The overall goal of this project is to improve our understanding of reaction kinetics and mechanism of platelet events of significance in cerebrovascular disease. The work focuses on platelet reactions under controlled, well-defined levels of fluid mechanical shearing stress- applied by use of a rotational viscometer. Prior work in this program has developed a methodology of analysis of the evolution in time of the platelet and platelet aggregate size distribution. By use of a population balance mathematical model, parameters are derived which indicate the state of activation of platelets and the stability of platelet aggregates. The proposed work will (1) improve the methodology by use of flow cytometry, and (2) apply the methodology in pilot studies comparing normal human subjects to patients with cerebrovascular disease.
Specific aims are outlined below. 1. Develop a flow cytometric methodology for studying platelet activation and aggregation in whole blood. Prior work and pilot studies show that platelets and platelet aggregates can be identified and distinguished from other blood cells by a fluorescent-conjugated monoclonal antibody to platelet membrane glycoprotein receptor Ib(GPIb). Other antibodies can be used to identify activated platelets. 2. Subject human blood specimens to known, controlled levels of shear stress in a rotational viscometer and determine the response by use of the flow cytometric methods. 3. Analyze the results by means of the population balance mathematical model to determine the collision efficiency and breakage rate parameters as functions of shear rate and shear exposure time. 4. Compare the results of items 2 and 3 with other results obtained using platelet-rich plasma (PRP) specimens, and platelet suspensions containing various inhibitors and agonists. 5. Evaluate the methods outlined in items 1-3 above for possible clinical utility. The hypothesis is that methods developed in this work can identify on a quantitative basis, the thrombotic tendency in human subjects. In these studies, normal subjects, stroke patients, and patients, and patients with a history of transient ischemic attacks will be evaluated.
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