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  • Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-as ... 20723025

    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.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.The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets.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.
    Document Type:
    Reference
    Product Catalog Number:
    06-195

  • Platelet matrix metalloprotease-1 mediates thrombogenesis by activating PAR1 at a cryptic ligand site. 19379698

    Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex. 26028144

    The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Platelet alpha2beta1 integrin activation: contribution of ligand internalization and the alpha2-cytoplasmic domain. 12738679

    The alpha2beta1 integrin is a major collagen receptor on platelets. Although it has been proposed that alpha2beta1, like alphaIIbbeta3, undergoes agonist-induced activation, neither the potential contributions of alpha2beta1 receptor/ligand internalization to the increase in ligand binding nor the roles of the alpha2 and beta1 cytoplasmic domains in activation of this integrin have been previously explored. Activation of alpha2beta1 was assessed with fluorescein isothiocyanate-labeled soluble type I collagen binding to platelets by flow cytometry. Although collagen internalization in response to agonist activation of platelets was significant, agonist-induced collagen binding still occurred under conditions that block internalization, with minimal changes in cell surface alpha2beta1 expression. Introduction of cell-permeable peptides containing the alpha2 cytoplasmic tail, and especially the membrane proximal KLGFFKR domain, induced alpha2beta1 activation in resting platelets, whereas a cell-permeable peptide containing the beta1 cytoplasmic tail was without effect. Thus, collagen binding to stimulated platelets is increased due to alpha2beta1 activation, in addition to internalization, and the GFFKR motif appears to play an important role in the activation process.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Platelet adhesion to multimerin 1 in vitro: influences of platelet membrane receptors, von Willebrand factor and shear. 19175495

    BACKGROUND: Multimerin 1 (MMRN1) is a large, homopolymeric adhesive protein, stored in platelets and endothelium, that when released, binds to activated platelets, endothelial cells and the extracellular matrix. OBJECTIVES: The goals of our study were to determine if (i) MMRN1 supports adhesion of resting and/or activated platelets under conditions of blood flow, and (ii) if MMRN1 enhances platelet adhesion to types I and III collagen. PATIENTS/METHODS: Platelet adhesion was evaluated using protein-coated microcapillaries, with or without added adhesive proteins and receptor antibodies. Platelets from healthy controls, Glanzmann thrombasthenia (GT) and severe von Willebrand factor (VWF)-deficient donors were tested. RESULTS: MMRN1 supported the adhesion of activated, but not resting, washed platelets over a wide range of shear rates. At low shear (150 s(-1)), this adhesion was supported by integrins alphavbeta3 and glycoprotein (GP) Ibalpha but it did not require integrins alphaIIbbeta3 or VWF. At high shear (1500 s(-1)), adhesion to MMRN1 was supported by beta3 integrin-independent mechanisms, involving GPIbalpha and VWF, that did not require platelet activation when VWF was perfused over MMRN1 prior to platelets. MMRN1 bound to types I and III collagen, independent of VWF, however, its enhancing effects on platelet adhesion to collagen at high shear were VWF dependent. CONCLUSIONS: MMRN1 supports platelet adhesion by VWF-dependent and -independent mechanisms that vary by flow rate. Additionally, MMRN1 binds to, and enhances, platelet adhesion to collagen. These findings suggest that MMRN1 could function as an adhesive ligand that promotes platelet adhesion at sites of vascular injury.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1976
    Product Catalog Name:
    Anti-Integrin αVβ3 Antibody, clone LM609 (Anti-Integrin αVβ3 Antibody, clone LM609)

  • The Platelet Glycoprotein GPIbbeta intracellular domain participates in von Willebrand factor induced-filopodia formation independently of the Ser 166 phosphorylation sit ... 19694944

    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 GPIbalpha and GPIbbeta intracellular domains contribution to this signaling, we generated 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 14-3-3zeta binding sites in GPIbalpha did not prevent filopodia extension. In contrast, deletion of the juxtamembrane (Leu(150) to Arg(160)) or central (Ala(159) to Pro(170)) intracellular segment of GPIbbeta resulted in a 21 and 23% reduction in the number of cells extending filopodia, respectively. This occured without decreasing adhesion efficiency or GPIb-IX association with filamin A or 14-3-3zeta. Alanine scanning mutagenesis of the Leu(150) to Pro(170) segment identified Arg(164), Leu(165), Leu(167), Thr(168) and Pro(170) as important residues for efficient filopodia formation. Surprisingly, mutation of the Ser(166) PKA phosphorylation site did not alter adhesion and shape change. A role for the GPIbbeta subunit was reinforced by the decreased capacity to extend filopodia upon adhesion on VWF of platelets from knock in mice expressing a GPIbbeta intracellular deletion mutant. Conclusions: Altogether, our results strongly support participation of GPIbbeta and its intracellular region in GPIb-dependent platelet activation and shape change triggered by a VWF matrix.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1678
    Product Catalog Name:
    Anti-Filamin A Antibody, clone PM6/317 (Anti-Filamin A Antibody, clone PM6/317)

  • Can platelet BACE1 levels be used as a biomarker for Alzheimer's disease? Proof-of-concept study. 22775589

    To date there is no validated peripheral biomarker to assist with the clinical diagnosis of Alzheimer's disease (AD). Platelet proteins have been studied as AD biomarkers with relative success. In this study, we investigated whether platelet BACE1 levels differ between AD and cognitively normal (CN) control patients. Using a newly developed ELISA method, we found that BACE1 levels were significantly lower in AD compared to CN subjects. These data were supported by the observation that several BACE1 isoforms, identified by Western blotting, were also lower in AD platelets. This proof-of-concept study provides evidence for testing platelet BACE1 levels as a peripheral AD biomarker using a novel, sensitive and inexpensive method.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5308
    Product Catalog Name:
    Anti-BACE Antibody, CT, clone 61-3E7 (Anti-BACE Antibody, CT, clone 61-3E7)

  • Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa. 21370733

    The aim of this study was to investigate the effect of platelet activating factor (PAF) on the quality characteristics of cryopreserved canine spermatozoa. Cryopreserved semen of 5 mixed-breed dogs was treated with different concentrations of exogenous PAF (1 x 10(-3) M, 1 x 10(-4) M, 1 x 10(-5) M and 1 x 10(-6) M) and examined at different time intervals (0, 30, 60 and 120 min). Cryopreserved semen treated without PAF was used as the control. Sperm quality was evaluated for motility (computer-assisted semen analysis, CASA), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (SYBR-14/PI assay and Hoechst 33258). Also, ATP content of spermatozoa was determined using a bioluminescence assay. Treatment of cryopreserved semen with 1 x 10(-3) M PAF at 120 min of incubation resulted in significantly higher total sperm motility compared with the control. It was observed that PAF-improved total sperm motility was concurrent with enhanced sperm motility patterns after treatment of cryopreserved semen. Treatment of cryopreserved semen with PAF did not improve either sperm mitochondrial function or plasma membrane integrity, as monitored by different fluorescent membrane markers. Furthermore, ATP content of cryopreserved spermatozoa was significantly higher when PAF was used at a concentration of 1 x 10(-3) M compared with the control and other PAF treatments, regardless of the incubation time. The findings of this study indicated that treatment with 1 x 10(-3) M PAF at 120 min of incubation rendered better quality of cryopreserved canine semen, which was associated with improved sperm motility parameters and ATP content. It can be suggested that exogenous PAF addition is beneficial as a supplement for canine semen extender used for cryopreservation.
    Document Type:
    Reference
    Product Catalog Number:
    20-106
    Product Catalog Name:
    pNPP (p-Nitrophenyl Phosphate) (pNPP (p-Nitrophenyl Phosphate))

  • Trp207Gly in platelet glycoprotein Ibalpha is a novel mutation that disrupts the connection between the leucine-rich repeat domain and the disulfide loop structure and ca ... 17083647

    BACKGROUND: Bernard-Soulier syndrome (BSS) is a severe inherited bleeding disorder that is caused by a defect in glycoprotein (GP)Ib-IX-V complex, the platelet membrane receptor for von Willebrand factor. PATIENTS: The diagnosis of BSS was made in two members of a Bukharian Jewish family who had life-long thrombocytopenia associated with mucocutaneous bleeding manifestations. METHODS AND RESULTS: Flow cytometry and Western blot analyses showed only trace amounts of GPIb and GPIX on the patients' platelets. Sequence analysis of the GPIbalpha gene revealed a homozygous T > G transversion at nucleotide 709 predicting Trp207Gly substitution in the mature protein. Introduction of the mutation into a mammalian expression construct abolished the surface expression of GPIbalpha in transfected baby hamster kidney cells. The crystal structure of the N-terminus of GPIbalpha (PDB: 1SQ0) indicates that Trp207 is completely buried and located in a disulfide loop structure that interacts with the leucine-rich repeat (LRR) domain. CONCLUSION: A novel mutation, Trp207Gly, causes BSS and predicts disruption of the interaction between a disulfide loop and the LRR domain that is essential for the integrity of GPIbalpha structure.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1202
    Product Catalog Name:
    Anti-CD42a Antibody, platelet lb-IX, clone FMC-25 (Anti-CD42a Antibody, platelet lb-IX, clone FMC-25)