Date of Award

Summer 2017

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Dermot Kenny

Funder/Sponsor

Health Research Board and RCSI Career Development Fund

Keywords

Platelets, Glycosylation, VWF, ABO(H) Antigens

Abstract

Background: Glycoprotein (GP)Ib, expressed on the surface of platelets, and its ligand, von Willebrand Factor (VWF) both express covalently linked ABO(H) blood group carbohydrate determinants. VWF is secreted by endothelial cells and circulates in the plasma in a coiled confirmation that is cryptic to platelet binding until it anchors to collagen exposed at sites of vascular damage. Blood group O is associated with an increased risk of bleeding whereas non-O is associated with increased risk of myocardial infarction.

Aims: We set out to characterise platelet function from donors with blood group O and non-O.

Methods: Parallel plate perfusion chamber assays were used to characterise platelet interactions with VWF. Novel surfaces were developed for the perfusion assays that allowed the capture of autologous VWF on the chamber surface. Platelet interaction with both captured autologous VWF and exogenous pooled, plasma derived VWF was assessed at 1500s-1.

P-Selectin expression, annexin V binding and number of disappearing platelets were used to determine functional responses to shear using a cone and plate viscometer. Platelet aggregation in response to Arachidonic Acid, Collagen, ADP, Epinephrine, Ristocetin and TRAP-6 was assessed using a 96-well plate assay.

Conclusion: We have established the basis of an assay that will provide information as to how platelets interact with autologous VWF. We have provided evidence that VWF can be captured from flowing blood by a nano-topographical interface of 50nM. VWF captured in this manner is able to support platelet adhesion and thrombus build up. Using an alternative antibody based system to coat our flow chambers with autologous VWF we have shown that platelets from donors with blood group O have more platelet tracks, stably adhered and translocating platelets on VWF compared to non-O donors. In contrast, when our flow chambers were coated with exogenous VWF, Type O platelets travelled further and faster across the pooled, plasma-derived VWF surface, taking longer to form stable bonds. Coupled with the observation that ristocetin and TRAP-6 induced aggregation are both lower in donors with blood Type O, these results provide evidence that ABO(H) glycans modulate platelet function and may explain the increased risk of myocardial infarction associated with non-O blood groups.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

8.41 MB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2017.

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