The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein–coupled receptor signaling in platelets

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein–coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ1 revealed that Gβ1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ1-GST protein, and PP1c co-immunoprecipitated with Gβ1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ1) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα−/− murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ1 complexes with myristoylated Gβ1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ1 protein enlists PP1c to modulate GPCR signaling in platelets.

The process of thrombosis entails activation of platelets at the site of ruptured atherosclerotic plaques. At these sites, platelets are likely exposed to several soluble agonists including thrombin, adenosine diphosphate (ADP), and thromboxane A2 (TXA2). These agonists engage distinct G protein-coupled receptors (GPCRs) 4 on platelets and initiate inside-out signaling (1,2). This signaling process activates integrin ␣ IIb ␤ 3 , enables the binding of plasma fibrinogen, and facilitates platelet aggregation. Binding of fibrinogen to activated ␣ IIb ␤ 3 in turn generates outside-in signals that modulate the rearrangement of platelet cytoskeleton and stabilizes the growing platelet clot (3).
An essential mediator of the GPCR signaling is the G protein G␣␤␥ heterotrimers. Agonist binding to GPCR triggers the exchange of G␣ subunit-associated guanosine 5Ј-diphosphate (GDP) with guanosine 5Ј-triphosphate (GTP). This event induces a conformational change in the G␣ subunit, which may lead to the separation or dissociation of G␣ and G␤␥ subunits, and thereby enable the G␣ and G␤␥ subunits to engage with distinct downstream effectors (4). Propagation of signals involves the assembly and disassembly of multiprotein complexes that are regulated in part by protein phosphorylation in a spatial and temporal fashion. Although phosphorylation is regulated by both protein kinases and protein phosphatases, our understanding of how serine/threonine (Ser/Thr) protein phosphatases integrate with the GPCR signaling pathways has lagged behind that of the Ser/Thr protein kinases.
Protein phosphatase 1 (PP1) is a Ser/Thr phosphatase that regulates an entire range of cellular functions including cell division, cell metabolism, and cell death (5). The major isoforms of the catalytic subunit of PP1 (PP1c␣, PP1c␤, and PP1c␥) share ϳ90% amino acid similarity and do not exist freely in cells. Instead, they associate with a wide range of proteins referred to as phosphatase interacting proteins (PIP) in a spatial This work was supported by National Institutes of Health Grants HL081613 and GM112806. K. V. V. is supported by the Mary R. Gibson Foundation. The authors declare that they have no conflicts of interest with the contents of this article. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs, the United States government, or the National Institutes of Health. 1 Both authors contributed equally to this work and are designated as co-first authors. 2

G␤ 1 is a new PP1c interacting protein
To identify PP1c interacting partners in the GPCR signaling pathways, we utilized a yeast two-hybrid (Y2H) system with the full-length PPP1Ca (PP1c␣) as a bait and screened the human bone marrow library. One of the interacting clones was identified as GNB1 (G␤ 1 subunit). G␤ 1 is a component of the heterotrimeric G proteins that associate with GPCRs. To validate the findings from yeast two-hybrid studies, we performed co-immunoprecipitation assays in epitope-tagged cells. G␤ 1 co-immunoprecipitated with PP1c␣, PP1c␤, and PP1c␥ 1 from human kidney embryonal 293 cells transfected with HA-tagged PP1c␣, PP1c␤, PP1c␥ 1 , or FLAG-tagged G␤ 1 plasmids (Fig.  1A). To ascertain the nature of PP1c-G␤ 1 interaction, we performed interaction studies with purified PP1c and G␤ 1 -GST proteins (Fig. 1B). In a glutathione-S-transferase (GST) pulldown assay, purified PP1c bound to G␤ 1 -GST fusion protein but not to GST protein (Fig. 1C), suggesting that G␤ 1 can interact directly with PP1c. These studies identify G␤ 1 as a novel PP1c interacting protein.

Presence of PP1c-G␤ 1 protein complex in platelets and its modulation during platelet activation
To examine if PP1c-G␤ 1 protein complex exists endogenously in platelets, we performed co-immunoprecipitation (Co-IP) assays with platelet lysate. G␤ 1 was immunoprecipitated using anti-G␤ 1 antibody from lysate obtained from unstimulated platelets and platelets treated with thrombin or ADP. Immunoprecipitated samples were immunoblotted with anti-PP1c antibody. Immunoblots of G␤ 1 immunoprecipitates from the resting (Res) platelets detected PP1c. However, the interaction of G␤ 1 with PP1c was decreased following platelet stimulation with GPCR agonist thrombin or ADP (Fig. 2, A and B). These studies suggest that PP1c-G␤ 1 protein complex may dissociate following the engagement of GPCRs.
One of the early consequences of GPCR signaling is the activation of the family of phospholipase C beta (PLC␤). Platelets deficient in both PLC␤ 2 and PLC␤ 3 showed decreased aggregation to low doses of thrombin and ADP (8). Among the PLC␤ isoforms, we noticed that only PLC␤ 3 contained one of the consensus PP1c binding motifs ((K/R)X(V/I)X(F/W)) 27 KFIKW 31 . Because the subcellular localization of PP1c is regulated by its ability to partner with distinct proteins, some of which may bear the PP1c binding motifs, we evaluated whether PP1c could interact with PLC␤ 3 in platelets using co-immunoprecipitation assays. Compared with the resting platelets, we observed an increased interaction of PLC␤ 3 with PP1c from thrombin-or ADP-stimulated platelets (Fig. 2, C and D). These studies suggest that the engagement of GPCR could lead to an association of PP1c with PLC␤ 3 . PLC␤ 3 is phosphorylated, and in particular Ser 1105 phosphorylation of PLC␤ 3 by protein kinase A (PKA) inhibits PLC␤ 3 activity (9). Therefore, we tested if agonist-induced association of PLC␤ 3 with PP1c effected Ser 1105 PLC␤ 3 phosphorylation. Basal phosphorylation of Ser 1105 PLC␤ 3 was evident in unstimulated platelets, and agonist stimulation led to a decrease in the phosphorylation of Ser 1105 PLC␤ 3 (Fig.  2, E and F). Taken together, these studies suggest that thrombin or ADP stimulation of platelets induces the dissociation of PP1c-G␤ 1 complex, which correlates with the association of PP1c with PLC␤ 3 and the subsequent dephosphorylation of the inhibitory Ser 1105 in PLC␤ 3 .

Genetic and pharmacological inhibition of G␤ 1 -reduced GPCR signaling
Compared with the G␣ subunits, the contribution of G␤ and G␥ subunits in platelet GPCR signaling-dependent functions is unclear. Studies with G␤ 1 Ϫ/Ϫ platelets were not pursued because G␤ 1 Ϫ/Ϫ mice die within 2 days of birth (10). Megakaryocytes, from which platelets are derived, have emerged as an alternate system to study GPCR-induced signaling. Specifically, this model system is amenable to genetic manipulation and is most responsive to thrombin receptor protease-activated receptor 4 (PAR4)-dependent signaling (11,12). Megakaryocytes cultured from the bone marrow of the wild-type mice were treated with murine GNB1 siRNA. Depletion of G␤ 1 was evident in megakaryocytes (Fig. 3A). Compared with the control siRNA-treated megakaryocytes, G␤ 1 -depleted megakaryocytes displayed decreased PAR4 activating peptide (PAR4-AP)-stimulated soluble fibrinogen binding (Fig.  3B). Fibrinogen binding was specific to integrin ␣ IIb ␤ 3 because addition of EDTA, a cation chelator, decreased PAR4-AP-dependent fibrinogen binding to base line (unstimulated). For the rest of the manuscript, all functional studies were performed with GPCR agonist thrombin. Next, we tested the effect of gallein (13), a small-molecule inhibitor of G␤␥, on human platelet aggregation. Thrombin-induced platelet aggregation was moderately reduced by gallein treatment (Fig. 3, C and D). Collectively, these studies suggest that genetic depletion of G␤ 1 in megakaryocytes and pharmacological blockade of G␤␥ signaling in platelets can decrease PAR4/thrombin-induced inside-out integrin ␣ IIb ␤ 3 signaling.

Disruption of PP1c-G␤ 1 complexes reduced thrombin-induced platelet aggregation
We noticed a variant of the RVXF PP1c binding motif in G␤ 1 at amino acids 78 through 82, 78 KLIIW 82 . To investigate if this region in G␤ 1 can support the interaction of PP1c, we constructed full-length G␤ 1 plasmids with alanine substitutions at amino acids Lys, Ile, and Trp corresponding to positions 78, 80, and 82, respectively. In addition, a G␤ 1 plasmid with all three amino acids (KIW) mutated to alanine (triple mutant) K27A/ I29A/W31A was also generated. FLAG-tagged alanine mutants of G␤ 1 were co-transfected with HA-tagged PP1c ␣, ␤, and ␥ 1 in HEK 293 cells and lysate subjected to co-immunoprecipitation assays. Cells expressing the wild-type G␤ 1 retained PP1c in the co-immunoprecipitation assay. Unexpectedly, PP1c co-immu-noprecipitated with G␤ 1 containing the alanine substitutions at amino acids Lys, Ile, and Trp. However, G␤ 1 with the triple alanine mutations failed to support PP1c interaction (Fig. 5A). PP1c was adequately expressed under all transfection conditions (Fig. 5A, lowest panel). These studies reveal that simultaneous disruption of amino acids Lys, Ile, and Trp within the variant PP1c binding region on G␤ 1 can diminish PP1c interaction. Thus, this region in G␤ 1 may support an interaction with PP1c.
Based on the interaction studies in HEK 293 cells, we generated a synthetic cell permeable myristoylated (Myr) G␤ 1 peptide ( 75 QDGKLIIWDSY 85 ) and a scrambled control peptide. Thus, the G␤ 1 peptide contained the variant PP1c binding motif and encompassed amino acids 75-85 of the intact protein. We investigated whether the cell-permeable G␤ 1 peptide was capable of disrupting the endogenous PP1c-G␤ 1 complex by co-immunoprecipitation studies. PP1c was detected in G␤ 1 immunoprecipitates obtained from DMSO and the scrambled peptide-treated platelets. Treatment of platelets with the G␤ 1 peptide reduced the interaction of PP1c with G␤ 1 (Fig. 5B). These studies indicate that the myristoylated cell-permeable G␤ 1 peptide can decrease the interaction of PP1c with G␤ 1 complex in platelets.
To evaluate the consequence of perturbing PP1c-G␤ 1 complex on platelet GPCR signaling, we analyzed platelet aggregation in response to low-dose (0.02 unit/ml) thrombin. Pretreat-

PP1c-G␤ 1 complex regulates platelet physiology
ment of human platelets with cell-permeable G␤ 1 peptide but not the scrambled peptide moderately decreased platelet aggregation in response to thrombin (Fig. 6, A and B). G␤ 1 peptide treatment delayed the time to achieve 50% thrombin-induced aggregation. These studies indicate that the complex of PP1c and G␤ 1 participates in platelet function.

Discussion
Platelet activation, in part, via GPCR signaling is critical for hemostasis and thrombosis. Heterotrimeric G proteins G␣ and G␤␥ subunits that associate with GPCRs transduce signals by engaging multiple effectors, including the protein kinases. Here, we show that G␤ 1 subunit interacts with the catalytic subunit of protein phosphatase 1 and modulates GPCR signaling in platelets. Specifically, GPCR signaling-dependent functions such as soluble fibrinogen binding and/or platelet aggre-gation was reduced if (a) G␤ 1 subunit was depleted by siRNA in murine megakaryocytes, (b) PP1c␣ was deleted by Cre-lox approach in murine platelets, (c) G␤␥ signaling was blocked by a small-molecule inhibitor in human platelets, and (d) PP1c-G␤ 1 protein complex was disrupted by a cell-permeable G␤ 1 peptide bearing the PP1c binding motif in human platelets. These studies suggest the importance of coupling G proteininduced inside-out ␣ IIb ␤ 3 signaling with phosphatase-driven signal transduction during platelet activation.
G␣ and G␤␥ subunits engage in multiprotein complexes in a spatial and temporal fashion to facilitate transmission of signals from the GPCR. For example, after GPCR activation, the G␤␥ subunits engage proteins including arrestin, G protein receptor kinase (GRK), PLC␤, PI3K␥, etc. (16). In a yeast two-hybrid screen, we noticed a novel interaction of PP1c␣ with the G␤ 1 subunit. Co-immunoprecipitation assays in epitope-tagged

PP1c-G␤ 1 complex regulates platelet physiology
cells revealed that all isoforms of PP1c could interact with G␤ 1 , whereas in vitro studies using purified proteins suggested that the interaction of G␤ 1 with PP1c was direct and independent of the G␣ and G␥ subunits (Fig. 1). Similar to the ability of G␤ 1 to associate with PP1, G␤-like (G␤L) protein can interact with other subtypes of Ser/Thr phosphatases such as PP4, PP2A, and PP6 in transfected 293 cells (17). Such interactions likely facilitate the recruitment of these phosphatases to the IB kinase (IKK) complex and regulate NF-B signaling. Besides the G␤ subunit, G␣ subunits (G␣ 12 and G␣ 13 ) can also interact with PP2A (18) and PP5 (19) and stimulate phosphatase activity in COS cells.
What is the significance of Ser/Thr phosphatase containing multiprotein complexes? Unlike Ser/Thr kinases that are diversified by gene duplication, the diversity of PP1 holoenzyme is achieved by the ability of PP1c to engage with a variety of proteins in a dynamic fashion to form separate PP1c-protein complexes. Indeed, over 200 distinct preassembled or signal-in-

PP1c-G␤ 1 complex regulates platelet physiology
duced phosphatase complexes have been identified, wherein, phosphatase-interacting proteins generally enable targeting of the catalytic subunit of the phosphatase to distinct subcellular locations and thereby provide specificity to the holoenzyme (6,20). Our studies identify the existence of two phosphatase complexes (PP1c-G␤ 1 and PP1c-PLC␤ 3 ) in platelets, not previously described in any cell types (Fig. 2). PP1-G␤ 1 complex, predominately noticed as a preformed complex in nonstimulated platelets, may facilitate the targeting of PP1 to the vicinity of GPCR and contribute to GPCR signaling. Indeed, we noticed that genetic depletion of G␤1 in murine megakaryocytes reduced GPCR signaling (Fig. 3). However, depletion of G␤1 is unlikely to ensure an unregulated PP1c that can indiscriminately dephosphorylate substrates. Rather, it is widely believed that PP1c does not exist freely in cell and is regulated by a multiprotein complex with PIP. Therefore, under conditions of G␤1 depletion, PP1c might be interacting with other isoforms of G␤ or other PIP and respond to thrombin treatment. Pharmacological inhibition of G␤␥ or disruption of PP1c-G␤ 1 complex also reduced GPCR signaling in human platelets (Figs. 3 and 6). Loss of PP1c␣ or PP1c␥ (an isoform that also interacts with G␤ 1 ) reduced low-dose thrombin-induced platelet aggregation (Fig.  4) (7). Although we have not investigated the molecular target(s) of PP1c-G␤ 1 complex in platelets, previous studies in other cell types have noticed PP1c␣-(21), PP1c␤- (22), and PP1c␥-mediated (23) dephosphorylation of GPCRs as one of the features for the GPCR resensitization process. It is important to note that besides PP1, PP2A (24), PP2B (25), and PP2C (26) are also reported to regulate GPCR phosphorylation.
PP1c-PLC␤ 3 complex observed in agonist-stimulated platelets might target PP1 to regulate PLC␤ 3 phosphorylation. In line with this argument, we noticed that Ser 1105 PLC␤ 3 phosphorylation was reduced in agonist-stimulated platelets (Fig. 2). Because previous studies suggest that Ser 1105 phosphorylation of PLC␤ 3 dampens its activity (9,27), the dephosphorylation of Ser 1105 PLC␤ 3 upon agonist stimulation in platelets may represent one mechanism of PLC␤ 3 activation to transmit GPCR signals. It is important to note that the expression of PLC␤ 2 is higher than PLC␤ 3 in human platelets (28). Moreover, deficiency of PLC␤ 2 in human patients was associated with reduced GPCR-induced calcium mobilization, impaired aggregation, and bleeding diathesis (28 -30). Although PLC␤ 2 isoform is well recognized for relaying GPCR signals, it is possible that PLC␤ 3 may have a redundant role in signal propagation downstream of GPCR. Indeed, platelets deficient in both PLC␤ 2 and PLC␤ 3 showed decreased aggregation to low doses of thrombin and ADP and reduced calcium mobilization and were unable to form stable clots in vivo (8).
Our studies bear some resemblance to previous observations in platelets and heterologous cells wherein PP1c␣ and PP1c␥ formed a complex with the PP1 regulatory protein spinophilin (31), and loss of PPP1R9B (spinophilin) reduced platelet aggregation to thrombin and thromboxane A2 (32). In another study, PP1c associated with a protein complex containing at least 14 -3-3␥ and the regulator of G protein signaling 18 (RGS18) in platelets when the cyclic nucleotide levels were elevated (33). PP1c likely facilitated the dephosphorylation of RGS18 Ser 9 and Ser 218 and the subsequent dissociation of RGS18 from 14 -3-3␥ complex that enabled RGS18 to dampen platelet activation. The detailed understanding of PP1c multiprotein complexes is not limited to academic interest because this knowledge may enable the development of peptides or small molecules that can bind to PP1 and thus disrupt specific protein complexes for functional studies and/or therapeutic modalities. Indeed, a cellpermeable PP1 disrupting peptide (PDP3) containing the PP1c binding motifs of nuclear inhibitor of PP1 (NIPP1) dephosphorylated mitotic substrates of PP1 and sensitized tumors to chemotherapy, which was induced by protein kinase inhibitors (34). In a similar approach, our studies revealed that G␤ 1 cell-permeable peptide disrupted PP1c-G␤ 1 interaction in platelets and reduced thrombin-induced platelet aggregation (Figs. 5 and 6).
There are some intrinsic limitations with this study. G␤ has five isoforms (16,35), and one or more of the G␤ isoforms may have facilitated PAR4-dependent signaling in G␤ 1 -depleted megakaryocytes. In the case of G␤ 1 peptide study, potential coupling of additional phosphatase subtypes PP2A or PP2B with G␤ 1 and/or other G␤ isoforms or G␣ subunits may have facilitated signaling in human platelets. Multiple redundant signaling pathways triggered by the high dose of thrombin could have activated PP1c␤ and PP1c␥ isoforms in the absence of PP1c␣ and sustained platelet activation. These caveats may have contributed to the moderate functional phenotypes we noticed with the different approaches. In addition, the use of peptides that contain PP1c binding motifs can disrupt additional PP1c-PIP interactions. Nevertheless, our observations from several lines of investigation suggest that PP1c-G␤ 1 complex along with PP1c-PLC␤ 3 complex may facilitate GPCR signaling as shown in a proposed model (Fig. 7).
In conclusion, using platelets as a model system, our studies provide a molecular basis for the functional coupling of PP1c with GPCR function/signaling and thus identify a novel phosphatase complex (PP1c-G␤ 1 ) as potential target for therapeutic

PP1c-G␤ 1 complex regulates platelet physiology
intervention in thrombotic and inflammatory conditions. Because GPCR, PP1c, and G␤ 1 are also ubiquitously expressed proteins that are involved in signal transduction in a variety of cell types, our findings will likely have relevance beyond platelets.

Yeast two-hybrid screening
Yeast two-hybrid screening was performed using the Mate & Plate human bone marrow library from Clontech (Palo Alto, CA) (catalog no. 630477). Full-length PP1c␣ (amino acids 1-473) was generated by PCR using primers 5Ј-GCC-GAATTCATGGCGGATTTAGATAAAC-3Ј (forward) and 3Ј-CGGGGATCCCTATTTCTTTGCTTGCTTTG-5Ј (reverse) and ligated between EcoR1 and BamH1 sites of the bait vector pGBKT7. The pGBKT7-PP1c␣ vector was transformed into the yeast strain AH109 using lithium acetate. To study the interaction, AH109 yeasts containing the bait vector were mated with Y187 yeasts containing the cDNA libraries cloned in a prey vector pGADT7. The mated yeasts were plated on selection media lacking leucine (L) and tryptophan (W) and incubated at 30°C until the colonies appear. Potential false positive colonies that grew on dropout medium (ϪLW) were ruled out by also qualitatively analyzing the expression of ␤-galactosidase (␤-gal) marker gene activity by nitrocellulose filter lift assays. Briefly, colonies were transferred to Whatman No. 3 MM filter paper, permeated by brief immersion in liquid nitrogen, and incubated on filter paper saturated with Z-buffer containing 1 mg/ml X-gal at 30°C for 0.5-8 h. ␤-gal-positive transformants were re-plated on synthetic medium lacking adenine, leucine, and tryptophan supplemented with histidine (-ALWH) to screen for Ade2 reporter gene activity assay. Plasmids from the positive colonies were recovered and amplified in Escherichia coli DH5␣ cells and selected for ampicillin and kanamycin resistance. Colonies which showed resistance for both the markers were analyzed by DNA sequencing using the T7 primers. Approximately 5 ϫ 10 6 transformants were screened. Plasmids recovered from yeast were re-transformed into native AH109 yeast with the bait construct or nonspecific control selected on high stringency plates with SD/ϪAde/ϪHis/ϪLeu/ ϪTrp/X-␤-galactosidase to confirm the interaction.

Human platelet isolation
Blood from healthy donors was drawn in an acid citrate dextrose anticoagulant or 3.8% citrate after all the donors signed the informed consent, which was approved by the Institutional Review Board of Baylor College of Medicine. Blood was centrifuged at 189 g for 15 min at room temperature to obtain platelet-rich plasma (PRP). In some experiments, platelet-rich plasma supplemented with 75 nM prostaglandin E 1 was recentrifuged at 524 g for 10 min. The resultant platelet pellet was resuspended in Tyrode's buffer and centrifuged. Washed platelets were then suspended in Tyrode's buffer and counts adjusted to 2.5 ϫ 10 8 platelets/ml.

GST pulldown assays
GST or G␤ 1 GST (Abnova) (4 g) pre-coupled with glutathione beads was mixed with 1 g of recombinant polyhistidine-tagged PP1c (Santa Cruz Biotechnology) for 3 h at 4°C. Beads were washed three times and the interacting proteins separated by SDS-PAGE and immunoblotted with anti-PP1c antibody.

Generation of DNA constructs and transfection assays
FLAG-tagged cDNA encoding full-length GNB1 and cloned in pCDNA3.1 was purchased from the cDNA Resource Center, Bloomsburg University. Generation of G␤ 1 constructs with single alanine mutations (K27A, I29A, W31A) and triple alanine mutations (K27A/I29A/W31A) was performed using site-directed mutagenesis kit (QuikChange II XL) (Agilent Technologies). All constructs were sequenced and verified to contain the desired mutations. Full-length cDNAs for PP1c ␣, ␤, and ␥ cDNA were subcloned into pCMV vector between EcoR1 and Not1 sites to generate HA-tagged PP1c. HEK 293 cells were co-transfected with HA-tagged PP1c ␣, ␤, ␥ and FLAG-tagged full-length G␤ 1 using Lipofectamine supplemented with PLUS reagent (Life Technologies). In some experiments, FLAGtagged G␤ 1 alanine mutants were used. Cells transfected with empty FLAG and HA vector (EV) served as control. After 48 h, cells were washed with phosphate-buffered saline (PBS) and lysed with radioimmune precipitation assay buffer supplemented with 1 mM Na 3 VO 4 and 1 g/ml leupeptin.

Generation of platelet-specific PP1c␣ ؊/؊ mice
All experiments with mice were performed following approval by the Baylor College of Medicine Institutional Animal Care and Use Committee. PP1c␣-loxP flox (fl) mice were generated in collaboration with Lexicon Genetics as described previously (14). Briefly, exon 3 of PP1c␣ was flanked with loxP sequence that can be recognized by Cre recombinase. PP1c␣ flox/flox homozygote mice were crossed with a megakaryocyte/ platelet-specific Pf4 Cre knock-in mice (The Jackson Laboratory) (15) to obtain platelet-specific PP1c␣ knock-out mice (PP1c␣ Ϫ/Ϫ ). All studies were performed using male and female littermates (PP1c␣ fl/fl and Cre positive, referred to as PP1c␣ Ϫ/Ϫ ) and PP1c␣ fl/fl and Cre negative, referred to as wild type (WT ϩ/ϩ ).

Co-immunoprecipitation assays and immunoblotting
Lysate from transfected 293 cells were immunoprecipitated with anti-FLAG antibody. Samples were separated on a SDS-PAGE and immunoblotted with anti-HA and anti-FLAG antibodies. Platelets (resting) or following stimulation with thrombin (0.5 unit/ml) or ADP (10 M) for 2 min under non-stirring conditions were lysed with radioimmune precipitation assay buffer and subjected to immunoprecipitation assays. In some experiments, platelets were treated with 0.2% vehicle DMSO, 200 M myristoylated G␤ 1 peptide ( 75 QDGKLIIWDSY 85 -NH 2 ) (Selleck Chemicals) or Myr-control scrambled peptide (DQI-LYDSKGWI) for 30 min at 37°C before lysis. Platelet lysates were immunoprecipitated with control IgG, anti-G␤ 1 (Santa Cruz) or anti-PP1c (Santa Cruz) antibodies and immunoblotted with anti-PP1c or anti-PLC␤ 3 antibodies, respectively. In some experiments, lysate of platelets simulated with agonist was separated on a SDS-PAGE gel and immunoblotted with anti-phospho PLC␤ 3 (Ser 1105 ) (Cell Signaling) and anti-PLC␤3 (Cell Signaling) antibodies, followed by HRP-conjugated sec-ondary antibodies, and blots were developed using SuperSignal chemiluminescent substrate (Thermo Scientific). Densitometry was performed using ImageJ software (National Institutes of Health).

Murine megakaryocytes, knockdown, and flow cytometry
Megakaryocytes were obtained from the bone marrow culture of the C57BL/6J black mice as we have described previously (12). Megakaryocytes on day 5 were transfected with 100 nM control siRNA or GNB1 siRNA using Mirus transfecting agent (Mirus Bio) for 48 h at 37°C. On day 7, megakaryocyte suspension was lysed and immunoblotted with anti-G␤ 1 and actin antibodies. An aliquot (50 l) of megakaryocyte suspension was mixed with 1 mM PAR4-AP (AYPGKF) (protein sequencing Advanced Technology Core Laboratories, Baylor College of Medicine) along with nonblocking anti-␣ IIb antibody, 7-amino-actinomycin D (7-AAD) (BD Biosciences), and Alexa 488 conjugated fibrinogen (20 g/ml final concentration) (Thermo Scientific) in the presence and absence of 10 mM EDTA for 20 min. Using the Coulter EPICS XL Flow Cytometry System (Beckman Coulter), Alexa fibrinogen binding was measured as mean fluorescence intensity (MFI) in FL1 channel only from a gated population of large megakaryocytes (size) that expressed ␣ IIb ␤ 3 (FL2) and were viable (negative for 7-AAD in the FL3).

Aggregation assays and soluble fibrinogen binding
Platelet aggregation was analyzed under stirring conditions using eight-channel PAP-8C aggregometer (Bio/Data Corp.) following one of the treatments described below. Washed human platelets were preincubated with DMSO (0.2%), 10 M and 20 M gallein (Tocris Bioscience) for 30 min, and then challenged with thrombin (0.05 unit/ml). Washed murine platelets (WT and PP1c␣ Ϫ/Ϫ ) were stimulated with low (0.02 unit/ml) and high (1 unit/ml) thrombin. In other studies, washed human platelets were treated with DMSO, 200 M G␤ 1 , or scrambled peptide for 30 min and then challenged with 0.02 unit/ml thrombin. To study the binding of soluble fibrinogen, washed resting (basal)-and thrombin (0.02 unit/ml)-stimulated murine platelets diluted to 2.5 ϫ 10 7 /ml were incubated with Alexa 488 conjugated fibrinogen (Thermo Scientific) and analyzed by flow cytometry as we have described previously (7).

Statistics
Data are expressed as mean Ϯ S.E. and the statistical significance of the data analyzed using GraphPad Prism. For analysis of two groups, paired Student's t test was used, and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test were used for comparison of differences among multiple groups. p Ͻ 0.05 was considered statistically significant.
Author contributions-S. P. performed all studies related to human platelets and murine megakaryocytes and analyzed all data. T. K. performed the yeast two-hybrid studies, generated the platelet-specific PP1c knock-out mice, conducted murine platelet studies, and analyzed data. A. C. N. provided the PP1c␣ flox mice and edited the paper. K. V. V. conceived, designed, and interpreted the study and wrote the paper.