Mapping the interaction of VWF and factor XIIa and its potential as a new anticoagulant target
File(s)
Author(s)
Henne, Patricia
Type
Thesis or dissertation
Abstract
Von Willebrand Factor and Factor XII have well established roles in haemostasis and
coagulation. While VWF is a mediator of platelet capture to sites of vascular injury under high
shear stress, FXII initiates the intrinsic pathway of coagulation. Interestingly, multiple studies
have shown that absence or inhibition of FXII does not impair haemostatic function but has a
thrombo-protective effect, rendering it a potentially safe anticoagulant target.
In this study, I investigated a novel binding interaction between VWF and FXII. Using substrate
based activation assays, I showed that VWF is a potent and previously unrecognised activator
of FXII. Additionally, I demonstrated that both inactive FXII and activated FXII bind to VWF but
not to VWF lacking the A1 domain suggesting that the FXII binding site lies within the A1
domain. By carrying out further binding studies using static binding assays and surface
plasmon resonance with isolated and full-length VWF A1 domain mutants, I identified amino
acids E1452 and D1459 on the α-6 helix of the A1 domain as the FXII binding site in VWF.
The physiological relevance of the VWF/FXII interaction was examined using plate-based
thrombolysis as well as perfusion assays investigating the formation of platelet rich blood clots
under conditions of flow. Using adapted thrombolysis assays, I showed that clot lysis times
were significantly reduced when either VWF, FXIIa or activity of both proteins was blocked.
Furthermore, I demonstrated that the interaction between VWF and FXIIa facilitates VWF
mediated platelet capture under conditions of high shear and that interference with FXII/VWF
binding has a prohibitive effect on overall thrombus formation.
Based on these results, I hypothesise binding of VWF to FXII is an important component of
thrombus formation and inhibition of this interaction might be a novel and safe way to prevent
thrombosis without increasing the risk of bleeding side effects.
coagulation. While VWF is a mediator of platelet capture to sites of vascular injury under high
shear stress, FXII initiates the intrinsic pathway of coagulation. Interestingly, multiple studies
have shown that absence or inhibition of FXII does not impair haemostatic function but has a
thrombo-protective effect, rendering it a potentially safe anticoagulant target.
In this study, I investigated a novel binding interaction between VWF and FXII. Using substrate
based activation assays, I showed that VWF is a potent and previously unrecognised activator
of FXII. Additionally, I demonstrated that both inactive FXII and activated FXII bind to VWF but
not to VWF lacking the A1 domain suggesting that the FXII binding site lies within the A1
domain. By carrying out further binding studies using static binding assays and surface
plasmon resonance with isolated and full-length VWF A1 domain mutants, I identified amino
acids E1452 and D1459 on the α-6 helix of the A1 domain as the FXII binding site in VWF.
The physiological relevance of the VWF/FXII interaction was examined using plate-based
thrombolysis as well as perfusion assays investigating the formation of platelet rich blood clots
under conditions of flow. Using adapted thrombolysis assays, I showed that clot lysis times
were significantly reduced when either VWF, FXIIa or activity of both proteins was blocked.
Furthermore, I demonstrated that the interaction between VWF and FXIIa facilitates VWF
mediated platelet capture under conditions of high shear and that interference with FXII/VWF
binding has a prohibitive effect on overall thrombus formation.
Based on these results, I hypothesise binding of VWF to FXII is an important component of
thrombus formation and inhibition of this interaction might be a novel and safe way to prevent
thrombosis without increasing the risk of bleeding side effects.
Version
Open Access
Date Issued
2019-12
Online Publication Date
2020-08-12T12:33:06Z
Date Awarded
2020-07
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Laffan, Michael
McKinnon, Thomas
Sponsor
British Heart Foundation
Publisher Department
Department of Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)