This revised proposal seeks to understand the mechanisms of platelet adhesion and activation by collagen. Collagen is one of the most thrombogenic components of the blood vessel wall. Therefore, the mechanisms by which platelets adhere to and are activated by collagen is important to our understanding of thrombosis and hemostasis. Dr. Santoro has previously established the role of the alpha2beta1 integrin as a receptor for collagen on platelets and other cell types. The DGEA sequence within the alpha1 chain of collagen has been tentatively identified as a recognition sequence for alpha2beta1. The DGEA sequence occurs twice within the alpha1 chain of collagen. As the first Specific Aim of this proposal, Dr. Santoro will utilize antipeptide antibodies directed at the DGEA and adjacent flanking sequences and site-directed mutagenesis of recombinant alpha1 collagen chains as independent and complimentary approaches to dissect the role of the DGEA sequences in alpha2beta1 recognition. The second Specific Aim relates to the identification of a non-DGEA sequence in recognition of the alpha2 (I) chain of collagen in alpha2beta1 integrin recognition. This chain of collagen lacks a DGEA sequence but still interacts with the receptor. Protein fragmentation, synthetic peptide, inhibitory antibodies and mutant recombinant protein approaches will be utilized to identify the recognition sequence.
The third Aim of this proposal relates to identification of a co-receptor that participates in concert with alpha2beta1, to produce signalling when platelets interact with collagen. There is substantial evidence that secretory pathway is not initiated by interaction of platelets with collagen via alpha2beta1 alone. Monoclonal antibody approaches will be implemented as a means of identifying this co-receptor. Taken together, these studies will potentially lead to an understanding of the molecular mechanisms by which platelets and other cells adhere and respond to collagen.
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