Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemical and Biomolecular Engineering

First Advisor

Scott L. Diamond

Abstract

GPVI is the first responder towards collagen surface and therefore, the role of GPVI in facilitating primary platelet deposition is well studied and unquestionable. However, whether it plays a part in secondary platelet deposition by binding with fibrin, thus facilitating further platelet activation remains a question. Depending on the experiment method and rationale behind it, different results and conclusion could be achieved, thus it calls for a method that could better recapitulate human blood system under in vitro setting with appropriate methods to inhibit GPVI.Indirect method incorporates Syk and Src family kinases (SFK) inhibitors; these molecules interfere with signaling from GPVI, α2β1, αIIbβ3, and GPIb-IX-V to reduce thrombotic risk or induce bleeding episodes. Collagen-mediated clustering of platelet GPVI results in phosphorylation of SFKs such as Lyn and Fyn, and active Lyn is constitutively bound to GPVI to allow rapid signaling. During clotting under flow, the generation of fibrin can have diverse influences on platelet signaling by sequestering thrombin and potentially activating GPVI signaling within the clot interior. These inhibitors tackle the thrombus formation at earlier stages since the platelets reach the activation surface. Direct inhibition of GPVI, which involves using an artificial anti-GPVI fragment, was used to avoid undesirable inhibition and compare the difference between inhibition of subsequent pathways. Using microfluidics, the effects of these inhibitors can be explored under defined hemodynamic flows and procoagulant surface triggers. Additionally, the drug may be present in the blood at desired time of clotting by perfusion switching to drug-treated blood. This experimental design allows exploration of platelet response at different stages of clotting through the measurement of drug potency to modulate clotting on different procoagulant surface conditions, interactions between various coagulation factors in plasma, and the kinetics of several competitive reactions to facilitate platelet recruitment, granule release and fibrin formation.

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