Flow cytometric studies of platelet responses to shear stress
Doctor of Philosophy
The objective of this study was to improve our understanding of platelet involvement in vascular disease. A cone-and-plate viscometer was used to simulate the fluid mechanical conditions in narrowed arteries. Platelet responses induced by shear stress were quantitated by flow cytometry. This methodology allows the study of platelets in whole blood rather than in platelet suspensions in buffer or plasma, a situation that reflects more closely the physiologic status of platelets circulating in vivo. Using this technique, platelets and platelet aggregates were distinguished from the other blood cells by a fluorescently-labeled monoclonal antibody (MoAb), 6D1, specific for the platelet glycoprotein (GP) Ib. Use of the MoAb anti-CD62, directed against the granule membrane protein-140, yielded sensitive determinations of platelet activation. This methodology was applied to test the efficacy of two novel antiplatelet drugs infused in patients undergoing angioplasty, in preventing the ex vivo shear-induced platelet aggregation (SIPA). Both of these drugs, c7E3-Fab and Integrelin, are GPIIb-IIIa antagonists, and were found to be potent inhibitors of SIPA. In contrast, aspirin, a widely used antiplatelet agent, does not affect SIPA. The same methodology was used to evaluate platelet responses to shear stress in groups of stroke patients and normal subjects. Activated platelets and neutrophil-platelet aggregates were found to circulate in patients' blood in numbers significantly higher than those of normal subjects'. Patients' blood was also more susceptible to SIPA. These enhanced platelet responses were also observed in the patients many days after the onset of stroke suggesting that they are not consequences of ischemia. Flow cytometry was also applied to quantitate shear-induced vWf binding to platelets. The results demonstrate that vWf binding is specific, and although it involves both GPIb and GPIIb-IIIa, it has an absolute requirement for GPIb. This study is the first to directly show that "large" vWf multimers bind to platelets in response to shear more readily than "small" vWf fractions. These findings may contribute to our understanding of mechanisms involved in the pathogenesis of thrombotic disorders, and provide insights for the development of new therapies for vascular diseases.