Identification of the mechanisms by which the coagulation protease thrombin activates platelets is critical for understanding haemostasis and thrombosis. Thrombin activates cells at least in part by cleaving protease-activated G-protein-coupled receptors (PARs). PAR3 and PAR4 are thrombin receptors expressed in mouse platelets. Inhibition of thrombin binding to mPAR3 (ref. 4) and knockout of the mPAR3 gene inhibited mouse platelet activation at low but not high concentrations of thrombin. Thus PAR3 is important for thrombin signalling in mouse platelets. Expression of human PAR3 in heterologous expression systems reliably resulted in responsiveness to thrombin. Curiously, despite its importance for the activation of mouse platelets by thrombin, mouse PAR3 (mPAR3) did not lead to thrombin signalling even when overexpressed. We now report that mPAR3 and mPAR4 interact in a novel way: mPAR3 does not itself mediate transmembrane signalling but instead functions as a cofactor for the cleavage and activation of mPAR4 by thrombin. This establishes a paradigm for cofactor-assisted PAR activation and for a G-protein-coupled receptor's acting as an accessory molecule to present ligand to another receptor.

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via proteaseactivated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was ϳ10 4 -fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-␣-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was ϳ10 4 -fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.

The GlobalFiler™ PCR Amplification Kit is a single multiplex assay that amplifies a set of 24 markers, which encompass the European Standard Set and CODIS (Combined DNA Index System) recommended composite set of loci. In addition to more loci and a 6-dye chemistry format, the Master Mix has been formulated to allow higher sample loading volume for trace DNA samples. The GlobalFiler™ Kit has been optimized to deliver high performance on casework samples, while also delivering fast thermal cycling, with an amplification time of approximately 80 min. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.Electronic supplementary materialThe online version of this article (10.1007/s00414-018-1817-5) contains supplementary material, which is available to authorized users.

G protein-coupled receptors can trigger metalloproteinase-dependent shedding of proteins from the cell surface. We now report that G protein-coupled receptors can themselves undergo regulated metalloproteinasedependent shedding. The N-terminal exodomain of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, displayed regulated shedding in endothelial cells, which normally express this receptor. Cleavage occurred at a site predicted to render the receptor unresponsive to thrombin. A chimeric protein in which the N-terminal exodomain of PAR1 was fused to an unrelated transmembrane segment was shed as efficiently as PAR1, shedding of both proteins was stimulated by phorbol ester and by a PAR1 agonist. TNF␣ protease inhibitor-2 (TAPI-2), phenanthroline, and tissue inhibitor of metalloproteinase-3 (TIMP-3) but not TIMP-1 or -2 inhibited such shedding. These and other data suggest that the information that specifies PAR1 shedding resides within its N-terminal exodomain rather than its heptahelical segment, that activation of protein kinase C or of PAR1 itself can stimulate PAR1 shedding in trans, and that ADAM17/TACE or a metalloproteinase with similar properties mediates PAR1 shedding. Regulated shedding reduced the amount of cell surface PAR1 available for productive cleavage by thrombin by half or more, but thus far we have been unable to demonstrate an effect of PAR1 shedding on cellular responsiveness to thrombin. Nonetheless, regulated shedding of G protein-coupled receptors represents a new mechanism by which signaling by this important class of receptors might be modulated.

BackgroundMetal-on-metal hip resurfacing arthroplasty (MoM HRA) may offer potential advantages over total hip arthroplasty (THA) for certain patients with advanced osteoarthritis of the hip. However, the cost effectiveness of MoM HRA compared with THA is unclear.Questions/purposesThe purpose of this study was to compare the clinical effectiveness and cost-effectiveness of MoM HRA to THA.MethodsA Markov decision model was constructed to compare the quality-adjusted life-years (QALYs) and costs associated with HRA versus THA from the healthcare system perspective over a 30-year time horizon. We performed sensitivity analyses to evaluate the impact of patient characteristics, clinical outcome probabilities, quality of life and costs on the discounted incremental costs, incremental clinical effectiveness, and the incremental cost-effectiveness ratio (ICER) of HRA compared to THA.ResultsMoM HRA was associated with modest improvements in QALYs at a small incremental cost, and had an ICER less than $50,000 per QALY gained for men younger than 65 and for women younger than 55. MoM HRA and THA failure rates, device costs, and the difference in quality of life after conversion from HRA to THA compared to primary THA had the largest impact on costs and quality of life.ConclusionsMoM HRA could be clinically advantageous and cost-effective in younger men and women. Further research on the comparative effectiveness of MoM HRA versus THA should include assessments of the quality of life and resource use in addition to the clinical outcomes associated with both procedures.Level of EvidenceLevel I, economic and decision analysis. See Guidelines for Authors for a complete description of levels of evidence.