Abstract
dc:description.abstractIn both thrombosis and haemostasis, two distinct populations of activated platelets form, pro-aggregatory and pro-coagulant platelets. Pro-aggregatory platelets have active integrins and aggregate together via fibrinogen to form the haemostatic plug which prevents bleeding. Pro-coagulant platelets expose phosphatidylserine (PS) which acts as a surface for binding of the tenase (FIXa/VIIIa) and prothrombinase (FXa/Va) complexes that generate a burst of thrombin production responsible for occlusive clot formation. All current anti-platelet drugs are associated with bleeding side effects since they inhibit pro-aggregatory platelet activity. No drug exists which selectively targets the pro-coagulant platelet population, though this could be an alternative anti-thrombotic approach associated with fewer bleeding side effects if pro-aggregatory activity remains. In order to test such an approach, a selective inhibitor of pro-coagulant platelet activity must be developed. Critical to platelet PS exposure is activity of the main platelet scramblase, TMEM16F. This thesis aimed to develop inhibitors of TMEM16F and platelet scramblase activity. R5421 was the only previously described inhibitor of platelet scramblase activity. R5421, however, is shown to be a non-selective inhibitor of platelet scramblase activity, also affecting platelet pro-aggregatory activity by inhibiting αIIbβ3 integrin activation, α-granule release and cytosolic Ca2+ signalling in response to a range of agonists. Ligand-based in silico methods were applied to identify new inhibitors of platelet scramblase activity. The pesticide thiodicarb was identified as a novel inhibitor of platelet scramblase activity but also acts non-selectively. Since R5421 has wide ranging off-target effects and attempts to improve its selectivity were unsuccessful, other lead compounds were pursued. The polyphenolic compounds tannic acid and epigallocatechin gallate (EGCG’) inhibit TMEM16F activity when over-expressed in cell lines. Both tannic acid and EGCG’ are shown to inhibit platelet scramblase activity. An empirical screen of polyphenolic compounds defined an emerging pharmacophore for inhibition of platelet scramblase activity. All catechin gallate compounds inhibited platelet scramblase activity non-selectively. Structure-based in silico methods were applied to identify new inhibitors of platelet scramblase activity, first using the fungal nhTMEM16 structure and later the mTMEM16F structure. These screens identified novel inhibitors of platelet scramblase activity which do not affect platelet αIIbβ3 integrin activation, α-granule release or cytosolic Ca2+ signalling. Further work is required to confirm whether these compounds act directly on TMEM16F or inhibit platelet scramblase activity through a different mechanism.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2020
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Millington-Burgess, Sarah
- Advisor dc:contributor.advisor
-
- Harper, Matthew
Subjects
dc:subject × 3Rights
dc:rightsIdentifiers
dc:identifier.*- Author Identifier
- 0000-0002-4740-637X
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/318284