Abstract Number: OC 49.1
Meeting: ISTH 2022 Congress
Background: The procoagulant platelet subpopulation is involved more in pathological thrombosis than haemostasis.
Aims: This study aimed to identify unique pathways through transcriptomics that predispose platelets to become procoagulant after activation, to facilitate targeting this subpopulation.
Methods: Washed platelets from healthy donors (n=6) were sorted into procoagulant (GSAO+/P-selectin+) and activated non-procoagulant (GSAO-/P-selectin+) subpopulations (Hua, Blood 2015), after thrombin 2U/mL plus collagen-related peptide 4µg/mL stimulation, using flow cytometry cell sorting. RNA was extracted, sequenced (single-end, 75 base-pair reads), with differentially expressed (DE, fold-change >/=2, false discovery rate/FDR < 0.05) genes identified using DESeq2 and edgeR tools. DE genes were input into Ingenuity Pathway Analysis (IPA). Functional validation was undertaken for candidate gene, Dynamin 1. Procoagulant platelet proportions were assessed by flow cytometry and confocal microscopy with inhibitors MiTMAB and dynasore.
Results: DESeq2 analysis yielded 1024 DE genes with a higher proportion upregulated in procoagulant platelets. Subpopulations segregated after unsupervised hierarchical clustering (Figure 1A-B). qPCR of a subset of genes (n=30) from different donors (n=10) confirmed differential expression within subpopulations. IPA identified integrin signalling, endocytosis and actin cytoskeleton-related pathways as highly enriched in procoagulant platelets (Figure 1C). Dynamin 1, involved in both endocytosis and actin rearrangement, was 4.9-fold increased (FDR < 0.05, both DE tools), confirmed by qPCR (3.4-fold increased, p < 0.01, Figure 1D). Dynamin inhibition by MiTMAB led to a dose-dependent, selective reduction in agonist-induced procoagulant platelet formation by flow cytometry (Figure 1E), and platelet “balloon” morphology by confocal microscopy (vehicle 21.6±0.7%, MiTMAB 1.7±0.3%, p=0.0003), while >99% of platelets remained activated (P-selectin+). Alternative dynamin inhibitor, dynasore, confirmed dose-dependent reduction in procoagulant platelet formation (n=6, p < 0.0001). Together, these suggest functional validation of transcriptomic differences.
Conclusion(s): The procoagulant subpopulation is transcriptionally distinct compared to activated non-procoagulant platelets. Functional validation of RNA-sequencing results by dynamin inhibition strongly suggests transcriptional differences are involved in determination of functionally distinct platelet subpopulations.
To cite this abstract in AMA style:Tohidi-Esfahani I, Whittaker S, Lee C, Campbell H, Lai K, Liang H, Kockx M, Larance M, CHEN V. Procoagulant platelets have a distinct transcriptome that identifies the large GTPase dynamin as a driver in their formation [abstract]. https://abstracts.isth.org/abstract/procoagulant-platelets-have-a-distinct-transcriptome-that-identifies-the-large-gtpase-dynamin-as-a-driver-in-their-formation/. Accessed September 27, 2023.
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