The platelet glycoprotein Ib-IX-V complex anchors lipid rafts to the membrane skeleton: implications for activation-dependent cytoskeletal translocation of signaling molecules

Summary.  Background:  The glycoprotein (GP) Ib-IX-V complex attaches platelets to areas of endothelial damage by binding von Willebrand factor (VWF), an interaction that transmits intracellular activation signals. These signals require that the complex associates with both lipid rafts and the membrane cytoskeleton, but it is not clear whether the same GPIb-IX-V subpopulation associates with both structures. Objectives: To determine which subpopulation of GPIb-IX-V associates with lipid rafts, and the consequences of that interaction. Methods:  We analyzed the content of proteins (particularly the GPIb-IX-V complex) and lipids in rafts from detergent lysates of platelets before and after removal of the actin cytoskeleton alone or both the actin cytoskeleton and membrane skeleton (by successive centrifugations of 15 800 ×  g and 100 000 ×  g ). Results:  In unstimulated platelets, little raft-associated GPIb-IX-V sedimented with the actin skeleton; most was removed by sedimentation of the membrane skeleton. The Src family kinase Lyn followed the same pattern. In VWF-activated platelets, almost all of the GPIb-IX-V complex and Lyn in rafts sedimented with the actin cytoskeleton, consistent with a previously described crosslinking of the membrane and actin skeletal structures following platelet activation. Disruption of the GPIbα–filamin linkage with N -ethylmaleimide prevented depletion of raft-associated GPIb-IX-V by skeletal sedimentation. Not all raft-associated proteins and lipids followed this pattern. Conclusion:  These results suggest that the raft association and cytoskeletal linkage of the GPIb-IX-V complex are interrelated, and both are required for optimal receptor function, perhaps because raft association attracts signaling proteins and membrane skeletal association allows these proteins to move en masse to new locations.     

The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin.

We have identified platelet glycoprotein (GP) Ibalpha as a counterreceptor for P-selectin. GP Ibalpha is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibalpha, and GP Ibalpha-expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin-dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibalpha. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibalpha required neither calcium nor carbohydrate core-2 branching or alpha(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibalpha, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.     

Localization of the kappa opioid receptor in lipid rafts

Lipid rafts are microdomains of plasma membranes enriched in cholesterol and sphingolipids in the outer layer. We determined whether kappa opioid receptors (KOR) in human placenta and FLAG (DYKDDDDK)-tagged human KOR (FLAG-hKOR) expressed in Chinese hamster ovary (CHO) cells are localized in lipid rafts and whether changes in cholesterol contents affect hKOR properties and signaling. Lipid rafts were prepared from placenta membranes and CHO cells expressing FLAG-hKOR using the Na2CO3 method and fractionation through a sucrose density gradient. The majority of the KOR in the placenta and FLAG-hKOR in CHO cells, determined by [3H]diprenorphine binding and/or immunoblotting with an anti-FLAG antibody, was present in low-density fractions, coinciding with high levels of caveolin-1 and cholesterol, markers of lipid rafts, which indicated that the KOR is localized in lipid rafts. Pretreatment with 2% methyl beta-cyclodextrin (MCD) reduced cholesterol content by approximately 48% and changed the cells from spindle-shaped to spherical. MCD treatment disrupted lipid rafts, shifted caveolin-1 and FLAG-hKOR to higher density fractions, increased the affinity of (-)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488H) for the hKOR, and greatly increased U50,488H-induced [35S]guanosine 5'-O-(3-thio)triphosphate binding and p42/44 mitogen-activated protein kinase phosphorylation. Cholesterol replenishment reversed all the MCD effects. Caveolin-1 immunoprecipitated with Galphai proteins and MCD treatment reduced caveolin-1 associated with Galphai proteins, which may contribute to the enhanced agonist-induced G protein activation. Caveolin-1 also immunoprecipitated with FLAG-hKOR, but MCD treatment had no effect on the association. Thus, the KOR is located in lipid rafts and its localization in the microdomains greatly affects coupling to G proteins.     

von Willebrand factor binding to platelet glycoprotein Ib-IX-V stimulates the assembly of an α-actinin-based signaling complex

Summary. Background: Pathological shear stress induces platelet aggregation that is dependent on von Willebrand factor (VWF) binding to glycoprotein (Gp)Ib‐IX‐V and phosphatidylinositol 3‐kinase activation. We tested the hypothesis that pathological shear stress stimulates phosphatidylinositol 3,4,5‐trisphosphate (PIP₃) synthesis by directing the assembly of a molecular signaling complex that includes class IA phosphatidylinositol 3‐kinase (PI 3‐KIA). Methods: Platelets were subjected to 120 dynes cm^?2 shear stress in a cone‐plate viscometer. Resting and sheared platelets were lyzed, immunoprecipitations of PI 3‐KIA performed, or lipids extracted for PIP₃ measurements. α‐Actinin was incubated with phosphatidylinositol 4,5‐bisphosphate (PIP₂), immunoprecipitated, and used as a substrate for in vitro PI 3‐KIA activity. Results: Pathological shear stress induces biphasic PIP₃ production. In resting platelets, PI 3‐KIA associates with α‐actinin and PIP₂. After exposure to shear stress, α‐actinin and PIP₂ rapidly disassociate from PI 3‐KIA. PI 3‐KIA then gradually re‐associates with PIP₂ and α‐actinin, and this complex becomes linked to GpIbα through the cytoskeleton. PIP₃ production and the observed changes in the association between α‐actinin, PIP₂, and PI 3‐KIA are inhibited when VWF binding to GpIbα is blocked. In a cell‐free system, α‐actinin binds PIP₂ and when the α‐actinin–PIP₂ complex is added to platelet PI 3‐KIA, PIP₃ production is stimulated. Conclusions: These results suggest that pathological shear‐induced VWF binding to GpIb‐IX‐V stimulates PIP₃ production through the assembly of an α‐actinin‐based complex that colocalizes PI 3‐KIA with substrate PIP₂.     

Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation.

Platelet activation by thrombin is critical for hemostasis and thrombosis. Structure-function studies with a recently cloned platelet thrombin receptor suggest that a hirudin-like domain in the receptor's extracellular amino terminal extension is a thrombin-binding determinant important for receptor activation. We now report that a peptide antiserum to this domain is a potent and specific antagonist of thrombin-induced platelet activation. This study demonstrates that the cloned platelet thrombin receptor is necessary for platelet activation by thrombin, and provides a strategy for developing blocking monoclonal antibodies of potential therapeutic value.     

Comparative analysis of various platelet glycoprotein IIb/IIIa antagonists on shear-induced platelet activation and adhesion

Platelet accretion into arterial thrombus in stenotic arterial vessels involves shear-induced platelet activation and adhesion. The Cone and Plate(let) Analyzer (CPA) is designed to simulate such conditions in vitro under a rotating high shear rate in whole blood. In the present study, we evaluated various experimental conditions (including aspirin, temperature, and calcium concentration) and investigated the effects of small molecules along with peptide glycoprotein IIb/IIIa antagonists on platelet adhesion using the CPA system. Concentration-dependent effect of glycoprotein IIb/IIIa antagonists on shear-induced platelet adhesion showed marked differences in potencies: IC50 = 34, 35, 91, 438, and 606 nM for DPC802 (a specific glycoprotein IIb/IIIa antagonist), roxifiban, sibrafiban, lotrafiban, and orbofiban (free acid forms), respectively, and IC50 values of 43, 430, and 5781 nM for abciximab, tirofiban, and eptifibatide, respectively. Parallel study was also conducted to evaluate the effect of glycoprotein IIb/IIIa inhibitors using optical aggregometry. The potency of fibans in blocking shear-induced platelet adhesion correlated well with their binding affinity to the resting and activated glycoprotein IIb/IIIa receptors, as well as their "off-rates". Nevertheless, none of these fibans was able to effectively block shear-induced platelet adhesion at targeted clinical dosing regimens except for abciximab. These data suggest that glycoprotein IIb/IIIa antagonists that show similar efficacy in the inhibition of platelet aggregation in a static in vitro assay may differ substantially in a shear-based system of platelet adhesion. The clinical significance of this phenomenon awaits further investigation.     

Negative regulators of platelet activation and adhesion

Summary

Platelets are small anucleated cells that constantly patrol the cardiovascular system to preserve its integrity and prevent excessive blood loss where the vessel lining is breached. Their key challenge is to form a hemostatic plug under conditions of high shear forces. To do so, platelets have evolved a molecular machinery that enables them to sense trace amounts of signals at the site of damage and to rapidly shift from a non‐adhesive to a pro‐adhesive state. However, this highly efficient molecular machinery can also lead to unintended platelet activation and cause clinical complications such as thrombocytopenia and thrombosis. Thus, several checkpoints are in place to tightly control platelet activation and adhesiveness in space and time. In this review, we will discuss select negative regulators of platelet activation, which are critical to maintain patrolling platelets in a quiescent, non‐adhesive state and/or to limit platelet adhesion to sites of injury.

     

Localization of human platelet autoantigens to the cysteine-rich region of glycoprotein IIIa.

The object of this study was to further localize autoantigenic structures on IIb-IIIa and, if possible, to precisely identify the epitopes recognized by human autoantibodies. In this paper, we identify a 50-kD chymotryptic fragment of IIIa that is recognized by a high percentage of human autoantibodies, typified by the prototype IgG autoantibody RA, which binds to IIIa on intact platelets as well as in an immunoblot assay under nonreduced conditions. Using an immunoblot assay, a carboxy-terminal region of this fragment (33 kD) that contains the cysteine-rich domains of IIIa was found to carry the epitope(s) recognized by the prototype autoantibody RA. The amino-terminal amino acid sequence of the reduced 33-kD fragment, the smallest fragment that retains the RA epitope, is XPSQQDEXSP, and that of the reduced 50-kD fragment is IVQVTFD. This indicates that the 33-kD fragment consists of approximately 175 amino acids beginning at residue 479 and extending at least through residues 636-654, while the 50-kD fragment spans the same region but begins at residue 427. It is apparent that the 33-kD fragment is generated from the 50-kD fragment by additional chymotryptic hydrolysis but remains associated because of the multiple disulfide bonds that are characteristic of this cysteine-rich domain. Sera from 48% of patients with chronic ITP and 2 of 8 patients with acute ITP contain antibodies that bind to the 50-kD fragment in an ELISA. Antibodies of the same specificity are also found in one-third of patients with either secondary immune thrombocytopenia or apparent non-immune thrombocytopenia. We conclude that the 50-kD cysteine-rich region of IIIa is a frequent target of autoantibodies in ITP, but that such antibodies may also be present in cases of thrombocytopenia that cannot be linked to an apparent autoimmune process.     

Studies on the binding of an alloimmune and two murine monoclonal antibodies to the platelet glycoprotein IIb-IIIa complex receptor

The platelet-binding characteristics of three different antibodies that completely block adenosine diphosphate (ADP)-induced platelet fibrinogen binding and react with glycoproteins IIb, IIIa, or both, were studied. Two of the antibodies are murine monoclonal antibodies (10E5 and 7E3), and the third is an alloimmune antibody produced by a patient with Glanzmann's thrombasthenia who has received multiple transfusions (E.S.). The two monoclonals differ in that 7E3, but not 10E5, binds to dog platelets as well as to human platelets. In mutual competition studies, 7E3 did not interfere with 10E5 binding, indicating that both antibodies could bind to the complex simultaneously. E.S. IgG produced only minor inhibition of 10E5 binding, but nearly complete inhibition of 7E3 binding, suggesting that its epitope lies closer to the 7E3 epitope than the 10E5 epitope. Ethylenediaminetetraacetic acid (EDTA) pretreatment of intact platelets at 22 degrees C and pH 7.75 did not affect 10E5 binding, but significantly inhibited 7E3 binding. Similar treatment of intact or solubilized platelets at high pH and temperature produced splitting of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex and loss of both 10E5 and 7E3 binding. The 10E5 bound equally well to unactivated and activated platelets, and even rapid fixation of whole blood produced only a modest decrease in 10E5 binding. Preincubation of platelets with either E.S. IgG or 10E5 partially prevented EDTA from splitting the complex, and both monoclonal antibodies were able to bind to such complexes despite the EDTA treatment. These studies indicate that the binding of antibodies to several different epitopes on the GPIIb-IIIa complex can result in similar functional properties in blocking fibrinogen binding and platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid rafts are required in Gαi signaling downstream of the P2Y12 receptor during ADP-mediated platelet activation

ADP is important in propagating hemostasis upon its secretion from activated platelets in response to other agonists. Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids, and have been implicated in the stimulatory mechanisms of platelet agonists. We sought to determine the importance of lipid rafts in ADP-mediated platelet activation via the G protein-coupled P2Y1 and P2Y12 receptors using lipid raft disruption by cholesterol depletion with methyl-beta-cyclodextrin. Stimulation of cholesterol-depleted platelets with ADP resulted in a reduction in the extent of aggregation but no difference in the extent of shape change or intracellular calcium release. Furthermore, repletion of cholesterol to previously depleted membranes restored ADP-mediated platelet aggregation. In addition, P2Y12-mediated inhibition of cAMP formation was significantly decreased upon cholesterol depletion from platelets. Stimulation of cholesterol-depleted platelets with agonists that depend upon Galpha(i) activation for full activation displayed significant loss of aggregation and secretion, but showed restoration when simultaneously stimulated with the Galpha(z)-coupled agonist epinephrine. Finally, Galpha(i) preferentially localizes to lipid rafts as determined by sucrose density centrifugation. We conclude that Galpha(i) signaling downstream of P2Y12 activation, but not Galpha(q) or Galpha(z) signaling downstream of P2Y1 or alpha2A activation, respectively, has a requirement for lipid rafts that is necessary for its function in ADP-mediated platelet activation.     

A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.     

Globular adiponectin induces platelet activation through the collagen receptor GPVI-Fc receptor γ chain complex

Summary.  Background:  The adipocyte-derived cytokine, adiponectin (Ad), exerts potent vascular effects, although the direct effects of Ad on blood platelets are unclear. Objective:  The influence of globular Ad (gAd) on blood platelet function was investigated. Research design and methods:  We measured platelet aggregation and tyrosine phosphorylation signaling events in human and mouse platelets. The ability of gAd to activate Glycoprotein VI (GPVI) activity was determined with a NFAT luciferase reporter assay. Results:  gAd, but not full length Ad, induced rapid aggregation and granule secretion of human and mouse platelets through a pathway that is ablated under conditions of Src kinase inhibition, indicating a tyrosine kinase-dependent mechanism. Consistent with this, gAd stimulates rapid tyrosine phosphorylation of several proteins in human and mouse platelets. The pattern of increase in tyrosine phosphorylation was similar to that induced by collagen, with the tyrosine kinase Syk and PLCγ2 being identified among the list of tyrosine phosphorylated proteins. As collagen activates platelet through the GPVI-Fc receptor γ-chain (FcRγ) complex, we used FcRγ null platelets (which also lack GPVI) to explore the mechanism by which gAd stimulates platelets. Stimulation of tyrosine phosphorylation and platelet aggregation by gAd was abolished in FcRγ null platelets and markedly reduced in the absence of PLCγ2. Further, GPVI was confirmed as a collagen receptor for gAd by increased luciferase activity in Jurkat T-cells transfected with GPVI. Conclusions:  We identify gAd as a novel ligand for GPVI that stimulates tyrosine kinase-dependent platelet aggregation. Our data raise the possibility that gAd may promote unwanted platelet activation at sites of vascular injury.     

Transmembrane domains are critical to the interaction between platelet glycoprotein V and glycoprotein Ib‐IX complex

Summary. Background: The glycoprotein (GP) Ib‐IX‐V complex, the von Willebrand factor receptor on the platelet surface, is critically involved in hemostasis and thrombosis. The GPV subunit interacts with GPIb‐IX to form the GPIb‐IX‐V complex, but the underlying molecular basis remains unclear. It was observed earlier that efficient expression of GPV in the plasma membrane requires co‐expression of GPIb‐IX. Objectives and methods: Hypothesizing that GPIb‐IX stabilizes GPV through direct interaction and consequently enhances GPV surface expression, we aim in this study to identify structural elements in the complex that mediate the interaction between GPV and GPIb‐IX by analyzing mutational effects on GPV surface expression in transfected Chinese hamster ovary cells. Results: Enhancement of GPV surface expression by GPIb‐IX requires transmembrane domains of both GPV and GPIbα, as replacing the GPV transmembrane domain with an unrelated poly‐leucine‐alanine sequence abolished the enhancing effect of GPIb‐IX. Additional mutagenesis analysis of the GPV transmembrane helix identified three helical sides containing conserved polar residues as critical to efficient GPV surface expression. Similarly, replacing residues in three sides (Gly495/Ala502/Leu509, Phe491/Trp498/Val505, and Y492/L499/L506) of the GPIbα transmembrane domain with leucines preserved the surface expression level of GPIb‐IX but significantly altered that of GPV. Conclusions: Our results demonstrate for the first time the importance of transmembrane domains for efficient surface expression of GPV and suggest that GPV and GPIbα transmembrane domains interact with each other, contributing to assembly of the GPIb‐IX‐V complex.     

GpIIb/IIIa is the main receptor for initial platelet adhesion to glass and titanium surfaces in contact with whole blood

Platelets are the first cells to adhere to a surface in contact with blood and are capable of mediating several different responses after contact with different protein-coated surfaces. They are the main source of growth factors such as platelet-derived growth factor and are therefore important in the healing process. In this study, initial platelet adhesion to and spread on hydrophilic and hydrophobic (methylized) glass and titanium with similar wettability were investigated. Whole coagulating blood was used to simulate the in vivo situation shortly after implantation, in which bleeding precedes inflammation and wound healing. Several different antibodies directed against platelet integrins and receptors (CD9, FcgammaRII, GPIIb/IIIa, vitronectin receptor, GPIb/V/IX) were used in an attempt to block platelet adhesion to the surfaces. Immunofluorescence results show that initial platelet adhesion to all the surfaces we investigated can be almost completely inhibited (approximately 95%) by clone M148, an antibody against the GPIIb/IIIa complex (integrin alpha(IIb)beta(3); CD41/CD61), but not with other antibodies to the separate parts of the integrin. Antibodies known to inhibit fibrinogen binding to GPIIb/IIIa after adenosine diphosphate- and collagen- induced aggregation had very little effect on initial platelet adhesion. None of the other integrins were found to have such an effect on initial platelet adhesion. Antibody clone M148 was furthermore found to inhibit platelet spreading. This study shows that regardless of wettability and the biomaterial used, initial adhesion of platelets appears to be mediated by GPIIb/IIIa binding to surface adsorbed fibrinogen.     

Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate.

Platelet adhesion and thrombus formation on subendothelium, studied at a shear rate of 2,600 s-1, were inhibited by two synthetic peptides known to interact with GPIIb-IIIa. One peptide (HHLGGAKQAGDV) corresponds to the carboxyl terminal segment of the fibrinogen gamma-chain (gamma 400-411) and the other (RGDS) contains the amino acid sequence Arg-Gly-Asp (RGD) common to fibronectin, von Willebrand factor, vitronectin and the alpha-chain of fibrinogen. Neither platelet adhesion nor thrombus formation were decreased in a patient with severe congenital fibrinogen deficiency and this was equally true when his blood was further depleted of the small amounts of fibrinogen present utilizing an anti-fibrinogen antibody. In normal subjects, adhesion and thrombus formation were inhibited by the Fab' fragments of a monoclonal anti-GPIIb-IIIa antibody (LJ-CP8), which interferes with the interaction of platelets with all four adhesive proteins in both the fluid and solid phase. However, another anti-GPIIb-IIIa antibody (LJ-P5) that had minimal effects on the interaction of platelets with fibrinogen, but inhibited to varying degrees platelet interaction with other adhesive proteins, was equally effective. The findings demonstrate that, at a shear rate of 2,600 s-1, adhesive proteins other than fibrinogen are involved in GPIIb-IIIa-mediated platelet adhesion and thrombus formation on subendothelium. In addition, since LJ-P5 inhibited the binding of soluble von Willebrand factor and vitronectin, these adhesive proteins may be involved in platelet thrombus formation. In contrast to the results obtained at a shear rate of 2,600 s-1, fibrinogen could play a role in mediating platelet-platelet interactions with weak agonists or lower shear rates.     

A role for lipid rafts in B cell antigen receptor signaling and antigen targeting

The B cell antigen receptor (BCR) serves both to initiate signal transduction cascades and to target antigen for processing and presentation by MHC class II molecules. How these two BCR functions are coordinated is not known. Recently, sphingolipid- and cholesterol-rich plasma membrane lipid microdomains, termed lipid rafts, have been identified and proposed to function as platforms for both receptor signaling and membrane trafficking. Here we show that upon cross-linking, the BCR rapidly translocates into ganglioside G(M1)-enriched lipid rafts that contain the Src family kinase Lyn and exclude the phosphatase CD45R. Both Igalpha and Lyn in the lipid rafts become phosphorylated, and subsequently the BCR and a portion of G(M1) are targeted to the class II peptide loading compartment. Entry into lipid rafts, however, is not sufficient for targeting to the antigen processing compartments, as a mutant surface Ig containing a deletion of the cytoplasmic domain is constitutively present in rafts but when cross-linked does not internalize to the antigen processing compartment. Taken together, these results provide evidence for a role for lipid rafts in the initial steps of BCR signaling and antigen targeting.