Federico Pre-Doc Fellowship: The Role of Pyk2 in Platelet-:eukocyte Interactions and the Arterial Response to Injury

Polymorphonuclear neutrophils (PMNs) represent the largest leukocyte subpopulation in blood and are often the first blood cells to accumulate in inflammatory lesions. The interaction of PMNs with platelets is a pivotal link between inflammation and thrombosis. In our investigations, we have shown that platelet-PMN interactions at the site of vascular damage may also contribute to the arterial injury response and the development of intimal hyperplasia. PMN-platelet adhesion is initiated by P-selectin, a cell adhesive protein expressed at high density on activated platelets, and stabilized by activation of and ligand binding to beta2 integrins present on PMNs. We have recently demonstrated that Src family kinases are activated in PMNs upon platelet contact and are required for firm adhesion mediated by integrin alphaMbeta2. Moreover, genetic and pharmacologic approaches to target this signaling system inhibit the development of intimal hyperplasia in . an experimental model. However, the molecular mechanisms responsible for this signaling cascade are not established. The broad objective of this proposal is to characterize the molecular events that are initiated in PMNs upon interaction with platelets and to asses their contribution to the vascular injury response. Pyk2, which is expressed in cells of myeloid origin, is a non-receptor tyrosine kinase that is closely related to p125 focal adhesion kinase and undergoes tyrosine phosphorylation in beta2-integrin-dependent manner. Based on our preliminary data, we hypothesize that the interaction of PMNs with P~selectin on activated platelets triggers Src family kinase-dependent activation of Pyk2 that is required for full ~2 integrin activation and firm leukocyte recruitment at sites of arterial injury. In Specific Aim One, we propose to test this hypothesis in isolated PMNs and platelets from wild-type and pyk2-deficient mice. In Specific Aim Two, we propose to test this hypothesis by determining the role of Pyk2 in the arterial response and neointimal formation using a well charachterized mouse model of arterial denudation injury. Our results should provide specific insight into signaling systems involved in the interface of inflammation and thrombosis and may provide novel targets for treatment and prevention of vascular disorders.