PB0642 - The use of plasminogen activator inhibitor-1 specific Affimers as a tool to modulate fibrin clot properties and thrombosis risk

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Rawan M. Altalhi, PhD

PhD student
University of Leeds
Leeds, England, United Kingdom

Co-Author(s)

CT

Christian Tiede

BioScreening Technology Group Manager
University of Leeds
Leeds, England, United Kingdom
FP

Fladia Phoenix

Research Technician
University of Leeds
Leeds, England, United Kingdom
DT

Darren Tomlinson

Associate Professor
University of Leeds
Leeds, England, United Kingdom
Khalid M. Naseem, PhD

Professor
University of Leeds
Leeds, England, United Kingdom
RA

Ramzi Ajjan, PhD

Professor
University of Leeds
Leeds, England, United Kingdom
NP

Nikoletta Pechlivani

Postdoctoral researcher
University of Ledds
LEEDS, England, United Kingdom

Background: Hypofibrinolysis is associated with increased risk of thrombotic disease. Diabetes is characterized by hypofibrinolysis and elevated levels of plasminogen-activator inhibitor (PAI)-1 which is stabilised by binding to vitronectin. We hypothesise that inhibition of PAI-1 activity using PAI-1/vitronectin-bound conformational peptides, named Affimers, improves hypofibrinolysis and consequently decreases the risk of thrombosis.

Aims: Isolate PAI-1/vitronectin binding Affimers and characterise their role in modulating fibrinolysis.

Methods: A large phage display library of Affimers (>3x1010) was used to screen for PAI-1/vitronectin binders. Functional analysis of Affimers was undertaken using turbidimetric and thromboelastometric assays employing plasma and whole blood systems.

Results: 104 PAI-1/vitronectin Affimers were isolated of which 15 demonstrated distinct sequences. These Affimers were subcloned and expressed in E.coli and one Affimer, named RV8, reduced fibrin clot lysis time in plasma samples of patients with Type 2 diabetes (T2DM) without affecting fibrin network structure. RV8 decreased lysis time by 27% in T2DM pooled plasma when used at 5 µg/ml, and by 5-27% when samples from 12 different T2D individuals were tested. Whole blood ROTEM analysis (n=3 samples) revealed a 49% reduction in lysis time without affecting the maximum clot firmness (MCF) which remained similar to control.

Conclusion(s): Our data confirm the viability of this novel approach of isolating PAI-1 binding Affimers, modulating fibrin clot lysis and in turn identifying potential therapeutic targets to reduce thrombosis risk. The effects of RV8 appears to be more pronounced in whole blood, possibly due to the contribution of platelet-released PAI-1 to modulation of fibrinolysis.