Regulation of Syk activity in GPVI-mediated platelet activation

Activation of platelets is essential for hemostasis. Following damage to the vascular endothelium collagen is exposed, to which platelets stably adhere. After adhesion on collagen, a signaling cascade is initiated, mediated by Glycoprotein VI (GPVI), which results in platelet activation. A major signaling protein in GPVI signaling is Spleen tyrosine kinase (Syk). It undergoes phosphorylation and activation following GPVI stimulation. Syk's central role in this physiological process suggests regulation of its activity is required to maintain the platelets response to collagen within physiological limits. The regulation of Syk activation is the focus of this work. Previously published reports implicate the phosphatases SHP-1, SHP-2 and TULA-2 in the negative regulation of Syk. Therefore, we tested these phosphatases possible role in platelets. We show that SHP-1 can dephosphorylate Syk in vitro, but is unable to bind Syk. Also, Syk is hypophosphorylated in GPVI-stimulated SHP-1 deficient platelets and platelet functional responses are minimally affected compared to wild-type platelets. SHP-2 is unable to bind Syk and Syk is not a good substrate for SHP-2 in vitro. TULA-2 dephosphorylated Syk in vitro and associated with Syk in platelets. In TULA-2 deficient platelets, Syk and PLCγ2 were hyperphosphorylated compared to wild-type platelets. Deletion of TULA-2 resulted in enhanced GPVI-dependent platelet functional responses and a prothrombotic phenotype. c-Cbl has been shown to be a negative regulator of GPVI signaling, possibly by regulating Syk phosphorylation. Thus, SHP-1, SHP-2 and TULA-2’s role in c-Cbl regulation of GPVI was also investigated. We show that TULA-2 is able to bind c-Cbl in platelets. SHP-1 and SHP-2 do not. Furthermore, we show a striking similarity between the phenotype of TULA-2 and c-Cbl deficient platelets. However, in vitro binding studies show TULA-2 is able to bind Syk independently of c-Cbl. Thus, the exact role of c-Cbl in regulating Syk dephosphorylation is unclear. In conclusion, we show SHP-1 and SHP-2 are probably not involved in the negative regulation of Syk. However, TULA-2 is the major phosphatase responsible for the negative regulation of Syk in GPVI signaling. This serves to negatively regulate GPVI-mediated platelet function and prevent uncontrolled platelet activation that could lead to thrombosis.

Grantor dc:publisher

Temple University. Libraries

Year dc:date.issued

2010

Author dc:creator

• Thomas, Dafydd Huw

Advisor dc:contributor.advisor

• Daniel, James L.

Committee members dc:contributor.committeemember

• Ashby, Barrie
• Kunapuli, Satya P.
• Tsygankov, Alexander Y.
• Wang, Hong, 1956 September 19-
• Bergmeier, Wolfgang

• Health Sciences, Pharmacology
• Collagen
• Gpvi
• Phosphatase
• Platelets
• Syk
• Tula

Statement dc:rights

• IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available.

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

eng

Dc Identifier Other

864884803

OAI identifier oai:identifier

oai:scholarshare.temple.edu:20.500.12613/4114