Microfluidic Assays of vWF-Mediated Platelet Adhesion Under Supraphysiological Shear
Restricted (Penn State Only)
- Author:
- Watson, Connor
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 12, 2024
- Committee Members:
- Pak Kin Wong, Major Field Member
Keefe Manning, Chair & Dissertation Advisor
Lacy Alexander, Outside Unit & Field Member
Scott D Simon, Special Member
Francesco Costanzo, Major Field Member
Christopher Siedlecki, Major Field Member
Daniel Hayes, Program Head/Chair - Keywords:
- Microfluidics
Platelets
Blood
von Willebrand Factor
Thrombosis
Mechanical Circulatory Support - Abstract:
- Cardiovascular disease can be characterized by altered hemodynamics, increased thrombogenic potential of the vasculature, hypercoagulability or platelet dysfunction. Severe cases often require heart transplantation, but due to high demand and an undersupplied donor pool, bridge-to-transplant devices are needed. Blood-wetted devices, mainly pumps, valves, and stents, typically extend patient lifespan but exhibit a number of side effects, namely thrombosis, bleeding events, and hemolysis. The supraphysiological shear forces applied to blood by mechanical circulatory support (MCS) result in activated platelets, and the elongation, cleavage, and damage of plasma-borne von Willebrand Factor (vWF). Identifying the mechanisms by which supraphysiological shear rates trigger shear-induced platelet aggregation (SIPA) and rapid occlusive thrombus formation are critical to improving clinical outcomes in an enormous patient population. Microfluidic studies have explored vWF-mediated thrombus formation under wide ranging conditions, investigating pathways of primary hemostasis, thrombosis under physiological shear rates, and initiation of SIPA by vWF unfurling. While these studies are promising, gaps in knowledge remain that require significant progress towards identifying interactions between biochemical and physical mechanisms that regulate platelet adhesion. Determining the influence of fluid dynamics on these mechanisms and the behavior of platelet receptors at supraphysiological shear rates is critical to translating benchtop studies to improved clinical outcomes. While microfluidic channels have become a widespread method of conducting adhesion and flow assays, these models are not yet standardized and technique varies widely between research groups. To this end, a microfluidic platform was developed to conduct assays of platelet adhesion at the site of a sudden expansion, a well-studied geometry that creates flow separation. Flow within this platform was characterized and tuned to approximate clinically-relevant shear rates. Adhesion of vWF and platelet adhesion was quantified under these conditions. Enzymatic treatment demonstrated that sustained thrombus formation was dependent on high molecular weight vWF multimers, validating the platform as a suitable method for investigating platelet adhesion within these flow parameters. The mechanical contribution of red blood cells, an understudied component of arterial thrombosis, was studied in the same sudden expansion model. Platelet adhesion was shown to be entirely dependent on margination by red cells, resulting in elevated thrombus formation as hematocrit increased. Thrombus formation was shown to be tied to the local fluid dynamics of the geometry, as deceleration within the step region mediated platelet adhesion at lower hematocrit levels. The SIPA behavior observed in these studies was tied to function of the integrin αIIbβ3. An additional microfluidic channel was developed, implementing a stenotic progression from a healthy to a severely occluded vessel. Prescribed flow rates in these stenotic channels generated shear rates ranging from arterial to a maximum characteristic of MCS devices. Major platelet receptors αIIbβ3, GPIb, and GPVI were inhibited to identify the respective role in SIPA. While distinct roles were identified tying each receptor to platelet adhesion or aggregation at varied shear rates, a novel role of GPVI was identified, regulating intrathrombus stability at elevated shear rates. These results support the hypothesized efficacy of GPVI as an antiplatelet target, free of interference with primary hemostasis. These stenotic channels were modified to separate the effect of shear rate gradients (S.R.G.s) from that of shear rate on SIPA. Elevated S.R.G.s at the same shear rate result in increased thrombus formation, highlighting the importance of considering local fluid dynamics when modeling vWF-mediated platelet adhesion. Taken in sum, these studies investigate a comprehensive set of conditions in which platelet adhesion and aggregation are determined by a complex interplay of blood biochemistry, flow conditions, and platelet reactivity. The platelet mechanisms outlined in these findings may be leveraged towards improved predictive models of thrombosis and novel antiplatelet agents.