The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

Background Hemostasis 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 Findings Using 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/Significance These 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.


Introduction
Agonist-induced platelet activation is critical for maintaining hemostasis at the site of vascular injury. Activation of platelets also contributes to the process of thrombosis following the rupture of atherosclerotic plaques. Thrombin generated at the site of injury binds to protease activated receptors (PAR)1 or PAR4 on human or mouse platelets, triggers protein phosphorylation on serine/ threonine (Ser/Thr) residues and facilitates integrin a IIb b 3 activation [1]. In particular, Ser/Thr kinases such as mitogen-activated protein kinase (MAPK) [2], protein kinase G (PKG) [3,4], protein kinase B/ AKT [5,6], protein kinase C a (PKC a) [7], PKC h [8] and glycogen synthase kinase 3b (GSK3b) [9] play a key role in a IIb b 3 activation by thrombin or PAR4 activating peptide (PAR4-AP). Importantly, thrombin-induced and Ser/Thr kinase-mediated activation of signaling circuitry in platelets is reversible. Since protein phosphatases can fine-tune kinase-mediated signaling processes, we hypothesized that Ser/Thr phosphatases participate in hemostasis/ thrombosis by regulating agonist-induced platelet activation.
Platelets express several Ser/Thr phosphatases, and a pool of the catalytic subunits of protein phosphatase 1 (PP1c) and protein phosphatase 2A (PP2Ac) associates with integrin a IIb b 3 [10,11]. Generic Ser/Thr phosphatase inhibitors like okadaic acid and calyculin A inhibited agonist-induced platelet aggregation, secretion [12][13][14], adhesion and spreading to immobilized fibrinogen [15]. Since these pharmacological agents inhibit several closely related phosphatases (namely, PP1, PP2A and PP4) [16], it is difficult to interpret the specific contribution of protein phosphatase 1 (PP1) and/or its isoforms in agonist-induced integrin a IIb b 3 signaling. Our goal was to address a role for PP1 in platelets using a genetic approach.
PP1 is a major eukaryotic Ser/Thr protein phosphatase that regulates a variety of functions like glycogen metabolism, muscle contraction, transcription, translation and cell division [17,18]. PP1 is a multimeric enzyme formed by the assembly of catalytic and regulatory subunits. PP1c is a 35-38 kDa protein that exists as three isoforms: a, b/d and c sharing greater than 90% amino acid sequence similarity. Two splice variants of PP1cc (PP1cc1 and PP1cc2) have also been identified. All PP1c isoforms are ubiquitously expressed, except for PP1cc2 that is testis specific. Cellular PP1 activity is regulated by multiple factors: 1) reversible phosphorylation of the regulatory subunits 2) dissociation of the regulatory and the catalytic subunits 3) allosteric regulation of the regulatory subunits and 4) inducible expression of the regulatory subunits [17].
Among the various isoforms of PP1c, mice bearing a targeted deletion of the gene for PP1cc [Ppp1cc 2/2 ] are viable and are considered in this study [19]. Using PP1cc 2/2 platelets, we report a moderate decrease in low dose thrombin-induced a IIb b 3 activation, soluble fibrinogen binding, platelet aggregation and thrombus formation. GSK3b-Ser9 phosphorylation that promotes platelet function was also decreased in thrombinstimulated PP1cc 2/2 platelets. GSK3b inhibitor partially abrogated the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cc-/-platelets. These studies suggest a positive role for PP1cc in thrombin-induced platelet functional responses.

Materials
Unless stated, all reagents were from Sigma-Aldrich (St. Louis, MO). Fluorescein isothiocyanate (FITC)-conjugated antibodies (anti-mouse CD41 (a IIb ) and CD62 (P-selectin) were from BD Bioscience (San Jose, CA). Phycoerythin-conjugated JON/A (recognizes activated murine a IIb b 3 ) and thrombin were gifts from Dr. B. Nieswandt (University of Wurzburg, Germany) and Dr. J. Fenton (New York State Department of Health, Albany), respectively. ADP and collagen were from Helena Laboratories (Beaumont, TX). GSK3b inhibitor VIII was from Calbiochem EMD4Biosciences (Darmstadt, Germany). Protease-activated receptor 4-activating peptide (PAR4AP) AYPGKF was synthesized by the Protein Core Facility at Baylor College of Medicine. Collagen-related peptide (CRP) was synthesized at Baylor College of Medicine and cross-linked by glutaraldehyde. Alexa 488conjugated fibrinogen was from Invitrogen, (Carlsbad, CA) while human fibrinogen was from Enzyme Research Laboratories Inc. (South Bend, IN). Antibodies to phospho-AKT Ser473, phospho-GSK3b Ser9 and AKT were obtained from Cell signaling (Boston, MA). Antibodies to thea, and c isoforms of PP1c, PAR3 and PAR4 were purchased from Santacruz Biotechnology (Santacruz, CA). b isoform of PP1c was from Upstate Biotechnology/ Millipore (Billerica, MA).

Mice Platelet Preparation
All animal studies were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine. CD-1 mice carrying the PP1cc targeted deletion [19] and subsequently backcrossed for 10 generations onto Balb/C background were obtained from Dr. S. Varmuza (University of Toronto). For most studies, 8-14 weeks old wild type (WT) and PP1cc 2/2 littermate mice matched for age and gender were used. Blood was collected from the inferior vena cava of isoflurane-anesthetized mice into acid-citric acid-dextrose (ACD) at a ratio of 1:10 (vol/vol). Whole blood was further diluted with an equal volume of Dulbecco's phosphate buffered saline (D-PBS) (Invitrogen), [2.6 mM KCl, 1.4 mM KH 2 PO 4 , 137 mM NaCl, 8 mM Na 2 HPO 4 ] containing ACD (1:9 parts ACD/D-PBS). Platelet rich plasma (PRP) was obtained by centrifugation of the diluted blood at 68 g for 10 minutes at room temperature. PRP was further centrifuged at 754 g for10 minutes and the resulting platelet pellet was washed once in D-PBS and resuspended in D-PBS containing 0.005 U/ml apyrase to avoid desensitization of ADP receptors. Platelets were counted using a Coulter counter (Beckman-Coulter (Z1), Miami, FL) and adjusted to a final concentration of 2.5610 8 /ml and then allowed to rest for at least 1 hour.

Immunoblotting Studies
Resting platelets were lysed with 1% Triton X-100 containing lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors. Forty mg of protein lysate were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies to different isoforms of PP1c, PAR3, PAR4 and actin. In some experiments, washed platelet treated with varying concentrations of thrombin for 2 minutes were lysed and immunoblotted with antibodies to phospho-AKT Ser 473, phospho-GSK3b Ser9, and AKT. Binding of primary antibodies were detected by HRP-conjugated secondary antibodies developed using ECL-system (Amersham Biosciences).

Platelet Adhesion and Clot Retraction
Ninety six well plates were coated with 100 mg/ml of fibrinogen and blocked with 5 mg/ml of Bovine Serum Albumin (BSA). Control wells were coated with only BSA. Fibrinogen or BSA coated wells were incubated with 1610 7 washed platelets in D-PBS supplemented with 1.8 mM CaCl 2 and 0.49 mM MgCl 2 for varying time periods at 37uC. Unbound platelets were washed and the attached platelets were quantified by assaying for acid phosphatase activity at 405 nM. The number of platelets attached was obtained by using a standard curve for absorbance versus cell number. Fibrin-clot retraction, was initiated by the addition of 1 U/ml thrombin to 150 ml of mouse platelet-rich plasma containing (2.5610 8 platelets/ml) supplemented with 3 mM CaCl 2 . After incubating at RT for varying time periods, the amount of liquid not incorporated into the clot is measured. The volume of the clot was determined by subtracting the measured volume from 150 ml (initial volume). Clot volume was expressed as a percentage of the initial volume.

In Vivo Platelet Thrombus Formation
Microvascular thrombosis was examined in the cremaster venules using a previously well described model of light/dyeinduced endothelial injury by intravital microscopy [20,21]. Briefly, male mice anesthetized with 50 mg/kg phenobarbital sodium were placed on a custom plexiglas tray maintained at 37uC with a homeothermic blanket. To assist in breathing, a tracheotomy was performed. Later, an internal jugular vein and common carotid artery were cannulated to facilitate administration of various preparations and monitoring of blood pressure/ heart rate, respectively. The cremaster microvascular bed was prepared, perfused with bicarbonate-buffered saline solution (pH range, 7.35-7.45) at 35uC and transferred to the stage of an upright intravital video microscope (BX-50; Olympus, Tokyo, Japan). After equilibration for 30 minutes, 10 mL/kg 5% FITClabeled dextran (150 kD) was injected intravenously through the jugular vein. A suitable venule was selected after survey of the vascular bed with a 46 objective (NA 0.13) lens. Following the measurement of the diameter and blood flow velocity of the vessel (Doppler velocimeter; Microcirculation Research Institute, College Station, TX), a photochemical injury was initiated by exposing approximately 100 mm of the vessel to filtered excitation light at 0.6 W/cm 2 (from a 175W xenon lamp; Sutter Instrument, Novato, CA; and an HQ-FITC filter cube; Chroma Technology, Brattleboro, VT). Epi-illumination was applied continuously, and the time of onset of platelet aggregates (thrombus onset) and the time of flow cessation (for at least 60 seconds) were monitored and recorded using a 406 water-immersion objective (NA 0.8) by an observer who was blinded to the genotype of the animals. Thrombi were induced in 1-2 venules per animal, and the results for each animal were averaged.

Tail-Bleeding Times
Tails of anesthetized mice (WT and PP1cc 2/2 ) were severed with a scalpel 1 mm from the tip and immediately immersed in a tube containing PBS solution at 37uC. Thirty seconds later, the tail was immersed into a new tube with PBS and this process of transferring the tail to a new tube was repeated until the bleeding stopped. The time from the incision to cessation of blood (as revealed by visual examination of blood in the tubes) were recorded as tail bleeding times.

Statistical Analysis
Results are expressed as means +/2 SEM. Experimental conditions were compared by using paired student's t test.

PP1cc 2/2 Platelets Exhibited Comparable Expression of Other PP1c Isoforms
Immunoblotting studies with an antibody specific for the c isoform of PP1c confirmed the absence of PP1cc protein in PP1cc 2/2 platelets ( Figure 1C). Furthermore, there was no evidence for a compensatory up-regulation of a and b isoforms of PP1c in the platelets from PP1cc 2/2 mice ( Figure 1A and 1B). These blots were stripped and reprobed with actin to ensure equal protein loading (Figure 1 A, Band C lower panel).

PP1cc 2/2 Platelets Exhibited a Mild Agonist-Specific Impairment in Fibrinogen Binding and Aggregation
To determine if the loss of Ppp1cc gene affected platelet reactivity washed platelets from the WT and PP1cc 2/2 mice were stimulated with platelet agonists and analyzed for soluble fibrinogen binding. Low concentrations of strong agonists like thrombin, collagen and dilute suspension of platelets were used to ensure that platelets did not aggregate in this assay. ADP, collagen and CRP induced-soluble fibrinogen binding were comparable between the WT and PP1cc 2/2 platelets (Figure 2A). In contrast, addition of low doses of thrombin to PP1cc 2/2 platelets resulted in a modest but significant decrease in the soluble fibrinogen binding. A ,42% (p = 0.02) and ,45% (p = 0.01) decreased fibrinogen binding in response to 0.01 U/ml and 0.02 U/ml of thrombin was observed in PP1cc 2/2 platelets ( Figure 2B). However, the difference in fibrinogen binding between WT and  PP1cc 2/2 platelets was lost with increasing concentrations of thrombin (not shown). Addition of cation chelator EDTA known to block a IIb b 3 function inhibited fibrinogen binding in WT and PP1cc 2/2 platelets to values obtained in unstimulated platelets.
Many, but not all responses of thrombin in human platelets could be reproduced by thrombin receptor activating peptide [22][23][24]. To explore whether PP1cc participated in thrombin signaling downstream of mouse thrombin receptor PAR4, we stimulated platelets with protease-activated receptor 4 activating peptide (PAR-4AP) and assessed fibrinogen binding. Compared with wild type platelets, PP1cc 2/2 platelets showed only marginally decreased soluble fibrinogen binding in response to PAR-4AP (P = 0.05) ( Figure 2C).
Soluble fibrinogen binding under stirring conditions can lead to platelet aggregation. Next, we determined whether platelet aggregation in response to agonist was altered in PP1cc 2/2 platelets. Compared with WT platelets, PP1cc 2/2 platelets demonstrated ,60% decreased aggregation to 0.02 U/ml thrombin. Aggregation in response to ADP and collagen was comparable between the WT and PP1cc 2/2 platelets ( Figure 2D). Since moderate differences in soluble fibrinogen binding and aggregation between the WT and PP1cc 2/2 platelets were noticed only at low thrombin concentrations and not with other agonists, we conducted the rest of the studies in this report with only low dose thrombin.

, Thrombin Receptors and Normal Granule Secretion
To identify potential mechanisms for the decreased reactivity in thrombin stimulated PP1cc 2/2 platelets, we analyzed the expression of thrombin receptors PAR3 and PAR4. Because reliable antibodies that detect PAR4 by immunofluoresence were not readily available, we subjected platelet lysate to immunoblotting experiments with anti-PAR3 and PAR4 antibodies. Comparable expression of PAR3 and PAR4 receptors was observed between the WT and PP1cc 2/2 platelets ( Figure 3A). Besides PARs, GPIba serves as high affinity thrombin binding site [25]. However, GPIba expression was not different in WT and PP1cc 2/2 platelets (data not shown). Next, we studied the activation status of integrin a IIb b 3 in thrombin-stimulated WT and PP1cc 2/2 platelets by flow cytometry using a murine a IIb b 3 activation-specific JON/A antibody. Under resting conditions, there was no difference in JON/A binding between the WT and PP1cc 2/2 platelets. In contrast, addition of thrombin (0.02 U/ ml) to PP1cc 2/2 platelets resulted in a moderate but significant decrease in JON/A binding ( Figure 3B). Expression levels of a IIb b 3 in the resting and thrombin stimulated platelets were comparable in the WT and PP1cc 2/2 mice ( Figure 3C). This indicates that the decreased JON/A binding in thrombin stimulated PP1cc 2/2 platelets were not due to the altered integrin expression. Potential alterations in platelet granule secretion events in PP1cc 2/2 mice could account for the decreased a IIb b 3 activation at low doses of thrombin. However, secretion of P-selectin (a granule content) was not altered between WT and PP1cc 2/2 platelets ( Figure 3D). Furthermore, addition of exogenous fibrinogen did not rescue the decreased aggregation of thrombin stimulated PP1cc 2/2 platelets (not shown). ADP scavenger apyrase did not abolish thrombininduced fibrinogen binding difference between the WT and PP1cc 2/2 platelets ( Figure 2B). This observation suggests that the fibrinogen binding difference between the WT and PP1cc 2/2 platelets is not dependent on dense granule ADP. Thus, these studies suggest that decreased a IIb b 3 activation and not altered receptor expression or granule secretion may have contributed to the decreased fibrinogen binding in thrombin-treated PP1cc 2/2 platelets.

PP1cc 2/2 Platelet Exhibited Normal Outside in Integrin a IIb b 3 Signaling
We assessed if functions mediated by outside-in integrin a IIb b 3 signaling were altered in the PP1cc 2/2 mice. In contrast to the data from soluble fibrinogen, adhesion of PP1cc 2/2 platelets to immobilized fibrinogen was comparable to the WT platelets ( Figure 4A). Thrombin-induced clot retraction of platelet rich plasma samples was also not altered between the WT and PP1cc 2/2 mice ( Figure 4B). Collectively, these studies imply that outside-in integrin a IIb b 3 signaling processes were not altered by the lack of PP1cc in platelets.

PP1cc 2/2 Mice Exhibited Delayed Thrombus Formation in a Light-Dye Injury Thrombosis Model
To establish if the defect in PP1cc 2/2 platelets observed in vitro might affect thrombus formation in vivo, we studied the responses of WT and PP1cc 2/2 mice to a light/dye-induced injury model that has been previously shown not to denude the endothelium [20]. The initiation time for thrombus formation was not different between the WT and PP1cc 2/2 mice (Fig. 5A). In contrast, we noticed a moderate but significant (P = 0.03) delay in the time for complete cessation of blood flow (occlusion time) in PP1cc 2/2 mice (Fig. 5A). Microvascular diameter and wall shear rate was comparable between the WT and PP1cc 2/2 mice (23.360.3 mm vs. 24.660.5 mm and 515628 sec 21 vs. 516629 sec 21 , respectively). Similarly, blood pressure and heart rate was not significantly different between the WT and PP1cc 2/2 mice (82.861.9 mmHg vs. 81.5621. mmHg and 436614 min 21 and 465612 min 21 , respectively) suggesting that the delayed thrombus formation in PP1cc 2/2 mice was independent of these above mentioned factors. Thus, in this thrombosis model, PP1cc participates modestly in stabilizing thrombus formation. In contrast, tail bleeding times were not different between the WT and PP1cc 2/2 mice ( Figure 5B).

Thrombin-Stimulated PP1cc 2/2 Platelets Exhibited Decreased Glycogen Synthase Kinase3 b -Serine 9 Phosphorylation in an AKT Independent Manner
Platelet functional responses to thrombin are mediated in part by activation of signaling molecules in the PI3K-AKT-GSK-3b circuitry. Interestingly, there exist some similarly in the phenotype of PP1cc 2/2 mice when compared to AKT2 2/2 , AKT1 2/2 and GSK3b +/2 mice. All four null mice predominantly showed thrombin or PAR4 specific impairment in platelet function except AKT1 2/2 mice, which also exhibited defects in platelet responses to collagen [6]. Therefore, we investigated if PP1cc regulates thrombin-induced signaling to AKT. Activation of AKT as measured by phosphorylation of AKT-Ser473 was comparable between the thrombin stimulated WT and PP1cc 2/2 platelets ( Figure 6A and 6B). Next, we assessed the activation of GSK3b (AKT substrate) because PP1 can regulate GSK-3b Ser9 phosphorylation, independent of AKT, under in vitro conditions [26]. GSK3b is a suppressor of platelet function and agonistinduced Ser9 phosphorylation decreases GSK3b kinase activity and promotes platelet function [9]. Consistent with the earlier report [9], thrombin stimulation in WT platelets resulted in sustained phosphorylation of GSK-3b-Ser9. Unexpectedly, thrombin-stimulated PP1cc 2/2 platelets showed a maximum of ,1.9 fold decreased GSK-3b-Ser9 phosphorylation (6A and 6C). These observations imply a role for PP1cc in GSK-3b-Ser9 phosphorylation downstream of thrombin signaling by an AKT independent pathway. It should be emphasized that although a majority of PAR4-stimulated GSK3b-Ser9 phosphorylation was previously shown to be dependent on PI3K/AKT signaling, the same study also reported a fraction of GSK3b-Ser9 phosphorylation that was independent of PI3K/AKT pathway [9]. Thus, a pathway involving PP1cc might represent one of the non-AKT dependent mechanisms that could potentially regulate GSK3b-Ser9 phosphorylation.
Since GSK3b-Ser9 phosphorylation inhibits the constitutive GSK3b activity, we examined whether the decreased GSK3b-Ser9 phosphorylation (active GSK3b) upon thrombin stimulation could contribute to the decreased fibrinogen binding in PP1cc 2/2 platelets. Compared with WT platelets, PP1cc 2/2 platelets exhibited ,40% decreased fibrinogen binding upon stimulation with 0.02 U/ml thrombin in the presence of DMSO (control). However, in the presence of GSK3b inhibitor VIII, PP1cc 2/2 platelets exhibited ,23% decreased fibrinogen binding compared to the WT platelets, suggesting that the difference in fibrinogen binding between the thrombinstimulated WT and PP1cc 2/2 platelets were partially abolished by GSK3b inhibitor. Thus, these studies imply that GSK3 activity, in part, contributes to the decreased fibrinogen binding in PP1cc 2/2 platelets.

Discussion
Compared with Ser/Thr kinases, a specific role for PP1cc in platelet activation is unclear. Although a compensatory upregulation of PP1ca and PP1cb isoforms was not particularly evident in PP1cc 2/2 platelets, a potential redundancy between the individual PP1c isoforms in PP1cc 2/2 platelet function is to be expected and may underlie the mild platelet phenotype seen in PP1cc 2/2 mice. Despite this constrain, our studies uncovers two novel aspects of PP1cc with regards to the platelet biology. 1) PP1cc regulates low dose thrombin-induced integrin a IIb b 3 activation, soluble fibrinogen binding, platelet   aggregation and in vivo thrombus formation. 2) PP1cc is needed for a sustained thrombin-stimulated GSK3b-Ser9 phosphorylation in platelets.
Targeted disruption of the Ppp1c gene in mice led to impaired spermiogenesis and male infertility [19]. Although PP1cc is more intensely studied in the area of reproductive biology, our studies indicate that PP1cc can also regulate platelet activation in an agonist-specific manner. In particular, a subtle role for PP1cc was noted in a IIb b 3 activation, fibrinogen binding and aggregation response to low dose thrombin but not to ADP, collagen and CRP (Figures 2). Perhaps, activation of multiple redundant signaling pathways by higher concentrations of thrombin and/or other agonists might favor activation of PP1ca or PP1cb in the absence of PP1cc to sustain activation of platelets. More importantly, thrombus formation induced by light-dye injury was significantly delayed in the cremasteric venules of the PP1cc 2/2 mice ( Figure 5A). Since PP1cc 2/2 is not a platelet specific knockout, contribution of PP1cc from other cell types within the vasculature towards in vivo thrombosis cannot be ruled out. Nevertheless, this in vivo study is consistent and correlates well with the in vitro platelet data. In contrast to the thrombosis model, normal hemostasis was not altered in PP1cc 2/2 mice as revealed by normal tail bleeding times ( Figure 5B). It is not known whether the differential role played by PP1cc in hemostasis versus thrombosis models was due to the intrinsic differences in the type and extent of injury in the two models, or due to the compensatory mechanisms, which was sufficient to drive hemostasis but not thrombosis.
Previous studies have identified that outside-in integrin a IIb b 3 signaling mechanisms facilitated the dissociation of a IIb b 3 bound PP1c, with the ability to dephosphorylate platelet myosin light chain (MLC) [10]. Following the engagement of integrin a IIb b 3 , PP1cc was shown to be involved in the dephosphorylation of ADP-induced nPKCg activation [27]. These observations imply that PP1cc might regulate platelet functions mediated by outsidein integrin a IIb b 3 signaling processes. However, outside-in integrin a IIb b 3 signaling dependent functions like adhesion and spreading (not shown) to immobilized fibrinogen and clot retraction were not altered in PP1cc 2/2 mice. Since all PP1c isoforms can interact with a IIb b 3 (unpublished observation), one possibility is that the PP1cc isoform does not participate in the functional responses initiated by outside-in integrin a IIb b 3 signaling. Indeed, platelet MLC is dephosphorylated by PP1cb/d (myosin phosphatase) but not PP1cc [28]. Another possibility relates to an easy compensation by other PP1c isoforms following engagement of integrin a IIb b 3 to alter platelet outside-in signaling functions. Finally, the platelet functional data from PP1cc 2/2 mice was not completely consistent with the studies in human platelets treated with Ser/Thr protein phosphatase inhibitors. The latter studies employed calyculin A and okadaic acid and observed inhibition of platelet aggregation and secretion to various agonists and adhesion to immobilized fibrinogen [12][13][14][15]. These differences may stem from the fact that the platelet phenotype obtained in response to the pharmacological agents might be due to inhibition of multiple phosphatases as opposed to inhibition of only PP1cc in this study.
Decreased platelet responses to thrombin observed in the PP1cc 2/2 mice was not due to altered expression of PAR3, PAR4, a IIb b 3 or decreased platelet counts. In fact, we noticed a moderate but significant increase in platelet numbers in PP1cc 2/2 mice [Mean6SEM 596.78610 3 /mm 3 617.94 in WT compared with 693.71610 3 /mm 3 619.87 in PP1cc 2/2 mice; N = 12, P = 0.0003]. However, we cannot rule out the possibility that the lack of PP1cc isoform could alter PAR4 activation associated proximal signaling events such as binding of GTP to the G proteins that couple to PAR4. Perhaps, multiple substrates of PP1cc may exist in the signaling pathways that control a IIb b 3 activation downstream of thrombin signaling. Absence of PP1cc may alter the phosphorylation of one or more of these cellular proteins, thereby suppressing the platelet function. In fact, we noticed that the GSK3b-Ser9 phosphorylation was significantly reduced in thrombin stimulated PP1cc 2/2 platelets ( Figures 6A  and 6C). GSK3b is Ser/Thr kinase and phosphorylation of Ser9 residue inhibits its constitutively active kinase activity [29]. Although an initial study using a more generic and non selective GSK3 inhibitor implied a positive role for GSK3b in platelet function [30], a more recent comprehensive genetic analysis using GSK3b heterozygote mice revealed that GSK3b is a negative regulator of platelet function [9]. Results from the latter study indicated that thrombin stimulated GSK3b Ser 9 phosphorylation and removed the suppressive role of GSK3b on platelet function. Thus, a decreased GSK3b-Ser9 phosphorylation upon thrombin stimulation is predicted to maintain a constitutive GSK3b activity, thereby inhibiting the platelet function. Indeed, our studies revealed that the PP1cc 2/2 platelets stimulated with thrombin had decreased GSK3b-Ser9 phosphorylation along with decreased a IIb b 3 activation, fibrinogen binding, aggregation and delayed thrombus formation. Moreover, pharmacological inhibition of GSK3b partially abolished the difference in fibrinogen binding between WT and PP1cc 2/2 platelets ( Figure 6D). These observations suggest that additional mechanisms that underpin the decreased fibrinogen binding in thrombin-stimulated PP1cc 2/2 platelets exist and remain to be identified.
The molecular basis for the relationship between the loss of PP1cc and decreased GSK3b-Ser9 phosphorylation is currently unclear. PP1cc does not appear to directly dephosphorylate Ser9 on GSK3b, because GSK3b-Ser9 phosphorylation under basal conditions was not significantly increased in PP1cc 2/2 platelets. Although AKT has a major role in GSK3b Ser9 phosphorylation, PP1cc does not appear to regulate GSK3b phosphorylation via AKT, since we observed comparable level of AKT-Ser473 phosphorylation in thrombin stimulated WT and PP1cc 2/2 platelets. A potential for AKT independent regulation of GSK3b-Ser9 phosphorylation was also noted in the previous study [9]. Since PKC, PKA or p90Rsk can also phosphorylate GSK3b at Ser(9) [31], it is likely that PP1cc could affect GSK3b Ser9 phosphorylation by regulating the activation of one or more of these Ser/Thr kinases. Alternatively, PP1cc may inhibit other Ser/Thr phosphatases such as PP2A [32], which may participate in dephosphorylating Ser9 on GSK3b under conditions of thrombin stimulation thereby ensuring increased GSK3b-Ser9 phosphorylation.
In summary, our studies indicate that under conditions of low thrombin stimulation, platelet PP1cc is required for GSK3b-Ser9 phosphorylation, a IIb b 3 activation, soluble fibrinogen binding, platelet aggregation and stabilization of thrombus formation. The agonist specific role for PP1cc is intriguing and future studies will be needed to address how PP1cc couples specifically to signaling by PAR receptors and not to other G-protein coupled receptors.