Functional Analysis of Platelet Disabled-2 Phosphorylation in Platelet Function and Thrombin Signaling

Platelets are anucleated cells that represent the second most numerous blood cells in the peripheral blood. Appropriate control of platelet activation is important for maintaining normal human physiological haemostasis. Thrombin-induced platelet activation plays a pivotal role in the regulation of haemostsis and thrombosis. Despite thrombin-induced platelet activation has been studied for many years, the mechanistic insight of thrombin signaling is not yet completely understood. Among the platelet proteins, the adaptor protein Disabled-2 (DAB2) is implicates in fibrinogen endocytosis and modulation of platelet function in vitro. Recent studies in our laboratory using knockout mouse as a model system revealed that DAB2 deficiency impairs platelet function and thrombin signaling resulting in a prolonged bleeding time and defect in thrombus formation. Because the expression and isoform of DAB2 and the molecular basis of thrombin signaling are quite different between mouse and human platelets, it is not clear whether human DAB2 plays a similar role as mouse DAB2 in platelets. Notably, we found that thrombin induces DAB2 phosphorylation at Ser723 in human platelets. Because Ser723 is not present in the amino acid sequences of mouse/rat DAB2, the phosphorylation by thrombin is human species-specific and is likely evolved during evolution. Until now, it remains unclear how Ser723 is phosphorylated and what is the significance of DAB2-Ser723 phosphorylation in platelet function and thrombin signaling. We will therefore address these issues in this grant proposal. In the first and second year, we will generate and characterize a phospho-specific antibody that recognizes the Ser723 phosphorylation site of human DAB2. This antibody will be used to investigate DAB2 phosphorylation in human platelet in response to various agonists including thrombin. We will then investigate the functional impacts of DAB2-Ser723 phosphorylation in human platelet using the R9 cellular permeable peptide delivery system. Accordingly, DAB2-Ser723 phosphorylation will be abrogated by delivering R9-DAB2 peptides spanning the phosphorylation site into platelets followed by series of platelet functional assays. In the third year, we will take advantage of lentivirus-transduced gene expression and bone marrow transplantation techniques to generate mouse platelets carrying human DAB2. These mice will be subject to in vivo platelet functional analysis such as bleeding time and thrombus formation. In addition, human DAB2-expressing platelets will be collected from the mice for in vitro functional assays. This study thereby will shed new insight for the underlying mechanisms of thrombin signaling and platelet function and should contribute to our understanding for the functional role of DAB2 expression and Ser723 phosphorylation in human platelets. The accomplishment of this proposal will also provide new insight for platelet biology.

Project ID:PC10301-0137
External Project ID:NSC102-2628-B182-009-MY3