Glycoprotein VI Signaling In Platelets Research Articles

Platelet glycoprotein VI-dependent thrombus stabilization is essential for the intraportal engraftment of pancreatic islets.

Platelet activation and thrombus formation have been implicated to be detrimental for intraportal pancreatic islet transplants. The platelet-specific collagen receptor glycoprotein VI (GPVI) plays a key role in thrombosis through cellular activation and the subsequent release of secondary mediators. In aggregometry and in a microfluidic dynamic assay system modeling flow in the portal vein, pancreatic islets promoted platelet aggregation and triggered thrombus formation, respectively. While platelet GPVI deficiency did not affect the initiation of these events, it was found to destabilize platelet aggregates and thrombi in this process. Interestingly, while no major difference was detected in early thrombus formation after intraportal islet transplantation, genetic GPVI deficiency or acute anti-GPVI treatment led to an inferior graft survival and function in both syngeneic mouse islet transplantation and xenogeneic human islet transplantation models. These results demonstrate that platelet GPVI signaling is indispensable in stable thrombus formation induced by pancreatic islets. GPVI deficiency resulted in thrombus destabilization and inferior islet engraftment indicating that thrombus formation is necessary for a successful intraportal islet transplantation in which platelets are active modulators.

Plant-based Food Cyanidin-3-Glucoside Modulates Human Platelet Glycoprotein VI Signaling and Inhibits Platelet Activation and Thrombus Formation

Background: Platelets play an important role in hemostasis, thrombosis, and atherosclerosis. Glycoprotein VI (GPVI) is a major platelet receptor that interacts with exposed collagen on injured vessel walls. Our previous studies have shown that anthocyanins (a type of natural plant pigment) attenuate platelet function; however, whether anthocyanins affect collagen-induced GPVI signaling remains unknown.Objective: The objective of this study was to explore the effects of cyanidin-3-glucoside (Cy-3-g, one of the major bioactive compounds in anthocyanins) on platelet activation and thrombosis and the GPVI signaling pathway.Methods: Platelets from healthy men and women were isolated and incubated with different concentrations (0, 0.5, 5, and 50 μM) of Cy-3-g. The expression of activated integrin αIIbβ3, P-selectin, CD63, and CD40L, fibrinogen binding to platelets, and platelet aggregation were evaluated in vitro. Platelet adhesion and aggregation in whole blood under flow conditions were assessed in collagen-coated perfusion chambers. Thrombosis and hemostasis were assessed in 3-4-wk-old male C57BL/6J mice through FeCl3-induced intravital microscopy and tail bleeding time. The effect of Cy-3-g on collagen-induced human platelet GPVI signaling was explored with Western blot.Results: Cy-3-g attenuated platelet function in a dose-dependent manner. The 0.5-μM dose of Cy-3-g inhibited (P < 0.05) human platelet adhesion and aggregation to collagen at both venous (-54.02%) and arterial (-22.90%) shear stresses. The 5-μM dose inhibited (P < 0.05) collagen-induced human platelet activation (PAC-1: -48.21%, P-selectin: -50.63%), secretion (CD63: -73.89%, CD40L: -43.70%), fibrinogen binding (-56.79%), and aggregation (-17.81%). The 5-μM dose attenuated (P < 0.01) thrombus growth (-66.67%) without prolonging bleeding time in mice. The 50-μM dose downregulated (P < 0.05) collagen-induced GPVI signaling in human platelets and significantly decreased phosphorylation of Syk-linker for activation of T cells (LAT)-SLP76 (Syk: -39.08%, LAT: -32.25%, SLP76: -40.00%) and the expression of Lyn (-31.89%), Fyn (-36.27%), and phospholipase C-γ2 (-39.08%).Conclusions: Cy-3-g inhibits human platelet activation, aggregation, secretion, and thrombus formation, and downregulates the collagen-GPVI signaling pathway. Supplementation of Cy-3-g may have protective effects against atherothrombosis.

Clustering of glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets

Essentials Dimeric high‐affinity collagen receptor glycoprotein VI (GPVI) is present on resting platelets.Spatio‐temporal organization of platelet GPVI‐dimers was evaluated using advanced microscopy.Upon platelet adhesion to collagenous substrates, GPVI‐dimers coalesce to form clusters.Clustering of GPVI‐dimers may increase avidity and facilitate platelet activation SummaryBackgroundPlatelet glycoprotein VI (GPVI) binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen, and occurs constitutively on resting platelets.ObjectiveTo identify higher‐order oligomerization (clustering) of pre‐existing GPVI dimers upon interaction with collagen as a mechanism to initiate GPVI‐mediated signaling.Methods GPVI was located by use of fluorophore‐conjugated GPVI dimer‐specific Fab (antigen‐binding fragment). The tested substrates include Horm collagen I fibers, soluble collagen III, GPVI‐specific collagen peptides, and fibrinogen. GPVI dimer clusters on the platelet surface interacting with these substrates were visualized with complementary imaging techniques: total internal reflection fluorescence microscopy to monitor real‐time interactions, and direct stochastic optical reconstruction microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI dimer clusters, glycoprotein Ib, integrin α2β1, and phosphotyrosine.ResultsUpon platelet adhesion to all collagenous substrates, GPVI dimers coalesced to form clusters; notably clusters formed along the fibers of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate, collagen III being the most effective. Clusters on fibrinogen‐adhered platelets were much smaller and more numerous; whether these are pre‐existing oligomers of GPVI dimers or fibrinogen‐induced is not clear. Some GPVI dimer clusters colocalized with areas of phosphotyrosine, indicative of signaling activity. Integrin α2β1 was localized to collagen fibers close to GPVI dimer clusters. GPVI clustering depends on a dynamic actin cytoskeleton.ConclusionsPlatelet adhesion to collagen induces GPVI dimer clustering. GPVI clustering increases both avidity for collagen and the proximity of GPVI‐associated signaling molecules, which may be crucial for the initiation and persistence of signaling.

ELMO1 Is a Novel Negative Regulator of Glycoprotein VI Signaling in Platelets

PI3-kinase (phosphoinositide 3-kinase) is an important signaling molecule that is activated downstream of various receptors upon platelet activation. PI3-kinase activation leads to the generation of PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate) subsequently leading to the recruitment of PH (pleckstrin homology) domain containing proteins to the plasma membrane. Our laboratory screened for proteins that interacted with PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate) using PIP3 beads. One of the proteins that interacted with PIP3 was ELMO1 (Engulfment and cell motility-1). ELMO1 is a scaffold protein with no catalytic activity and is well known to regulate actin cytoskeletal rearrangement via Rac1 in other cells. However, it is not known whether ELMO1 is expressed in platelets and if so, does it regulate platelet functional responses. Here, we show that ELMO1 is present in both human and murine platelets. We used ELMO1-deficient (ELMO1-/-) mice to study its role in platelets. ELMO1-/- murine platelets showed enhanced platelet aggregation and dense granule secretion in response to the GPVI agonist, CRP (Figure 1 A & B), compared to the wildtype controls although there was no difference in GPVI expression levels between the two. There was no difference observed in response to AYPGKF- or 2-MeSADP. These data suggest that ELMO1 plays a specific role downstream of GPVI pathway but GPCRs. Moreover, ELMO1-/- platelets exhibited enhanced clot retraction and spreading indicating its role in Glycoprotein IIb/IIa (GPIIb/IIIa) mediated outside-in signaling. Furthermore, whole blood from ELMO1-/- mice perfused over collagen under arterial shear conditions exhibited enhanced thrombus formation. In an in vivo pulmonary thromboembolism model, ELMO1-/- mice showed reduced survival compared to the wildtype control. ELMO1-/- mice also showed shorter time to occlusion and increased thrombus stability using the ferric-chloride injury model indicating the role of ELMO1 in thrombus formation in vivo. At the molecular level, Rac1 activity was enhanced in ELMO1-/- murine platelets compared to the wildtype control in response to CRP (Figure 1C). Together, these data suggest that ELMO1 regulates Rac1 activity upon GPVI-mediated thrombus formation and it may play a negative regulator role in both inside-out and outside-in signaling, which might involve Rac1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Alteration of platelet GPVI signaling in ST-elevation myocardial infarction patients demonstrated by a combination of proteomic, biochemical, and functional approaches.

The platelet-specific collagen receptor glycoprotein VI (GPVI) is critical for the formation of arterial thrombosis in vivo. We analyzed GPVI-activated platelets from ST-elevation myocardial infarction (STEMI) patients and matched stable coronary artery disease (SCAD) controls in order to provide novel clues on the degree of involvement of GPVI signaling in the acute event. Firstly, platelets were isolated from systemic venous blood and activated with the GPVI specific agonist CRP (collagen-related peptide). STEMI and SCAD samples were compared by a phosphoproteomics approach. Validations were by immunoblotting in systemic and intracoronary blood from independent cohorts of patients. Twenty-six differentially regulated proteins were identified when comparing CRP-activated systemic platelets from STEMI and SCAD patients, 4 of which were selected for validation studies: PLCɣ2, G6f, SLP-76, and Dok-2. Immunoblot analyses showed these four proteins had higher tyrosine phosphorylation levels in response to CRP in platelets from STEMI patients, being these levels more pronounced at the culprit site of coronary artery occlusion. Moreover, platelet aggregation studies showed a higher response to GPVI agonists in STEMI patients compared to SCAD controls. In conclusion, we show an altered activation state of GPVI signaling in STEMI patients, confirming this receptor as a promising anti-thrombotic target for myocardial infarction.

CLP36 Is a Negative Regulator of Glycoprotein VI Signaling in Platelets

At sites of vascular injury, exposed subendothelial collagens not only trigger sudden platelet adhesion and aggregation, thereby initiating normal hemostasis, but also can lead to acute ischemic diseases, such as myocardial infarction or stroke. The glycoprotein (GP) VI/Fc receptor γ-chain complex is a central regulator of these processes because it mediates platelet activation on collagens through a series of tyrosine phosphorylation events downstream of the Fc receptor γ-chain-associated immunoreceptor tyrosine-based activation motif. GPVI signaling has to be tightly regulated to prevent uncontrolled intravascular platelet activation, but the underlying mechanisms are not fully understood. We studied the role of PDZ and LIM domain family member CLP36 in platelet physiology in vitro and in vivo. We report that CLP36 acts as a major inhibitor of GPVI immunoreceptor tyrosine-based activation motif signaling in platelets. Platelets from mice either expressing a low amount of a truncated form of CLP36 lacking the LIM domain (Clp36(ΔLIM)) or lacking the whole protein (Clp36(-/-)) displayed profound hyperactivation in response to GPVI agonists, whereas other signaling pathways were unaffected. This was associated with hyperphosphorylation of signaling proteins and enhanced Ca(2+) mobilization, granule secretion, and integrin activation downstream of GPVI. The lack of functional CLP36 translated into accelerated thrombus formation and enhanced procoagulant activity, assembling a prothrombotic phenotype in vivo. These data reveal an inhibitory function of CLP36 in GPVI immunoreceptor tyrosine-based activation motif signaling and establish it as a key regulator of arterial thrombosis.

A novel histidine tyrosine phosphatase, TULA-2, associates with Syk and negatively regulates GPVI signaling in platelets

AbstractT-cell ubiquitin ligand-2 (TULA-2) is a recently discovered histidine tyrosine phosphatase thought to be ubiquitously expressed. In this work, we have investigated whether TULA-2 has a key role in platelet glycoprotein VI (GPVI) signaling. This study indicates that TULA-2 is expressed in human and murine platelets and is able to associate with Syk and dephosphorylate it. Ablation of TULA-2 resulted in hyperphosphorylation of Syk and its downstream effector phospholipase C-γ2 as well as enhanced GPVI-mediated platelet functional responses. In addition, shorter bleeding times and a prothrombotic phenotype were observed in mice lacking TULA-2. We therefore propose that TULA-2 is the primary tyrosine phosphatase mediating the dephosphorylation of Syk and thus functions as a negative regulator of GPVI signaling in platelets.

A Novel Histidine Tyrosine Phosphatase, TULA-2, Associates with Syk and Negatively Regulates GPVI Signaling in Platelets.

Glycoprotein VI (GPVI) is the primary platelet receptor for collagen signaling. Following damage to the vascular endothelium, the GPVI receptor interacts with the exposed sub-endothelial collagen. This interaction initiates a signaling cascade involving phosphorylation of the dual ITAM motif of the FcRγ chain by Fyn and Lyn, followed by the recruitment, phosphorylation and activation of Syk. This leads to the eventual activation of PLCγ2 and the release of calcium from intracellular stores to cause platelet activation. While a lot is known about the activation processes involved in GPVI signaling less is known about its negative regulation. The T-cell ubiquitin ligand (TULA) family of proteins has been implicated in the negative regulation protein tyrosine kinase (PTK)-dependent signaling pathways. More recently, it has been shown the TULA family member, TULA-2, exhibits phosphatase activity towards PTKs, including Syk, and this activity is responsible for the negative regulation of T-cell receptor signaling (Mikhailik et. al. 2007, Agrawal et. al. 2008). Thus, we investigated the role of TULA-2 in the negative regulation of the GPVI signaling cascade. We show that TULA-2 is expressed in both human and murine platelets. Deletion of TULA-2 in murine platelets manifests itself functionally as enhanced aggregation in response to the GPVI agonist convulxin as well as enhanced dense granule secretion when compared to wild type platelets. No difference was witnessed in response to the PAR4 agonist AYPGKF. TULA-2-deficient platelets also exhibit sustained hyperphosphorylation of Syk at tyrosines 525 and 526 as well as hyperphosphorylation of PLCγ2 at tyrosines 753 and 759, indicative of enhanced kinase and phospholipase activity respectively. GST-pulldown experiments suggest that Syk and TULA- 2 are able to associate in resting and convulxin stimulated platelets and in-vitro phosphatase assays demonstrate that TULA-2 can dephosphorylate Syk at tyrosines 525 and 526. Taken together, these data suggest that TULA-2 is a negative regulator of GPVI signaling and this regulation is mediated by an association of TULA-2 with Syk, allowing the dephosphorylation of Syk at catalytically important tyrosine residues.

Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines.

Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcRgamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). To determine why GPVI-FcRgamma signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVI-FcRgamma signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI-FcRgamma has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery.

Glycoprotein VI agonists have distinct dependences on the lipid raft environment.

It has been reported that the association of glycoprotein VI (GPVI) with lipid rafts regulates GPVI signaling in platelets. Secreted adenosine 5'-diphosphate (ADP) potentiates GPVI-induced platelet aggregation at particular agonist concentrations. We have investigated whether the decrease in GPVI signaling, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-beta-cyclodextrin and measured signaling events downstream of GPVI activation. Lipid raft disruption decreases aggregation induced by low concentrations of convulxin, but this decrease is almost eliminated in the presence of ADP antagonists. Signaling indicators, such as protein phosphorylation and calcium mobilization, were not affected by raft disruption in collagen or convulxin stimulated platelets. Interestingly, however, raft disruption directly reduced GPVI signaling induced by collagen-related peptide. Lipid rafts do not directly contribute to signaling by the physiologic agonist collagen. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates GPVI signaling.