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Platelets undergo profound rapid F-actin cytoskeleton remodeling and experience morphological and functional changes in response to receptor-mediated signaling from damaged blood vessel walls. The evolutionarily conserved proteins coronin 1 and adenylyl cyclase-associated protein 1 (CAP1) regulate the F-actin cytoskeleton and participate in signaling events. They are abundant in platelets but until recently their roles were poorly understood. Subcellular fractionation found coronin 1 is mainly cytosolic, but a significant amount associates with membranes in an actin- independent manner and does not translocate to or from the membrane upon platelet stimulation or inhibition. Coronin 1, 2 and 3 associate with the Triton insoluble cytoskeleton upon platelet stimulation. Immunostainings of spread platelets revealed that coronin 1, 2 and 3 demonstrate strong accumulation at F- actin nodules and display diffuse cytoplasmic localisation with discontinuous accumulation at the cell cortex. This is consistent with the role of coronins as integrators of extracellular signals with actin remodeling. Ablation of coronin 1 in platelets is associated with impaired translocation of integrin β2 to the cell surface upon stimulation with thrombin. However, morphological and functional defects are absent including Arp2/3 complex translocation, VASP phosphorylation, spreading ability, secretion, basal receptor levels, αIIbβ3 activation and haemostasis. While integrin β2 translocation appears specifically or predominantly dependent on coronin 1, the lack of other phenotypes suggests a high extent of functional overlap and redundancy among coronins 1, 2 and 3 in platelets. Subcellular fractionation found that ~20% of CAP1 is membrane-associated in an F-actin independent manner. Immunostainings demonstrated that in basal platelets CAP1 is mostly cortical whereas stimulation results in translocation to the cytosol in a significant proportion of platelets which can be inhibited by prostacyclin or nitric oxide. This places CAP1 at a crossroad of signalling pathways that controls platelet activation by contributing to actin remodelling at the cell cortex and actin nodules during platelet spreading.

DNA and protein sequence data sets have exploded in size over the past several years and have stimulated the development of new methods for closing the sequence/structure gap. Bench laboratory methods, while being the most accurate, are still far too time consuming and limited to breech this gap. Thus computational methods have become the standard. The work described here details the development of a system to automatically select a structural template, align the template and query sequence and model the query. The system was implemented fully for antibodies but maintains a modular design that can accommodate a wider variety of molecule types. Extensive testing was performed using antibodies and the results of this analysis reveal the potential limitations of homology modeling and point toward ways to improve on the template selection method.