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Prepublished online as a Blood First Edition Paper on December 19, 2002; DOI 10.1182/blood-2002-09-2807.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Medicine and the Program in
Genetics, State University of New York, Stony Brook; the Massachusetts
General Hospital Cancer Center, Harvard Medical School, Charlestown;
and the Department of Medicine, Veteran's Administration Medical
Center, Northport, NY.
Human blood platelets are anucleate cells whose response to
extracellular stimuli results in actin cytoskeleton rearrangements, thereby providing the critical initial step in the regulation of
hemostasis. The serine protease Remodeling of actin filaments represents the
penultimate event in activation-dependent cellular migration and
morphogenesis, cytoskeletal changes that are dramatically recapitulated
in activated blood platelets. Various extracellular stimuli rapidly
transform the normally quiescent discoid platelet to a contractile
sphere extruding lamellae and filopodia, physiologic structures that modulate the first key steps in hemostasis by regulating platelet adhesiveness and aggregation. In these cells, specific GTPases appear
to be responsible for distinct types of actin assembly that would lead
to the formation of either filopodia (presumably involved in
fibrin-platelet or platelet-platelet interactions) or lamellae
(involved in adhesion for plugging vascular leaks).1 Rho
GTPases form a subgroup of the Ras superfamily that is largely involved
in the regulation of cytoskeletal organization in response to
extracellular stimuli.2 Like all members of the
superfamily, the activity of Rho GTPases is determined by the ratio of
their guanosine triphosphate-guanosine diphosphate (GTP/GDP)-bound
forms, which are regulated by the opposing effects of guanine
nucleotide exchange factors (GEFs) The serine protease Molecular studies
Platelet preparation and immunoprecipitation analyses
The F-actin content for individual experiments was monitored by parallel determination using flow cytometry.9 In brief, aliquots of GFPs or PRP (stimulated or unstimulated) were fixed in 3.7% formaldehyde followed by incubation with 30 U/mL fluorescein isothiocyanate (FITC)-phalloidin (Molecular Probes) on a rotator for 60 minutes at 25°C. After washing with phosphate-buffered saline (PBS), the fluorescent geometric means of 1 × 104 platelets were determined by fluorescence-activated cell sorter (FACS), and results at individual time points were expressed as the ratio to unstimulated labeled platelets. Complex assembly and activation state of GTPases GTP-charged complex assembly was completed using a bacterially expressed glutathione-S-transferase (GST) chimer containing a C-terminal PAK1 binding domain (PBD) sequence from the p21-activated kinase (p21PAK, PAK1).10 Commercially available GST-PBD beads with a capacity of 0.5 µg/µL (Upstate Biotechnology) were used interchangeably with a functionally similar product generated from pGEX2TK-PBD or mutant GST-PBD incapable of binding GTP-bound forms of rac1 or cdc42 (PGEX2TK-PBDL83L86) (both kindly provided by Dr L. van Aelst, Cold Spring Harbor Laboratory, NY). Plasmids expressing GST-PBD, mutant GST-PBD, or wild-type GST (pGEX2X) were used for recombinant protein generation in Escherichia coli host strain BL21 with glutathione Sepharose beads essentially as previously described10,11 and were used within 24 hours of preparation. Preparations were standardized using bicinchoninic acid protein determination assays (Pierce) and were confirmed by SDS-PAGE using albumin standards. Platelets (5 × 107) resuspended in 200 µL HBMT/2 mM MgCl2 were activated with individual agonists and immediately solubilized in ice-cold 4 × RIPA/8 mM MgCl2 (supplemented with protease inhibitors outlined above), followed by gentle rocking for 60 minutes at 4°C in the presence of 5 µg GST-PBD, GST, or mutant GST-PBD beads. After 3 washes in ice-cold 1 × RIPA buffer, samples were analyzed by SDS-PAGE and immunoblot analysis. For standardization, equivalent aliquots of unstimulated platelets were lysed in the identical RIPA buffer supplemented with 1 mM dithiothreitol and 1 mM MgCl2 and were loaded with either 100 µM GTP S or 100 µM GDP S (Sigma)
for 60 minutes at 25°C, before affinity purification onto
GST-PBD.
GST-PBD specificity assays for some experiments were completed by COS1 transfections (1 × 107 cells) using dominant-negative rac1N17 and cdc42N17 or constitutively active rac1V12 and cdc42V12 mutants (kindly provided by Dr D. Bar-Sagi, Stony Brook, NY). At 72 hours, cells were directly solubilized and incubated with 5 µg GST-PBD in RIPA buffer as outlined above, followed by SDS-PAGE and immunoblot analysis using rac1 or cdc42 antibodies. Platelet immunofluorescence GFP resuspended in TSE (10 mM Tris [pH 7.4], 150 mM NaCl, 10 mM EDTA) were immediately fixed in 2% paraformaldehyde (PFA)-PBS followed by centrifugation at 1500g onto glass slides (unactivated) or were directly centrifuged onto glass slides at 1500g before fixation using 2% PFA/PBS (activated). Agonist-activated platelets were stimulated in suspension using 10 nM thrombin or 20 µM PAR142-47 for 60 seconds, followed by immediate fixation and centrifugation onto glass slides. Cells were permeabilized using 0.1% Triton/HS (20 mM HEPES [pH 7.5], 150 mM NaCl, 10% glycerol), blocked by washing 3 times with 0.1% bovine serum albumin (BSA)-PBS at 25°C, and stained using anti-IQGAP2 (1:50) for 60 minutes at 25°C, followed by FITC-conjugated antimouse IgG (1:500) (Jackson Immunolabs) for 60 minutes at 25°C. Fluorescent images were obtained using a Nikon Diaphot inverted confocal microscope equipped with fluorescence optics.Transfection studies and immunofluorescence analysis COS1 cells were grown in Dulbecco minimal essential medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate. Transient transfections with full-length IQGAP2 cDNA7 were completed using diethylaminoethyl (DEAE)-dextran. Immunofluorescence staining was completed 48 to 72 hours after transfection in 8-well chamber slides after fixation and permeabilization using ice-cold 100% acetone for 60 seconds. After a 16-hour blocking step at 4°C using PBS/0.1% BSA, cells were stained for 60 minutes at 25°C with murine anti-IQGAP2 (1:100), followed by immunofluorescence detection for 60 minutes at 25°C using 1:750 dilution of the FITC-conjugated antimouse antibody (Jackson Immunolabs). Parallel immunostaining demonstrated no cross-reactivity or fluorescence bleed-through of the species-specific secondary antibodies. F-actin staining was completed using 66 nM Alexa Fluor 568 phalloidin (Molecular Probes) for 60 minutes at 25°C.
The gene encoding IQGAP2 is identified within the PAR gene
cluster.
Characterization of platelet IQGAP2 IQGAP2 was initially classified as a liver-specific GTPase activating protein (GAP) because its primary translation product predicted the presence of a RasGAP-GTPase binding domain (GBD) found in the orthologous Schizosaccharomyces pombe sar1 gene.7 Nonetheless, IQGAP2 lacks inherent GAP activity toward Ras or other GTPases in vitro, presumably related to lack of an arginine finger residue within its GBD known to be essential for GAP activity.21 Rather, IQGAP2 interacts with GDP- or GTP-bound rac1 and cdc42 (but not RhoA) with comparable affinities in vitro,7 though its function remained largely enigmatic. The colocalization of IQGAP2 with the thrombin receptors PAR1 and PAR3 coupled with thrombin's ability to activate an overlapping
subset of Rho GTPasesprompted subsequent investigations into a
potential role for IQGAP2 in platelet-activation events. Northern
analyses using megakaryocyte-like HEL cells demonstrated the identical
approximately 6.5-kb IQGAP2 transcript as that found in HepG2
cells, and RT-PCR demonstrated IQGAP2 mRNA in HEL cells and human
platelets (not shown). These transcript analyses were consistent with
results of protein expression studies that demonstrated a single
approximately 180-kDa protein in HEL and platelet lysates analyzed
under reducing and nonreducing conditions and identical to the
immunoreactive species seen in HepG2 cell lysates7;
furthermore, an anti-IQGAP2 IgG specifically immunoprecipitated the
protein from platelet lysates (Figure 1D).
Quiescent gel-filtered platelets demonstrated diffuse cytoplasmic IQGAP2 staining without a clear cell-surface expression pattern, results that were dramatically different from those seen using glass- or thrombin-activated platelets. Gentle cytocentrifugation of platelets onto glass slides results in platelet activation with the generation of lamellae and filopodia. In these cells, IQGAP2 staining was most readily identifiable in platelet filopodia, with less prominent staining evident in the contractile platelet body (Figure 2G-I). Furthermore, selective activation using thrombin or PAR142-47 demonstrated a pattern most consistent with cytoskeletal IQGAP2 translocation (Figure 2J-L). IQGAP2 interacts with actin and arp2/3 The pattern of IQGAP2 redistribution in activated platelets suggested that IQGAP2 might have a physiologic role in cytoskeletal actin assembly, consistent with its predicted domain structure that encompasses an N-terminal actin-binding calponin homology (CH) domain.22 Transient transfection of a full-length IQGAP2 cDNA in COS1 cells demonstrated diffuse cytoplasmic staining and intense cell-surface membrane staining 48 hours after transfection. Simultaneous phalloidin staining demonstrated that IQGAP2 colocalized with filamentous (F)-actin in both lamellipodia and filopodia (Figure 2A-F). Thus, the patterns of IQGAP2 expression mirrored those expected for a rac1/cdc42 effector proteinthat is, expression appeared
localized to areas of filopodial/lamellipodial extensions (demonstrable
in platelets and transfected COS1 cells). These patterns are consistent
with the previously identified GTPase specificity as determined by in
vitro nitrocellulose pull-down assays,7 and they suggested
a function for IQGAP2 in cytoskeleton actin assembly similar to that
previously described for its closely related homologue,
IQGAP1.23
Actin assembly is initiated on the exposure of actin filament barbed
ends, occurring by uncapping of barbed ends of preexisting filaments,
by severing filaments, or by de novo nucleation.24,25 The
arp2/3 complex is a 7-member protein complex found in human platelets
in nearly equivalent stoichiometric ratios, and it is the only known
cellular factor that nucleates the formation of actin
filaments.26 Arp2/3 promotes the addition of actin
monomers to the barbed ends of actin filaments,26 a
reaction that requires a scaffolding protein such as the
Wiskott-Aldrich syndrome protein (WASp)27,28 or the
Listeria monocytogenes ActA
protein.29 To determine whether IQGAP2 scaffolding
functions involved biochemical interactions with F-actin and arp2/3 in
a physiologic platelet model system, confirmatory association studies
were completed using either gel-filtered platelets or platelet-rich
plasma, with essentially identical results (Figure
3). Platelet activation with
IQGAP2 scaffolding functions require [GTP]rac1/cdc42 Although IQGAP2 has been shown to bind rac1 and cdc42 using in vitro nitrocellulose binding assays, the functional role(s) of such associations in a primary cell (such as platelets) endogenously expressing all these proteins remains unknown. Human platelets contain approximately 2 to 3 µM rac1 and 0.2 to 0.3 µM cdc42,9 both of which are primarily (more than 90%) found in the cytosolic fractions of quiescent platelets.9,30 In unstimulated platelets, IQGAP2-specific immunoprecipitation studies demonstrated that cytosolic IQGAP2 was complexed to rac1 but not to cdc42, presumably related to the relative concentrations of these 2 GTPases in human platelets.9 Reciprocal immunoprecipitation analyses using either anti-rac1 or anti-cdc42 antibodies confirmed these initial observations (data not shown). Despite evidence for this IQGAP2/rac1 complex in quiescent platelets, most (more than 90%) of rac1 appeared to be free of IQGAP2, as demonstrated by immunoblot analysis of the post-IQGAP2 immunoprecipitate sample. In parallel experiments using thrombin-stimulated platelets, immunoprecipitation studies failed to demonstrate increased rac1 or cdc42 recruitment to the cytoplasmic IQGAP2-arp2/3-actin complex (not shown). Because IQGAP2 associates with GDP- and GTP-bound GTPases,7 this lack of quantitative difference could be explained by the exchange of GDP- for GTP-bound rac1/cdc42, with no total change in overall stoichiometric associations.To more specifically dissect the molecular mechanisms whereby rac1 and
cdc42 could effect IQGAP2-associated arp2/3-actin complex assembly, the
activation state of bound GTPases was studied using a recombinantly
expressed PAK1 binding domain (PBD). This PBD, containing a
highly conserved 14-amino acid cdc42/rac1 interactive binding region
(CRIB) sequence,31 specifically binds and
distinguishes active (GTP-bound) from inactive (GDP-bound) forms of
rac1 and cdc42,10 and it has been used as a sensitive
means of demonstrating GTPase charging in thrombin-stimulated
platelets9 or formyl-methionyleucylphenylalanine (fMLP)-activated neutrophils.10 To determine whether
PBD could be used to study IQGAP2/GTPase interactions, we cotransfected IQGAP2 with dominant-negative rac1N17 and cdc42N17 or constitutively active rac1V12 and cdc42V12 mutants into COS1 cells, followed by
affinity purification using GST-PBD agarose beads (or GST beads alone).
As shown in Figure 4A, IQGAP2 was
specifically identified only in the presence of [GTP]rac1 or
[GTP]cdc42, suggesting that the PBD could specifically bridge a
[GTP]GTPase/IQGAP2 complex. Although the efficiency for this
interaction was no more than 5% of cellular IQGAP2, it suggested a
means of discriminating between the activation-dependent states of
IQGAP2/GTPase interactions.
Pull-down assays of thrombin-stimulated platelets using GST-PBD agarose beads were then pursued, with end points focusing on the ability of IQGAP2 to interact with actin and arp2/3. Consistent with the evidence for biochemical interactions outlined above, thrombin stimulation again demonstrated assembly of the entire IQGAP2/arp2/3-actin complex in a time-course essentially identical to that seen using IQGAP2-specific immunoprecipitations. The complex was assembled within 15 seconds, with fall-off 60 seconds after agonist stimulation (Figure 4B). Minimal to no complex assembly was evident using mutant GST-PBD or GST beads alone (not shown), and the assay failed to pull-down the [GTP]cdc42 effector protein WASp or gelsolin, again supporting the IQGAP2-dependent specificity of the complex assembly scaffold. Furthermore, preincubation of platelets with cytochalasin D abrogated thrombin-induced IQGAP2/actin interaction and F-actin content (Figure 4C-D). Maximal IQGAP2 association was evident at the most immediate time point, with subsequent fall-off by 60 seconds, and entirely consistent with the rapid movement of IQGAP2 into the cytoskeletal fraction (see below). Although low-level IQGAP2 remained evident at 60 seconds and beyond, as demonstrated by long exposure, arp2/3 and actin were found at disproportionately higher concentrations. At this point, we could not exclude the possibility that another, as yet uncharacterized, rac1/cdc42 effector protein was a component of the scaffolding complex (though it did not appear to be platelet WASp). IQGAP2 scaffolding functions are  21) receptor
complex,32 though activation is mediated by intracellular signaling through a platelet glycoprotein VI/FcR-chain receptor tyrosine kinase complex.33 ADP-induced platelet
aggregation is mediated through the activation of PY21 and
PY212 purinergic receptors.34 Unexpectedly,
the ability to assemble the complex was uniquely agonist dependent,
with no evidence that fully aggregative doses of either
fibrillar collagen (10 µg/mL) or ADP (10 µM) could effect
IQGAP2/arp2/3-actin complex formation (Figure 4B).
Divergent IQGAP2 effector pathways mediated by [GTP]rac1 or [GTP]cdc42 binding Given that these assays do not distinguish [GTP]rac1-from [GTP]cdc42-induced complex assembly, dissection of the GTPases regulating agonist-dependent IQGAP2 scaffold formation was pursued in greater detail. Consistent with the results outlined in Figure 4, complex assembly correlated with GTP-charging capabilities of individual agonists, demonstrating changes restricted to thrombin-stimulated platelets (Figure 5). Interestingly, rac1 and cdc42 demonstrated distinctly divergent responses on thrombin stimulation. The cytosolic concentration of [GTP]rac1 increased substantially (maximally accounting for approximately 45% of cellular [GTP]rac1), first evident by 5 seconds and reaching peak concentrations at 15 seconds, with rapid subsequent decay. In contrast, cytosolic concentrations of [GTP]cdc42 rapidly decreased and became asymptotically undetectable beyond 30 seconds. These initial responses were clearly evident at 2 seconds (preceding [GTP]rac1 charging) and were consistent with the previously described rapid translocation of nearly 30% of [GTP]cdc42 to the platelet actin cytoskeleton.9,30 We hypothesized that if the activation of IQGAP2 required the binding of [GTP]rac1 or [GTP]cdc42, its translocation to the actin cytoskeleton would only be evident in thrombin-stimulated platelets. This was confirmed by isolating the Triton-X insoluble actin cytoskeletons of agonist-treated platelets and probing for the presence of IQGAP2 (Figure 5C). The rapid translocation of IQGAP2 was readily seen only on thrombin stimulation and as evaluated by parallel studies using total platelet lysates, estimated to account for approximately 20% of total cellular IQGAP2. Neither collagen- nor ADP-stimulated platelets demonstrated IQGAP2 cytoskeletal translocation, even with extension of the experiments to 5 minutes to ensure maximal platelet activation.
A functional transcriptosome composed of its receptor(s) and intracellular effector protein has been identified that uniquely regulates an agonist-specific human platelet activation pathway. Recent evidence35 from human genomic analyses has identified discrete chromosomal regions that demonstrate clustered expression patterns in healthy and malignant tissues. Such patterns of expression were suggested as a means of identifying novel genes that may be differentially associated with a particular (malignant) phenotype, but developmental functional relationships within these regions were not anticipated. Computational genomic approaches have suggested that so-called gene adjacencies may be used to predict functional coupling, though the model systems for such contextual-based approaches are best exemplified in prokaryotes.36,37 To our knowledge, this is the first example of such a functional relationship in humans, raising the possibility that comparably developed computational methods may be adapted for human genomic analysis. Although IQGAP2 is contextually adjacent to 2 thrombin receptors, it remains unestablished if its scaffolding functions are achievable through the activation of all 3 PARs individually, only the contiguous PAR1 and PAR3, or the dual PAR1/PAR4 receptor system evident in human platelets.8,12,14 Previous observations have outlined distinct roles for
WASp25 and Scar38 as endogenous effectors of
actin polymerization mediated through arp2/3 assembly and activation.
IQGAP2 thus represents an additional protein implicated in such a
function, though direct evidence for an actin nucleating function
remains to be demonstrated. Alternatively, IQGAP2 activation may
generate specific cross-linked F-actin structures unrelated to a
function in the initiation of actin polymerization.23,39
In the case of WASp, the activating signal appears to be
cdc42-dependent.25 It is clear, however, that
WASp-deficient platelets demonstrate essentially normal patterns of
collagen and thrombin-responsiveness, consistent with the role of
another scaffolding protein, such as IQGAP2 in cytoskeletal actin
reorganization.40 In preliminary data, we have
demonstrated by RT-PCR and immunoblot analysis that IQGAP1 is also
expressed in human platelets. IQGAP1 and IQGAP2 are approximately 60%
homologous, but they display distinct abilities to bind GTPases Components of cell-signaling pathways are likely to assemble into
multimolecular complexes held together by scaffolding proteins, as has
been specifically demonstrated for What is the significance of IQGAP2 interacting with rac1 and
cdc42 GTPases, and could differential binding mediate distinct effector
functions? The integration of data presented here and elsewhere
demonstrates that rac1 and cdc42 display divergent fates during
platelet activation, with most rac1 remaining in the cytoplasm and a
larger fraction of cdc42 translocating to the actin
cytoskeleton.9,47 In contrast to the data of Azim et
al,9 our data did not demonstrate significant cytosolic
increases of [GTP]cdc42 but are more consistent with rapid
translocation to the cytoskeleton. In this respect, the results from
both laboratories are in general agreement and entirely consistent with
prior evidence for an
We thank Drs L. Van Aelst, D. Bar-Sagi, N. Nasser, and I. Spector for reagents and helpful discussions, Dr Machesky for anti-arp antibodies, Dr D. Gnatenko for assistance with densitometric calculations, and David Colflesh (University Microscopy Imaging Center) for assistance with the confocal microscopy.
Submitted September 16, 2002; accepted November 29, 2002.
Prepublished online as Blood First Edition Paper, December 19, 2002; DOI 10.1182/blood-2002-09-2807.
Supported by grants from the National Institutes of Health (NHL49141 and HL53665), a Veteran's Administration Medical Center Research Enhancement Award Program (REAP), and an American Heart Association Postdoctoral Fellowship and NIH DK62040 grants (V.S.). W.F.B. is an Established Investigator of the American Heart Association.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Wadie F. Bahou, Division of Hematology, HSCT15-040, State University of New York, Stony Brook, NY 11794-8151; e-mail: wbahou{at}notes.cc.sunysb.edu.
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Top
Abstract
Introduction
3' off the negative
strand.
DNA are shown in panels B and C. (D) Platelet
immunoprecipitations were completed using 1 × 108
RIPA-solubilized gel-filtered platelets (GFP), and anti-IQGAP2
monoclonal antibody (mAb) (lane 2) or preimmune mouse IgG (lane 3),
followed by immunoblot analysis using the IQGAP2-specific mAb (arrow).
The corresponding SDS-solubilized GFP lysate is shown in lane
1.
