To cite this article: van der Wal DE, Du VX, Lo KSL, Rasmussen JT, Verhoef S, Akkerman JWN. Platelet apoptosis by cold-induced glycoprotein Iba clustering. J Thromb Haemost 2010; 8: 2554-62.Summary. Background: Cold-storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Iba-distribution indicative of receptor clustering and initiates thromboxane A 2 -formation. GPIba is associated with 14-3-3 proteins, which contribute to GPIba-signaling and in nucleated cells take part in apoptosis regulation. Objectives and methods: We investigated whether GPIba-clustering induces platelet apoptosis through 14-3-3 proteins during cold (4 h 0°C)-rewarming (1 h 37°C). Results: During cold-rewarming, 14-3-3 proteins associate with GPIba and dissociate from Bad inducing Baddephosphorylation and activation. This initiates pro-apoptosis changes in Bax/Bcl-x L and Bax-translocation to the mitochondria, inducing cytochrome c release. The result is activation of caspase-9, which triggers phosphatidylserine exposure and platelet phagocytosis by macrophages. Responses are prevented by N-acetyl-D-glucosamine (GN), which blocks GPIba-clustering, and by O-sialoglycoprotein endopeptidase, which removes extracellular GPIba. Conclusions: Cold-rewarming triggers apoptosis through a GN-sensitive GPIba-change indicative of receptor clustering. Attempts to improve platelet transfusion by cold-storage should focus on prevention of the GPIba-change.
Post-transcriptional gene silencing is a promising therapy for the monogenic, autosomal dominant, Huntington’s disease (HD). However, wild-type huntingtin (HTT) has important cellular functions, so the ideal strategy would selectively lower mutant HTT while sparing wild-type. HD patients were genotyped for heterozygosity at three SNP sites, before phasing each SNP allele to wild-type or mutant HTT. Primary ex vivo myeloid cells were isolated from heterozygous patients and transfected with SNP-targeted siRNA, using glucan particles taken up by phagocytosis. Highly selective mRNA knockdown was achieved when targeting each allele of rs362331 in exon 50 of the HTT transcript; this selectivity was also present on protein studies. However, similar selectivity was not observed when targeting rs362273 or rs362307. Furthermore, HD myeloid cells are hyper-reactive compared to control. Allele-selective suppression of either wild-type or mutant HTT produced a significant, equivalent reduction in the cytokine response of HD myeloid cells to LPS, suggesting that wild-type HTT has a novel immune function. We demonstrate a sequential therapeutic process comprising genotyping and mutant HTT-linkage of SNPs, followed by personalised allele-selective suppression in a small patient cohort. We further show that allele-selectivity in ex vivo patient cells is highly SNP-dependent, with implications for clinical trial target selection.
Huntington’s disease (HD) is caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin gene that results in expression of a mutant huntingtin protein (mHTT) containing an expanded polyglutamine tract in the amino terminus. A number of therapeutic approaches that aim to reduce mHTT expression either locally in the CNS or systemically are in clinical development. We have previously described sensitive and selective assays that measure human HTT proteins either in a polyglutamine-independent (detecting both mutant expanded and non-expanded proteins) or in a polyglutamine length-dependent manner (detecting the disease-causing polyglutamine repeats) on the electrochemiluminescence Meso Scale Discovery detection platform. These original assays relied upon polyclonal antibodies. To ensure an accessible and sustainable resource for the HD field, we developed similar assays employing monoclonal antibodies. We demonstrate that these assays have equivalent sensitivity compared to our previous assays through the evaluation of cellular and animal model systems, as well as HD patient biosamples. We also demonstrate cross-site validation of these assays, allowing direct comparison of studies performed in geographically distinct laboratories.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.