AbstractInitial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s−1 induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s−1. Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation.
Platelet adhesion to subendothelial matrices in the damaged vessel wall is the prime event in the arrest of bleeding. Recruitment of platelets to sites of vascular injury is hampered by the rapid flow of blood in arteries and arterioles. In these vessels, the interaction between von Willebrand factor (VWF), a multimeric plasma glycoprotein, and the platelet glycoprotein (GP) Ib-IX-V receptor complex is critical for initial platelet adhesion.1 This interaction requires unfolding of the VWF A1 domain and allows platelets to decelerate until they attach firmly in a process assisted by platelet integrins.1,2 Defects in both the GPIb-IX-V complex (Bernard Soulier syndrome) and VWF (von Willebrand disease) result in a bleeding diathesis, which underscores the importance of this interaction in hemostasis.3,4
The GPIb-IX-V complex consists of four transmembrane subunits; GPIbα, GPIbβ, GPIX and GPV that are expressed in a 2:4:2:1 stoichiometry.5 Each platelet contains approximately 25,000 copies of GPIbα, the subunit that binds to the VWF A1 domain.6 The extracellular domain (residues 1–485) of GPIbα consists of an N-terminal flank, seven leucine-rich repeats, a C-terminal flank, a sulphated region and a highly glycosylated macroglycopeptide domain. Residues 486–514 form the transmembrane domain and the cytoplasmic tail consists of 96 amino acid residues (residues 515–610),7–9 which contain binding sites for multiple intracellular proteins, including filamin A10 and the adaptor protein 14-3-3ζ.11 The region that interacts with the VWF A1 domain resides within the concave face of the leucine-rich repeat domain of GPIbα.12,13 Despite the fundamental importance in initiating platelet adhesion, the molecular mechanism regulating the VWF-GPIbα interaction remains incompletely understood.
Binding of VWF to GPIbα requires the dynamic conditions of flowing blood. The unique biomechanical properties of VWF and GPIbα allow the interaction to strengthen upon increasing hemodynamic drag.14 An explanation for this counterintuitive finding is that VWF needs to change its conformation to allow GPIbα access to the A1 domain. Elevated shear force and immobilization on a surface trigger this conformational change in vivo, a process mimicked by the antibiotic ristocetin in vitro.15 The interaction between VWF and GPIbα is also regulated through changes in GPIbα. This molecule’s adhesive properties depend on translocation to cholesterol-rich membrane domains known as lipid rafts,16,17 which may increase the local density of GPIbα receptors and stimulate their signaling properties. Indeed, studies with Chinese hamster ovary cells in which GPIbα was artificially dimerized have suggested that receptor clustering increases the overall strength of the VWF-GPIbα interaction.18,19
During efforts to optimize the storage conditions of platelet concentrates used for transfusion, we recently demonstrated that GPIbα clusters in lipid rafts when platelets are kept at low temperature.20 Analysis of Förster resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy (FLIM) revealed that cooling of platelets triggers [GPIbα-GPIbα] associations in lipid rafts within a range of 1–10 nm. In the present study, we assessed whether clustering of GPIbα occurs under physiological conditions, investigated its influence on VWF interaction and identified the responsible molecular mechanism.
Citrated blood (10.9 mM f.c.) was obtained from a patient with von Willebrand disease type 3. Permission was obtained from the local medical ethics committee. The patient had no detectable plasma VWF (<0.1%), 1% plasma factor VIII, <1% factor VIII activity, no ristocetin-induced platelet aggregation, and a normal platelet count and volume.21
Materials, antibodies, platelet preparation and incubations
A detailed description of the materials, antibodies, platelet preparation and incubations used in this study can be found in the Online Supplementary Methods.
Platelet adhesion and rolling under flow conditions
A parallel plate perfusion chamber22 was used to investigate platelet adhesion and rolling. Further details are available in the Online Supplementary Methods.
Exposure to shear force
Platelets were exposed to shear force by perfusion through a microcapillary (inner diameter 760 μm, blocked with 4% bovine serum albumin). Washed platelets were resuspended in HT buffer (2.5×10 cells/L, pH 7.3) supplemented with 4% human albumin. Platelet suspensions were prewarmed to 37°C for 5 min and perfused through the microcapillary at indicated shear rates for 5 sec. The length of the microcapillaries was matched with the shear rate, which means that the platelet suspensions had similar shear exposure times at different shear rates. Indicated shear rates are the maximal shear rates to which platelets were exposed near the wall of the microcapillary. The wall shear rate (γw) inside a microcapillary is described as
Where Q is the volumetric flow rate and r is the inner radius of the microcapillary.
Platelet agglutination was measured in a Chrono-log Lumi-Aggregometer (model 700, Chrono-log Corporation, Haverton, PA, USA) with Aggrolink 8.0 software. Washed platelets in HT buffer were pre-incubated with the prostacyclin (PGI2) analog iloprost and dRGDW (5 min, 37°C) and stimulated with VWF (10 μg/mL) and ristocetin (0.3 mg/mL) while stirring (900 rpm). Data are expressed as percentage of maximal agglutination, with light transmission through HT buffer set at 100%.
Flow cytometric analysis, immunoprecipitations and western blots
A detailed description of the flow cytometric analysis, immunoprecipitations and western blots can be found in the Online Supplementary Methods.
Analysis of GPIbα distribution by Förster resonance energy transfer and fluorescence lifetime imaging microscopy
GPIbα distribution was analyzed by FRET/FLIM as described elsewhere.20 In brief, 6B4-Fab fragments conjugated to either Alexa Fluor-488 or Alexa Fluor-594 (6B4-488 and 6B4-594, respectively) were incubated with fixed platelet samples under conditions in which each Fab labeled ~50% of the total number of receptors. GPIbα translocation to lipid rafts was determined by labeling GPIbα with 6B4-488 and monosialo-tetrahexosylganglioside (GM1) with Cholera toxin subunit B conjugated to Alexa Fluor-594 (CTB-594; 5 μg/mL). The fluorescence lifetimes of the donor fluorophore (6B4-488) were determined in the absence and presence of acceptor fluorophore (6B4-594 or CTB-594) and used to calculate the FRET efficiency, defined as
where τ is the donor fluorophore’s lifetime in nanoseconds in the absence (τD) and presence (τD/A) of the acceptor fluorophore. To determine variation in FRET efficiency, the lifetimes of three randomly chosen quadrants were quantified.
Data are means ± SEM. Statistical analysis was based on GraphPad Prism 5 (San Diego, CA, USA). Differences between control platelets and incubations were analyzed by the Mann-Whitney test. P-values less than 0.05 (* or * ) and between incubations (|−*−|)) were considered statistically significant.
Platelet adhesion to von Willebrand factor under conditions of flow triggers GPIbα clustering
Platelet adhesion at shear rates above 1,000 s depends on the interaction between surface-bound VWF and GPIbα.1 We analyzed the effect of this interaction on the spatial distribution of GPIbα on the platelet plasma membrane with FRET/FLIM. Whereas GPIbα molecules were dispersed in resting platelets (Figure 1A,B), indicated by a FRET efficiency of 0.9±0.2%, GPIbα clustered upon adhesion to VWF (FRET efficiency 10.3±0.9% ). Clustering was not caused by close contact between adjacent platelets, as FRET efficiency did not differ between single platelets and platelets that adhered as small aggregates (Online Supplementary Figure S1A,B). As the platelet-VWF interaction is influenced by flow conditions, we analyzed the GPIbα distribution of platelets adhered to VWF at different shear rates. Adhesion to VWF at 300 s left GPIbα dispersed, but perfusion at 750 s and higher induced clustering (Figure 1C). The observed increase in clustering was not the result of more efficient adhesion, as the number of platelets binding to VWF was similar at each shear rate (Online Supplementary Figure S1C). To investigate whether changes in GPIbα distribution were specific for adhesion to VWF, platelets were perfused over collagen. Adhesion to collagen at a low shear rate (300 s) in the presence or absence of VWF resulted in FRET efficiencies similar to those observed in resting platelets (Figure 1D). Perfusion over collagen at 1,600 s in the absence of VWF had little effect on GPIbα distribution. In contrast, addition of VWF prior to perfusion at 1,600 s increased FRET efficiency to 8.3±0.6%, indicating that clustering of GPIbα requires the presence of VWF.
Exposure to high shear leads to reversible von Willebrand factor-independent GPIbα clustering
The change in GPIbα distribution measured on surface-attached platelets might be the result of shear, of rolling/attachment or both. To understand the contribution of shear, we perfused platelets in VWF-free buffer through a microcapillary tube at different shear rates in the absence of an adhesive surface. FRET/FLIM analysis showed that a shear rate of 300 s left GPIbα dispersed. Exposure to 1,600 s had a minor effect on GPIbα distribution, whereas a shear rate of 10,000 s increased FRET efficiency to 9.1±0.6% (Figure 2A). Addition of exogenous VWF prior to perfusion at a shear rate of 10,000 s did not further increase clustering. Platelet α-granules also contain VWF,23 which might be released during platelet isolation and thereby influence GPIbα clustering. To investigate this possibility, experiments were repeated using a nanobody against VWF which prevents its binding to GPIbα. Exposure of platelets to shear force in the presence of this nanobody led to the same increase in FRET efficiency as control platelets (Figure 2B). GPIbα clustering induced by a shear rate of 10,000 s was also similar in platelets from a patient with VWD type 3 (Online Supplementary Figure S2A), indicating that GPIbα clusters independently of the presence of VWF.
GPIbα clustering was reversible, as exposure to shear followed by incubations under static conditions resulted in a gradual decline to the range found in resting platelets (Figure 2C). Platelet exposure to a shear rate of 10,000 s did not result in P-selectin expression, αIIbβ3 activation, VWF binding (Figure 2D), cytoskeleton reorganization or altered whole protein tyrosine phosphorylation (Online Supplementary Figure S2B,C). Conversely, clustering was not induced by stimulation with cross-linked collagen-related peptide (CRP), thrombin receptor activating peptide (TRAP) or the thromboxane A2 receptor (TPα) agonist U46619 under static conditions (Figure 2E). Ristocetin-induced VWF binding did induce clustering of GPIbα. Transient GPIbα clustering did not affect the ability of platelets to respond to agonists, because stimulation with TRAP or CRP before and after exposure to shear resulted in similar levels of P-selectin expression and αIIbβ3 activation (Figure 2F).
Platelet interaction with von Willebrand factor is stimulated by clustered GPIbα
To clarify whether changes in GPIbα distribution contributed to platelet responsiveness to VWF, agglutination was measured in platelets with shear-induced clustered GPIbα. VWF and a suboptimal concentration of ristocetin were used and aggregation was prevented by pre-incubation with iloprost, a stable analog of prostacyclin, and dRGDW. Neither agent affected shear-induced GPIbα clustering (data not shown). Maximal agglutination of platelets with pre-clustered GPIbα was four-fold higher than with controls (Figure 3A,B).
VWF enables platelets to roll over the damaged vessel wall until they attach firmly in an integrin-dependent manner. The effect of GPIbα clustering was measured by platelet perfusion over a VWF-coated surface in the presence of dRGDW to block αIIbβ3-mediated attachment. Induction of GPIbα clustering prior to perfusion reduced the rolling velocity by 40% (Figure 3C,D). These data show that GPIbα clustering facilitates the platelet-VWF interaction.
GPIbα translocates to lipid rafts and forms clusters through 14-3-3ζ binding
Platelet binding to VWF depends on reallocation of GPIbα in membrane domains enriched in sphingomyelin and cholesterol, known as lipid rafts.16,17 To understand the role of raft allocation in GPIbα clustering, GPIbα was labeled with 6B4-488 (donor) and the raft marker GM1 with CTB conjugated to Alexa Fluor-594 (CTB-594; acceptor). The surface of resting platelets showed little co-localization but adhesion to VWF or exposure to high shear (10,000 s) induced GPIbα-GPIbα as well as GPIbα-GM1 associations, suggesting that clustering and raft translocation go hand in hand (Figure 4A). Disruption of lipid rafts by cholesterol depletion with methyl-β-cyclodextrin (mβCD) effectively abrogated GPIbα clustering. The time-dependent decay of shear-induced GPIbα clusters closely followed raft translocation, again suggesting a tight interrelationship (Figure 4B).
Binding of the adaptor protein 14-3-3ζ to the cytoplasmic tail of GPIbα is essential for platelet interaction with VWF.24,25 To assess the role of 14-3-3ζ in GPIbα clustering, we pre-incubated platelets with MPαC. This membrane permeable peptide represents the critical 14-3-3ζ binding site on GPIbα, which includes the constitutively phosphorylated Ser-609 residue on its cytoplasmic tail.24,26,27 As expected, the peptide prevented VWF-induced 14-3-3ζ binding to GPIbα (Online Supplementary Figure S2). Figure 4C shows that pre-incubation with MPαC had little effect on lipid raft translocation induced by platelet adhesion to VWF, but completely abrogated clustering of GPIbα. Control peptide MαC, which lacks phosphorylation at Ser-609, had no effect on GPIbα redistribution. FRET/FLIM analysis of platelets exposed to a shear rate of 10,000 s procuded similar results (Figure 4D), demonstrating that 14-3-3ζ binding to the cytoplasmic tail of GPIbα is essential for its clustering. To elucidate the importance of 14-3-3ζ-induced clustering, we determined the rolling velocity of platelets perfused over VWF in the presence of MPαC (Figure 4E). While control peptide MαC had no effect, the rolling velocity of platelets pre-incubated with MPαC increased more than two-fold. Moreover, inhibition of 14-3-3ζ-induced GPIbα clustering impaired stable adhesion to VWF during whole blood perfusion (Figure 4F).
Arachidonic acid mediates 14-3-3ζ-induced GPIbα clustering
Platelet interaction with VWF or incubation at low temperature activates the stress kinase P38-mitogen-activated protein kinase (P38MAPK), which liberates arachidonic acid (AA) from membrane phospholipids through cytosolic phospholipase A2.20,28,29 Incubation with inhibitors at 37°C indicated that P38MAPK-mediated AA release might support GPIbα-GPIbα interactions during exposure to shear. The P38MAPK inhibitor SB203580 and the cytosolic phospholipase A2 inhibitor AACOCF3 inhibited the rise in FRET efficiency induced by high shear. The low FRET efficiency observed under static conditions increased 12-fold upon addition of AA. The intracellular accumulation of free AA might therefore contribute to GPIbα clustering (Figure 5A). The FRET efficiency of shear-treated platelets decreased upon subsequent incubations under static conditions (Figure 2C).
In platelets, liberated AA is metabolized by cyclo-oxygenase-1 and lipo-oxygenase to thromboxane A2 and other eicosanoids, which could account for the reversibility of shear-induced GPIbα clustering. Indeed, accumulation of AA by inhibition of these enzymes with indomethacin and 5,8,11-eicosatriynoic acid prevented GPIbα clusters from dispersing after exposure to high shear (Figure 5B). AA release triggered by platelet stimulation with CRP or TRAP in the presence of indomethacin and 5,8,11-eicosatriynoic acid also induced clustering of GPIbα (Figure 5C). Control studies confirmed that SB203580 blocked shear-induced P38MAPK phosphorylation/activation whereas the other treatments left the enzyme undisturbed (Figure 5D). Treatments that inhibited AA release prevented shear-induced 14-3-3ζ-GPIbα association and blockade of AA degradation and the separate addition of AA preserved the complex (Figure 5E). These data indicate that liberated AA binds 14-3-3ζ and facilitates its translocation to the cytoplasmic tail of GPIbα.20,28
The inhibitors of AA release and degradation affected platelet rolling velocity over a VWF surface. Inhibition of AA release induced by shear increased the rolling velocity, whereas treatments that preserved accumulation of AA resulted in reduced velocities (Figure 5F). These data indicate that the AA-mediated transfer of 14-3-3ζ to the GPIbα cytoplasmic tail induces GPIbα clustering which supports platelet interaction with VWF at high shear.
Our results demonstrate that the exposure of platelets to high shear leads to clustering of GPIbα and enhances the interaction with VWF. Shear-induced clustering is reversible and not associated with granule release or activation of αIIbβ3. Clustering requires lipid raft translocation and critically depends on AA-mediated 14-3-3ζ binding to the cytoplasmic tail of GPIbα (Figure 6).
Previous studies found a role for receptor clustering in the GPIbα-VWF interaction.18,19 Experiments with Chinese hamster ovary cells showed that intracellular dimerization of a modified GPIbα construct increases the overall bond strength with VWF. We found that GPIbα formed clusters in platelets adhered to VWF after perfusion at a shear rate of 750 s or higher. At these shear rates, platelet adhesion strongly depends on the interaction between GPIbα and VWF, because only this interaction is sufficiently fast and strong to withstand the associated hemodynamic drag.1 Our results indicate that GPIbα clustering contributes to GPIbα-mediated platelet adhesion to VWF, as inhibition of clustering strongly attenuated both platelet rolling velocity and adhesion.
Initial adhesion to VWF increases the hemodynamic drag on platelets substantially and results in the formation of membrane tethers that are pulled from the cell surface.30,31 Upon adhesion to VWF, clustering was observed at a shear rate of 750 s or higher. In the absence of an adhesive surface, GPIbα clustering required exposure to 10,000 s, a shear rate found in stenotic arteries. By definition, only those platelets nearest to the vessel wall are subjected to this shear rate. Shear exposure is, therefore, probably limited to part of the platelet population perfused through a microcapillary. Nevertheless, this short exposure to shear stress induced clustering of GPIbα, illustrating the high sensitivity of platelets to mechanical stress. The combined force of shear exposure and tensile stress exerted on GPIbα when bound to VWF apparently cooperate in facilitating GPIbα clustering. Shear-induced clustering did not coincide with platelet activation, which is in line with earlier reports of VWF-dependent adhesion32 or aggregation of discoid platelets33 at high shear rates.
The effects of clustering on the interaction with VWF probably reflect avidity modulation, where an increased local density of GPIbα molecules increases the number of ligand-receptor bonds. Based on crystal structure studies, it is less likely that GPIbα clustering facilitates binding of two GPIbα molecules to a single A1 domain.12,34 Under the influence of elevated hemodynamic drag, VWF-bound GPIbα can subsequently undergo a conformational change that further strengthens the interaction.14
Disruption of lipid rafts by cholesterol depletion strongly impairs platelet adhesion to VWF under conditions of flow, indicating that GPIbα localization to these regions is essential for its function.16 Lipid rafts are viewed as platforms that can physically concentrate receptors, adaptor proteins and effector enzymes, which lead to amplification of signaling events. We observed little GPIbα localization in rafts on the surface of resting platelets, which increased about three-fold upon ligation to immobilized VWF or exposure to high shear in solution. Disruption of rafts prevented GPIbα from clustering. Although translocation was essential for this process, clustering depended critically on the interaction between 14-3-3ζ and GPIbα. The importance of the [14-3-3ζ-GPIbα] association for the interaction of platelets with VWF is well established,24,25 but the exact mode of action remains poorly defined. It has been suggested that this association participates in αIIbβ3 integrin activation in GPIb-IX-expressing Chinese hamster ovary cells.35,36 We show that inhibition of 14-3-3ζ binding to GPIbα impaired adhesion to VWF and increased rolling velocity. The presence of iloprost and dRGDW excluded involvement of αIIbβ3 integrin in platelet rolling on VWF. Together, these data indicate that the 14-3-3ζ association with GPIbα directly improves platelet interaction with VWF by allowing receptor clustering. The dimeric nature of 14-3-3ζ supports this finding.37 Indeed, a similar mechanism has been described in muscle cells, in which clustering of the acetylcholine receptor depends on 14-3-3ζ.38
P38MAPK is a kinase that is responsive to stress stimuli, including alterations in thermal28 and shear conditions.39 We found that platelet exposure to shear in solution leads to P38MAPK phosphorylation. Phosphorylated P38MAPK subsequently activates cytosolic phospholipase A2 to release AA from membrane phospholipids.40 Our study reveals a central role for AA in GPIbα clustering. Inhibition of AA release during exposure to shear prevented the transfer of 14-3-3ζ to the cytoplasmic tail of GPIbα. Moreover, addition of AA enhanced clustering and inhibition of AA metabolism resulted in irreversible clustering. The findings that AA binding to 14-3-3ζ induces 14-3-3ζ multimerization41 and that AA-bound 14-3-3ζ directly associates with GPIbα,28 both support the concept that the adaptor protein provides a platform for GPIbα clustering. In addition, lipid rafts are enriched in AA,42 which may explain the dependence of GPIbα cluster formation on these membrane domains. Rolling experiments established the importance of AA-mediated GPIbα clustering, as inhibitors of AA release reduced platelet interaction with VWF, while its accumulation enhanced this initial step in adhesion.
Aspirin, a widely used antithombotic drug, also interferes with AA conversion by inhibiting COX-1 activity. Our studies suggest that the use of aspirin may prolong the presence of GPIbα clusters, which contradicts the antithrombotic effects of this drug. However, the inhibitory effects of aspirin are attributed to inhibition of thromboxane A2-enhanced platelet activation,43 which is important for more advanced steps in thrombus formation. Interestingly, several studies have demonstrated that GPIbα-dependent platelet adhesion actually increases upon aspirin intake.44,45 These unexplained findings may be the result of aspirin-enhanced GPIbα clustering.
In conclusion, we have defined a central role for GPIbα clustering in platelet interaction with VWF under conditions of flow. Clustering of GPIbα requires translocation to lipid rafts and AA-mediated 14-3-3ζ binding to its cytoplasmic tail. These findings illustrate the mechanosensitive properties of platelets and give a new perspective on the molecular mechanism of arterial thrombus formation.
This study was supported by a grant from the Landsteiner Foundation of Blood Transfusion Research (LSBR grant n. 0807). Prof. Dr. JWN Akkerman is supported by the Netherlands Thrombosis Foundation. Dr. RT Urbanus is a research fellow of the Dutch Heart Foundation (grant n. 2010T068).
- The online version of this article has a Supplementary Appendix.
- Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.
- Received March 1, 2013.
- Accepted June 4, 2013.
- Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell. 1996; 84(2):289-97. PubMedhttps://doi.org/10.1016/S0092-8674(00)80983-6Google Scholar
- Schneider SW, Nuschele S, Wixforth A, Gorzelanny C, exander-Katz A, Netz RR. Shear-induced unfolding triggers adhesion of von Willebrand factor fibers. Proc Natl Acad Sci USA. 2007; 104(19):7899-903. PubMedhttps://doi.org/10.1073/pnas.0608422104Google Scholar
- Li C, Martin SE, Roth GJ. The genetic defect in two well-studied cases of Bernard-Soulier syndrome: a point mutation in the fifth leucine-rich repeat of platelet glycoprotein Ib alpha. Blood. 1995; 86(10):3805-14. PubMedGoogle Scholar
- Weiss HJ, Rogers J, Brand H. Defective ristocetin-induced platelet aggregation in von Willebrand’s disease and its correction by factor VIII. J Clin Invest. 1973; 52(11):2697-707. PubMedhttps://doi.org/10.1172/JCI107464Google Scholar
- Luo SZ, Mo X, Afshar-Kharghan V, Srinivasan S, Lopez JA, Li R. Glycoprotein Ibalpha forms disulfide bonds with 2 glycoprotein Ibbeta subunits in the resting platelet. Blood. 2007; 109(2):603-9. PubMedhttps://doi.org/10.1182/blood-2006-05-024091Google Scholar
- Du X, Beutler L, Ruan C, Castaldi PA, Berndt MC. Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane. Blood. 1987; 69(5):1524-7. PubMedGoogle Scholar
- Dong JF, Li CQ, Lopez JA. Tyrosine sulfation of the glycoprotein Ib-IX complex: identification of sulfated residues and effect on ligand binding. Biochemistry. 1994; 33(46):13946-53. PubMedhttps://doi.org/10.1021/bi00250a050Google Scholar
- Lopez JA, Chung DW, Fujikawa K, Hagen FS, Papayannopoulou T, Roth GJ. Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha 2-glycoprotein. Proc Natl Acad Sci USA. 1987; 84(16):5615-9. PubMedhttps://doi.org/10.1073/pnas.84.16.5615Google Scholar
- Korrel SA, Clemetson KJ, Van HH, Kamerling JP, Sixma JJ, Vliegenthart JF. Structural studies on the O-linked carbohydrate chains of human platelet glycocalicin. Eur J Biochem. 1984; 140(3):571-6. PubMedGoogle Scholar
- Andrews RK, Fox JE. Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX complex that binds to purified actin-binding protein. J Biol Chem. 1992; 267(26):18605-11. PubMedGoogle Scholar
- Du X, Harris SJ, Tetaz TJ, Ginsberg MH, Berndt MC. Association of a phospholipase A2 (14-3-3 protein) with the platelet glycoprotein Ib-IX complex. J Biol Chem. 1994; 269(28):18287-90. PubMedGoogle Scholar
- Huizinga EG, Tsuji S, Romijn RA, Schiphorst ME, de Groot PG, Sixma JJ. Structures of glycoprotein Ibalpha and its complex with von Willebrand factor A1 domain. Science. 2002; 297(5584):1176-9. PubMedhttps://doi.org/10.1126/science.107355Google Scholar
- Dumas JJ, Kumar R, McDonagh T, Sullivan F, Stahl ML, Somers WS. Crystal structure of the wild-type von Willebrand factor A1-glycoprotein Ibalpha complex reveals conformation differences with a complex bearing von Willebrand disease mutations. J Biol Chem. 2004; 279(22):23327-34. PubMedhttps://doi.org/10.1074/jbc.M401659200Google Scholar
- Kim J, Zhang CZ, Zhang X, Springer TA. A mechanically stabilized receptor-ligand flex-bond important in the vasculature. Nature. 2010; 466(7309):992-5. PubMedhttps://doi.org/10.1038/nature09295Google Scholar
- Dong JF, Berndt MC, Schade A, McIntire LV, Andrews RK, Lopez JA. Ristocetin-dependent, but not botrocetin-dependent, binding of von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with shear-dependent interactions. Blood. 2001; 97(1):162-8. PubMedhttps://doi.org/10.1182/blood.V97.1.162Google Scholar
- Shrimpton CN, Borthakur G, Larrucea S, Cruz MA, Dong JF, Lopez JA. Localization of the adhesion receptor glycoprotein Ib-IX-V complex to lipid rafts is required for platelet adhesion and activation. J Exp Med. 2002; 196(8):1057-66. PubMedhttps://doi.org/10.1084/jem.20020143Google Scholar
- Geng H, Xu G, Ran Y, Lopez JA, Peng Y. Platelet glycoprotein Ib beta/IX mediates glycoprotein Ib alpha localization to membrane lipid domain critical for von Willebrand factor interaction at high shear. J Biol Chem. 2011; 286(24):21315-23. PubMedhttps://doi.org/10.1074/jbc.M110.202549Google Scholar
- Kasirer-Friede A, Ware J, Leng L, Marchese P, Ruggeri ZM, Shattil SJ. Lateral clustering of platelet GP Ib-IX complexes leads to up-regulation of the adhesive function of integrin alpha IIbbeta 3. J Biol Chem. 2002; 277(14):11949-56. PubMedhttps://doi.org/10.1074/jbc.M108727200Google Scholar
- Arya M, Lopez JA, Romo GM, Cruz MA, Kasirer-Friede A, Shattil SJ. Glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3): effects of receptor clustering and von Willebrand factor adhesion. J Thromb Haemost. 2003; 1(6):1150-7. PubMedhttps://doi.org/10.1046/j.1538-7836.2003.00295.xGoogle Scholar
- Gitz E, Koekman CA, van den Heuvel DJ, Deckmyn H, Akkerman JW, Gerritsen HC. Improved platelet survival after cold storage by prevention of glycoprotein Ibalpha clustering in lipid rafts. Haematologica. 2012; 97(12):1873-81. PubMedhttps://doi.org/10.3324/haematol.2012.066290Google Scholar
- Nichols WL, Hultin MB, James AH, Manco-Johnson MJ, Montgomery RR, Ortel TL. von Willebrand disease (VWD): evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA). Haemophilia. 2008; 14(2):171-232. PubMedhttps://doi.org/10.1111/j.1365-2516.2007.01643.xGoogle Scholar
- Sakariassen KS, Aarts PA, de Groot PG, Houdijk WP, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med. 1983; 102(4):522-35. PubMedGoogle Scholar
- Wencel-Drake JD, Painter RG, Zimmerman TS, Ginsberg MH. Ultrastructural localization of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section. Blood. 1985; 65(4):929-38. PubMedGoogle Scholar
- Dai K, Bodnar R, Berndt MC, Du X. A critical role for 14-3-3zeta protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications. Blood. 2005; 106(6):1975-81. PubMedhttps://doi.org/10.1182/blood-2005-01-0440Google Scholar
- Yuan Y, Zhang W, Yan R, Liao Y, Zhao L, Ruan C. Identification of a novel 14-3-3zeta binding site within the cytoplasmic domain of platelet glycoprotein Ibalpha that plays a key role in regulating the von Willebrand factor binding function of glycoprotein Ib-IX. Circ Res. 2009; 105(12):1177-85. PubMedhttps://doi.org/10.1161/CIRCRESAHA.109.204669Google Scholar
- Bodnar RJ, Gu M, Li Z, Englund GD, Du X. The cytoplasmic domain of the platelet glycoprotein Ibalpha is phosphorylated at serine 609. J Biol Chem. 1999; 274(47):33474-9. PubMedhttps://doi.org/10.1074/jbc.274.47.33474Google Scholar
- Du X, Fox JE, Pei S. Identification of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ib alpha. J Biol Chem. 1996; 271(13):7362-7. PubMedhttps://doi.org/10.1074/jbc.271.13.7362Google Scholar
- van der Wal DE, Gitz E, Du VX, Lo KS, Koekman CA, Versteeg S. Arachidonic acid depletion extends survival of cold-stored platelets by interfering with the [glycoprotein Ibalpha - 14-3-3zeta] association. Haematologica. 2012; 97(10):1514-22. PubMedhttps://doi.org/10.3324/haematol.2011.059956Google Scholar
- Kramer RM, Roberts EF, Um SL, Borsch-Haubold AG, Watson SP, Fisher MJ. p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2. J Biol Chem. 1996; 271(44):27723-9. PubMedhttps://doi.org/10.1074/jbc.271.44.27723Google Scholar
- Dopheide SM, Maxwell MJ, Jackson SP. Shear-dependent tether formation during platelet translocation on von Willebrand factor. Blood. 2002; 99(1):159-67. PubMedhttps://doi.org/10.1182/blood.V99.1.159Google Scholar
- Reininger AJ, Heijnen HF, Schumann H, Specht HM, Schramm W, Ruggeri ZM. Mechanism of platelet adhesion to von Willebrand factor and microparticle formation under high shear stress. Blood. 2006; 107(9):3537-45. PubMedhttps://doi.org/10.1182/blood-2005-02-0618Google Scholar
- Ruggeri ZM, Orje JN, Habermann R, Federici AB, Reininger AJ. Activation-independent platelet adhesion and aggregation under elevated shear stress. Blood. 2006; 108(6):1903-10. PubMedhttps://doi.org/10.1182/blood-2006-04-011551Google Scholar
- Nesbitt WS, Westein E, Tovar-Lopez FJ, Tolouei E, Mitchell A, Fu J. A shear gradient-dependent platelet aggregation mechanism drives thrombus formation. Nat Med. 2009; 15(6):665-73. PubMedhttps://doi.org/10.1038/nm.1955Google Scholar
- Uff S, Clemetson JM, Harrison T, Clemetson KJ, Emsley J. Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation. J Biol Chem. 2002; 277(38):35657-63. PubMedhttps://doi.org/10.1074/jbc.M205271200Google Scholar
- Gu M, Xi X, Englund GD, Berndt MC, Du X. Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model. J Cell Biol. 1999; 147(5):1085-96. PubMedhttps://doi.org/10.1083/jcb.147.5.1085Google Scholar
- Mangin P, David T, Lavaud V, Cranmer SL, Pikovski I, Jackson SP. Identification of a novel 14-3-3zeta binding site within the cytoplasmic tail of platelet glycoprotein Ibalpha. Blood. 2004; 104(2):420-7. PubMedhttps://doi.org/10.1182/blood-2003-08-2881Google Scholar
- Fu H, Subramanian RR, Masters SC. 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol. 2000;40617-47. Google Scholar
- Lee CW, Han J, Bamburg JR, Han L, Lynn R, Zheng JQ. Regulation of acetylcholine receptor clustering by ADF/cofilin-directed vesicular trafficking. Nat Neurosci. 2009; 12(7):848-56. PubMedhttps://doi.org/10.1038/nn.2322Google Scholar
- Sumpio BE, Yun S, Cordova AC, Haga M, Zhang J, Koh Y. MAPKs (ERK1/2, p38) and AKT can be phosphorylated by shear stress independently of platelet endothelial cell adhesion molecule-1 (CD31) in vascular endothelial cells. J Biol Chem. 2005; 280(12):11185-91. PubMedhttps://doi.org/10.1074/jbc.M414631200Google Scholar
- Canobbio I, Reineri S, Sinigaglia F, Balduini C, Torti M. A role for p38 MAP kinase in platelet activation by von Willebrand factor. Thromb Haemost. 2004; 91(1):102-10. PubMedGoogle Scholar
- Brock TG. Arachidonic acid binds 14-3-3zeta, releases 14-3-3zeta from phosphorylated BAD and induces aggregation of 14-3-3zeta. Neurochem Res. 2008; 33(5):801-7. PubMedhttps://doi.org/10.1007/s11064-007-9498-3Google Scholar
- Pike LJ, Han X, Chung KN, Gross RW. Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis. Biochemistry. 2002; 41(6):2075-88. PubMedhttps://doi.org/10.1021/bi0156557Google Scholar
- Patrono C, Garcia Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med. 2005; 353(22):2373-83. PubMedhttps://doi.org/10.1056/NEJMra052717Google Scholar
- Turner NA, Moake JL, Kamat SG, Schafer AI, Kleiman NS, Jordan R. Comparative real-time effects on platelet adhesion and aggregation under flowing conditions of in vivo aspirin, heparin, and monoclonal antibody fragment against glycoprotein IIb–IIIa. Circulation. 1995; 91(5):1354-62. PubMedGoogle Scholar
- Grabowski EF. Platelet aggregation in flowing blood at a site of injury to an endothelial cell monolayer: quantitation and real-time imaging with the TAB monoclonal antibody. Blood. 1990; 75(2):390-8. PubMedGoogle Scholar