A Microfluidic Approach For Investigating The Role Of Blood Flow In Thrombosis

Microfluidic devices provide a powerful platform for the study of blood biology due to their control of flow geometry, shear flow conditions, and blood biochemistry. This thesis describes a series of microfluidic approaches to investigate a range of pathological flow conditions and their effects on thrombus formation. An impingement-post microfluidic device was designed to shear platelet-free plasma at pathological rates to aggregate von Willebrand factor (VWF) into insoluble fibers held in place by a micropost. VWF fibers were non-amyloid and resistant to ADAMTS13 and tissue plasminogen activator. Factors XIIa and XIa were captured in VWF fibers during aggregation, and could initiate fibrin formation on VWF. When whole blood was perfused over VWF fibers, platelets rolled, bound, and activated in a shear-dependent manner on the fiber surface. To study the structure of thrombi formed in regions of flow separation/reattachment in diseased arteries, another microfluidic device was designed to perfuse blood creating a stagnation point on a thrombotic surface. For clotting over collagen/tissue factor surfaces, platelet thrombi exhibited core-shell architecture with a P-selectin-positive, fibrin-rich core and a P-selectin-negative outer shell. VWF was present throughout the clot structure at both low and high shear rates, but platelet and VWF deposition was markedly decreased when N-acetylcysteine was added. In contrast, inhibition of fibrin polymerization did not change the overall structure of the thrombi, suggesting VWF is more important to clot stability at stagnation points. N-acetylcysteine was found to reduce deposition of VWF on collagen, but was not able to dissolve VWF fibers formed at pathological shear rates in the impingement-post device. Shear-induced platelet activation (SIPA) on VWF was analyzed by stretching VWF with bound platelets using increasing shear rate to measure platelet activation. As VWF fibers stretched in shear flow, the proportion of activated platelets (measured by P-selectin exposure) also increased. Platelets bound to sorbed, non-stretchable VWF displayed the same activation pattern, suggesting fluid shear is more important in SIPA than VWF stretching. GPVI-bound soluble fibrin inhibits platelet activation by collagen. Developing new microfluidic models of thrombosis may lead to a better understanding of the mechanisms involved and produce diagnostic tests for thrombotic diseases.

Author dc:creator

• Herbig, Bradley Andrew

Advisor dc:contributor.advisor

• Scott L. Diamond

Statement dc:rights

• Bradley Andrew Herbig

Language dc:language

en

Repository record dc:identifier.uri

https://repository.upenn.edu/handle/20.500.14332/29925

OAI identifier oai:identifier

oai:repository.upenn.edu:20.500.14332/29925