Factors that regulate thrombin generation in the blood : studies in healthy subjects and patients with thrombotic diseases
thesisposted on 06.05.2015, 13:19 by Mohammed A. Alsahli
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.