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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Hematology, Brigham and Women's
Hospital, and the Division of Immunology, Children's Hospital, Harvard
Medical School, Boston, MA; and the Department of Biochemistry,
Cambridge University, Cambridge, United Kingdom.
How platelet shape change initiated by a collagen-related peptide
(CRP) specific for the GPVI/FcR Blood platelets play a critical role in hemostasis.
After blood vessel injury and disruption of the endothelial layer,
platelets adhere to collagen in the basement membrane through
glycoprotein (GP) Ia-IIa (integrin The intracellular signaling pathway leading to activation of platelets
by thrombin begins with the activation of phospholipase C- Activation of platelets by thrombin results in the formation of
filopodia and in cell spreading by the extension of
lamellae.5 Filopodia and lamellae are composed of bundles
of long filaments and orthogonal arrays of short filament networks,
respectively. The extension of lamellae observed in platelets activated
by thrombin requires the severing of actin filaments present in the
resting cell, the formation and activation of barbed-end nucleation
sites, and the addition of actin monomers onto these nucleation sites to double the F-actin content.5 We have argued, based on
the structural changes that normally occur in platelets and the lack thereof in the platelets of gelsolin-deficient mice, that 75% of the
actin nucleation activity derives from Ca++-activated,
gelsolin-based filament fragmentation and the subsequent uncapping of
these filaments by membrane ppIs.6,7 Production of ppIs
requires the activation of the small GTPase Rac,8 and a
rapid and robust activation of Rac follows ligation of the thrombin receptor.9 Phosphoinositide (PI) 3-kinase is not required
for the platelet spreading mediated by thrombin,10 but it
is involved in platelet spreading over fibrinogen-coated surfaces
mediated by adenosine diphosphate (ADP)11 and in platelet
actin assembly initiated by the fibrinogen receptor, the integrin
In contrast, less is known about platelet shape change induced by
GPVI/FcR SLP-76 is an adaptor protein predominantly expressed in hematopoietic
cells, notably in T cells and in myeloid cells.16-18 It
contains multiple N-terminal tyrosine phosphorylation sites, a central proline-rich region that constitutively associates with the
SH3 domains of the adaptor protein Grb2,16 and a
C-terminal SH2 domain. After ligation of the T-cell antigen
receptor, SLP-76 is rapidly tyrosine phosphorylated by
ZAP-7019-21 and associates with Vav,22 a
guanine nucleotide exchange factor for Rac. In addition, SLP-76
interacts through its SH2 domain with Fyb/SLAP-130.23,24 SLP-76-deficient mice have severe impairment of T-cell
development.18,25 They also manifest a bleeding diathesis
resulting in significant perinatal mortality,15,25 similar
to Syk-deficient mice.13 Surviving adults show massive
bleeding of intraperitoneal fluid and diffuse edema of the neck,
thorax, and intestinal regions.
PI 3-kinase also plays a role in GPVI/FcR The aim of the current study was to investigate the mechanisms of
platelet shape changes induced by GPVI/FcR Human and mouse platelet preparation and stimulation
Video microscopy
Electron microscopy Platelets were attached to the surfaces of 5-mm round CRP-coated (6 µg/mL) coverslips by centrifugation at 330g for 5 minutes at 37°C. Platelets either were fixed by the addition of 1% (vol/vol) glutaraldehyde for 10 minutes to view the topology of the cells or they were extracted with 0.75% Triton X-100 in 60 mmol/L Pipes, 25 mmol/L HEPES, 10 mmol/L EGTA, and 2 mmol/L MgCl2 (PHEM buffer) containing protease inhibitors and 2 µmol/L phallacidin for 2 minutes. Cytoskeletons were fixed with 1% glutaraldehyde in PHEM buffer as previously described.5 The coverslips were washed in water, rapidly frozen, freeze-dried, and coated with 1.4 nm tantalum-tungsten and 4 nm carbon. Replicas were picked up on carbon-formvar-coated copper grids and photographed at 100 kV in a JEOL 1200-EX electron microscope.Tyrosine phosphorylation of PLC- 2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) bound
to protein G-Sepharose beads. The immune complexes were solubilized in
SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer33 containing 5% -mercaptoethanol. After they
were boiled for 5 minutes, proteins were separated by SDS-PAGE on an
8% polyacrylamide gel and transferred to an Immobilon-P membrane
(Millipore). The membrane was incubated in a blocking solution (100 mmol/L NaCl, 20 mmol/L Tris/HCl, pH 7.4) containing 1% BSA, then
probed with a 1:1 mixture of 4G10 (Upstate Biotechnology) and PY20
(Transduction Laboratories) anti-phosphotyrosine monoclonal antibodies.
Detection was performed with an enhanced chemiluminescence
system (Pierce).
Intracellular free calcium concentration ([Ca++]i) measurements [Ca++]i measurements were performed on indo1-AM-labeled platelets, as previously described.34 Briefly, indo1 fluorescence was recorded using a spectrofluorometer (LS50; Perkin-Elmer Cetus Instruments, Norwalk, CT). Excitation and emission wavelengths were 331 and 410 nm, respectively. [Ca++]i was calibrated according to Grynkiewicz et al35: [Ca++]i = Kd × (F Fmin)/(Fmax F),
where F is the measured fluorescence intensity, and Fmin
and Fmax are the fluorescence intensities obtained without
external Ca++ and at saturating Ca++,
respectively. Kd for indo1 was taken to be 250 nmol/L.
Measurement of F-actin content Resting or activated platelets in suspension were fixed in 3.4% (vol/vol) formaldehyde and permeabilized with 0.1% Triton X-100 in the presence of 10 µmol/L fluorescein isothiocyanate (FITC)-phalloidin (Sigma). Bound FITC-phalloidin was quantitated by FACS analysis using a Becton Dickinson flow cytometer. In total, 10 000 events were analyzed for each sample.Measurement of filament ends Resting or CRP-activated platelets in suspension were extracted with 0.1% Triton in PHEM buffer containing protease inhibitors and 2 µmol/L phallacidin. Then, 185 µL 100 mmol/L KCl, 2 mmol/L MgCl2, 0.5 mmol/L adenosine triphosphate, 0.1 mmol/L EGTA, 0.5 mmol/L dithiothreitol, and 10 mmol/L Tris, pH 7.0 were added to 100 µL platelet lysate, and the polymerization rate assay was started by the addition of 15 µL monomeric pyrene-labeled rabbit skeletal muscle actin to a final concentration of 1 µmol/L. Pyrene-actin fluorescence was recorded using a spectrofluorometer (LS50; Perkin-Elmer Cetus Instruments). Excitation and emission wavelengths were 366 and 386 nm, respectively. Activity inhibited by 2 µmol/L cytochalasin B is defined as barbed-end actin assembly. Activity not inhibited by cytochalasin B is defined as pointed-end actin assembly. The number of actin filament ends was determined as previously described.5 There were 1.5 × 107 platelets per assay. Initial barbed- and pointed-end addition rates in 1 µmol/L actin solution are 10 and 1 monomers s 1, respectively.
Morphology of platelets activated by CRP A reproducible series of morphologic changes occurs when human platelets contact a CRP-coated surface (Figure 1). As initial contact with the surface is made, platelets convert from discs to round or spherical forms. This event is rapidly followed by the extension of filopodia from the rounded cell. Filopodial growth is a dynamic process, detectable first as a rippling and/or a bulging at the surface of platelets followed by the extension of filopodia. Filopodia continue to extend and withdraw as the cells flatten onto the surface. The central bodies of platelets activated on CRP remain 3-dimensional and display dynamic membrane activity, including the formation of unique blunt motile filopodia that rotate around the cell. Simultaneous with filopodia formation and movement, there is a partial filling of the spaces between filopodia with small lamellae-like extensions of membrane.
High-resolution electron microscopy reveals the underlying structure of
the activated human platelet cytoskeleton and the arrangement of actin
filaments within the specific cellular processes described (Figure
2). Filopodia
SLP-76-deficient platelets fail to form filopodia and spread lamellae in response to CRP The availability of mice lacking specific signaling or actin regulatory proteins allows evaluation of their roles in these responses. Platelets obtained from WT mice behaved as do the human blood platelets when exposed on CRP-coated surfaces (Figure 3A). CRP induced predominantly the formation of filopodia in mouse platelets, and platelets adhering to CRP-coated surfaces remained filopodial even after incubation for long periods of time. SLP-76-deficient platelets exposed to CRP failed to spread and for the most part retained their disc shapes, though a few short filopodia extended from their surfaces (Figure 3B,C). In contrast, spreading of SLP-76-deficient platelets in response to thrombin was unaffected relative to WT platelets (Figure 3D-F). Resting SLP-76-deficient platelets have a discoid shape similar to that of WT platelets (data not shown).
PI 3-kinase inhibition delays the mobilization of platelet Ca++ induced by CRP PI 3-kinase is required for platelet spreading on fibrinogen-coated surfaces mediated by ADP.11 We therefore investigated the role of PI 3-kinase in the platelet shape changes mediated by CRP. We compared tyrosine phosphorylation of PLC- 2 and
Ca++ mobilization induced by CRP in mouse SLP-76-deficient
platelets and in human platelets preincubated with 50 nmol/L wortmannin or 25 µmol/L LY294002 to inhibit PI 3-kinase. Similar results were
found with wortmannin and LY294002; only the results obtained with
wortmannin are presented.
CRP failed to induce tyrosine phosphorylation of PLC-
CRP-ligation of GPVI/FcR
Platelet shape change is associated with an increase in cellular
F-actin content.5 Actin assembly begins immediately after the addition of CRP and reaches a maximum of 70% of the total actin
within 60 seconds (Figure 6). Both CRP
and thrombin stimulated a 2-fold increase in the F-actin content of
both mouse WT and human platelets. Although PI 3-kinase inhibition does
not prevent filopodial growth as discussed (Figure 5A), wortmannin or
LY294002 prevents measurable actin assembly by CRP, suggesting that the bulk of the actin assembly measured derives from the lamellar assembly
that fills the spaces between filopodia. Only thrombin, but not CRP,
increased the F-actin content of SLP-76-deficient platelets. Actin
assembly is markedly reduced in SLP-76-deficient platelets activated
with CRP, consistent with the essential role of SLP-76 in early steps
of the signaling pathway of GPVI/FcR
In the case of thrombin receptor, actin assembly after ligation
correlates with the temporal exposure of actin filament barbed ends.5 CRP treatment of platelets also increases the
number of barbed ends to a maximum of 400 per cell. Wortmannin
decreases this number by 80% but fails to completely prevent
barbed-end exposure after CRP (Figure 7).
Wortmannin-insensitive nucleation sites may therefore drive the
assembly of actin into filopodia, but, under these conditions, the
actin disassembly and assembly rates are balanced.
CRP-induced lamellae formation and actin assembly require gelsolin expression The efficient extension of lamellae in platelets activated by thrombin requires gelsolin.6,7 In similar fashion, filopods grow from the surfaces of gelsolin-deficient platelets when they contact a CRP-coated surface, but these platelets reproducibly fail to extend lamellae between the filopodia (Figure 5B). Table 1 shows that mouse platelets lacking gelsolin and activated on CRP-coated surfaces spread poorly, and lamellae extension was reduced by 85% compared to WT platelets. Before filopods formed, the surfaces of gelsolin-deficient platelets rippled and undulated, and small blebs were formed and retracted. As for thrombin, actin assembly was also markedly reduced in gelsolin-deficient platelets activated with CRP (Figure 6).
Platelet shape changes induced by trimeric G-protein-coupled
receptors such as the thrombin receptor are widely
studied.5,8 These changes result in lamellar extension and
cell spreading by a mechanism requiring gelsolin for actin filament
severing and actin assembly onto sites equivalent to filament
barbed-end sites.6,7 In the current work, we studied how
platelets change shape when activated by GPVI/FcR To understand the signaling pathways leading to platelet shape changes
downstream of GPVI/FcR Our study shows that the formation of lamellae normally mediated by CRP
is blocked by wortmannin (Figure 5, Table 1). Hence, though PI 3-kinase
is not required for the platelet spreading mediated by
thrombin,10 it is required for lamellae spreading on
CRP-coated surfaces. This requirement is similar to platelet activation
by the fibrinogen receptor, the integrin Formation of lamellae induced by GPVI/FcR Filopodial growth induced by CRP is not inhibited in human platelets treated with wortmannin or in mouse gelsolin-deficient platelets (Figure 5). Wortmannin or LY294002 treatment, however, prevents a large amount of the actin assembly induced by CRP (Figure 6), suggesting that the bulk of measurable actin assembly derives from the lamellar assembly that fills the spaces between filopodia, but not from the formation of filopodia themselves. Although wortmannin decreases the number of nucleation sites by 80%, it fails to completely stop barbed-end exposure after CRP (Figure 7). Therefore, the wortmannin-insensitive nucleation sites may drive the assembly of filopodia under conditions in which actin filament disassembly and assembly rates balance. Our observations that SLP-76-deficient platelets fail to spread and
increase their F-actin content may also suggest that SLP-76 plays a
role in the reorganization of the actin cytoskeleton triggered by
engagement of GPVI/FcR In summary, SLP-76 is essential for the signaling pathways leading to
platelet shape changes initiated by GPVI/FcR
We thank Jenny Bandura and Laurice Salib for technical assistance. We thank Drs Thomas P. Stossel, Karin M. Hoffmeister, and Eric Krump for helpful discussions and critical reading of the manuscript and Drs Richard W. Farndale and C. Graham Knight (Cambridge University, UK) for preparation and shipping of CRP. H.F. dedicates this work to the memory of his father, Maurice Falet (1940-1999), and of his friend, Eric Krump (1966-1999).
Submitted May 5, 2000; accepted July 28, 2000.
Supported by National Institutes of Health grants HL-56262 and HL-56949 (J.H.H.) and AI-35714 (R.S.G.) and by grants from Baxter HealthCare and Centeon Corporations.
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: John H. Hartwig, Division of Hematology, Brigham and Women's Hospital, 221 Longwood Avenue, LMRC 301, Boston, MA 02115; e-mail: hartwig{at}calvin.bwh.harvard.edu.
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