Date of Award

2012

Document type

Thesis

Degree Name

MSc by research (Master of Science by research)

First Supervisor

Dr Sarah O'Neill

Second Supervisor

Professor Niamh Moran

Keywords

Cardiovascular Diseases, Blood Platlets

Abstract

The pathological role of platelets in cardiovascular disease is well established. The major platelet cell adhesion molecule, am,(33 has been a target for research into anti-thrombotic agents. It is a cell-surface cell adhesion molecule belonging to the integrin family and it plays a critical role in platelet function. It is present on the plasma membrane of circulating platelets in an inactive or quiescent state. Following vascular injury or thrombotic triggers, it ‘activates’ by undergoing a profound change of conformation. However, little is known about the molecular mechanisms underlying this activation. Previous work has identified a regulatory sequence (KVGFFKR) within the cytoplasmic tail that controls integrin activation (Stephens et al., 1998). Subsequently a number of novel platelet cytoplasmic proteins were discovered that interact with this regulatory region. They include CIB1 (Naik et al., 1997), AUP-1 (Kato et al., 2002), PP1 (Vijayan et al., 2004), PP2A (Gushiken et al., 2008), ICln and RN181 proteins (Larkin et al., 2004). Recent work by, confirmed all these proteins and also identified a number of novel platelet proteins that may regulate integrin function by competing for binding to the a-integrin regulatory region (Raab et al., 2010). The aims of this study are to characterize some of these proteins and to investigate their capacity to control integrin function in human platelets.

The proteins that were chosen for this study are cyclic AMP protein kinase a (regulatory subunit; PKARla) and 2 members of LIM protein family: PINCH-1 and the paxillin homologue Hic-5.

Western blotting was used to verify the presence of these proteins in human platelets and to examine their capacity to be altered by platelet activation. We demonstrated that platelets express all 3 proteins. In addition, I show evidence for activation-dependent modification of these proteins in response to pro-thrombotic stimuli. PKARla and Hic-5 show evidence of post-translational modification, whereas PINCH-1 shows evidence of proteolytic degradation in response to platelet activation. Our preliminary evidence (Raab et al., 2010) suggested binding of PKARla, PINCH-1 and Hic-5 to the integrin anbP3 via a motif in the platelet membrane. However not all those findings were consistent with the results that we got. By using co-immunoprecipitation we found that the proteins PKARla and PINCH-1 do not bind with the integrin directly. On the other hand, our observations for Hic-5 suggest that it may be a novel integrin regulating protein.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

File Size

1.69 MB

Comments

A thesis submitted to the Royal College of Surgeons for the degree of Master of Science, 2012.

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