Re-evaluation of the in vitro activity of the protease activated receptor-1 (PAR1) pepducin PZ-128

Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2020
Thesis identifier
  • T15772
Person Identifier (Local)
  • 201985866
Qualification Level
Qualification Name
Department, School or Faculty
  • Myocardial infarction and stroke are some of the leading causes of death globally, with antiplatelet agents commonly used for the clinical management of these conditions. Unfortunately, not all patients respond to existing antiplatelet therapies, which can result in vascular event recurrence and death. This has led to the need to develop alternative ways to inhibit platelet activity and prevent thrombus formation. Protease-activated receptor 1 (PAR1) is a key target due to its role in thrombin-mediated platelet activation.;PZ-128 is a cell penetrable pepducin that targets PAR1 and has entered phase II clinical trials, however, adverse effects in trial subjects have emerged that warrant reappraisal of its basic pharmacology. The aim of this thesis was to re-evaluate PZ-128 pharmacology using in vitro models to assess cellular toxicity and confirm if PZ-128 possesses the ability to promote cellular activity beyond its existing role as a PAR1 inhibitor. The methodology used for this purpose included the study of intracellular Ca2+ mobilisation, cell viability assessment, pAkt and pERK quantification, and receptor localisation studies.;TsA201 and HEK293 cells were used as they express endogenous levels of PAR1 and can be transfected with PAR1 tagged plasmids. Studies with PZ-128 (palmitate-KKSRALF-NH2) were carried out in parallel with a control pepducin (Sc-128) which is comprised of the same amino acids as PZ-128 which have been rearranged in sequence (palmitate-FSRLKAK-NH2). Treatment with thrombin (EC50 0.577 U/mL) or the synthetic PAR1 agonist TFLLR-NH2 (EC50 8.89 μM) confirmed PAR1 expression and receptor activity using Fluo-4 AM calcium flux assays. MTT assay was used to assess cell viability, with significant cell death observed in PZ-128-treated cells following 100 μM exposure for 24 hours, which coincided with loss of pAkt.;Interestingly, PZ-128 (25-100 μM), but not Sc-128, induced a dose-dependent increase in intracellular Ca2+ levels that was sustained and distinct from thrombin and TFLLR-NH2 responses. PZ-128 (30 μM) also caused sustained ERK phosphorylation (15-60 min). As GPCR internalisation events are common after receptor activation, the ability of PZ-128 to impact PAR1 localisation was assessed. Thrombin (1 U/mL for 30 minutes) efficiently internalised PAR1-GFP in transfected cells, however, no internalisation was observed in response to Sc-128 or PZ-128.;These results suggest that PZ-128 possesses activity that may compromise the cytosolic Ca2+ homeostasis and promote proliferative activity through activation of ERK MAPK pathways. Re-evaluation of existing published pharmacology of PZ-128 is needed to truly understand its full biological activity.;Cyclic analogues of PZ-128 have since been developed in collaboration with the Liskamp laboratory with the aim of advancing pepducin-based targeting of PAR1. While initial pilot studies were started in this thesis (HL-07 and HL-10), full characterisation was out with the scope of this project and is currently ongoing. Improvements to the design of PZ-128 may limit the cellular activity observed and will form the basis of future development.
Advisor / supervisor
  • Cunningham, Margaret Rose
Resource Type
Date Created
  • 2020
Former identifier
  • 9912936693502996