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Embargoed until 2017-04-30
Copyright: Hua, Minh
Embargoed until 2017-04-30
Copyright: Hua, Minh
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Abstract
The procoagulant platelet is characterised by its ability to support functional prothrombinase and links primary to secondary haemostasis. Excess procoagulant activity is linked to pathological thrombosis, however the identity of the procoagulant platelet has been elusive to date. Many of the biochemical and morphological features of the procoagulant platelet are common to the cyclophilin-D (Cyp-D) necrosis pathway. Platelet apoptosis, on the other hand, is thought to be involved in regulating a platelet’s lifespan. Whilst the procoagulant platelet has features akin to cell death, their precise nature is unknown, as progress in this area has been hampered by the lack of a suitable and specific marker.
GSAO [4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide] can label cells with a compromised plasma membrane when conjugated to reporter groups. This thesis shows that highly activated platelet subset undergoes Cyp-D mediated necrosis and provides the surface for coagulation assembly, thrombin generation and fibrin formation. The necrotic phenotype can be identified in flow cytometry analysis by co-expression of GSAO and P-selectin, with the major ligand for GSAO in necrotic platelets being thromboxane synthase-1 via a dithiol covalent linkage.
This thesis demonstrates that necrotic platelets are functionally procoagulant, by means of global coagulation assays. Correlation studies surface FXa and platelet dependent peak thrombin generation indicates that GSAO can be used a s a surrogate marker for the procoagulant platelet. In vivo, platelet necrosis is present in the occlusive 10% ferric chloride injury model and is minimally present in the non-occlusive laser injury model, demonstrating that necrotic platelets are formed in thrombi, but only in the context of certain stimuli.
This thesis demonstrates that cyclophilin D is a promising target for inhibition of procoagulant platelet formation. Platelet activation and necrosis pathways are uncoupled and an enhanced benefit is seen when both pathways are targeted in combination.
GSAO is a valuable probe that can investigate biological factors potentiating procoagulant platelet formation. It may be useful as a biomarker for prediction of thrombotic risk in pathological disease states.