Signaling Role for Phospholipase Cγ2 in Platelet Glycoprotein Ibα Calcium Flux and Cytoskeletal Reorganization

Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca2+ release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP3) and that treating platelets with the IP3 receptor antagonist APB-2 or the PLC inhibitor U73122 blocked cytosolic Ca2+ flux and platelet shape change. Normal shape change was observed in Gαq–/– mouse platelets, excluding a role for PLCβ isoforms in this process. However, decreased shape change and Ca2+ mobilization were observed in mice lacking PLCγ2, demonstrating that this isotype played an important, albeit incomplete, role in GPIb signaling. The signaling pathways utilized by GPIb involved one or more members of the Src kinase family as platelet shape change and Ca2+ flux were inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change and Ca2+ release occurred independently of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, because these platelet responses were normal in human platelets treated with the anti-FcγRIIA blocking monoclonal antibody IV.3 and in mouse platelets deficient in the FcRγ chain. Taken together, these studies define an important role for PLCγ2 in GPIb signaling linked to platelet shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and cytoskeletal reorganization involves a signaling pathway distinct from that utilized by ITAM-containing receptors.

Platelets are specialized blood cells that display unique adhesive properties relevant to hemostasis and thrombosis. Platelet adhesion is critically dependent on the synergistic interaction between multiple platelet receptors and adhesive ligands, with the involvement of specific receptor-ligand pairs dependent on the prevailing blood flow conditions (1). Under conditions of high shear stress, von Willebrand factor (vWf) 1 binding to the platelet adhesive receptor, glycoprotein (GP) Ib/V/IX is critical for the initiation of platelet-vessel wall and platelet-platelet interactions and as such is a key adhesive event promoting thrombus growth (2). In addition to its adhesive function, the GPIb-V-IX complex also plays an important role in regulating the organization of the platelet cytoskeleton (3). For example, it has long been recognized from the study of platelets deficient in GPIb-V-IX (Bernard-Soulier syndrome) that this complex is important for maintaining the compact structure of the membrane skeleton, due at least in part, to the physical linkage between GPIb and actin-binding protein (ABP) 280 (ABP-280 or filamin-1) (4). More recently it has been demonstrated that vWf engagement of GPIb can induce cytoskeletal reorganization (5) and that these cytoskeletal changes play a potentially important role in regulating the adhesive function of GPIb (6). The physiological importance of vWf in inducing cytoskeletal reorganization has previously been highlighted from the study of von Willebrand disease pigs (7). Platelets from these animals adhere to sites of vascular injury; however, they exhibit defective cytoskeletal remodeling leading to reduced filopodial extension and poor spreading.
Despite the potential importance of GPIb in initiating platelet activation, the mechanisms by which GPIb transduces signals linked to cytoskeletal remodeling remains incompletely understood. We have previously demonstrated a potentially important role for intracellular Ca 2ϩ mobilization in this process (5); however, to date, the proximal signaling molecules utilized by GPIb-V-IX to induce cytosolic Ca 2ϩ flux have not been identified. The GPIb-V-IX receptor consists of four protein subunits belonging to the leucine-rich repeat superfamily, GPIb␣, GPIb␤, GPIX, and GPV (8). The receptor intracellular region does not have catalytic activity, nor does it bind GTPbinding proteins or become phosphorylated by tyrosine kinases. Previous studies have suggested that GPIb␣ may signal directly as a consequence of its association with the cytoskeletal structural protein, actin-binding protein (ABP-280) (3), and/or signaling molecules such as 14-3-3 (9 -11), calmodulin (12), Src kinase (13), and phosphoinositide (PI) 3-kinase (14). Alternatively, GPIb may transduce signals indirectly through the physical association with the ITAM-bearing receptors, FcR␥ chain (15,16), or Fc␥RIIA (17,18). FcR␥ and Fc␥RIIA receptor signaling is initiated by Src kinase-dependent tyrosine phosphorylation of the receptors' ITAM motif, leading to the recruitment of p72 syk , PI 3-kinase, and the adaptor proteins (LAT, SLP-76, and vav), which ultimately promote the activation of phospholipase C␥2 (PLC␥2). The subsequent hydrolysis of phosphatidylinositol 4,5-diphosphate, leading to IP 3 generation and intracellular calcium release is a critical event for efficient platelet activation (19,20).
A consensus model for GPIb signaling has yet to emerge, despite intense investigation from a number of groups. In fact, there remains considerable controversy over a number of basic aspects with regards to GPIb signaling, including the contribution of direct and indirect pathways for platelet activation. One possible reason for the discrepancies in results from different studies reflects the variability in experimental approaches used to examine this process. For example, a wide variety of ligands (venom peptides, soluble human or bovine vWf, recombinant vWf fragments, and immobilized vWf), artificial modulators (ristocetin and botrocetin), cell types (human and mouse platelets, GPIb-V-IX-transfected CHO and K562 cell lines), and functional assays (suspension-based aggregation studies, shear-induced platelet aggregation, and static or flow-based adhesion assays) have been utilized to examine GPIb signaling. In many of these studies it is unclear what the direct contribution of the cytoplasmic tails of the GPIb-V-IX complex are for signal generation relative to indirect signaling mediated through the FcR␥ chain and Fc␥RIIA receptor, soluble agonist release, and integrin ␣ IIb ␤ 3 outside-in signaling. Furthermore, it is not always clear that the end-point used to examine GPIb signaling is in fact a direct functional response linked to this receptor.
We have previously established that the vWf-GPIb interaction per se is sufficient to induce cytosolic calcium spikes that are necessary for platelet cytoskeletal remodeling, independent of integrin ␣ IIb ␤ 3 (5). In the current study we have utilized platelet shape change and cytosolic calcium transients as bona fide GPIb-dependent responses to examine the proximal signaling events linked to this receptor. Through the use of various pharmacological inhibitors and knock-out mouse models linked to PLC signaling, our studies have demonstrated an important role for Src kinase-dependent activation of the PLC␥2 isoform in GPIb-dependent calcium flux and cytoskeletal remodeling. In contrast to many other studies, our results do not support an important role for the FcR␥ chain and Fc␥RIIA receptors in GPIb signaling.
Cell Line-Chinese hamster ovary (CHO) cells, expressing the GPIb-IX complex, were established as described previously (26).
Platelet Preparation-Blood was drawn from healthy volunteers who had not taken an anti-platelet medication in preceding 2 weeks and collected in an acid citrate dextrose anticoagulant. Platelets were isolated by sequential centrifugation of the blood and washed as previously described (22). Platelets were finally resuspended in Tyrode's buffer (12 mM NaHCO 3 , 0.3 mM NaH 2 PO 4 , 5 mM Hepes, pH 7.3, 137 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 5.5 mM glucose) containing human serum albumin and apyrase (0.02 unit/ml) and kept at 37°C. Aspirinated platelets were obtained by treating cells with 1 mM aspirin for 15 min, prior to final suspension in Tyrode's buffer. Washed mouse platelets were prepared according to the method described by Moog et al. (27). Mouse blood was taken from the abdominal aorta of anesthetized animals and collected in an acid citrate dextrose anticoagulant.
Platelet Aggregation Studies-Aggregation was performed using a dual-channel Payton aggregometer (Payton Associates, Scarborough, Ontario, Canada). Platelets (3 ϫ 10 8 platelets/ml) were stimulated at 37°C by collagen (1.25 g/ml) in the presence of human fibrinogen (275 g/ml), in a final volume of 500 l. Aggregation was initiated by stirring the platelet mixture at 1100 rpm. When vWf-induced platelet agglutination was examined, aspirinated platelets (3 ϫ 10 8 platelets/ml) were pre-treated with cFab 7E3 (20 g/ml) for 10 min or with EDTA (5 mM), then stirred in the presence of human vWf (20 g/ml) and ristocetin (1 mg/ml). In control studies, the pharmacological activity of the IP 3 receptor inhibitor, 2-APB, was confirmed by complete inhibition of thrombin (1 unit/ml)-induced platelet aggregation at 50 M. The ability of IV.3 (5 g/ml) to block Fc␥RIIA function was confirmed by inhibition of heat-aggregated immunoglobulins (800 g/ml)-induced platelet aggregations.
Cell Adhesion and Morphology Analysis-Human platelet adhesion studies were performed as previously described (5). Briefly, platelets (2 ϫ 10 7 /ml) in Tyrode's buffer were treated with c7E3 Fab (20 g/ml), then allowed to adhere to a HvWf (10 g/ml) matrix for 30 min at 37°C, in the presence of 2 units apyrase. Where indicated, platelets were also preincubated for 10 min with the inhibitors of phospholipase C (U73122 (10 M)), IP 3 receptor (2-APB (20 M)), Src kinases (PP1 (10 M) and PP2 (10 M)), and Fc␥RIIA blocking Ab IV.3 (5 g/ml), prior to application to vWf matrices. In some studies, the treated platelets were then allowed to adhere to vWf in the presence of Ca 2ϩ ionophore A23187 (40 nM) to induce extracellular Ca 2ϩ influx. Non-adherent cells were removed, and adherent cells were fixed with 3.7% formaldehyde for 20 min. In control studies, platelets were fixed in suspension then applied onto a vWf matrix. To examine the cell morphology, the coverslips were mounted onto glass slides with Mowiol 4-88 solution (France Biochem, Meudon, France), and platelets subjected to differential interference contrast microscopy (Leica TCSNT) (63ϫ objective). Alternatively, the fixed adherent platelets were stained with TRITC-phalloidin (2 g/ml) for 30 min and then subjected to fluorescence microscopy (UV illumination at 570 nm, Leica DMDL microscope) (63ϫ objective). The number of adherent cells was scored in five to eight random fields. Platelet shape change was defined by the transformation of platelet morphology from discoid with no filopodial projection to spherical with filopodial projections greater than 0.2 m in length.
When adhesion assays were performed using mouse platelets (3 ϫ 10 7 /ml) or CHO cells (1 ϫ 10 6 /ml), identical experimental conditions were used except that botrocetin (2 g/ml) was present to support cell adhesion to HvWf, and the integrin-vWf interaction was prevented by the addition of EDTA (5 mM) or c7E3 Fab (20 g/ml), respectively.
Immunoprecipitation and Western Blotting Studies-Washed platelets (2 ϫ 10 8 /ml) were treated with c7E3 Fab (20 g/ml) and allowed to adhere to immobilized HvWf (10 g/ml) for 20 min at 37°C. Where indicated, platelets were also pretreated with the inhibitor of Src family kinases, PP2 (5 M), prior to adhesion. The adherent platelets were lysed in a radioimmunoprecipitation assay buffer (10 mM Tris, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158 mM NaCl, 2 mM EDTA, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, and 2 mM benzamidine), scrapped off, and centrifuged at 15,000 ϫ g for 5 min. The supernatant was incubated for 2 h at 4°C with an anti-PLC␥ 2 polyclonal Ab (4 g) in the presence of 30 l of 50% protein A-Sepharose beads. The beads were washed and subjected to 7.5% SDS-PAGE under a reducing condition, immunoblotted with an anti-phosphotyrosine mAb PY-20 or an anti-PLC␥ 2 Ab, and developed with Enhanced Chemiluminescence (Dupont) as previously described (29).
Quantitation of IP 3 Levels-Washed platelets (2 ϫ 10 9 /ml) were treated with cFab 7E3 (20 g/ml) for 10 min, then aggregated with vWf (20 g/ml) in the presence of ristocetin (1 mg/ml) for the indicated period of time. Platelets were then lysed, and IP 3 in the total cell lysates quantitated using a commercial IP 3 assay kit (Amersham Biosciences, UK) according to the manufacturer's instruction. The IP 3 levels were determined using a standard curve established using known amounts of IP 3 .
Analysis of Cytosolic Ca 2ϩ Flux-The platelet intracellular Ca 2ϩ changes were monitored according to the previously detailed method (30). Washed human platelets were loaded with two membrane-permeable Ca 2ϩ indicator dyes, Oregon Green 488 BAPTA-AM-1 (1.25 M) and Fura Red AM (1 M), for 20 min at 37°C. The Ca 2ϩ dye-loaded platelets were treated with c7E3 Fab (20 g/ml) and then allowed to adhere to a vWf (10 M) matrix in the presence of ristocetin (1 mg/ml), under static conditions. The changes in fluorescence ratio of Oregon Green to Fura Red was then measured after 10 min of adhesion using confocal microscopy (1 frame/s) and converted to intracellular Ca 2ϩ concentrations as previously described (31). The Ca 2ϩ concentrations in resting platelets were also determined by measuring the fluorescence ratio (mean ratio ϭ 0.6, n ϭ 390) in platelets applied onto a non-reactive (10% human serum-coated) surface. A transient Ca 2ϩ signal was defined as a change in fluorescence ratio greater than two standard deviations (2 ϭ 0.38, n ϭ 390) above the mean fluorescence ratio (the mean fluorescence ratio in resting platelets). Where indicated, platelets were preincubated with the inhibitors of PLC, U73122 (1 M); of IP 3 receptor, 2-APB (20 M); of Src kinases, PP2 (5 M); or of Fc␥RIIA, IV.3 mAb (5 g/ml), prior to adhesion to vWf.
When these studies were performed on mouse platelets, adhesion assays were carried out in the presence of botrocetin (1 g/ml). EDTA (1 mM) was also present to prevent the integrin ␣ IIb ␤ 3 -vWf interaction and extracellular Ca 2ϩ influx.
Statistical Analysis-The statistical significance of differences between means was evaluated using Student's t test for paired samples, and p values of less than 0.05 were considered to be significant.

The vWf-GPIb Interaction Induces IP 3 Formation-We have previously demonstrated that vWf engagement of GPIb induces transient Ca 2ϩ spikes that initiate cytoskeletal remodeling (5).
In platelets, release of Ca 2ϩ from intracellular stores is primarily mediated by inositol 1,4,5-trisphosphate (IP 3 ), generated through the hydrolysis of the membrane phospholipid, phosphatidylinositol 4,5-diphosphate, by one or more phospholipase C (PLC) isoforms. To date, there is limited evidence that the vWf-GPIb interaction can induce PLC activation and IP 3 generation in platelets (16). To examine this possibility, we initially quantified IP 3 levels in platelets aggregated by vWf (10 g/ml) and ristocetin (1 mg/ml), in the presence of the antiintegrin ␣ IIb ␤ 3 antibody c7E3 Fab. IP 3 levels were determined on platelet aggregates rather than from adhesion assays, be-

FIG. 1. The vWf-GPIb interaction induces IP 3 formation. A,
washed human platelets (2 ϫ 10 9 /ml) treated with the anti-integrin ␣ IIb ␤ 3 mAb (c7E3 Fab, 20 g/ml) or from an individual with Glanzmann's thrombasthenia, were aggregated with HvWf (20 g/ml) and ristocetin (1 mg/ml) for the indicated times. Platelets were then lysed and cellular IP 3 levels determined using a commercial assay kit. Results represent the mean Ϯ S.E. from four independent experiments for normal platelets and one experiment for the individual with Glanzmann's thrombasthenia. B, relative increase in IP 3 levels induced by vWf or thrombin. Washed platelets were treated with buffer (Resting), vWf/ristocetin for 10 s (in the presence of c7E3 Fab), or with thrombin (1 unit/ml) for 30 s. The IP 3 levels were determined as described for A. Results presented are from one experiment, representative of four. C, relative increase in cytosolic calcium in response to vWf or thrombin stimulation. Washed platelets (1 ϫ 10 9 /ml) were loaded with Ca 2ϩ indicator dyes, then pretreated with c7E3 Fab (20 g/ml) for 10 min. Cells (2 ϫ 10 7 /ml) were then applied to a purified vWf matrix (10 g/ml) under static conditions in the presence of ristocetin (1 mg/ml, vWf). The Ca 2ϩ fluorescence change in individual platelets within a random field were monitored for 4 min using a confocal microscope at a scanning rate of one frame every second, and Ca 2ϩ concentrations were determined as detailed under "Experimental Procedures." The results demonstrate the relative increase in Ca 2ϩ levels in platelets adherent to vWf compared with non-stimulated platelets in suspension (Resting) or following activation with thrombin (1 unit/ml, Thrombin). These results are from one experiment, representative of five.
cause we were unable to harvest sufficient quantities of platelet lysates from the latter assays to reliably detect changes in IP 3 . Stimulation of platelets in suspension with vWf/ristocetin resulted in a transient increase in IP 3 levels from a resting concentration (1.1 Ϯ 1.4 pmol/10 9 platelets) up to a peak of 5.2 pmol/10 9 platelets after 10 s of stimulation. By 30 s, IP 3 levels had returned to basal levels (Fig. 1A). A similar transient increase in IP 3 was also observed in Glanzmann thrombasthenic platelets, confirming the lack of involvement of integrin ␣ IIb ␤ 3 (Fig. 1A). In all experiments, the increase in IP 3 induced by the vWf-GPIb interaction was weak relative to other agonists, with thrombin inducing peak IP 3 levels as high as 90 FIG. 2. The IP 3 receptor antagonist, APB-2, inhibits GPIb-induced platelet shape change and intracellular Ca 2؉ mobilization. A and B, washed human platelets (2 ϫ 10 7 /ml) were pretreated for 10 min with c7E3 Fab (20 g/ml) in the presence or absence of the IP 3 receptor antagonist APB-2 (20 M). Cells were either fixed in suspension (Non-Adherent) or allowed to adhere to a vWf matrix (10 g/ml, Adherent and Adherent ϩ APB-2) under static conditions for 30 min. The ability of ionophore A23187 to reverse the inhibitory effects of APB-2 was examined by stimulating APB-2-treated platelets with low dose ionophore A23187 (40 nM) during platelet adhesion on vWf (Adherent ϩ APB-2 ϩ Iono). Platelet morphology was assessed by differential interference contrast microscopy (ϫ63 objective) (A), and the percentage of cells undergoing shape change was quantitated from five random fields from three independent experiments performed in duplicate (B). The results in B represent the mean Ϯ S.E. percentage of total adherent cells. C, Ca 2ϩ dye-loaded platelets (2 ϫ 10 7 /ml) were incubated with either vehicle (0.25% Me 2 SO, Control) or APB-2 (20 M) for 10 min in the presence of c7E3 Fab (20 g/ml). The platelets were then applied onto a vWf matrix (10 g/ml) in the presence of ristocetin (1 mg/ml). The intracellular Ca 2ϩ changes were monitored, and Ca 2ϩ concentrations were determined as detailed for C. Typical Ca 2ϩ flux profiles for three individual platelets (representative of over 100) are shown. D, percentage of total adherent cells undergoing Ca 2ϩ oscillations. Note that in these experiments up to 5% of cells exhibited spontaneous transient calcium spikes prior to adhesion to the vWf matrix (Non-Adherent). These data represent the mean Ϯ S.E. from three independent experiments. pmol/10 9 platelets (Fig. 1B and data not shown).
To investigate the relationship between IP 3 generation and calcium mobilization during platelet adhesion on vWf, platelet Ca 2ϩ levels were monitored using a quantitative dual-dye ratiometric assay, as detailed under "Experimental Procedures." As demonstrated in Fig. 1C, the Ca 2ϩ concentration in resting   FIG. 3. The PLC antagonist, U73122, inhibits GPIb-induced platelet shape change and intracellular Ca 2؉ mobilization. A and B, washed human platelets (3 ϫ 10 7 /ml) were pretreated for 10 min with c7E3 Fab (20 g/ml) in the presence or absence (Ctrl) of U73122 (10 M) or the inactive U73122 analogue U73343 (10 M). Cells were allowed to adhere to an HvWf matrix for 15 min at 37°C in the presence of 2 g/ml botrocetin. Adherent platelets were fixed and stained with TRITC-phalloidin and imaged using fluorescence microscopy. Representative phalloidin-staining images are shown (A). The bar graphs demonstrate the total adherent platelets in five random fields and percentage of total adherent platelets with filopodial extensions (B). Results represent the mean Ϯ S.E. obtained from four independent experiments (***, p Ͻ 0.0001). The treated platelets were stimulated with 1.25 g/ml Type I collagen or 5 M serotonin, and the platelet shape change was detected by the reduction in light transmission through the platelet suspension using a platelet aggregometer (C). Results are from one experiment, representative of four. D, Ca 2ϩ dye-loaded platelets (1 ϫ 10 7 /ml) were incubated with either vehicle (0.25% Me 2 SO, Control), or U73122 (1 M) for 10 min in the presence of c7E3 Fab (20 g/ml). The platelets were then applied onto a vWf matrix (10 g/ml) in the presence of ristocetin (1 mg/ml). The intracellular Ca 2ϩ changes were monitored, and Ca 2ϩ concentrations were determined as detailed for Fig. 1C. Typical Ca 2ϩ flux profiles for three individual platelets (representative of over 100) and percentage of total adherent cells undergoing Ca 2ϩ oscillations are shown. These data represent the mean Ϯ S.E. from three independent experiments. platelets was low. Following adhesion to vWf, ϳ60% of adherent platelets elicited transient Ca 2ϩ spikes ranging from 50 -200 nM (Fig. 1C). Consistent with the low IP 3 levels, GPIb-dependent Ca 2ϩ signals were small relative to those induced by thrombin (up to 2000 nM).
Platelet adhesion on vWf, in the presence of integrin ␣ IIb ␤ 3 antagonists, results in morphological alterations characterized by sphere forming of the cell body and extension of multiple filopodia (5). To further establish the relationship between IP 3 generation, Ca 2ϩ flux, and platelet shape change on vWf, the effects of the IP 3 receptor antagonist, 2-aminoethoxydiphenyl borate (APB-2), were examined. As demonstrated in Fig. 2, APB-2 decreased the proportion of platelets undergoing shape change from 85% in control platelets to less than 20% in treated platelets (Fig. 2, A and B). Pretreating platelets with APB-2 (20 M) completely abolished GPIb-induced Ca 2ϩ spikes (Fig. 2C). In control studies, we confirmed that APB-2 was not having nonspecific inhibitory effects on platelets, because stimulating APB-2-treated platelets with low dose Ca 2ϩ ionophore A23187 (40 nM) restored platelet shape change (Fig. 2, A and B). This effect of ABP-2 was not caused by its blockade of extracellular Ca 2ϩ entry, as GPIb-induced Ca 2ϩ mobilization occurred in the presence of the extracellular Ca 2ϩ chelator EDTA (5) and similar inhibitory effects of APB-2 were observed under these conditions (data not shown).
Involvement of PLC in Platelet Cytoskeletal Reorganization Triggered by the vWf-GPIb-V-IX Interaction-To investigate further the potential involvement of PLC in GPIb signaling, we examined the effects of the PLC antagonist, U73122 (32), on GPIb-induced Ca 2ϩ mobilization and platelet shape change. As shown in Fig. 3A, U73122 reduced the proportion of platelets undergoing filopodial extension from 95% down to less than 5% of platelets (Fig. 3B). The effect on filopodial formation was observed at similar concentrations to those required to inhibit shape change induced by collagen or serotonin, two agonists known to activate the PLC␥ and PLC␤ isoforms, respectively (Fig. 3C). U73122 (1 M) totally prevented GPIb-induced intracellular Ca 2ϩ flux (Fig. 3D). In further control studies, we demonstrated that the structurally related inactive U73122 analogue, U73343, had no effect on vWf-induced platelet shape change, and furthermore, the inhibitory effects of U73122 on platelet shape change could be completely reversed with low dose ionophore A23187 (40 nM) (data not shown).
CHO cells transfected with the GPIb-IX complex are able to adhere to a vWf matrix and extend filopodia in a manner similar to platelets (5). Incubation with U73122 prevented cytoskeletal reorganization in GPIb-IX-transfected CHO cells, whereas control U73343 had no effect (Fig. 4, A and B). Such comparable inhibition in different cellular systems supports the involvement of PLC activation in GPIb-IX signaling leading to cytoskeletal reorganization.
Cytoskeletal Reorganization Induced by vWf-GPIb Interaction Does Not Require the PLC␤ Isotype-To investigate the potential PLC isotypes involved in GPIb-dependent cytoskeletal reorganization, adhesion assays were performed on mouse platelets deficient in the ␣ subunit of the G q heterotrimeric protein (23). Platelets express G q but not its structural homologue G 11 , and as a result G␣ q -deficient platelets do not activate PLC␤ (23). G␣ q -deficient and wild type platelets displayed no difference in their capacity to adhere to a vWf matrix and undergo shape change, indicating that the PLC␤-dependent pathway was not required for vWf-GPIb signaling (Fig. 5, A-C).
In control studies, we confirmed that vWf-induced cytoskeletal reorganization in mouse platelets was PLC-dependent, because it was abolished by the PLC inhibitor U73122 (data not shown).
The vWf-GPIb Interaction Induces PLC␥2 Tyrosine Phosphorylation through Src Family Tyrosine Kinases-Previous studies have reported PLC␥2 tyrosine phosphorylation in vWf-aggregated platelets (15,18), although the functional significance of this event remains unclear. Moreover, it is not clear whether PLC␥2 tyrosine phosphorylation occurs downstream of GPIb or requires vWf binding to integrin ␣ IIb ␤ 3 . In platelets aggregated by HvWf (10 g/ml) and ristocetin (1 mg/ml), in the presence of the anti-integrin ␣ IIb ␤ 3 antibody c7E3 Fab (Fig. 6A), a rapid increase in PLC␥2 tyrosine phosphorylation was observed at 10 s, which slowly decreased over time with kinetics that resemble the production of IP 3 . In comparison to collagen, phosphorylation induced by vWf was weak (Fig. 6A), consistent with low level phosphoinositide turnover in these platelets. To investigate the ability of GPIb to induce tyrosine phosphorylation

FIG. 4. The PLC antagonist, U73122, inhibits GPIb-induced filopodial formation in CHO cells. CHO cells ex-
pressing GPIb-IX were incubated in the absence (Ctrl) or presence of U73122 (10 M) or U73343 (10 M) then allowed to adhere for 30 min to HvWf-coated slides in the presence of 2 g/ml botrocetin and 5 mM EDTA. After fixation and labeling with TRITC-phalloidin, adherent cells were examined by fluorescence microscopy. Representative phalloidin-stained images are shown (A). The bar graphs demonstrate the total adherent CHO cells in eight random fields and percentage of total adherent cells with filopodial extension (B). Results represent the mean Ϯ S.E. obtained from three independent experiments (***, p Ͻ 0.0005).
of PLC␥2 during platelet adhesion on immobilized vWf, platelets were allowed to adhere to a vWf matrix in the presence of the integrin ␣ IIb ␤ 3 antagonist, c7E3 Fab. Platelet adhesion to vWf was associated with the tyrosine phosphorylation of PLC␥2. Pretreating platelets with the Src family kinase inhibitor, PP2, completely eliminated GPIb-induced PLC␥2 phosphorylation (Fig. 6B), confirming an important role for Src kinases in this process.
To investigate the functional significance of Src kinases for GPIb-induced cytoskeletal reorganization, platelet adhesion experiments were performed in the presence of PP1 and PP2. Although PP1 and PP2 had no significant effect on the number of platelets adhering to vWf, they resulted in a Ͼ75% reduction in the proportion of platelets undergoing shape change (Fig. 7,  A and B), and Ca 2ϩ responses (Fig. 7D). Dose-response studies revealed that PP1 and PP2 inhibited shape change induced by vWf and collagen over a concentration range previously demonstrated to be Src kinase-selective ( Fig. 7 and data not shown). Taken together, these studies are consistent with a potential role for Src kinase-mediated activation of PLC␥ isoforms in GPIb-induced cytoskeletal remodeling.
Cytoskeletal Reorganization Induced by vWf-GPIb Interaction Is Regulated by PLC␥2-PLC␥1 and PLC␥2 isotypes have been detected in platelets, and the latter is clearly implicated in signaling by ITAM-bearing receptors (GPVI and Fc␥RIIA). Indeed, PLC␥2-deficient platelets have a major defect in collagen signaling (25,33,34). To investigate the potential role of PLC␥2 in GPIb signaling, adhesion studies were performed on PLC␥2deficient platelets. As demonstrated in Fig. 8B, these platelets adhered as efficiently to vWf as PLC␥2ϩ/ϩ controls, however, PLC␥2-deficient platelets exhibited a significant reduction in their capacity to extend filopodia (Fig. 8, A and C). Studies examining Ca 2ϩ flux demonstrated ϳ35% of adherent wild-type mouse platelets displaying GPIb-induced Ca 2ϩ transients similar to those observed for human platelets, with peak Ca 2ϩ concentrations ranging from 175 to 500 nM (Fig. 8D). In PLC␥2deficient mouse, a decreased number of adherent platelets were able to sustain Ca 2ϩ oscillations. Interestingly, this defect in platelet shape change and Ca 2ϩ mobilization (Fig. 8, D and E) was not as profound as those observed in platelets treated with Src kinase, PLC, or IP 3 receptor antagonists, suggesting the possible involvement of other PLC isoforms in GPIb signaling.
The Fc␥RIIA Receptor and FcR␥ Chain Are Not Required for GPIb-induced Platelet Shape Change-The demonstration that GPIb-induced cytoskeletal reorganization is regulated by Src kinases and PLC␥2, raises the possibility that GPIb utilizes FIG. 5. G␣ q -deficient mouse platelets undergo normal cytoskeletal reorganization. Washed G␣ q ϩ/ϩ and G␣ q Ϫ/Ϫ platelets (3 ϫ 10 7 /ml) were allowed to adhere for 15 min to an HvWf matrix in the presence of 2 g/ml botrocetin and 20 g/ml c7E3 Fab. Adherent cells were fixed and examined by scanning electron microscopy. Representative G␣ q ϩ/ϩ and G␣ q Ϫ/Ϫ platelet scanning electron microscopy images are shown (A). The adherent platelets were also stained with TRITC-phalloidin, the total number of adherent platelets (B) and the number of filopodia extended on each platelet was analyzed in eight random fields and expressed as percentage of total adherent cells (C). These results are the mean Ϯ S.E. from three separate experiments.
FIG. 6. GPIb induces tyrosine phosphorylation of PLC␥2 through Src family tyrosine kinases. A, washed human platelets (2 ϫ 10 9 /ml) were pretreated with the anti-integrin ␣ IIb ␤ 3 mAb (c7E3 Fab, 20 g/ml) for 10 min prior to the initiation of platelet aggregation with HvWf (20 g/ml) and ristocetin (1 mg/ml) for the indicated times. Platelets were then lysed, immunoprecipitated with an anti-PLC␥2 antibody, and subjected to immunoblotting with either an anti-phosphotyrosine Ab (4G10) or an anti-PLC␥2 Ab. As control, washed platelets in suspension were stimulated in the aggregometer with 1.25 g/ml Type I collagen in the presence of fibrinogen. B, PLC␥2 phosphorylation was also studied in vWf-adherent platelets in the absence or presence of the Src kinase inhibitor PP2. Washed human platelets (2 ϫ 10 8 /ml) were incubated for 10 min with c7E3 Fab (20 g/ml). The cells were either lysed in the RIPA buffer while in suspension (Non-adherent) or allowed to adhere to a vWf matrix (10 g/ml) in the absence (Adherent) or presence of PP2 (5 M, Adherent ϩ PP2) for 20 min at 37°C. The adherent cells were then lysed, and PLC␥2 was immunoprecipitated from the lysates and subjected to immunoblotting with either an antiphosphotyrosine Ab (PY20) or an anti-PLC␥2 Ab. The results in A and B are from one experiment, representative of three.
ITAM-containing signaling receptors to induce platelet activation. In support of this hypothesis, the two major ITAM-containing receptors in platelets, Fc␥RIIA receptor and FcR␥ chain, have previously been demonstrated to be physically associated with GPIb (15,17). In addition, recent studies utilizing the Fc␥RIIA function-blocking mAb antibody, IV.3, have dem- FIG. 7. Src kinases are involved in GPIb-induced cytoskeletal reorganization and intracellular Ca 2؉ mobilization. A-C, washed human platelets (3 ϫ 10 7 /ml) were treated with 20 g/ml c7E3 Fab and 5 mM EDTA in the absence (Ctrl) or presence of 10 M PP1. The platelets were then allowed to adhere to HvWf in the presence of 2 g/ml botrocetin, fixed, stained with TRITC-phalloidin, and analyzed under fluorescence microscopy. Representative phalloidin-stained platelet images are shown (A). The number of total adherent platelets and cells with filopodial extensions were analyzed in five random fields (ϫ63 objective) and expressed as percentage of total. Results are the mean Ϯ S.E. from three independent experiments (*, p Ͻ 0.05) (B). The effects of PP1 on platelet shape change induced by collagen (1.25 g/ml) were assessed using an aggregometer (C). Results are from one of experiment, representative of three. D, Ca 2ϩ dye-loaded platelets (2 ϫ 10 7 /ml) were incubated with c7E3 Fab (20 g/ml) in the presence of vehicle (0.25% Me 2 SO, Control) or PP-2 (5 M) for 10 min. Platelets were then applied to a vWf matrix (10 g/ml) in the presence of ristocetin (1 mg/ml) under static conditions. Ca 2ϩ flux profiles in three representative platelets (of over 100) are shown. The accompanying bar graph demonstrates the percentage of total adherent platelets undergoing Ca 2ϩ oscillations in the absence (Adherent) or presence of PP2 (Adherent ϩ PP2). Note that, in this experiment, Ͻ5% of platelets in suspension exhibited transient spontaneous calcium oscillations (Non-Adherent). The results represent the mean Ϯ S.E. from three independent experiments. onstrated an important role for this receptor in regulating PLC␥2 tyrosine phosphorylation and platelet secretion in vWfaggregated platelets (18,35). To examine the involvement of Fc␥RIIA in GPIb-induced shape change, human platelets were pretreated with IV.3. As demonstrated in Fig. 9, IV.3 had no inhibitory effect on GPIb-induced platelet shape change (A and B) or Ca 2ϩ mobilization (C). In control studies, we confirmed that the IV.3 antibody effectively blocks Fc␥RIIA by its ability to completely abolish platelet aggregation induced by heataggregated immunoglobulin (IgG) (data not shown).
A recent study has suggested a potentially important role for the FcR␥ chain in promoting vWf-induced PLC␥2 tyrosine phosphorylation and platelet aggregation (16). To examine the necessity of FcR␥ chain for GPIb-induced platelet shape change and Ca 2ϩ mobilization, studies were performed on platelets derived from FcR␥ chain-deficient mice (FcR␥Ϫ/Ϫ) (24). As demonstrated in Fig. 10, platelets from wild-type (FcR␥ϩ/ϩ) and FcR␥Ϫ/Ϫ mice changed shape and extended filopodia in an identical manner (Fig. 10A). Analysis of the adherent platelets indicated that the percentage of adherent platelets undergoing shape change and the number of adherent platelets were indistinguishable between FcR␥ϩ/ϩ and FcR␥Ϫ/Ϫ mouse platelets (Fig. 10, B and C). A similar pattern of Ca 2ϩ transients was detected in FcR␥ϩ/ϩ and FcR␥Ϫ/Ϫ mouse platelets (Fig. 10D). PLC␥2-deficient mouse platelets have partial defects in GPIb-induced cytoskeletal reorganization and intracellular Ca 2؉ mobilization. Platelet adhesion on vWf was studied on PLC␥2ϩ/ϩ and PLC␥2 Ϫ/Ϫ mouse platelets as described in Fig. 5. Representative PLC␥2ϩ/ϩ and PLC␥2Ϫ/Ϫ platelet scanning electron microscopy images are shown (A). The number of total adherent platelets (B) and the number of filopodia per platelet were analyzed on the TRITC-phalloidin stained platelets in eight random fields and expressed as percentage of total adherent cells (C). These results are the mean Ϯ S.E. from three separate experiments (*, p Ͻ 0.05). D, Ca 2ϩ dye-loaded platelets (2 ϫ 10 7 /ml) from PLC␥2-wild-type (PLC␥2ϩ/ϩ) or PLC␥2-knockout (PLC␥2Ϫ/Ϫ) mice were allowed to adhere to a vWf (10 g/ml) matrix in the presence of EDTA (1 mM) and botrocetin (1 g/ml). The Ca 2ϩ flux profiles in three individual adherent platelets from wild-type or knock out mice were shown. The accompanying bar graph shows the percentage of total adherent platelets undergoing Ca 2ϩ oscillations from four random fields of six independent experiments (typically Ͼ100 cells/experiment). The results represent the mean Ϯ S.E.
In contrast to FcR␥ϩ/ϩ platelets, on a type 1 fibrillar collagen matrix, FcR␥-deficient platelets failed to undergo any Ca 2ϩ responses (Fig. 10D) and morphological changes following adhesion to collagen (data not shown). These findings do not support an important role for the FcR␥ chain for vWf-induced cytoskeletal reorganization.

DISCUSSION
The GPIb-V-IX receptor complex is unique among adhesion receptors in that it not only regulates the cytoskeletal architecture of resting platelets but can also induce cytoskeletal remodeling following engagement of its adhesive ligand (5). Recent evidence suggests that vWf-induced cytoskeletal reorganization may play a potentially important role in regulating the adhesive function of GPIb, relevant to shear-induced platelet activation (6). Despite the potential functional importance of vWf-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for PLCdependent phosphoinositide turnover, leading to IP 3 generation and subsequent calcium mobilization, for GPIb-dependent cytoskeletal remodeling. More specifically, through the analysis of PLC␥2-deficient platelets and pharmacological inhibition of Src kinases, our studies have demonstrated the involvement of PLC␥2, and potentially a second Src kinase-regulated form of PLC, in GPIb signaling. Finally, our studies do not support a major role for the ITAM-bearing receptors, Fc␥RIIA or FcR␥ chain, in GPIb-dependent cytoskeletal remodeling.
Several lines of experimental evidence support a functionally important role for PLC-mediated phosphoinositide turnover for GPIb-induced cytoskeletal change. First, we have demonstrated that the vWf-GPIb interaction is sufficient to induce a small increase in IP 3 , independent of integrin ␣ IIb ␤ 3 . Second, that pharmacological blockade of the IP 3 receptor completely blocks platelet shape change. Third, inhibition of PLC with U73122, but not its inactive structural analogue U73343, abolishes GPIb-dependent cytoskeletal changes. Finally, studies on mouse platelets lacking PLC␥2 demonstrated the involvement of this enzyme in GPIb-dependent cytoskeletal remodeling. These findings, combined with recent reports demonstrating that the vWf-GPIb interaction is sufficient to mobilize calcium from internal stores (5), provide strong evidence that GPIb can induce phosphoinositide turnover in platelets.
The ability of the vWf-GPIb interaction to induce a cytosolic calcium response has remained controversial, with some studies demonstrating transmembrane calcium influx (36,37), other studies suggesting calcium release from internal stores (38,39), whereas other studies have failed to detect a cytosolic calcium signal (16,42). The reason for these discrepancies is likely to reflect technical differences between the studies. From previous studies (5,38) and the present results it appears that GPIb is a weak activator eliciting weak transient Ca 2ϩ response in platelets. Furthermore, in contrast to soluble agonists, vWf does not induce a synchronized calcium response throughout the platelet population, with a low percentage of primary adherent platelets undergoing a calcium response at any one time. The low level, transient IP 3 generation in our studies is consistent with the calcium dynamics observed in primary adherent platelets. These findings may partly explain why other studies have failed to demonstrate IP 3 generation or a detectable Ca 2ϩ signal following vWf engagement of GPIb.
Our studies define an important, albeit not absolute requirement for PLC␥2 for GPIb-dependent cytoskeletal remodeling and Ca 2ϩ mobilization. The partial defect in filopodia formation in PLC␥2Ϫ/Ϫ platelets was accompanied by a partial decrease in Ca 2ϩ signaling suggesting the involvement of another PLC isotype. G␣ q -deficient mouse platelets have impaired receptor-coupled PLC␤ activation and do not aggregate in response to ADP, U46619, or thrombin nor change shape in response to ADP stimulation (23). Although platelets primarily express two PLC␤ isotypes (PLC␤2 and PLC␤3), the demonstration that G␣ q -deficient platelets undergo shape change indistinguishable from normal platelets rules out an important role for these enzymes in GPIb-shape change. Platelets also contain the PLC␤1 and PLC␦1 isotypes, however, their levels of expression are much lower than other PLC isoforms and their roles in platelets and mechanisms of activation remain unclear. FIG. 9. Fc␥RIIA receptor blockade does not modify GPIb-induced shape change and Ca 2؉ mobilization. Washed human platelets (2 ϫ 10 7 /ml) were fixed in suspension (Non-adherent) or applied to a vWf (10 g/ml) matrix under static conditions for 30 min at 37°C in the presence of c7E3 Fab (20 g/ml) alone (Adherent) or together with the anti Fc␥RIIA-blocking mAb, IV.3 (Adherent ϩ IV.3) (5 g/ml). Adherent platelets were fixed then subjected to scanning electron microscopy (A). The bar graph indicates the percentage of total adherent platelets undergoing shape change (B). These results represent the mean Ϯ S.E. from four independent experiments. C, Ca 2ϩ dye-loaded washed platelets (2 ϫ 10 7 /ml) were incubated with c7E3 Fab (20 g/ml) alone or together with mAb IV.3 (5 g/ml) for 10 min. These platelets were then allowed to adhere to a vWf (10 g/ml) matrix, in the presence of ristocetin. The percentage of total adherent platelets undergoing Ca 2ϩ oscillations was analyzed from four random fields from four independent experiments.
Platelets express both members of the PLC␥ family: PLC␥1 and PLC␥2; however the latter is expressed at higher levels than PLC␥1 (40). Nonetheless, it remains conceivable that this en-zyme may also contribute to GPIb signaling, particularly in light of our observations that inhibition of Src kinases or PLCs has a more profound effect on platelet shape change and Ca 2ϩ FIG. 10. FcR␥ chain is not required for GPIb-induced platelet shape change and Ca 2؉ mobilization. Adhesion studies were carried out for washed FcR␥ϩ/ϩ and FcR␥Ϫ/Ϫ mouse platelets, as described in Fig. 5. Representative scanning electron microscopy images for FcR␥ϩ/ϩ and FcR␥Ϫ/Ϫ are shown (A). The number of total adherent platelets (B) and the number of filopodia per platelet were analyzed on the TRITCphalloidin-stained platelets in eight random fields and expressed as percentage of total adherent cells (C). These results are the mean Ϯ S.E. from three separate experiments. D, platelets from normal or FcR␥Ϫ/Ϫ mice were washed and loaded with Ca 2ϩ dyes. The platelets (2 ϫ 10 7 /ml) were then allowed to adhere to a vWf (10 g/ml) matrix in the presence of EDTA (1 mM) and botrocetin (1 g/ml) or collagen type 1 matrix in the presence of EGTA/Mg 2ϩ (1 mM/2 mM), under static conditions. The Ca 2ϩ flux profiles in three individual adherent platelets from FcR␥ϩ/ϩ and FcR␥Ϫ/Ϫ mice are shown. The percentage of total adherent platelets undergoing Ca 2ϩ oscillations was analyzed from four random fields from four independent experiments (typically Ͼ100 cells/experiment). The results represent the mean Ϯ S.E. mobilization than that observed in PLC␥2-deficient platelets. The recent observation of a residual activation to collagen in PLC␥2-deficient mice (33,34) involving the PLC␥1 isotype raises the possibility of a similar involvement of PLC␥1 in GPIb/vWf-triggered activation (34). This possibility will require further investigation.
Our studies do not support an important role for the ITAMbearing receptors, Fc␥RIIa and the FcR␥ chain, for GPIb signaling, at least in the context of cytoskeletal remodeling. Evidence for an important role for Fc␥RIIA in GPIb signaling has been derived from studies demonstrating physical association between the receptors (15,17) and from functional studies demonstrating that the anti-Fc␥RIIA-blocking antibody, IV.3, prevents vWf-induced granule release and Fc␥RIIA tyrosine phosphorylation (18). However, our finding that IV.3 did not inhibit GPIb-dependent cytoskeletal change is not altogether surprising given previous findings that GPIb-V-IX-transfected CHO cells, which do not express Fc receptors (41), undergo cytoskeletal reorganization following adhesion to vWf. Furthermore, in contrast to human platelets, mouse platelets do not naturally express Fc␥RIIA, yet undergo normal shape change in response to vWf. Given the important role of Src kinases, and in particular the involvement of PLC␥2 in GPIb signaling, we sought evidence for the involvement of FcR␥ chain in vWfinduced cytoskeletal changes. It has previously been demonstrated that the FcR␥ chain becomes tyrosine-phosphorylated following vWf stimulation of platelets and that it co-immunoprecipitates with GPIb in GST-Syk pull-downs (16). Furthermore, FcR␥-deficient mouse platelets have decreased phosphorylation of Syk, PLC␥2, and linker for activation of T-cell (LAT) and defective platelet aggregation in response to vWf. This has lead to a model in which vWf engagement of GPIb promotes Src-dependent FcR␥ chain phosphorylation and assembly of a multicomponent signaling complex involving p72 Syk , SLP76, and LAT. However, recent studies (42) have questioned the functional significance of this pathway with respect to GPIb signaling, a finding consistent with our inability to detect differences in the shape change and calcium responses of FcR␥deficient mouse platelets.
The exact signaling pathway utilized by GPIb to induce PLC␥2 activation remains unclear but undoubtedly involves one or more members of the Src kinase family. The mode of activation of Src kinases by GPIb is not obvious, because the intracellular domains of the GPIb-V-IX complex are devoid of Tyr residues and SH2 domains. A recent study has demonstrated an indirect association between GPIb and Src through a complex involving the p85 subunit of PI 3-kinase (13). Other studies have reported co-precipitation of a non-receptor tyrosine kinase, possibly Src, with GPIb, although the molecular basis for this association has not been defined (43). There is also evidence that GPIb may signal through lipid rafts where GPIb has been found to co-localize with signaling enzymes and adaptor molecules (44), although others have not confirmed these findings (36). GPIb may also signal through the assembly of cytoskeletal signaling complexes, because it has previously been demonstrated that the vWf-GPIb interaction can induce the cytoskeletal association of Src and a range of other signaling enzymes (13). Resolution of this important issue will require more detailed analysis of the key structural domains of the GPIb/V/IX complex involved in signal transduction.