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- Banerjee, Meenakshi, et al.
(author)
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Prospective, International, Multisite Comparison of Platelet Isolation Techniques for Genome-Wide Transcriptomics : Communication from the SSC of the ISTH
- 2024
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In: Journal of Thrombosis and Haemostasis. - : John Wiley & Sons. - 1538-7933 .- 1538-7836.
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Journal article (peer-reviewed)abstract
- Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the ISTH SSCs, we assessed how three of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALLTM filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting the techniques are transferrable and reproducible. Moreover, all three isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbβ3 activation is reduced by anti-CD45 bead isolation compared to washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.
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- Boknäs, Niklas, et al.
(author)
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Contact activation : important to consider when measuring the contribution of tissue factor-bearing microparticles to thrombin generation using phospholipid-containing reagents
- 2014
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In: Journal of Thrombosis and Haemostasis. - : John Wiley & Sons. - 1538-7933 .- 1538-7836. ; 12:4, s. 515-518
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Journal article (peer-reviewed)abstract
- Background: A commercial MP reagent containing phospholipids is used for thrombin generation (TG) measurements to estimate the procoagulant activity of microparticles (MPs). Previous reports have shown that contact activation affects TG when TF levels are low, and that addition of phospholipids might augment this effect.Objectives: To quantify the impact of contact activation on TG in the presence of phospholipids and low/no TF, as is the case using a commercially available MP-reagent. Methods Thrombin generation was analyzed using MP- or platelet-rich plasma (PRP)-reagent in the presence and absence of corn trypsin inhibitor and anti-TF antibodies, respectively. To quantify the impact of different experimental parameters on contact activation, microparticle-depleted plasma was analyzed in the presence of different concentrations of phospholipids, TF and/or contact activating agents (kaolin).Results: Even with low contact activating blood collection tubes, substantial thrombin generation was observed with the MP-reagent, but this was completely inhibited by addition of corn trypsin inhibitor. Control experiments illustrate that the phospholipids in the reagent play a major role in enhancing TG initiated by FXIIa. Even with the PRP-reagent, which is recommended for determining the content of phospholipids from MPs, TG was partly dependent on contact activation.Conclusions: Contact activation plays a major role in TG when using reagents/samples containing phospholipids but little or no tissue factor. This needs to be considered and accounted for in future clinical studies using TG to assess the procoagulant activity of MPs.
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- Chanzu, Harry, et al.
(author)
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Platelet α-granule cargo packaging and release are affected by the luminal proteoglycan, serglycin
- 2021
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In: Journal of Thrombosis and Haemostasis. - : John Wiley & Sons. - 1538-7933 .- 1538-7836. ; 19:4, s. 1082-1095
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Journal article (peer-reviewed)abstract
- BackgroundSerglycin (SRGN) is an intragranular, sulfated proteoglycan in hematopoietic cells that affects granule composition and function.ObjectiveTo understand how SRGN affects platelet granule packaging, cargo release, and extra-platelet microenvironments.MethodsPlatelets and megakaryocytes from SRGN−/− mice were assayed for secretion kinetics, cargo levels, granule morphology upon activation, and receptor shedding.ResultsMetabolic, 35SO4 labeling identified SRGN as a major sulfated macromolecule in megakaryocytes. SRGN colocalized with α-granule markers (platelet factor 4 [PF4], von Willebrand factor [VWF], and P-selectin), but its deletion did not affect α-granule morphology or number. Platelet α-granule composition was altered, with a reduction in basic proteins (pI ≥8; e.g., PF4, SDF-1, angiogenin) and constitutive release of PF4 from SRGN−/− megakaryocytes. P-Selectin, VWF, and fibrinogen were unaffected. Serotonin (5-HT) uptake and β-hexosaminidase (HEXB) were slightly elevated. Thrombin-induced exocytosis of PF4 from platelets was defective; however, release of RANTES/CCL5 was normal and osteopontin secretion was more rapid. Release of 5-HT and HEXB (from dense granules and lysosomes, respectively) were unaffected. Ultrastructural studies showed distinct morphologies in activated platelets. The α-granule lumen of SRGN−/− platelet had a grainy staining pattern, whereas that of wild-type granules had only fibrous material remaining. α-Granule swelling and decondensation were reduced in SRGN−/− platelets. Upon stimulation of platelets, a SRGN/PF4 complex was released in a time- and agonist-dependent manner. Shedding of GPVI from SRGN−/− platelets was modestly enhanced. Shedding of GP1b was unaffected.ConclusionThe polyanionic proteoglycan SRGN influences α-granule packaging, cargo release, and shedding of platelet membrane proteins.
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