The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed in the injured vessel wall or atherosclerotic plaque rupture initiates platelet transient adhesion to the injured vessel wall which triggers intracellular signaling cascades leading to platelet activation and thrombus formation. GPIbα. GPIb-IX-VWF interaction-induced activations of Src family kinases and protein kinase C were clearly reduced in S609A mutation. Furthermore S609A mutation significantly inhibited GPIb-IX-VWF interaction-induced elevation of cytoplasmic Ca2+ levels in circulation cytometry analysis. Taken collectively these data show the association of 14-3-3ζ with the cytoplasmic website of GPIbα takes on an important part in GPIb-IX-VWF interaction-induced signaling. Keywords: 14-3-3ζ glycoprotein (GP) Ib-IX platelets von Willebrand element (VWF) 1 Intro The connection of platelet glycoprotein (GP) Ib-IX with von Willebrand element (VWF) exposed in the hurt vessel wall initiates platelet transient adhesion [1-3] and simultaneously causes intracellular signaling cascades Deflazacort  such as activation of multiple protein kinases elevation of intracellular Ca2+ levels and phosphatidylserine (PS) exposure leading to integrin Deflazacort αIIbβ3 activation and integrin-dependent platelet stable adhesion and thrombus formation . Although several typical events have been confirmed Deflazacort to play key tasks in GPIbα-VWF interaction-induced platelet signaling the molecule that initiates the GPIbα-VWF interaction-induced signaling leading to platelet activation remains unknown. Several intracellular molecules that have been confirmed to interact with the cytoplasmic website of the GPIb-IX complex are involved in platelet activation. Filamin A interacted with the cytoplasmic 557-579 sequence of GPIbα regulates tyrosine kinase signaling in platelets under high shear stress conditions [6-9]. Calmodulin binds directly to the juxtamembrane cytoplasmic sequences of GPIbβ and GPV in resting platelets but it dissociates from GPIb-IX when platelets are triggered . Phosphoinositide 3-kinase (PI3-kinase) interacts with the cytoplasmic website of GPIbα and is associated with GPIb-IX-mediated platelet functions . The connection of 14-3-3ζ with Deflazacort the cytoplasmic domains of GPIb-IX takes on a key part in the VWF binding function of GPIb-IX and subsequent platelet activation [12-14]. However the data concerning the part IL1-ALPHA of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling still remain controversial [15-17]. It has been reported that deletion of the 14-3-3ζ binding site in the C-terminal cytoplasmic website of GPIbα inhibited GPIb-IX-mediated αIIbβ3 activation and cell distributing on VWF surface . On the other hand the data from another group indicated that binding of 14-3-3ζ to GPIbα inhibited platelet distributing on VWF surface while disruption of 14-3-3ζ connection with GPIbα improved integrin-induced cytoskeletal reorganization . Therefore the part of 14-3-3ζ in GPIb-IX-VWF interaction-induced platelet activation needs to be further investigated. In the current study the data display that disruption of 14-3-3ζ connection with GPIbα from the S609A mutation induced inhibition of GPIb-IX-VWF interaction-induced signaling cascades. 2 Results 2.1 The S609A Mutation Disrupts the Association of 14-3-3ζ with GPIbα before and after VWF Binding to GPIbα 14 has been confirmed to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX . However the part of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling still remains controversial. We had founded two CHO cell lines expressing crazy type GPIb-IX (1b9) and mutant GPIb-IX replacing Ser609 of GPIbα with alanine (S609A) . In order to investigate the part Deflazacort of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling firstly the VWF binding functions of 1b9 and S609A were assessed by circulation cytometry. Consistent with the previous statement  a certain level of VWF binding to S609A or 1b9 was recognized in the present study and there was no statistical difference in the VWF binding function between 1b9 and S609A cells (Number S1). Then the associations of 14-3-3ζ with GPIbα were examined in 1b9 and S609A cells by coimmunoprecipitation analysis before and after VWF binding. GPIb-IX-expressing CHO cells were firstly stimulated by ristocetin which can induce the association of VWF with GPIbα in the presence or absence of VWF and then were solubilized in cell lysis buffer. The Deflazacort lysates were immunoprecipitated with SZ2 and protein G-conjugated sepharose 4B beads.