Tyrosine kinase pathways play a critical role in many cellular functions, including immune responses, integrin activation, granule release, growth and differentiation. Whereas the tyrosine kinase pathways involved in the Syk-LAT-PLC?2 pathway, be it downstream of GPVI, Fc?RIIA, or CLEC-2, are well established in platelets, mechanisms involved in the regulation of these pathways through dephosphorylation are not clear. Typically tyrosine phosphatases dephosphorylate these signaling molecules and some protein tyrosine phosphatases (PTPs), such as PTP1B, SHP1 and 2, and CD148 have been identified in platelets. We have identified that TULA2 is a PTP that regulates Syk inactivation in platelets. We have strong preliminary data that supports our hypothesis that as yet uncharacterized tyrosine phosphatases in platelets regulate ITAM and hemITAM signaling events at distinct stages. We propose to test our hypothesis through the following specific aims. 1) We propose that CD45, a PTP that is known to be not expressed in platelets, is expressed in platelets without the extracellular domain and regulates the activation of Src family kinases (SFKs) thereby regulating tyrosine kinase pathways. Recent studies have indicated a link between CD45+ platelets and acute myocardial infarction and restenosis after stent implantation in patients with coronary artery disease. We will demonstrate the existence of such isoform in platelets and evaluate its role in the regulation of tyrosine kinase pathways downstream of ITAM and hemITAM receptors, using CD45 null mice. Our preliminary data demonstrate the functional role of CD45 in the regulation of collagen-induced platelet activation and signaling events as well as points to the reduced SFK activation downstream of GPVI in CD45 null murine platelets. 2) We hypothesize that the tyrosine phosphatase PTPROt regulates Syk activation in platelets downstream of both ITAM and hemITAM receptors. We will test this hypothesis using PTPRO null murine platelets and will identify the mechanism of PTPROt regulation of Syk and SFKs by biochemical approaches. Our preliminary data show that PTPRO null murine platelets have defective responses to collagen. 3) We propose that the tyrosine phosphatase PTPN7 regulates MAP kinase pathways and proximal signaling events in platelets and thereby regulates platelet functional responses. We will evaluate the role of PTPN7 in platelets downstream of agonist receptors including GPVI and CLEC-2 using PTPN7 null mice. Our preliminary studies show that GPVI- mediated ERK2 phosphorylation as well as aggregation, secretion, and thromboxane generation are potentiated in PTPN7 null murine platelets relative to wild type littermates. Understanding these signaling cascades in platelets will help us identify specific therapeutic targets that regulate tyrosine kinase pathways towards the development of antithrombotic agents and anti-hemorrhagic agents.
An indepth understanding of the novel signaling molecules and pathways in platelets will help us understand how signaling pathways are regulated in other cells. Understanding these signaling cascades in platelets will help us evaluate and predict possible implications of the therapeutic agents that could interfere with these pathways.
