Platelet activation begins with the initial deposition of platelets on a damaged vessel wall, then continues asadditional platelets are recruited and adhere to each other. These events bring platelets into stable contactwith each other, forming junctions where protein:protein interactions can occur between adjacent platelets.The long term goal of this project is to understand how events at platelet junctions contribute to the plateletresponse to injury. Our hypothesis is that 1) relevant signaling continues after platelet aggregation hasbegun, 2) some of the signaling arises from interactions between molecules other than integrins on thesurface of adjacent platelets, and 3) these contact-dependent interactions at junctions can serve either aspositive regulators, promoting the growth and stability of the hemostatic mass to prevent re-bleeding, or asnegative regulators, limiting growth and stability so that vascular occlusion is avoided. During the mostrecent funding period we have identified ephrin B1 and semaphorin 4D on the platelet surface, and shownthat the binding of these ligands to their respective receptors (EphB1 and EphA4 for ephrinBI; CD72 andplexin B1 for sema4D) promotes thrombus growth. We have also determined that ESAM, a putative celladhesion molecule in the CTX family, translocates to junctions when platelets are activated and then acts asa negative regulator, so that loss of ESAM expression promotes, rather than impairs, extension of theplatelet mass. The studies described in this proposal are divided into four specific aims focusing on plateletjunctions and contact-dependent interactions.
Aim #1 will test our current model that ESAM is a negativeregulator of platelet:platelet interactions and explore the consequences of a loss of ESAM function onplatelet activation using an existing line of ESAM knockout mice.
Aim #2 will focus on the molecular basis forESAM's contribution, starting with our recent identification of two scaffold proteins, NHERF-1 and CAL, thatbind to the ESAM cytoplasmic domain.
Aim #3 is a comparative analysis of the three other CTX familymembers expressed in platelets (JAM-A, JAM-C and CD226) to determine whether their role is the same asESAM. Initial results obtained with JAM-A knockout mice, suggest that this may be the case.
Aim #4 isdevoted to the characterization of additional junction molecules in platelets, starting with ephrin A1 onplatelets and continuing with an unbiased search for novel proteins.
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