Expression and function of the mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcRgamma chain

Platelet glycoprotein (GP) VI has been proposed as the major collagen receptor for activation of human platelets. Human GPVI belongs to the immunoglobulin superfamily and is noncovalently associated with the FcRgamma chain that is involved in signaling through the receptor. In mice, similar mechanisms seem to exist as platelets from FcRgamma chain-deficient mice do not aggregate in response to collagen. However, the activating collagen receptor on mouse platelets has not been definitively identified. In the current study we examined the function and in vivo expression of GPVI in control and FcRgamma chain-deficient mice with the first monoclonal antibody against GPVI (JAQ1). On wild type platelets, JAQ1 inhibited platelet aggregation induced by collagen but not PMA or thrombin. Cross-linking of bound JAQ1, on the other hand, induced aggregation of wild type but not FcRgamma chain-deficient platelets. JAQ1 stained platelets and megakaryocytes from wild type but not FcRgamma chain-deficient mice. Furthermore, JAQ1 recognized GPVI (approximately 60 kDa) in immunoprecipitation and Western blot experiments with wild type but not FcRgamma chain-deficient platelets. These results strongly suggest that GPVI is the collagen receptor responsible for platelet activation in mice and demonstrate that the association with the FcRgamma chain is critical for its expression and function.     

The human megakaryocytic cell line UT‐7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor

We have demonstrated that a unique megakaryocytic cell line UT‐7/TPO could respond to one of the primary platelet signals through GP (glycoprotein) VI and a secondary signal of the AA (arachidonic acid) cascade. Unlike other megakaryocytic cell lines, UT‐7/TPO was found to express GPVI and its associate signal molecule of FcRγ (Fc receptor γ chain). When UT‐7/TPO was stimulated with the GPVI agonist convulxin, the [Ca²⁺]i (intracellular Ca²⁺) was elevated in a convulxin concentration‐dependent manner, and [Ca²⁺]i elevation was blocked by pretreatment with the Src family kinase inhibitor PP2 and the phospholipase inhibitor U73122. These results strongly indicate that endogenously expressed GPVI signal molecules are functional in UT‐7/TPO. Concerning the AA cascade, the expression of COX (cyclooxygenase)‐1 and TX (thromboxane) synthase was observed, and this cell line was able to produce TX by exogenous AA, followed by [Ca²⁺]i elevation mediated through the TX receptor. It is worth noting that convulxin stimulation did not cause TX generation, even through the GPVI pathway and the AA cascade are functional in this cell line. As there are many reports that convulxin‐stimulated platelets failed to produce TX, it is suggested that UT‐7/TPO has the same property as the platelets in regards to convulxin stimulation. Thus, UT‐7/TPO is useful for the observation of both the GPVI pathway and AA cascade without requiring either the induction of differentiation or GPVI transfection. Furthermore, this cell line provides a new tool for research on platelet activation signals.     

Lipid rafts orchestrate signaling by the platelet receptor glycoprotein VI

The platelet collagen receptor glycoprotein VI (GPVI) couples to the immune receptor adaptor Fc receptor gamma-chain (FcRgamma) and signals using many of the same intracellular signaling molecules as immune receptors. Studies of immune receptor signaling have revealed a critical role for specialized areas of the cell membrane known as lipid rafts, which are enriched in essential signaling molecules. However, the role of lipid rafts in signaling in nonimmune cells such as platelets remains poorly defined. This study shows that GPVI-FcRgamma does not constitutively associate with rafts, but is recruited to lipid rafts following receptor stimulation in both GPVI-expressing RBL-2H3 cells and human platelets. FcRgamma is required for GPVI association with lipid rafts, as mutant GPVI receptors that do not couple to FcRgamma were unable to associate with lipid rafts after receptor clustering. Following GPVI stimulation in platelets, virtually all phosphorylated FcRgamma was found in lipid rafts, but inhibition of FcRgamma phosphorylation did not block receptor association with lipid rafts. This work demonstrates that lipid rafts orchestrate GPVI receptor signaling in platelets in a manner analogous to immune cell receptors and supports a model of GPVI signaling in which FcRgamma phosphorylation is controlled by ligand-dependent association with lipid rafts.     

Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.     

The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors.

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.     

Effects of inhibitor of Src kinases, SU6656, on differentiation of megakaryocytic progenitors and activity of alpha1,6-fucosyltransferase

alpha1,6-fucosyltransferase (FUT8) attaches fucose residues via an alpha1,6 linkage to the innermost N-acetylglucosamine residue of N-linked glycans. Glycans with this type of structure are present in GpIIb/GpIIIa complex (CD41a) which is present on megakaryocytes (Mks) and platelets. CD41a is the earliest marker of megakaryocytopoiesis. The aim of this study was to analyse the morphology, phenotype, ploidy level and activity of FUT8 during induced differentiation/maturation of Mk progenitor cells in ex vivo culture. We used SU6656, a selective inhibitor of Src tyrosine kinases, as differentiation-inducing agent for Mks. The addition of SU6656 to the culture system of megakaryocytic progenitors from cord blood CD34(+) cells and Meg-01 cell line induced their maturation towards later stages of Mk differentiation with increased activity of FUT8. We suggest FUT8 as a candidate for an early marker of differentiation and possibly of the ploidy level of Mks. We confirm a special status of FUT8 in megakaryocytopoiesis.     

The platelet receptor GPVI mediates both adhesion and signaling responses to collagen in a receptor density-dependent fashion.

The platelet response to collagen is a primary event in hemostasis and thrombosis, but the precise roles of the numerous identified platelet collagen receptors remain incompletely defined. Attention has recently focused on glycoprotein VI (GPVI), a receptor that is expressed on platelets in association with a signaling adapter, the Fc receptor gamma chain (Fc Rgamma). Genetic and pharmacologic loss of GPVI function results in loss of collagen signaling in platelets, but studies to date have failed to demonstrate that GPVI-Fc Rgamma expression is sufficient to confer collagen signaling responses. These results have led to the hypothesis that collagen responses mediated by GPVI-Fc Rgamma may require the collagen-binding integrin alpha2beta1 as a co-receptor, but this model has not been supported by a recent study of mouse platelets lacking alpha2beta1. In the present study we have used a novel anti-GPVI monoclonal antibody to measure the level of GPVI on human platelets and to guide the development of GPVI-expressing cell lines to assess the role of GPVI in mediating platelet collagen responses. GPVI receptor density on human platelets appears tightly regulated, is independent from the level of alpha2beta1 expression, and significantly exceeds that on previously characterized GPVI-expressing RBL-2H3 cells. Using newly generated GPVI-expressing RBL-2H3 cells with receptor densities equivalent to that on human platelets, we demonstrate that GPVI expression confers both adhesive and signaling responses to collagen in a graded fashion that is proportional to the GPVI receptor density. These results resolve some of the conflicting data regarding GPVI-collagen interactions and demonstrate that 1) GPVI-Fc Rgamma expression is sufficient to confer both adhesion and signaling responses to collagen, and 2) GPVI-mediated collagen responses are receptor density-dependent at the receptor levels expressed on human platelets.     

2-Arachidonoylglycerol enhances platelet formation from human megakaryoblasts

Platelets modulate vascular system integrity, and their loss is critical in haematological pathologies and after chemotherapy. Therefore, identification of molecules enhancing platelet production would be useful to counteract thrombocytopenia. We have previously shown that 2-arachidonoylglycerol (2-AG) acts as a true agonist of platelets, as well as it commits erythroid precursors toward the megakaryocytic lineage. Against this background, we sought to further interrogate the role of 2-AG in megakaryocyte/platelet physiology by investigating terminal differentiation, and subsequent thrombopoiesis. To this end, we used MEG-01 cells, a human megakaryoblastic cell line able to produce in vitro platelet-like particles.

2-AG increased the number of cells showing ruffled surface and enhanced surface expression of specific megakaryocyte/platelet surface antigens, typical hallmarks of terminal megakaryocytic differentiation and platelet production. Changes in cytoskeleton modeling also occurred in differentiated megakaryocytes and blebbing platelets. 2-AG acted by binding to CB₁ and CB₂ receptors, because specific antagonists reverted its effect. Platelets were split off from megakaryocytes and were functional: they contained the platelet-specific surface markers CD61 and CD49, whose levels increased following stimulation with a natural agonist like collagen. Given the importance of 2-AG for driving megakaryopoiesis and thrombopoiesis, not surprisingly we found that its hydrolytic enzymes were tightly controlled by classical inducers of megakaryocyte differentiation.

In conclusion 2-AG, by triggering megakaryocyte maturation and platelet release, may have clinical efficacy to counteract thrombocytopenia-related diseases.     

Expression of the platelet receptor GPVI confers signaling via the Fc receptor gamma -chain in response to the snake venom convulxin but not to collagen

The mechanism of signal transduction underlying the activation of platelets by collagen has been actively investigated for over 30 years, but the receptors involved remain incompletely understood. Studies of human platelets, which are unresponsive to collagen, mouse knockout models, and platelet biochemical studies support the hypothesis that the recently cloned platelet surface protein GPVI functions as a signaling receptor for collagen. To directly test this hypothesis, we have expressed wild-type and mutant forms of GPVI in RBL-2H3 cells, which express the Fcepsilon receptor gamma-chain (Fc Rgamma), the putative signaling co-receptor for GPVI in platelets, but lack GPVI itself. Expression of GPVI in RBL-2H3 cells confers strong adhesive and signaling responses to convulxin (a snake venom protein that directly binds GPVI) and weak responsiveness to collagen-related peptide but no responsiveness to collagen. To elucidate the mechanism of GPVI intracellular signaling, mutations were introduced in the receptor's transmembrane domain and C-terminal tail. Unlike reported studies of other Fc Rgamma partners, these studies reveal that both the GPVI transmembrane arginine and intracellular C-tail are necessary for coupling to Fc Rgamma and for signal transduction. To our knowledge, these studies are the first to demonstrate a direct signaling role for GPVI and the first to directly test the role of GPVI as a collagen receptor. Our results suggest that GPVI may be necessary but not sufficient for collagen signaling and that a distinct ligand-binding collagen receptor such as the alpha(2)beta(1) integrin is likely to play a necessary role for collagen signaling as well as adhesion in platelets.     

Embryonic stem cell differentiation system for evaluating gene functions involved in physiological megakaryocytic differentiation

Megakaryocytic differentiation is accompanied by marked morphological changes induced by endomitosis and proplatelet formation. Molecular mechanisms underlying this unique cell differentiation process have been investigated by gain/loss-of-function studies using leukemic cell lines. However, these cell lines cannot completely mimic physiological megakaryocytic differentiation, including the morphological changes, and sometimes lead to contradictory results between cell lines. The goal of this study was to establish a novel cell differentiation system that completely mimics physiological megakaryocytic differentiation for analyzing gene function. To that end, we used homologous recombination to prepare an embryonic stem (ES) cell line containing a GFP-transgene driven by the PF4 promoter at the Hprt locus. Differentiation of these cells resulted in megakaryocytes and proplatelets, suggesting physiological megakaryocytic differentiation. However, the number of GFP-expressing cells was low (1.7% GFP(+) cells among CD41(+) cells). Insertion of full-length or small core β-globin insulators on either side of the transgene significantly increased the number of GFP-expressing cells (～60% GFP(+) cells among CD41(+) cells), and GFP-expression was specifically observed in megakaryocytic cells. Similar results were obtained with other ES cells containing a GPIIb-GFP transgene. Altogether, we have succeeded in efficiently expressing exogenous genes specifically in differentiating megakaryocytes and in establishing a novel ES cell differentiation system for analyzing gene function involved in physiological megakaryocytic differentiation.     

Convulxin binding to platelet receptor GPVI: competition with collagen related peptides

Convulxin (CVX), a potent platelet aggregating protein from the venom of the snake Crotalus durissus terrificus, is known to bind to the platelet collagen receptor, glycoprotein VI (GPVI). CVX binding to human platelets was investigated by flow cytometry, using fluorescein labeled convulxin (FITC-CVX). Scatchard analysis indicated high and low affinity binding sites with Kd values of 0.6 and 4 nM and Bmax values of 1200 and 2000 binding sites per platelet. FITC-CVX binding was inhibited by collagen related peptides (CRPs) comprising a repeated GPO sequence, namely GCO(GPO)(10)GCOGNH(2) and GKO(GPO)(10)GKOGNH(2), which also bind to receptor GPVI. These peptides (monomeric or cross-linked forms) gave a high affinity inhibition of 10-20% for concentrations between 10 ng/ml and 5 microg/ml, followed by a second phase of inhibition at concentrations greater than 5 microg/ml. It was shown also that the inhibition of FITC-CVX binding by CRPs was independent on the time of preincubation of platelets with CRPs, and the same percentage of inhibition was seen with various concentrations of convulxin. Confocal microscopy of the distribution of FITC-CVX binding sites on platelets showed an homogeneous distribution of FITC-CVX bound to GPVI, although some limited clustering may exist.     

Development of platelet inhibition by cAMP during megakaryocytopoiesis

Prostacyclin is a potent inhibitor of agonist-induced Ca2+ increases in platelets, but in the megakaryocytic cell line MEG-01 this inhibition is absent. Using human megakaryocytic cell lines representing different stages in megakaryocyte (Mk) maturation as well as stem cells and immature and mature megakaryocytes, we show that the inhibition by prostacyclin develops at a late maturation stage shortly before platelets are formed. This late appearance is not caused by insufficient cAMP formation or absent protein kinase A (PKA) activity in immature cells. Instead, the appearance of Ca2+ inhibition by prostacyclin is accompanied by a sharp increase in the expression of the catalytic subunit of PKA (PKA-C) but not by changes in the expression of the PKA-regulatory subunits Ialpha/beta, IIalpha, and IIbeta. Overexpression of PKA-C in the megakaryocytic cell line CHRF-288-11 potentiates the Ca2+ inhibition by prostacyclin. Thus, up-regulation of PKA-C appears to be a key step in the development of Ca2+ inhibition by prostacyclin in platelets.     

Expression of the fibrinogen genes in rat megakaryocytes

A variety of evidence suggests that megakaryocytes synthesize fibrinogen and comparative immunochemical and structural studies indicate that fibrinogen produced in or associated with megakaryocytes may be different than fibrinogen produced in the liver. Two studies have reported that the gamma' chain, which is produced from the gamma chain gene by alternative splicing, is absent from fibrinogen produced in the megakaryocyte. Since there is only a single gene for each of the three fibrinogen chains the reported structural differences suggest different mechanisms for production of hepatic and megakaryocytic fibrinogen. We have begun an investigation of the varying mechanisms for expression of the fibrinogen genes by examining the structure of fibrinogen mRNA's in the two tissues. Fibrinogen mRNA's of identical length are found in both liver and megakaryocytes. Furthermore, despite the reported absence of the gamma' chain in platelet-associated fibrinogen, we have used a probe specific for the alternative spliced region of the gamma' mRNA to clearly demonstrate this chain in megakaryocyte mRNA. These studies indicate that the gamma' mRNA is either not translated in platelets or that the gamma' chain is unable to associated with the alpha and beta chains to form a mature molecule.     

Spatially distinct production of reactive oxygen species regulates platelet activation

Platelets play a key role in hemostasis and changes in redox balance are known to alter platelet activation and aggregation. Interestingly, activation of platelets leads to production of reactive oxygen species (ROS), but the role(s) of these ROS remain unclear. Using flow cytometry and chemiluminescence, agonist-induced ROS generation was found to be spatially distinct with stimulation through the major collagen receptor GPVI inducing only intraplatelet ROS while thrombin induced production of extracellular ROS. Platelet activation by either the GPVI-selective agonist convulxin or thrombin was differentially regulated by ROS generation. Thus, surface expression of CD62P, CD40L, or activated integrin alphaIIbbeta3 was abrogated by pharmacologic antioxidants but externalization of phosphatidylserine was not inhibited. Furthermore, extracellular antioxidants SOD/catalase markedly inhibited thrombin-, but not convulxin-, induced CD62P expression and alphaIIbbeta3 activation. The data suggest that ROS selectively regulate biochemical steps in platelet activation and that distinct source(s) of ROS and discrete redox-sensitive pathway(s) may control platelet activation in response to GPVI or thrombin stimulation. Thus, targeting ROS with site-specific antioxidants may differentially regulate platelet activation via thrombin or collagen.     

Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin

The snake venom toxin convulxin activates platelets through the collagen receptor glycoprotein VI (GPVI)/Fc receptor gamma-chain (FcR gamma-chain) complex leading to tyrosine phosphorylation and activation of the tyrosine Syk and phospholipase Cgamma2 (PLCgamma2). In the present study, we demonstrate that convulxin is a considerably more powerful agonist than collagen or the GPVI-selective collagen-related peptide (CRP). Confirmation that the response to convulxin is mediated solely via Syk was provided by studies on Syk-deficient platelets. The increase in phosphorylation of the FcR gamma-chain is associated with marked increases in tyrosine phosphorylation of downstream proteins including Syk, linker for activation of T cells (LAT), SLP-76, and PLCgamma2. The transmembrane adapter LAT coprecipitates with SLP-76 and PLCgamma2, as well as with a number of other adapter proteins, some of which have not been previously described in platelets, including Cbl, Grb2, Gads, and SKAP-HOM. Gads is constitutively associated with SLP-76 and is probably the protein bridging its association with LAT. There was no detectable association between Grb2 and SLP-76 in control or stimulated cells, suggesting that the interaction of LAT with Grb2 is present in a separate complex to that of LAT-Gads-SLP-76. These results show that the trimeric convulxin stimulates a much greater phosphorylation of the FcR gamma-chain and subsequent downstream responses relative to CRP and collagen, presumably because of its ability to cause a greater degree of cross-linking of GPVI. The adapter LAT appears to play a critical role in recruiting a number of other adapter proteins to the surface membrane in response to activation of GPVI, presumably at sites of glycolipid-enriched microdomains, enabling an organized signaling cascade that leads to platelet activation.     

A novel viper venom metalloproteinase, alborhagin, is an agonist at the platelet collagen receptor GPVI

The interaction of platelet membrane glycoprotein VI (GPVI) with collagen can initiate (patho)physiological thrombus formation. The viper venom C-type lectin family proteins convulxin and alboaggregin-A activate platelets by interacting with GPVI. In this study, we isolated from white-lipped tree viper (Trimeresurus albolabris) venom, alborhagin, which is functionally related to convulxin because it activates platelets but is structurally different and related to venom metalloproteinases. Alborhagin-induced platelet aggregation (EC50, <7.5 microg/ml) was inhibitable by an anti-alphaIIbbeta3 antibody, CRC64, and the Src family kinase inhibitor PP1, suggesting that alborhagin activates platelets, leading to alphaIIbbeta3-dependent aggregation. Additional evidence suggested that, like convulxin, alborhagin activated platelets by a mechanism involving GPVI. First, alborhagin- and convulxin-treated platelets showed a similar tyrosine phosphorylation pattern, including a similar level of phospholipase Cgamma2 phosphorylation. Second, alborhagin induced GPVI-dependent responses in GPVI-transfected K562 and Jurkat cells. Third, alborhagin-dependent aggregation of mouse platelets was inhibited by the anti-GPVI monoclonal antibody JAQ1. Alborhagin had minimal effect on convulxin binding to GPVI-expressing cells, indicating that these venom proteins may recognize distinct binding sites. Characterization of alborhagin as a GPVI agonist that is structurally distinct from convulxin demonstrates the versatility of snake venom toxins and provides a novel probe for GPVI-dependent platelet activation.     

Functional expression of TRAIL and TRAIL-R2 during human megakaryocytic development

The expression and function of surface TRAIL and TRAIL receptors were investigated in primary megakaryocytic cells, generated in serum-free liquid phase from peripheral human CD34(+) cells. The surface expression of both TRAIL and "death receptor" TRAIL-R2 became detectable starting from the early phase of megakaryocytic differentiation (day 6 of culture) and persisted at later (days10-14) culture times. On the other hand, "death receptor" TRAIL-R1, "decoy receptors" TRAIL-R3, and TRAIL-R4 were barely detectable or undetectable at any time point examined. Addition of recombinant TRAIL at day 6 of culture increased the rate of spontaneous apoptosis of CD34(+)/CD41(dim) megakaryoblasts and it significantly decreased the total output of mature megakaryocytic cells evaluated after additional 4-8 days of culture. Conversely, addition in culture of TRAIL-R2-Fc chimera, which blocked the interaction between endogenous TRAIL and TRAIL-R2 on the surface of cultured megakaryocytic cells, increased the total megakaryocytic cell count. In addition, recombinant TRAIL promoted a small but reproducible increase of maturation in the surviving megakaryocytic cell population, evaluated by both phenotypic analysis and morphology. A similar pro-maturation effect was observed when TRAIL was added to bone marrow-derived CD61(+) megakaryocytic cells. Thus, our data suggest a role of TRAIL as a regulator of megakaryocytopoiesis.