Author ORCID Identifier
https://orcid.org/0009-0004-0483-9361
Date Available
12-9-2026
Year of Publication
2025
Document Type
Doctoral Dissertation
Degree Name
Doctor of Philosophy (PhD)
College
Medicine
Department/School/Program
Molecular and Cellular Biochemistry
Faculty
Sidney W. Whiteheart, PhD
Abstract
In their role as “vascular sentries,” platelets play a critical role in maintaining vascular integrity and hemostasis. However, heightened platelet reactivity is associated with the development and complications of late-stage cardiovascular disease (CVD). Platelets contribute to these processes via secretion of a variety of factors, including pro-inflammatory signals. Platelet secretion is a calcium-dependent process that relies on the fusion of secretory granule membranes with the plasma membranes, an event mediated by SNARE proteins. VAMP8 is the primary vesicle-associated (v-) SNARE in platelets, and modulating platelet secretion via deletion of VAMP8 has been shown to reduce platelet reactivity in the FeCl3-induced carotid injury model, an established preclinical model of arterial thrombosis. However, other inflammation-associated mechanisms such as platelet pyroptosis have remained unstudied in this model. Traditional measurement of thrombosis relies on Doppler probe-based measurements of blood flow but lacks temporal precision and spatial resolution, inhibiting its utility in identifying novel regulators of thrombosis.
To address these limitations, we developed a method utilizing laser speckle contrast imaging (LSCI) as a high-resolution method for measuring vessel occlusion and thrombus stability in the FeCl₃ model. Following topical FeCl₃-induced carotid injury, we performed LSCI measurements to assess thrombosis, yielding times to initial and stable occlusion to differentiate between thrombus formation and stability as well as area under the curve (AUC) as a coarse metric of overall thrombosis. The method was optimized by measuring thrombotic responses to various degrees of injury by manipulating FeCl3 concentration. To assess the suitability of this method for measuring thrombosis inhibition in vivo, both pharmacologic and genetic models were tested. Analysis of pyroptosis-associated protein Gasdermin-D (GSDMD) was also assayed to assess the ability of the system to detect unconventional regulators of thrombosis. To validate that effects on accelerated thrombosis were also detectable, LSCI was then used to assess thrombosis in a model of hypercholesterolemia. Finally, LSCI measurement of occlusive arterial thrombosis was performed as part of a study investigating the effects of VAMP8deletion in a model of hypercholesterolemia to determine how loss of platelet secretion affects thrombosis under pathologic conditions.
LSCI detected differences in occlusion in a FeCl3 dose-dependent manner and allowed prolonged observations to assess thrombus stability. Pharmacologic and genetic manipulations were assessed at 6% FeCl3, representing a mild injury with heterogeneous response, to determine the sensitivity of the method. Mice treated with a SYK inhibitor (Bi 100249420) displayed significantly delayed occlusion, as expected, supporting the use of LSCI for preclinical testing. Further, GSDMD-deficient mice showed both a delay in initial occlusion times and time to stable occlusion compared with wild-type controls, consistent with a mild yet measurable impairment in thrombus stability, which positions GSDMD as a novel regulator of thrombosis. Induction of hypercholesterolemia using a PCSK9 gain-of-function AAV model resulted in accelerated stable thrombosis, which was also detectable by LSCI even at a mild injury level.
As previously stated, the established protective effect of VAMP8 deletion was detectable using previous methods, but whether this effect would persist in pathological conditions was unclear. Using LSCI, we demonstrated that hypercholesterolemic VAMP8-deficient mice still exhibited protection from occlusive arterial thrombosis compared to hypercholesterolemic controls, providing further evidence that VAMP8 is a viable therapeutic target for cardiovascular disease. Moreover, atherosclerotic plaque development was significantly reduced and circulating markers of platelet reactivity were also altered in VAMP8-deficient mice, setting up a potential role for platelet secretory machinery in cardiovascular disease development.
Here, we demonstrate the benefits of LSCI measurement in the FeCl₃-induced carotid injury method using a variety of preclinical mouse models. Our findings not only identify GSDMD as a potential novel regulator of thrombosis but also support a role for the previously established regulator of platelet secretion, VAMP8 in hypercholesterolemia and atherosclerosis. This method enables nuanced evaluation of thrombosis dynamics and offers a clinically relevant approach for arterial thrombosis research in preclinical settings, which will be necessary to address these avenues of inquiry as the field moves forward.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2025.548
Funding Information
This work is supported by grants from the National Institutes of Health, National Heart, Lung and Blood Institute (HL56652, HL138179, and HL150818 to S.W.W.) and a Merit Grant from the VA to SWW from 2021-2025.
Recommended Citation
Driehaus, Elizabeth, "IMPROVING ASSESSMENT OF THROMBOSIS USING LSCI: INSIGHTS INTO SYSTEMIC PLATELET FUNCTION AND VASCULAR PATHOLOGY" (2025). Theses and Dissertations--Molecular and Cellular Biochemistry. 77.
https://uknowledge.uky.edu/biochem_etds/77
