BackgroundHemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear.Methodology/Principal FindingsUsing γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets.Conclusions/SignificanceThese studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.

Stable platelet-platelet and platelet-extracellular matrix interactions play a critical role in hemostasis and thrombosis. These interactions can be mediated by integrin ␣ IIb ␤ 3 signaling at the sites of vascular injury. The binding of ␣ IIb ␤ 3 to an extracellular ligand-like fibrinogen triggers outside-in signals in platelets via modulation of the activities of several kinases and phosphatases. These signals in turn regulate the cytoskeletal reorganization, which contributes to stable platelet adhesion and spreading events (1). Signaling molecules, including the tyrosine kinase c-Src (2), protein tyrosine phosphatase 1 B (PTP-1B) 3 (3), and the catalytic subunit of protein phosphatase 2A (PP2Ac) (4) either associate constitutively or are recruited to the ␣ IIb ␤ 3 complex in response to integrin engagement. Kinases and phosphatases that assemble within the protein complexes organized by the cytoplasmic tails of ␣ IIb and ␤ 3 could facilitate reversible phosphorylation of multiple effector proteins and mediate outside-in signaling process.Protein phosphatase 2A (PP2A) is a serine/threonine (Ser/ Thr) phosphatase that regulate cell growth, development, and apoptosis. PP2A holoenzyme contains the catalytic subunit C (PP2Ac) and the structural or scaffolding subunit A (PP2Aa). The A subunit of PP2A interacts with multiple regulatory B subunits and regulates the subcellular localization, substrate specificity, and the catalytic activity of PP2A (5). Although, PP2Ac exhibit ␣ and ␤ isoforms, a 10-fold abundant expression of PP2Ac␣ in most cell types (6) had led us to consider PP2Ac␣ in our previous integrin studies (4). These studies revealed that the depletion of PP2Ac␣ in 293 ␣ IIb ␤ 3 cells resulted in an enhanced adhesion to immobilized fibrinogen (4). However, an underlying mechanism is incompletely understood.Alternative model systems like 293 ␣ IIb ␤ 3 cells or Chinese Hamster Ovary (CHO) ␣ IIb ␤ 3 cells have proved useful in elucidating the signaling properties of integrin ␣ IIb ␤ 3 (7-9). In particular, such models are appealing if the role of the signaling molecules under study will be deciphered using a genetic approach. In the context of this study, embryonic lethality of PP2Ac␣-null mice (10), lack of a specific PP2Ac␣ pharmacological inhibitor, and the non feasibility of gene expression in anucleate platelets have limited the extensive use of platelets. The aim of this study in 293 ␣ IIb ␤ 3 cells was to elucidate the molecular events that underpin the PP2Ac␣ mediated negative regulation of ␣ IIb ␤ 3 cell adhesion. In particular; we wished to examine the regulation of c-Src activity (referred to as Src in this manuscript) by PP2Ac␣. c-Src signaling constitutes one of the early signaling events in the outside-in ␣ IIb ␤ 3 signaling process (11).In this study, we identified that loss of PP2Ac␣ activated Src via PTP-1B. Furthermore, Src and PTP-1B activity was required for the enhanced adhesive phenotype displayed by the PP2Ac␣-depleted 293 ␣ IIb ␤ 3 cells. * This work was supported, in whole or in part...

Although protein kinases and phosphatases participate in integrin αIIbβ3 signaling, whether integrin functions are regulated by the catalytic subunit of protein phosphatase 1 (PP1c) isoforms are unclear. We show that siRNA mediated knockdown of all PP1c isoforms (α, β and γ1) in 293 αIIbβ3 cells decreased adhesion to immobilized fibrinogen and fibrin clot retraction. Selective knockdown of only PP1cγ1 did not alter adhesion or clot retraction, while depletion of PP1cβ decreased both functions. Unexpectedly, knockdown of PP1cα enhanced αIIbβ3 adhesion to fibrinogen and clot retraction. Protein interaction studies revealed that all PP1c isoforms can interact with the integrin αIIb subunit. Phosphoprofiling studies revealed an enhanced activation of mitogen-activated protein kinase (MAPK) p38 in the PP1cα depleted cells. Enhanced adhesive phenotype displayed by the PP1cα depleted 293 αIIbβ3 cells was blocked by pharmacological inhibition of p38. Conversely, the decreased adhesion of PP1cα overexpressing cells was rescued by the expression of constitutively active p38α or p38γ. Thus, PP1c isoforms have distinct contribution to the outside-in αIIbβ3 signaling-dependent functions in 293 αIIbβ3 cells. Moreover, PP1cα negatively regulates integrin function by suppressing the p38 pathway.

154 Outside-in integrin αIIbγ3 signaling mediated by kinases and phosphatases regulate platelet adhesion and thrombus formation. The catalytic subunit of protein phosphatase 1 (PP1c), expressed as α, β and γ isoforms regulates a variety of cellular functions. We have previously demonstrated that fibrinogen-αIIbβ3 engagement during outside-in signaling dissociated integrin αIIb*β3 anchored PP1c, which was followed by PP1c activation and dephosphorylation of myosin light chain (MLC). However, it is unknown whether any functions regulated by outside-in integrin αIIbβ3 signaling are modulated by PP1c. To overcome the lack of specificity of pharmacological agents towards PP1c, we have pursued a genetic approach to inhibit PP1c in 293 cells overexpressing αIIbβ3. In this study, we demonstrated that PP1cα interacts with αIIbβ3 in platelets and 293 cells by co-immunoprecipitation and GST pull down assays. Knockdown of PP1cα by short interference RNA resulted in a 50-60% enhanced adhesion (p<0.003) of 293 cells to immobilized fibrinogen. Conversely, overexpression of PP1cα inhibited (p<0.002) αIIbβ3 adhesiveness to fibrinogen. Furthermore, the enhanced adhesiveness of PP1cα depleted 293 cells was also observed with von Willebrand factor, indicating that the differential adhesion due to the lack of PP1cα was not ligand specific. Signaling pathways implicated in the reorganization of cellular cytoskeleton, like extracellular-regulated kinase (ERK1/2) and AKT were activated in PP1cα depleted 293 cells. Such observations imply that PP1cα, knockdown of PP1cγ, which also associates with αIIbβ3, did not affect the adhesion to fibrinogen. Indeed, platelets from the PP1cγ null and wild type mice showed comparable adhesion to fibrinogen. Unexpectedly, depletion of PP1cβ isoform in 293 cells significantly (p<0.01) inhibited the ability of these cells to adhere to fibrinogen. Since MLC is a specific substrate of PP1cβ, it is likely that alterations in the MLC phosphorylation may underlie the loss of adhesiveness in PP1cβ depleted cells. Finally, depletion of all the PP1c isoforms also significantly (p<0.001) inhibited the adhesion of 293 cells to fibrinogen. These data illustrate that each isoform of PP1c contributes distinctly to the adhesive phenotype of integrin αIIbβ3 and implies that the control of outside-in integrin αIIbβ3 signaling response might be a composite of all PP1c isoforms. Perhaps, each isoforms have specific substrates that contribute to the process of adhesion, such that alterations in the phosphorylation of these substrates may underlie the distinct functional roles for PP1c. Disclosures: No relevant conflicts of interest to declare.

190 Signal transduction mediated by the kinases and phosphatases are critical for platelet activation at the site of vascular injury. Compared to the kinases, a role for phosphatases in platelet activation is less understood. Our previous studies have focused on the roles of serine/threonine protein phosphatase 1 (PP1) and 2A (PP2A) in regulating integrin αIIbβ3functions. However, platelets also express protein phosphatase 2B (PP2B) and its role in platelet function is unexplored. PP2B-Aα and PP2B-Aβ constitute two ubiquitous isoforms of the PP2B catalytic subunit. Due to the general concerns regarding the specificity of the PP2B inhibitors, we have utilized mice deficient in the β isoform of the catalytic subunit of PP2B (PP2B-Aβ) to explore the role of PP2B in platelet functions. Mice lacking PP2B-Aα are short lived and are not considered in this study. Loss of PP2B-Aβ did not cause any compensatory increase in the PP2B-Aα levels in platelets. Compared to the wild type (WT) platelets, PP2B-Aβ−/− platelets displayed increased aggregation in response to low doses of protease-activated receptor 4-activating peptide (PAR4-AP), ADP, collagen and collagen related peptide (CRP). Enhanced α granule secretion in response to the low doses of PAR4-AP and CRP was noticed in PP2B-Aβ−/− platelets, relative to the WT platelets. Functions regulated by the outside-in αIIbβ3 integrin signaling like adhesion to immobilized fibrinogen and fibrin clot retraction were enhanced in the PP2B-Aβ−/− platelets. These studies indicate that PP2B-Aβ negatively regulate platelet functions in vitro. Consistent with these observations, PP2B-Aβ−/− mice exhibited a shorter tail bleeding time compared to the WT mice. In a FeCl3 induced endothelial denudation injury model, PP2B-Aβ−/− mice showed decreased time to occlusion in the carotid artery, and reduced number of emboli compared to the WT mice. These studies indicate that PP2B-Aβ suppress multiple murine platelet functions that contribute to an occlusive thrombi. Unlike a positive thrombus promoting role for the PP1cγ that was noticed in our previous study, PP2B-Aβ suppressed murine platelet activation, suggesting that different subtypes of Ser/Thr phosphatases have distinct roles in murine platelet activation. Disclosures: No relevant conflicts of interest to declare.