Factors that regulate thrombin generation in the blood : studies in healthy subjects and patients with thrombotic diseases

Cell-derived microparticles (MPs) have been demonstrated to play a major role in haemostasis and thrombosis, inflammation, vascular reactivity and angiogenesis. They are released from virtually all body cell types during activation or apoptosis. This project aimed to characterise the procoagulant properties of MPs derived from platelets and other vascular cells and evaluate the mechanisms whereby platelets generate procoagulant MPs. A lack of standardisation of pre-analytical variables has hindered MP analysis. Systematic analysis revealed that platelet contamination should be efficiently removed from MP preparations using appropriate centrifugation protocols and that repeated freezing and thawing has no significant impact on the procoagulant activity of MPs providing the plasma is free of platelets and cell fragments, but does cause a slight decay of clotting factor activity. Microparticles derived from platelets, endothelial cells and macrophages had significant procoagulant phospholipid (PPL) activity with macrophage-derived>platelet-derived >endothelial cell-derived microparticles. However, the tissue factor (TF) activity was different for each cell type with strong activity for macrophage-derived MPs, moderate activity for endothelial cell-derived MPs, but platelet-derived MPs did not express any TF activity. It was demonstrated that platelet immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors; GPVI, FcγRIIA and CLEC-2, but not the G protein-coupled receptors (GPCRs), are the primary receptors eliciting platelet procoagulant response and MP formation. However, GPCRs play a crucial role in the feedback mechanism of platelet procoagulant response. Additionally, activation via CLEC-2 was demonstrated to induce platelet procoagulant responses and MP formation in a similar manner to GPVI. Heparin-induced thrombocytopenia (HIT) is caused by the interaction between HIT-immune complexes with platelet FcγRIIA leading to platelet activation, MP generation and thrombocytopenia. MPs generated by HIT-immune complexes in suspected HIT patients was demonstrated to exhibit similar procoagulant activity to those generated through the other two platelet ITAM-containing receptors. Using their procoagulant activity measured in thrombin generation assay, it provides a rapid functional diagnostic assay for HIT with good correlation and association with other clinical and laboratory investigations

Comparison of the Release of microRNA and extracellular vescicles from platelets in response to different agonists

On activation platelets release microRNA and extracellular vesicles (EV) into the circulation. The release of EV from platelets has been shown to be dependent on the agonist; here we investigated whether the microRNA profile or EV released from platelets was also agonist specific. Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (GPVI), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block COX-1 and apyrase to remove ADP. The released microRNA was profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterised by size (Nanoparticle Tracking Analysis; NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70. Platelet activation triggered the release of 57-79 different microRNA, dependent upon agonist, with a core of 46 microRNA observed with all agonists. There was a high level of correlation between agonists (r >0.98; p<0.0001 for all), and with the microRNA content of the parent platelets (r2 >0.98; p<0.0001). The 46 microRNA seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control and differentiation. MiR-223-3p was most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have antiinflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect. These data suggest that all tested agonists trigger the release of a similar microRNA profile whilst the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNA and pdEV

Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists

<p>On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific.</p> <p>Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70.</p> <p>Platelet activation triggered the release of 57–79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (<i>r</i>² > 0.98; <i>p</i> < 0.0001 for all), and with the microRNA content of the parent platelets (<i>r</i>² > 0.98; <i>p</i> < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect.</p> <p>These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.</p