Date

10-2015

Document Type

Dissertation

Degree Name

Ph.D.

Department

Department of Biomedical Engineering

Institution

Oregon Health & Science University

Abstract

Hemostasis is the physiological process of cessation of bleeding at sites of vascular injury. Primary hemostasis consists of the recruitment of blood platelets and the formation of platelet aggregates at the site of vessel damage. Secondary hemostasis occurs with activation of blood plasma coagulation proteins to generate insoluble fibrin around platelet aggregates to form a thrombus. Similar mechanisms of action can lead to the activation of platelets and the coagulation cascade in diseased blood vessels, resulting in pathological thrombus formation, while the failure to fully activate these pathways can lead to bleeding complications. Thus, there is a need for accurate, reproducible measurement systems for monitoring platelet function and coagulation, detecting disease states, and evaluating efficacy of novel antithrombotic therapeutics. This dissertation centers on the development of metrological techniques to study the mechanisms of thrombosis and hemostasis.

Common microscopy techniques are useful for mechanistic studies but are limited when quantitatively evaluating physical features of biological specimens. Here we present the development and utilization of label-free imaging techniques to investigate the physical parameters (e.g., volume and mass) of platelet aggregates and thrombi formed in response to exposure to combinations of procoagulant agonists under shear flow conditions. These techniques revealed that coagulation restricts platelet aggregate growth at high physiologic shear rates and the formation of fibrin significantly increases clot density while thrombus volume remains constant.

Accurate assessment of platelet function is critical for identifying platelet function disorders and measuring the effect of antiplatelet therapies. This dissertation presents the development of an assay that utilizes the biophysical property of platelet concentration, in conjunction with label-free, quantitative imaging techniques to assess platelet function under static conditions. The utility of this technique to evaluate antiplatelet therapies was demonstrated by the inhibition of glycoprotein (GP) IIb/IIIa abrogating platelet aggregation and significantly reducing sample volume and mass at high platelet concentrations on collagen-coated surfaces.

The developing hemostatic system in neonates is functionally distinct from adults. However, blood volume limitations have hindered the evaluation of platelet function in neonates. Here we describe the development of four small volume, whole blood, platelet function assays for assessing neonatal platelet adhesion, activation, and aggregation under static and fluid shear conditions. With these assays, we show that neonatal platelets activate to a lesser degree relative to adult platelets, GPIIb/IIIa mediates neonatal platelet adhesion and aggregation on collagen surfaces under fluid shear, and neonatal platelets adhere to collagen in a von Willebrand factor-dependent manner.

Thrombotic events are highly associated with cancer and metastasis; however, the role of coagulation in facilitating metastasis is ill-defined. This dissertation presents the development of a technique that evaluates cancer cell interactions with platelet aggregates and thrombi under fluid shear conditions. Utilization of this technique revealed that coagulation and fibrin formation promotes metastatic colon adenocarcinoma cell adhesion and arrest to thrombi, and demonstrated that the presence of polymorphonuclear leukocytes enhanced cancer cell-thrombi interactions.

Collectively, this dissertation presents the development of novel measurement systems to study the pathways that contribute to thrombosis and hemostasis.

Identifier

doi:10.6083/M46Q1W6R

Available for download on Tuesday, December 11, 2018

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