The glycoprotein Ibα–von Willebrand factor interaction induces platelet apoptosis

Summary. Background: The interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbα–VWF interaction. Objectives: To investigate whether the GPIbα–VWF interaction induces platelet apoptosis and the role of 14‐3‐3ζ in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild‐type (1b9) or mutant GPIb–IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin‐induced GPIbα–VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear‐induced apoptosis was eliminated by the anti‐GPIbα antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb–IX with GPIbα truncated at residue 551 or a serine‐to‐alanine mutation at the 14‐3‐3ζ‐binding site in GPIbα. Conclusions: This study demonstrates that the GPIbα–VWF interaction induces apoptotic events in platelets, and that the association of 14‐3‐3ζ with the cytoplasmic domain of GPIbα is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.     

Platelet apoptosis by cold‐induced glycoprotein Ibα clustering

Summary. Background: Cold‐storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Ibα‐distribution indicative of receptor clustering and initiates thromboxane A₂‐formation. GPIbα is associated with 14‐3‐3 proteins, which contribute to GPIbα‐signaling and in nucleated cells take part in apoptosis regulation. Objectives and methods: We investigated whether GPIbα‐clustering induces platelet apoptosis through 14‐3‐3 proteins during cold (4 h 0 °C)‐rewarming (1 h 37 °C). Results: During cold‐rewarming, 14‐3‐3 proteins associate with GPIbα and dissociate from Bad inducing Bad‐dephosphorylation and activation. This initiates pro‐apoptosis changes in Bax/Bcl‐x_L and Bax‐translocation to the mitochondria, inducing cytochrome c release. The result is activation of caspase‐9, which triggers phosphatidylserine exposure and platelet phagocytosis by macrophages. Responses are prevented by N‐acetyl‐d ‐glucosamine (GN), which blocks GPIbα‐clustering, and by O‐sialoglycoprotein endopeptidase, which removes extracellular GPIbα. Conclusions: Cold‐rewarming triggers apoptosis through a GN‐sensitive GPIbα‐change indicative of receptor clustering. Attempts to improve platelet transfusion by cold‐storage should focus on prevention of the GPIbα‐change.     

The membrane‐proximal intermolecular disulfide bonds in glycoprotein Ib influence receptor binding to von Willebrand factor

Summary. Background: In the platelet glycoprotein (GP)Ib–IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted to the N‐terminal domain of the GPIbα subunit. How the other subunits in the complex, GPIbβ and GPIX, regulate the GPIbα–VWF interaction is not clear. Objectives and methods: As GPIbα connects with two GPIbβ subunits via disulfide bonds, we tested whether these intersubunit covalent links were important to the proper VWF‐binding activity of the GPIb–IX complex by characterizing the structure and VWF‐binding activity of a mutant GPIb–IX complex that lacked the GPIbα–GPIbβ disulfide bonds. Results: Mutating both Cys484 and Cys485 of GPIbα to serine prevents GPIbα from forming covalent disulfide bonds with GPIbβ, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIbβ subunits to form a disulfide bond between themselves. As compared to Chinese hamster ovary (CHO) cells stably expressing the wild‐type GPIb–IX complex at a comparable level, CHO cells stably expressing the mutant GPIb–IX complex bind to significantly less soluble VWF in the presence of ristocetin and roll on the immobilized VWF under flow at a higher velocity. Conclusions: The disulfide bonds between GPIbα and GPIbβ are necessary for optimal GPIbα binding to VWF. The structural plasticity around the disulfide bonds may also help to shed light on the inside‐out mechanism underlying GPIbβ modulation of VWF binding.     

The platelet glycoprotein GPIbβ intracellular domain participates in von Willebrand factor induced‐filopodia formation independently of the Ser 166 phosphorylation site

See also Gardiner EE. A GPIb‐IX‐V complex signaling environment. This issue, pp 1075–6. Summary. Background: Circulating platelets are initially recruited at the site of vessel injury by von Willebrand factor (VWF) immobilized on collagen fibers. This process, mediated by the GPIb–V–IX complex, is accompanied by specific intracellular signaling leading to reorganization of the platelet actin cytoskeleton and extension of filopodia. Objectives/methods: To evaluate the GPIbα and GPIbβ intracellular domains contribution to this signaling, we generated Chinese hamster ovary (CHO) cells expressing a GPIb–IX complex with mutant forms of the two subunits and we measured their ability to extend filopodia upon adhesion on a VWF matrix. Results: Complete intracellular deletion or elimination of the filamin or the 14‐3‐3ζ binding sites in GPIbα did not prevent filopodia extension. In contrast, deletion of the juxtamembrane (Leu¹⁵⁰–Arg¹⁶⁰) or central (Ala¹⁵⁹–Pro¹⁷⁰) intracellular segment of GPIbβ resulted in a 21% and 23% reduction in the number of cells extending filopodia, respectively. This occurred without decreasing adhesion efficiency or GPIb–IX association with filamin A or 14‐3‐3ζ. Alanine scanning mutagenesis of the Leu¹⁵⁰–Pro¹⁷⁰ segment identified Arg¹⁶⁴, Leu¹⁶⁵, Leu¹⁶⁷, Thr¹⁶⁸ and Pro¹⁷⁰ as important residues for efficient filopodia formation. Surprisingly, mutation of the Ser¹⁶⁶ PKA phosphorylation site did not alter adhesion and shape change. A role for the GPIbβ subunit was reinforced by the decreased capacity to extend filopodia upon adhesion on VWF of platelets from knock‐in mice expressing a GPIbβ intracellular deletion mutant. Conclusions: Altogether, our results strongly support participation of GPIbβ and its intracellular region in GPIb‐dependent platelet activation and shape change triggered by a VWF matrix.     

Redox control of β2‐glycoprotein I–von Willebrand factor interaction by thioredoxin‐1

Summary. Background: β₂‐Glycoprotein I (β₂GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β₂GPI in thrombus formation is unknown. We have recently shown that β₂GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin‐1 and protein disulfide isomerase, and that reduction of β₂GPI can take place on the platelet surface. Methods: β₂GPI, reduced by thioredoxin‐1, was labeled with the selective sulfhydryl probe N^a‐(3‐maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β₂GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin‐activated VWF was studied in the presence of reduced β₂GPI. Results: We demonstrate that the Cys288–Cys326 disulfide in domain V of β₂GPI is the predominant disulfide reduced by thioredoxin‐1. Reduced β₂GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β₂GPI reduced by thioredoxin‐1, in comparison with non‐reduced β₂GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol‐dependent interaction of β₂GPI with VWF may contribute to the redox regulation of platelet adhesion.     

Platelet endothelial cell adhesion molecule‐1 regulates collagen‐stimulated platelet function by modulating the association of phosphatidylinositol 3‐kinase with Grb‐2‐associated binding protein‐1 and linker for activation of T cells

Summary. Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)γ‐chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3‐kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb‐2‐associated binding protein‐1 (Gab1), which is regulated by binding of the Src homology 2 domain‐containing protein tyrosine phosphatase‐2 (SHP‐2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcRγ‐chain signaling via recruitment of SHP‐2 to phosphorylated immunoreceptor tyrosine‐based inhibitory motifs in PECAM‐1. Objective: To investigate the possibility that PECAM‐1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI‐mediated platelet activation in platelets. Methods: The ability of PECAM‐1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM‐1‐associated SHP‐2 in collagen‐stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen‐stimulated PI3K signaling. We therefore propose that PECAM‐1‐mediated inhibition of GPVI‐dependent platelet responses result, at least in part, from recruitment of SHP‐2–p85 complexes to tyrosine‐phosphorylated PECAM‐1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.     

Platelet activation by dimeric β2-glycoprotein I requires signaling via both glycoprotein Ibα and apolipoprotein E receptor 2'

Summary. Background: Dimerization of β₂‐glycoprotein I (β₂‐GPI) by autoantibodies is thought to trigger the clinical manifestations observed in the antiphospholipid syndrome. Arterial thrombosis, a frequently occurring clinical manifestation of the antiphospholipid syndrome, is a process in which platelets play a crucial role. Previous work has shown that binding of dimeric β₂‐GPI to the platelet receptors apolipoprotein E receptor 2′ (ApoER2′) and glycoprotein Ibα (GPIbα) mediates increased platelet activation in an in vitro thrombosis model. Objective: The individual roles of ApoER2′ and GPIbα in mediating platelet activation by dimeric β₂‐GPI has hitherto been unclear. In this study, we have determined the roles of either receptor in platelet activation by dimeric β₂‐GPI. Methods: Platelet activation by dimeric β₂‐GPI was studied under conditions of flow. Intracellular signaling induced by dimeric β₂‐GPI was subsequently analyzed by means of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis. Results: The increase in platelet deposition onto a fibronectin surface under conditions of flow by dimeric β₂‐GPI was completely abolished by inhibition of the interaction of dimeric β₂‐GPI with either GPIbα or ApoER2′. Upon platelet stimulation with dimeric β₂‐GPI, GPIbα translocated to the cytoskeleton via the scaffold protein 14‐3‐3ζ. Concomitantly, ApoER2′ dissociated from the adapter protein Disabled1, presumably through phosphorylation of the cytoplasmic tail. Inhibition of one process could not inhibit the other. Conclusion: We show that dimeric β₂‐GPI signals via two distinct pathways in platelets, both of which are required for platelet activation. Abrogation of either signal results in loss of activation.     

Human target validation of phosphoinositide 3‐kinase (PI3K)β: effects on platelets and insulin sensitivity,using AZD6482 a novel PI3Kβ inhibitor

Nylander S, Kull B, Björkman JA, Ulvinge JC, Oakes N, Emanuelsson BM, Andersson M, Skärby T, Inghardt T, Fjellström O, Gustafsson D. Human target validation of phosphoinositide 3‐kinase (PI3K)β:effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kβ inhibitor. J Thromb Haemost 2012; 10: 2127–36. See also Jackson SP, Schoenwaelder SM. Antithrombotic phosphoinositide 3‐kinase β inhibitors in humans – a ‘shear’ delight! This issue, pp 2123–6. Summary. Background: Based on in vitro and animal data, PI3Kβ is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear. Objective: To strengthen the PI3Kβ target validation using the novel, short‐acting inhibitor AZD6482. Methods and results: AZD6482 is a potent, selective and ATP competitive PI3Kβ inhibitor (IC₅₀ 0.01 μm ). A maximal anti‐platelet effect was achieved at 1 μm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti‐thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3‐h infusion. The ex vivo anti‐platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin‐induced human adipocyte glucose uptake in vitro (IC₅₀ of 4.4 μm ). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 μm but reduced by about 60% at a plasma exposure of 27 μm . In man, the homeostasis model analysis (HOMA) index increased by about 10–20% at the highest plasma concentration of 5.3 μm . Conclusions: This is the first human target validation for PI3Kβ inhibition as anti‐platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at ‘supratherapeutic’ plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.     

Molecular basis of Bernard–Soulier syndrome in 27 patients from India

To cite this article: Sumitha E, Jayandharan GR, David S, Jacob RR, Sankari Devi G, Bargavi B, Shenbagapriya S, Nair SC, Abraham A, George B, Viswabandya A, Mathews V, Chandy M, Srivastava A. Molecular basis of Bernard–Soulier syndrome in 27 patients from India. J Thromb Haemost 2011; 9 : 1590–8. Summary. Background: Bernard–Soulier syndrome (BSS) is an extremely rare (1:1 million) bleeding disorder of platelet adhesion, caused by defects in the glycoprotein (GP)Ib/IX/V complex. Patients and methods: The diagnosis in 27 patients was based on low platelet count, presence of giant platelets and aggregometry studies. Flow cytometry to assess the surface GPIb/IX/V complex showed reduced (7.7–57%) expression. gDNA was screened for mutations in the GPIBA, GPIBB, GP9 genes using PCR‐conformation sensitive gel electrophoresis (CSGE). Results: Thirteen different disease‐causing mutations, including missense (54%), frameshifts (38%) and nonsense (8%) mutations, were identified in 27 patients. Eleven of them were novel including five novel frameshifts (GPIbα: p.Gln97_98fsX113, p.Pro402_403fsX52; GPIbβ: p.Arg17fsX14; GPIX: p.Gly24fsX43, p. Pro130fs, a nonsense mutation (GPIX, p.94, Gln>X) and five novel missense mutations (GPIbα: p.492, Tyr>His; GPIbβ: p.65, Pro>Arg, p.129, Gln>His, p.132, Leu>Pro; GPIX: p.55, Phe>Cys). Interestingly, four common mutations, Cys8Arg (n = 6) and Phe55Ser (n = 2), Phe55Cys (n = 2) in GPIX and a novel 22‐bp deletion in the GPIBB gene predicting p.Arg17fsX 14 (n = 10) were seen in 20 patients. Conclusion: The molecular data presented here is the largest series of BSS patients to be reported so far, adding significantly to the mutation database of this condition and also useful for its genetic diagnosis in India.     

Phosphoinositide 3‐kinase mediates CD40 ligand‐induced oxidative stress and endothelial dysfunction via Rac1 and NADPH oxidase 2

Summary. Objectives: CD40 ligand (CD40L) has been implicated as an inducer of reactive oxygen species (ROS) generation in endothelial cells, but definitive evidence for this and the in vivo relevance haves not been demonstrated fully. We thus investigated whether phosphoinositide 3‐kinase (PI3K) was linked to ROS generation and endothelial reactivity in response to CD40L. Methods and Results: CD40L treatment activated PI3K activity by regulating the association between PI3K p85 and the CD40 receptor. CD40L exposure also stimulated the GTPase Rac1, which is known to activate NADPH oxidases, and enhanced ROS formation, whereas PI3K inhibition or depletion by small interfering RNA (siRNA) prevented these responses. Subsequently, PI3K overexpression activated Rac1 and increased ROS generation. These responses were not observed in the presence of inactive Rac1 or siRNA against the NADPH oxidase subunit NOX2. Protein kinase Cζ mediates PI3K‐regulated NADPH oxidase activation by promoting cellular p47phox translocation. Importantly, PI3K inhibition prevented CD40L‐mediated ROS generation and endothelial dysfunction in a mouse model. In summary, PI3K mediates CD40L‐induced ROS production and subsequent endothelial dysfunction. Conclusions: Targeting PI3K may provide a new therapeutic approach in diseases associated with oxidative stress and endothelial dysfunction.     

Nitric oxide inhibits von Willebrand factor‐mediated platelet adhesion and spreading through regulation of integrin αIIbβ3 and myosin light chain

Summary. Background: von Willebrand factor (VWF)‐mediated platelet adhesion and spreading at sites of vascular injury is a critical step in hemostasis. This process requires two individual receptors: glycoprotein Ib (GPIb)‐V‐IX and integrin α_IIbβ₃. However, little is known about the negative regulation of these events. Objectives: To examine if the endogenous platelet inhibitor nitric oxide (NO) has differential effects on adhesion, spreading and aggregation induced by immobilized VWF. Results: S‐nitrosoglutathione (GSNO) inhibited platelet aggregation on immobilized VWF under static and flow conditions, but had no effect on platelet adhesion. Primary signaling events underpinning the actions of NO required cyclic GMP but not protein kinase A. Dissecting the roles of GPIb and integrin α_IIbβ₃ demonstrated that NO targeted α_IIbβ₃‐mediated aggregation and spreading, but did not significantly influence GPIb‐mediated adhesion. To understand the relationship between the effects of NO on adhesion and subsequent aggregation, we evaluated the activation of α_IIbβ₃ on adherent platelets. NO reduced the phosphorylation of extracellular stimuli‐responsive kinase (ERK) and p38, required for integrin activation resulting in reduced binding of the activated α_IIbβ₃‐specific antibody PAC‐1 on adherent platelets. Detailed analysis of platelet spreading initiated by VWF demonstrated key roles for integrin α_IIbβ₃ and myosin light chain (MLC) phosphorylation. NO targeted both of these pathways by directly modulating integrin affinity and activating MLC phosphatase. Conclusion: These data demonstrate that initial activation‐independent platelet adhesion to VWF via GPIb is resistant to NO, however, NO inhibits GPIb‐mediated activation of α_IIbβ₃ and MLC leading to reduced platelet spreading and aggregation.     

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.     

TNF receptor‐associated factor 4 (TRAF4) is a novel binding partner of glycoprotein Ib and glycoprotein VI in human platelets

Summary. Background: Reactive oxygen species generation is one consequence of ligand engagement of platelet glycoprotein (GP) receptors GPIb‐IX‐V and GPVI, which bind VWF/collagen and initiate thrombosis at arterial shear; however, the precise molecular mechanism coupling redox pathway activation to engagement of these receptors is unknown. Objective: The objective of this study was to identify novel binding partners for GPIb‐IX‐V and GPVI that could provide a potential link between redox pathways and early platelet signaling events. Methods and Results: Using protein array analysis and affinity‐binding assays, we demonstrated that the orphan TNF receptor‐associated factor (TRAF) family member, TRAF4, selectively binds cytoplasmic sequences of GPIbβ and GPVI. TRAF4, p47^phox [of the NADPH oxidase (Nox2) enzyme complex] and other redox relevant signaling proteins such as Hic‐5, co‐immunoprecipitate with GPIb/GPVI from human platelet lysates whilst MBP‐TRAF4 or MBP‐p47^phox fusion proteins specifically pull‐down GPIb/GPVI. GPIb‐ or GPVI‐selective agonists induce phosphorylation of the TRAF4‐associated proteins, Hic‐5 and Pyk2, with phosphorylation attenuated by Nox2 inhibition. Conclusion: These results describe the first direct association of TRAF4 with a receptor, and identify a novel binding partner for GPIb‐IX‐V and GPVI, providing a potential link between these platelet receptors and downstream TRAF4/Nox2‐dependent redox pathways.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

凝血酶信号传导过程中P13-K与MLCK激酶的作用

本研究比较凝血酶受体活化过程中不同浓度的wortmannin对血小板聚集率以及血小板膜表面活化标记物糖蛋白GPIb表达的影响,探讨脂酰肌醇-3-激酶（P13-K）与肌球蛋白轻链激酶（MLCK）在凝血酶信号传递机制中的作用。分别以凝血酶受体活化肽PAR1-AP（SFLLRN）与PAR4-AP（AYPGKF）诱导血小板活化。检测100nmol／Lwortmannin（抑制P13-K）与10μmol／Lwortmannin（抑制MLCK）作用过程中血小板聚集与血小板膜表面糖蛋白GPIb的改变。结果显示：wortmannin作用对两类凝血酶受体诱导血小板活化的过程均有不同程度的影响。100nmol／L的wortmannin部分抑制PAR1-AP引起的血小板聚集,对PAR4-AP诱导的聚集过程没有影响;10μmol／Lwortmannin明显抑制两类PAR肽引起的血小板活化,抑制程度达到60％-80％,仅出现少部分聚集。流式细胞仪检测显示,GPIba的动态分布过程中100nmot／Lwortmannin能部份抑制GPIbα向胞内移动,PAR1—AP刺激后5分钟内wortmannjn起效明显（1、2、5分钟P〈0．05）,对PAR4-AP的反应则稍延迟,在2到10分钟有意义（2、5、10分钟P〈0．05）。10μmol／Lwortmannin对整个PAR活化过程中的GPIba逆转没有任何影响,只对PAR1刺激过程中GPIbcL的恢复起作用,延缓GPIbα重返血小板膜表面的进程;而PAR4-AP作用后各时间段GPIba的动态分布不受10μmol／Lwortmannin任何影响。结论：P13-K与MLCK在凝血酶受体的活化过程中作用不同,P13-K在GPIbat内转的短暂过程中起着重要作用;而MLCK磷酸化仅仅对PAR1受体介导的GPIbα回复起促进作用。     

Identification of five novel 14-3-3 isoforms interacting with the GPIb–IX complex in platelets

Summary.  Background : Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib–IX complex initiates a signaling cascade leading to integrin α_IIbβ₃ activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3ζ with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. Objective : The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb–IX. Results : In this study, western blot analyses showed that platelets also contain the 14-3-3β, 14-3-3γ, 14-3-3ε, 14-3-3η and 14-3-3θ isoforms, but lack 14-3-3σ. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3ζ, bind to GPIb–IX. In addition, their interaction was found to critically require the same GPIbα domains (580–590 and 605–610) already identified as essential for 14-3-3ζ binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbβ. In contrast, deletion or mutation of the GPIbβ intracytoplasmic tail did not affect the interaction of GPIb–IX with the 14-3-3 isoforms, questioning the importance of this domain. Conclusions : Our study suggests that, to inhibit GPIb-induced integrin α_IIbβ₃ activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.     

RGD‐ligand mimetic antagonists of integrin αIIbβ3 paradoxically enhance GPVI‐induced human platelet activation

Summary. Background: The integrin α_IIbβ₃ is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of α_IIbβ₃, for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD‐ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. Objectives: To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. Methods: We assessed the effect of lotrafiban, and other α_IIbβ₃ antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro‐coagulant activity) and dense granule release. Results: In all cases, no effect of α_IIbβ₃ antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)‐associated FcR γ‐chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y₁₂ antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. Conclusions: These data provide a mechanism for enhancement of platelet activity by α_IIbβ₃ inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.     

Ciliary neurotrophic factor (CNTF) promotes skeletal muscle progenitor cell (MPC) viability via the phosphatidylinositol 3‐kinase–Akt pathway

Muscle progenitor cells (MPCs) are currently being investigated as cellular vectors to deliver neurotrophic factor (NF) for the promotion of re‐innervation after axonal injury. Ideally NF delivery in such a model would enhance axonal regeneration while simultaneously promoting MPC viability. To date, insulin‐like growth factor 1 (IGF‐1) is one of the few NFs known to promote both re‐innervation and MPC viability. We herein identify ciliary neurotrophic factor (CNTF) as a factor that promotes MPC viability in culture, and demonstrate CNTF to impart greater viability effects on MPCs than IGF‐1. We demonstrate that pharmacological inhibition via LY294002 results in abrogation of CNTF‐mediated viability, suggesting that the CNTF‐mediated MPC viability benefit occurs via the PI3–Akt pathway. Finally, we employ a genetic model, establishing MPC cultures from mice deficient in class I_A PI‐3 K (p85α^?/?) mice, and demonstrate that the viability benefit imparted by CNTF is completely abrogated in PI‐3 K‐deficient MPCs compared to wild‐type controls. In summary, our investigations define CNTF as a promoter of MPC viability beyond IGF‐1, and reveal that the CNTF‐mediated MPC viability effects occur via the PI3–Akt pathway. Copyright © 2012 John Wiley & Sons, Ltd.     

Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3‐kinase

Summary. Background and objective: Salvianolic acid A (SAA) is a water‐soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in Asian medicine. As platelets play pivotal roles in atherothrombogenesis, we studied the effect of SAA on platelet activation and its underlying mechanisms. Methods and Results: SAA dose‐dependently inhibited platelet aggregation induced by ADP, thrombin, collagen and U46619. It reduced ADP‐enhanced platelet P‐selectin expression and fibrinogen binding, which consequently hampered ADP‐induced platelet–leukocyte aggregation. SAA also inhibited platelet spreading on fibrinogen, a process mediated by outside‐in signaling. Under an arterial shear rate of 1000 s^?1, SAA decreased platelet adhesion on collagen surfaces by ～ 40%. Western blot analysis showed that SAA, like the phosphoinositide 3‐kinase (PI3K) inhibitors LY294002 and TGX‐221, potently inhibited PI3K, as shown by reduced Akt phosphorylation. The in vitro findings were further evaluated in the mouse model of arterial thrombosis, in which SAA prolonged the mesenteric arterial occlusion time in wild‐type mice (35 ± 2 min without SAA and 56 ± 4 min with SAA; P < 0.01). Interestingly, SAA could even counteract the shortened arterial occlusion time in Ldlr^tm1Her mutant mice (21 ± 2 min without SAA and 45 ± 4 min with SAA; P < 0.01). Conclusions: SAA inhibits platelet activation via the inhibition of PI3K, and attenuates arterial thrombus formation in vivo. Our data suggest that SAA may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.     

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.