The broad long-term objectives of this grant proposal are to enhance our understanding of blood cell adhesion phenomena and to translate that knowledge into improved diagnosis, treatment and prevention of disease. In previous grant periods, the biology of the beta3 integrin receptors GPIIb/llla (alphaIIbbeta3) and alphaVbeta3 was studied using monoclonal antibodies and analysis of patients with Glanzmann thrombasthenia, including the role of alphaIIbbeta3 in platelet function [which led to the development of monoclonal antibody c7E3 Fab (abciximab) as a new antiplatelet therapy] and the role of the alphaVbeta3 receptor in sickle cell adhesion. In addition, studies in a mouse model of sickle cell disease identified a potential direct role of leukocytes in microvascular obstruction. The recent availability of the crystal structure of alphaVbeta3, in conjunction with the P.l.'s studies on the epitope of 7E3, and advances in computer modeling of protein structure and murine embryonic stem cell biology, open new possibilities for understanding integrin structure and function. Thus, in Specific Aim 1, studies will be performed to: A. Define the 7E3 epitope on alphaIIbbeta3 and alphaVbeta3 using multiple techniques and to assess the alphaIIbbeta3 activation mechanism by studying the binding of 7E3 to receptors locked in various states of activation, B. Engineer the 7E3 epitope into mouse alphabbeta3 and alphaVbeta3 by modifying mouse beta3 and produce a mouse expressing the 7E3 epitope for preclinical studies of new indications for abciximab. C. Use a new computer model of alphaIIb to understand better alphaIIbbeta3 structure and function, and the abnormalities in select patients with Glanzmann thrombasthenia, and D. Express select beta3 Glanzmann thrombasthenia mutations in megakaryocytes derived from murine ES beta3-/- cells for functional analysis.
In Specific Aim 2, important aspects of sickle cell disease will be studied, focusing on the poorly understood biology of large vessel arterial stenosis and thrombosis as well as the potential roles of P-selectin and beta3 integrins in microvascular occlusion and organ damage. These will include: A. The acute and chronic response of """"""""Berkeley"""""""" sickle cell mice and appropriate controls to arterial injury. B. The impact of breeding either P-selectin deficiency or beta3 deficiency into Berkeley sickle cell mice on erythrocyte survival, organ damage, and microvascular occlusion.
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