Platelet activation typically begins with the exposure of the subendothelial collagen matrix and continues as additional platelets are recruited into the growing platelet mass by soluble agordsts. The signal transduction pathways that underlie these events have been the subject of intense scrutiny, with particular emphasis on pathways leading to integrin activation. Building upon observations made during the last funding cycle, the proposed studies will test the hypothesis that intracellular signaling continues even after platelet aggregation has occurred, helping to perpetuate the growth and stability of the platelet plug. Our primary focus will be upon Eph kinases, a family of receptor tyrosine kinases whose tigands, the ephrins, are also cell surface molecules. Interactions between Eph kinases on one cel! and ephfins on another can produce signaling in both. Although Eph kinases and ephrins are best known for their role during development, we have found that human platelets express at least two Eph kinases (EpEA4 and EphB1) and an ephrin (ephrin BI) that is a ligand for both. We have also shown that signaling through Eph kinases and ephrins promotes platelet adhesion, and that blockade of Eph/ephrin interactions will cause platelets to disaggregate. Our investigations of mechanism show that EphA4 forms signaling complexes during platelet aggregation that include the kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Consistent with their proposed role, the binding of ephrins to Eph kinases would not be expected to occur until after platelet aggregation has begun. Drawing on these observations, we now propose the following four specific aims.
Aim #1 will focus on the composition of the signaling complexes that form as a consequence of Eph/ephrin interactions during platelet aggregation, and examine the role of Eph and epbdn phosphorylation in this process.
Aim #2 will focus on the signaling pathways activated by Eptdephrin interactions in platelets, starting with two that we have already identified: activation of the Ras family member, Rap 1B, and phosphorylation of the cytoplasmic domain of the beta chain of alphalIb/beta3.
Aim #3 will test the proposed role of Eph kinases and ephrins in vivo using mouse models.
Aim #4 will examine the hypothesis that interactions between EphA4 and L1 affect the ability of L1 to support platelet aggregation by interacting in trans with L1 and beta3 integrins on the surface of adjacent platelets. Taken together, the proposed studies should provide new insights into the late events of platelet activation, an area of investigation that is relatively unexplored.
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