Platelet transcriptome analysis of mice with altered thrombin signaling

S. Schubert, F. Marini, C. Santos, H. Binder, W. Ruf (Mainz, Germany)

Date: 16.02.2017,
Time: 11:00 - 12:00

Objective: Platelets are terminally differentiated blood components required for hemostasis and contributing to thrombosis. The RNA profile of platelets may provide indirect insights into alterations of megakaryocytes and the bone marrow or systemic diseases that result in platelets acquiring additional RNA cargo. Platelets are therefore circulating sentinels for pathological changes and easily accessible for profiling of their RNA content by next generation sequencing (NGS). Our goal was to identify platelet signatures indicative of prothrombotic states. We therefore determined the platelet transcriptome of genetically modified mice that (1) have chronically impaired vascular control of thrombin associated with platelet hyper-reactivity due to a point mutation in thrombomodulin (TMProPro) or (2) altered thrombin responses due to loss of thrombin signaling (PAR4-/-) or binding to the extracellular domain of GPIba (GPIba-IL4 chimerea).

Methods: We determined total RNA profiles from wild-type, TMProPro, PAR4-/- and GPIba-IL4 mice (n=6). Leukocyte-depleted platelet total RNA was subjected to deep NGS (>35 million reads). Reads were mapped to ENSEMBL GRCm38.76 and data were analyzed by featureCounts of the Rsubread package. Differential expression was analyzed with DESeq2.

Results: Principal component analysis showed highly reproducible NGS profiles for replicates of each genotype and a clear separation between receptor mutants. Differential expression analysis identified 190 differentially expressed transcripts selective for TMProPro, 565 transcripts for PAR4-/- and 609 transcripts for GPIba-IL4 platelets. In addition, we found unique transcript changes common to TMProPro and PAR4-/- (93), TMProPro and GPIba-IL4 (158), and PAR4-/- and GPIba-IL4 (426) platelets, indicating common pathways influenced by thrombin signaling and binding to its receptors.

Conclusion: The transcriptome analysis of platelets from genetically modified mice yielded highly reproducible RNA profiles that provide insights into common and distinct pathways regulated by thrombin and its receptors expressed by the endothelium and platelets/megakaryocytes. The identified differentially expressed transcripts may be of utility to assess platelet function alterations due to chronically elevated thrombin levels and coagulation activation in diverse clinical settings predisposing to thrombo-embolic complications.