levels in comparison with healthy subjects inversely correlated with the levels of TF. Conclusions: Down‐regulation of miR‐19b and miR‐20a observed in patients with SLE and APS could contribute to increased TF expression and thus provoke the hypercoagulable state characteristic of these patients.

Studies are presented characterizing platelet CDCrel-1, a protein expressed to high levels by megakaryocytes and belonging to a family of conserved proteins, termed septin. Septin filaments originally were identified in yeast as essential for budding but have become increasingly associated with processes in higher eukaryotic cells involving active membrane movement such as cytokinesis and vesicle trafficking. Direct proof of an in vivo function for septins in higher eukaryotes is limited to the characterization of the Drosophila septin, termed PNUT. We present studies identifying platelet CDCrel-1 as a protein kinase substrate in the presence of known platelet agonists. The immunopurification of CDCrel-1 revealed it to be part of a macromolecular complex containing a protein involved in platelet secretion, syntaxin 4. Moreover, CDCrel-1 was localized in situ to areas surrounding platelet-storage granules. The relevance of CDCrel-1 to normal platelet function was established with the characterization of platelets from a CDCrel-1 ^Null mouse. As compared with platelets from wild-type littermates, CDCrel - 1 ^Null platelets aggregate and release stored [ ¹⁴ C]serotonin in the presence of subthreshold levels of collagen. These results provide new insights into the mechanisms regulating platelet secretion and identify platelet septins as a protein family contributing to membrane trafficking within the megakaryocyte and platelet.