Date of Award

Fall 2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

Steven A. Jones

Abstract

Platelet activation involves multiple events, one of which is the generation and release of nitric oxide (NO), a platelet aggregation inhibitor. Platelets simultaneously send and receive various agents that promote a positive and negative feedback control system during hemostasis. Although the purpose of platelet-derived NO is not fully understood, NO is known to inhibit platelet recruitment. NO's relatively large diffusion coefficient allows it to diffuse more rapidly than platelet agonists. It may thus be able to inhibit recruitment of platelets near the periphery of a growing thrombus before agonists have substantially accumulated in those regions.

Results from two studies in our laboratory differed in the extent to which platelet-derived NO decreased platelet adhesion. Frilot studied the effect of L-arginine (L-A) and NG-Methyl-L-arginine acetate salt (L-NMMA) on platelet adhesion to collagen under static conditions in a Petri dish. Eshaq examined the percent coverage on collagen-coated and fibrinogen-coated microchannels under shear conditions with different levels of L-A and Adenosine Diphosphate (ADP). Frilot's results showed no effect of either L-A or L-NMMA on surface coverage, thrombus size or serotonin release, while Eshaq's results showed a decrease in surface coverage with increased levels of L-A. A possible explanation for these contrasting results is that platelet-derived NO may be more important under flow conditions than under static conditions.

For this project, the effects of L-A. ADP and L-NMMA on platelet adhesion were studied at varying shear stresses on protein-coated glass slides. The surface exposed to platelet-rich-plasma in combination with each chemical solution was observed under AFM, FE-SEM and fluorescence microscopy. Quantitative and qualitative comparisons of images obtained with these techniques confirmed the presence of platelets on the protein coatings. AFM images of fibrinogen and collagen-coated slides presented characteristic differences. Adhered platelets were identified, particularly with the AFM. The effects of chemical additives were examined under the same microscopy techniques. The resulting fluorescent microscopy data suggests statistical differences between the percent surface coverage of different shear regions on the glass slides. No statistically significant change in surface coverage was found with the addition of ADP on fibrinogen-coated slides, but showed differences on collagen with all chemicals. However, in high shear regions. L-A produced a significant decrease in platelet adhesion and L-NMMA produced a statistically significant increase in platelet adhesion on fibrinogen and collagen-coated slides. The AFM images of the chemical additives provided no differences between one another except with ADP. The no shear and low shear conditions provided no variations between AFM images via visual confirmation and statistical significance. The only AFM image shear region differences were obtained from low to high shear regions and static to high shear regions comparisons.

The objective of this project was to determine whether the static conditions used by Frilot and the dynamic conditions used by Eshaq could explain the different effects of L-A observed in those studies. In addition, the ability of the fluorescent imaging technique to quantify platelet adhesion was examined by comparison of fluorescent imaging to AFM and FE-SEM. The results of this study were consistent with both the lack of an effect of chemical additives under static conditions reported by Frilot and the reduction of platelet adhesion in response to L-A reported by Eshaq.

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