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Richard W Farndale - One of the best experts on this subject based on the ideXlab platform.

  • dimers of the platelet collagen receptor Glycoprotein VI bind specifically to fibrin fibers during clot formation but not to intact fibrinogen
    Journal of Thrombosis and Haemostasis, 2021
    Co-Authors: Masaaki Moroi, Richard W Farndale, Isuru Induruwa, Stephanie M Jung
    Abstract:

    OBJECTIVE The platelet collagen receptor Glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced VIa the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI-dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions. METHODS AND RESULTS ELISA using biotinyl-fibrinogen immobilized on streptaVIdin-coated wells indicated that GPVI dimers do not bind intact fibrinogen. Clots were formed by adding thrombin to a mixture of near-plasma level of fibrinogen and recombinant GPVI ectodomain: GPVI dimer (GPVI-Fc2 or Revacept) or monomer (GPVI-His: single chain of Revacept GPVI domain, with His tag). Clot-bound proteins were analyzed by SDS-PAGE/immunoblotting. GPVI-dimer bound to noncrosslinked fibrin clots with classical one-site binding kinetics, with µM-level KD , and to crosslinked clots with higher affinity. Anti-GPVI-dimer (mFab-F) inhibited the binding. However, GPVI-His binding to either type of clot was nonsaturable and nearly linear, indicating very low affinity or nonspecific binding. In clots formed in the presence of platelets, clot-bound platelet-derived proteins were integrin αIIbβ3, present at high levels, and GPVI. CONCLUSIONS We conclude that dimeric GPVI is the receptor for fibrin, exhibiting a similar KD to those obtained for its binding to fibrinogen D-fragment and D-dimer, suggesting that fibrin(ogen)'s GPVI-binding site becomes exposed after fibrin formation or cleavage to fragment D. Analysis of platelets bound to fibrin clots indicates that platelet GPVI binds to fibrin fibers comprising the clot.

  • nonredundant roles of platelet Glycoprotein VI and integrin αiibβ3 in fibrin mediated microthrombus formation
    Arteriosclerosis Thrombosis and Vascular Biology, 2021
    Co-Authors: Paola E. J. Van Der Meijden, Richard W Farndale, Gina Perrella, Jingnan Huang, Isabella Provenzale, Frauke Swieringa, Floor C J I Heubelmoenen, Mark Roest, Mark R Thomas
    Abstract:

    Objective: Fibrin is considered to strengthen thrombus formation VIa integrin αIIbβ3, but recent findings indicate that fibrin can also act as ligand for platelet Glycoprotein VI. Approach and Resu...

  • nonredundant roles of platelet Glycoprotein VI and integrin αiibβ3 in fibrin mediated microthrombus formation
    Arteriosclerosis Thrombosis and Vascular Biology, 2020
    Co-Authors: Paola E. J. Van Der Meijden, Richard W Farndale, Gina Perrella, Jingnan Huang, Isabella Provenzale, Frauke Swieringa, Floor C J I Heubelmoenen, Mark Roest, Mark R Thomas
    Abstract:

    Objective Fibrin is considered to strengthen thrombus formation VIa integrin αIIbβ3, but recent findings indicate that fibrin can also act as ligand for platelet Glycoprotein VI. Approach and Results: To investigate the thrombus-forming potential of fibrin and the roles of platelet receptors herein, we generated a range of immobilized fibrin surfaces, some of which were cross-linked with factor XIIIa and contained VWF-BP (von Willebrand factor-binding peptide). Multicolor microfluidics assays with whole-blood flowed at high shear rate (1000 s-1) indicated that the fibrin surfaces, regardless of the presence of factor XIIIa or VWF-BP, supported platelet adhesion and activation (P-selectin expression), but only microthrombi were formed consisting of bilayers of platelets. Fibrinogen surfaces produced similar microthrombi. Markedly, tiggering of coagulation with tissue factor or blocking of thrombin no more than moderately affected the fibrin-induced microthrombus formation. Absence of αIIbβ3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of Glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca2+ signaling and aggregation, while inhibition of Syk further reduced these responses. In platelet suspension, Glycoprotein VI blockage or Syk inhibition prevented fibrin-induced platelet aggregation. Microthrombi on fibrin surfaces triggered only minimal thrombin generation, in spite of thrombin binding to the fibrin fibers. Conclusions Together, these results indicate that fibrin fibers, regardless of their way of formation, act as a consolidating surface in microthrombus formation VIa nonredundant roles of platelet Glycoprotein VI and integrin αIIbβ3 through signaling VIa Syk and low-level Ca2+ rises.

  • platelet collagen receptor Glycoprotein VI dimer recognizes fibrinogen and fibrin through their d domains contributing to platelet adhesion and activation during thrombus formation
    Journal of Thrombosis and Haemostasis, 2018
    Co-Authors: Isuru Induruwa, Richard W Farndale, Masaaki Moroi, Arkadiusz Bonna, J D Malcor, Joannamarie Howes, E A Warburton, Stephanie M Jung
    Abstract:

    Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi. GPVI-dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D-domains. Collagen, D-fragment and D-dimer may share a common or proximate binding site(s) on GPVI-dimer. GPVI-dimer-fibrin interaction supports spreading, activation and adhesion involVIng αIIbβ3. Summary Background Platelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin. Objectives To determine if GPVI-dimer, GPVI-monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site. Methods Recombinant GPVI monomeric extracellular domain (GPVIex ) or dimeric Fc-fusion protein (GPVI-Fc2 ) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involVIng collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti-GPVI-dimer or anti-αIIbβ3. Results Under static conditions, GPVIex did not bind to any fibrinogen substrate. GPVI-Fc2 exhibited specific, saturable binding to both D-fragment and D-dimer, which was inhibited by mFab-F (anti-GPVI-dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI-Fc2 binding to D-fragment or D-dimer was abrogated by collagen type III, Horm collagen or CRP-XL (crosslinked collagen-related peptide), suggesting proximity between the D-domain and collagen binding sites on GPVI-dimer. Under low shear, adhesion of normal platelets to D-fragment, D-dimer, fibrinogen and fibrin was inhibited by mFab-F (inhibitor of GPVI-dimer) and abolished by Eptifibatide (inhibitor of αIIbβ3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbβ3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab-F, supporting some independent GPVI-dimer involvement in this interaction. Conclusion Only dimeric GPVI interacts with fibrinogen D-domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.

  • clustering of Glycoprotein VI gpVI dimers upon adhesion to collagen as a mechanism to regulate gpVI signaling in platelets
    Journal of Thrombosis and Haemostasis, 2017
    Co-Authors: Natalie S Poulter, Richard W Farndale, Robert K Andrews, Elizabeth E Gardiner, Alice Y Pollitt, Dylan M Owen, H Shimizu, D Ishikawa, Dominique Bihan, Masaaki Moroi
    Abstract:

    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 SUMMARY: Background Platelet 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. Objective To 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 α2 β1 , and phosphotyrosine. Results Upon 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 α2 β1 was localized to collagen fibers close to GPVI dimer clusters. GPVI clustering depends on a dynamic actin cytoskeleton. Conclusions Platelet 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.

Bernhard Nieswandt - One of the best experts on this subject based on the ideXlab platform.

  • platelet Glycoprotein VI aids in local immunity during pneumonia derived sepsis caused by gram negative bacteria
    Blood, 2018
    Co-Authors: Theodora A M Claushuis, Bernhard Nieswandt, Louis Boon, Joris J T H Roelofs, Onno J De Boer, Cornelis Van T Veer, Tom Van Der Poll
    Abstract:

    Platelet collagen receptor Glycoprotein VI (GPVI) and podoplanin receptor C-type lectin-like receptor 2 (CLEC2) are receptors implicated in platelet activation that both signal VIa an immunoreceptor tyrosine–based activation motif. Platelets are necessary for host defense and prevention of hemorrhage during sepsis, but the role of platelet GPVI and CLEC2 herein is unknown. To investigate this, we infected mice depleted of platelet GPVI or CLEC2 by antibody treatment or GPVI −/− mice with the common human sepsis pathogen Klebsiella pneumoniae VIa the airways to induce pneumonia-derived sepsis. The GPVI ligand collagen and the CLEC2 ligand podoplanin were constitutively present in the lung, whereas the GPVI ligands fibrin and histone were induced during pneumonia. During late-stage infection, both mice depleted of GPVI and GPVI −/− mice showed increased bacterial growth in lungs, and GPVI −/− mice also showed increased bacterial growth in distant body sites. Despite higher bacterial loads, GPVI-depleted mice showed reduced platelet numbers, platelet activation, and platelet–leukocyte complex formation in the bronchoalveolar space. Consistently, in human whole blood, GPVI stimulation of platelets increased platelet–leukocyte complex formation and leukocyte activation, which was accompanied by enhanced phagocytosis of Klebsiella . GPVI-depleted mice showed increased lung hemorrhage during infection, but not to the extent observed in platelet-depleted mice, and lung bleeding was not significantly different between GPVI −/− and wild-type mice. CLEC2 depletion did not affect any of the responses during pneumonia. These results suggest that platelet GPVI, but not CLEC2, contributes to local host defense during pneumonia-derived sepsis by enhancing leukocyte function.

  • targeting Glycoprotein VI and the immunoreceptor tyrosine based activation motif signaling pathway
    Arteriosclerosis Thrombosis and Vascular Biology, 2014
    Co-Authors: David Stegner, Elizabeth J Haining, Bernhard Nieswandt
    Abstract:

    Coronary artery thrombosis and ischemic stroke are often initiated by the disruption of an atherosclerotic plaque and consequent intravascular platelet activation. Thus, antiplatelet drugs are central in the treatment and prevention of the initial, and subsequent, vascular events. However, novel pharmacological targets for platelet inhibition remain an important goal of cardiovascular research because of the negative effect of existing antiplatelet drugs on primary hemostasis. One promising target is the platelet collagen receptor Glycoprotein VI. Blockade or antibody-mediated depletion of this receptor in circulating platelets is beneficial in experimental models of thrombosis and thrombo-inflammatory diseases, such as stroke, without impairing hemostasis. In this reVIew, we summarize the importance of Glycoprotein VI and (hem)immunoreceptor tyrosine-based activation motif signaling in hemostasis, thrombosis, and thrombo-inflammatory processes and discuss the targeting strategies currently under development for inhibiting Glycoprotein VI and its signaling.

  • impact of Glycoprotein VI and platelet adhesion on atherosclerosis a possible role of fibronectin
    Journal of Molecular and Cellular Cardiology, 2010
    Co-Authors: Andreas Bultmann, Bernhard Nieswandt, Konstantinos Stellos, Tanja Schonberger, Silvia Wagner, Mario Peluso, Carla Weis, Ildiko Konrad, Steffen Massberg, Meinrad Gawaz
    Abstract:

    Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intraVItal microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in VIvo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in VItro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex VIvo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both VIa GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly VIa fibronectin-could relevantly contribute to platelet-triggered atheroprogression.

  • two phase antithrombotic protection after anti Glycoprotein VI treatment in mice
    Arteriosclerosis Thrombosis and Vascular Biology, 2006
    Co-Authors: Valerie Schulte, Peter H Reusch, Miroslava Pozgajova, David Vargaszabo, Christian Gachet, Bernhard Nieswandt
    Abstract:

    Objective— Collagen and thrombin are the strongest physiological platelet agonists, acting through different receptors, among which Glycoprotein VI (GPVI) and protease-activated receptors, respectively, are the essential ones. In mice, targeting of GPVI with the monoclonal antibody JAQ1 induces depletion of the receptor from circulating platelets, resulting in abolished collagen responses and long-lasting antithrombotic protection. Methods and Results— Mice were treated with JAQ1, and the early effects of this treatment were analyzed. In addition to the known abolition of the collagen reactiVIty, this treatment also affected platelet response to thrombin but not other agonists. In platelets from JAQ1-treated mice, thrombin-induced activation of integrin αIIbβ3, the surface expression of P-selectin, and the procoagulant actiVIty were decreased on days 1 and 2, then progressively recovered and returned to normal on day 5. In parallel, the mice were transiently protected from lethal tissue factor–induced pul...

  • the Glycoprotein VI phospholipase cγ2 signaling pathway controls thrombus formation induced by collagen and tissue factor in VItro and in VIvo
    Arteriosclerosis Thrombosis and Vascular Biology, 2005
    Co-Authors: Imke C. A. Munnix, Jocelyn M. Auger, Marijke J. E. Kuijpers, Paola E. J. Van Der Meijden, Bernhard Nieswandt, Amrei Strehl, Marc A M J Van Zandvoort, Mirjam Oude G A Egbrink, Johan W. M. Heemskerk
    Abstract:

    Objective— Both collagen and tissue factor can be initiating factors in thrombus formation. We investigated the signaling pathway of collagen-induced platelet activation in interaction with tissue factor–triggered coagulation during the thrombus-forming process. Methods and Results— In murine blood flowing over collagen, platelet exposure of phosphatidylserine and procoagulant actiVIty, but not adhesion, completely relied on each of the following signaling modules: Glycoprotein VI (GPVI), FcR γ-chain, Src kinases, adaptor protein LAT, and phospholipase Cγ2 (PLCγ2). On flow in the presence of tissue factor, these signaling components were essential for platelet aggregation and greatly enhanced fibrin clot formation. Collagen-stimulated thrombin generation relied on the presence and actiVIty of GPVI, FcR γ-chain, Src kinase, LAT, and PLCγ2. The physiological importance of this GPVI pathway was shown in a FeCl 3 -induced in VIvo murine thrombosis model. In both venules and arterioles, signaling through GPVI, FcR γ-chain, and Src kinases enhanced the formation of phosphatidylserine-exposing and fibrin-rich thrombi. Conclusions— The GPVI-PLCγ2 activation pathway regulates collagen-dependent coagulation in venous and arterial thrombus formation.

Meinrad Gawaz - One of the best experts on this subject based on the ideXlab platform.

  • Glycoprotein VI novel target in antiplatelet medication
    Pharmacology & Therapeutics, 2021
    Co-Authors: Oliver Borst, Meinrad Gawaz
    Abstract:

    Subendothelial collagen exposed to platelets after rupture of atherosclerotic plaques is the main trigger of platelet activation und acute arterial thrombotic occlusion leading to myocardial infarction or ischemic stroke. An efficacious antiplatelet therapy is essential to prevent atherothrombotic events. However, increasing potency of antiplatelet treatment is associated with an increased risk of bleeding limiting the clinical benefit for the patient since current antiplatelet strategies concomitantly affect hemostasis. Therefore, an unmet clinical need remains to develop antiplatelet strategies that selectively inhibit atherothrombosis without interfering with control of bleeding. Platelet Glycoprotein VI (GPVI) plays a crucial role in collagen-induced activation and aggregation of platelets. Since GPVI is platelet-specific and strongly involved in the pathogenesis of arterial thrombosis without great impact on normal hemostasis, GPVI moved into the focus of novel approaches of antithrombotic therapy strategies. This reVIew summarizes ligands, properties, function and downstream signaling of GPVI and discusses the potential of GPVI as target for novel therapeutic strategies in thrombotic and inflammatory diseases on the basis of recent scientific findings and currently ongoing clinical phase I and phase II trials.

  • plasma levels of soluble Glycoprotein VI sgpVI are associated with ischemic stroke
    Platelets, 2013
    Co-Authors: Thomas Wurster, Meinrad Gawaz, Konstantinos Stellos, Elisabeth Kremmer, Thomas O Joos, Oliver Poetz, Arthur Melms, Andreas Schuster, Eike Nagel, Boris Bigalke
    Abstract:

    Platelet collagen receptor Glycoprotein VI (pGPVI) is elevated in patients with acute coronary syndrome (ACS) and ischemic stroke. Recently, we developed a novel bead-based sandwich immunoassay to determine soluble GPVI (sGPVI), which has been validated in ACS patients. This study aimed to evaluate the plasma levels of sGPVI and pGPVI expression in patients with suspected stroke. We consecutively evaluated 176 patients, who were admitted to the stroke unit. Surface expression of pGPVI was determined by flow cytometry, sGPVI concentrations were determined using our sandwich immunoassay. Unlike patients with TIA, patients with stroke showed significantly decreased plasma levels of sGPVI compared to patients with non-ischemic (NI) events (TIA: mean [µg/L] ± standard deVIation): 6.1 ± 2.1 vs. NI: 8 ± 4; p = 0.192; stroke: 5.9 ± 2.3 vs. NI; p = 0.013), whereas for pGPVI, patients with TIA and ischemic stroke revealed a significantly increased platelet surface expression compared to NI patients (TIA: mean fluorescence intensity [MFI] ± standard deVIation): 20.9 ± 5.4 vs. NI: 17.6 ± 5.2; p = 0.021; stroke: 20.3 ± 6.2 vs. NI; p = 0.016). Using logistic regression analysis, both sGPVI (p = 0.002) and pGPVI (p = 0.012) are independently associated with ischemic stroke compared to other laboratory markers. To predict the indiVIdual risk for ischemic stroke using the plasma levels of sGPVI, receiver operating characteristic (ROC) analysis determined an optimal cutoff value of sGPVI at 6.5 µg/l, thus, patients with decreased plasma levels (<6.5 µg/l) have a 1.5-fold adjusted odds ratio (95%confidence interval, 1.4-2.7). Lower plasma levels of sGPVI are associated with the slightly elevated risk of stroke and may be a promising novel biomarker.

  • novel antiplatelet drug revacept dimeric Glycoprotein VI fc specifically and efficiently inhibited collagen induced platelet aggregation without affecting general hemostasis in humans
    Circulation, 2011
    Co-Authors: Martin Ungerer, Meinrad Gawaz, Kerstin Uhland, Kai Rosport, Andreas Bultmann, Richard Piechatzek, Peter Schlieper, Gotz Munch
    Abstract:

    Background— Blocking of Glycoprotein VI–dependent pathways by interfering in vascular collagen sites is commonly seen as an attractive target for an antiplatelet therapy of acute atherosclerotic diseases such as myocardial infarction or stroke. Revacept (soluble dimeric Glycoprotein VI-Fc fusion protein) has been shown to reduce platelet adhesion by blocking vascular collagen in plaques or erosion and to be safe in preclinical studies. A dose-escalating clinical phase I study was performed to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of Revacept in humans. Methods and Results— In a first-in-humans study, 30 healthy men received a single intravenous administration of 10, 20, 40, 80, or 160 mg Revacept. The serum concentration–time courses of each dosage of Revacept showed a narrow variation and a concentration and time dependence. Revacept did not significantly affect the bleeding time. Collagen-induced platelet aggregation was dose-dependently inhibited up to 48 hours at lower doses and for 7 days after higher dose levels. In contrast, ADP- or thrombin receptor activating peptide–dependent platelet aggregation remained unaltered. There were no relevant drug-related adverse events or drug-related changes in laboratory parameters (biochemistry, hematology, and coagulation parameters). There were no drug-related changes in blood pressure, pulse rate, or ECG parameters (including 24-hour Holter monitoring). No anti-Revacept antibodies were detected. Conclusion— This phase I study demonstrated that Revacept is a safe and well-tolerated new antiplatelet compound with a clear dose-dependent pharmacokinetic profile with specific, dose-related inhibition of platelet aggregation despite completely unaltered general hemostasis. Clinical Trial Registration— URL: [www.clinicaltrials.gov][1]. Unique identifier: NCT 01042964. URL: eudract.ema.europa.eu. Identifier: 2005–004656-12. # Clinical Perspective {#article-title-31} [1]: http://www.clinicaltrials.gov

  • impact of Glycoprotein VI and platelet adhesion on atherosclerosis a possible role of fibronectin
    Journal of Molecular and Cellular Cardiology, 2010
    Co-Authors: Andreas Bultmann, Bernhard Nieswandt, Konstantinos Stellos, Tanja Schonberger, Silvia Wagner, Mario Peluso, Carla Weis, Ildiko Konrad, Steffen Massberg, Meinrad Gawaz
    Abstract:

    Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intraVItal microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in VIvo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in VItro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex VIvo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both VIa GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly VIa fibronectin-could relevantly contribute to platelet-triggered atheroprogression.

  • expression of platelet Glycoprotein VI is associated with transient ischemic attack and stroke
    European Journal of Neurology, 2010
    Co-Authors: Boris Bigalke, Konstantinos Stellos, Tobias Geisler, Peter Seizer, Elisabeth Kremmer, Andreas E May, Stephan Lindemann, Arthur Melms, Andreas R Luft, Meinrad Gawaz
    Abstract:

    Background and purpose:  Platelet collagen receptor Glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation. We aimed to investigate GPVI in patients presenting with symptoms of acute cerebrovascular disease and to define GPVI as biomarker for acute stroke. Methods:  We consecutively evaluated 205 patients, who admitted the stroke unit with symptoms for stroke. Surface expression of the platelet activation markers (GPVI, CD62P, GPIb) was determined by two-color whole blood flow cytometry. Results:  Patients with transient ischemic attack (TIA) (n = 18; 8.8%) as well as with stroke (n = 133; 64.9%) showed a significantly enhanced GPVI expression (mean fluorescence intensity ± SD) on admission compared to patients with non-ischemic (NI) events (n = 54; 26.3%) (TIA: 20.9 ± 7.1 vs. NI: 16.2 ± 3.9; P = 0.002; stroke: 20.4 ± 5.7 vs. NI; P = 0.002). Neither CD62P nor GPIb surface expression showed a significant difference. Logistic regression analysis revealed that on admission GPVI was associated with stroke independent of conventional laboratory markers such as C-reactive protein, blood glucose, and creatine kinase. Using a receiver operating characteristic curve on GPVI, we have determined the cut off value of 18.2 for stroke. Thus, patients with enhanced GPVI expression levels (≥18.2) had a 2.4-fold relative risk for stroke. Patients with elevated platelet GPVI expression level had a poorer clinical outcome in cumulative event-free surVIval for stroke, myocardial infarction, and cerebro-/cardiovascular death at 3-month follow-up (log rank; P = 0.045). Conclusions:  These findings indicate that platelet GPVI surface expression is significantly enhanced in patients with TIA and stroke compared to patients with NI events. Determination of platelet-specific GPVI may be useful as an early biomarker for cerebral ischemia.

Boris Bigalke - One of the best experts on this subject based on the ideXlab platform.

  • plasma levels of soluble Glycoprotein VI sgpVI are associated with ischemic stroke
    Platelets, 2013
    Co-Authors: Thomas Wurster, Meinrad Gawaz, Konstantinos Stellos, Elisabeth Kremmer, Thomas O Joos, Oliver Poetz, Arthur Melms, Andreas Schuster, Eike Nagel, Boris Bigalke
    Abstract:

    Platelet collagen receptor Glycoprotein VI (pGPVI) is elevated in patients with acute coronary syndrome (ACS) and ischemic stroke. Recently, we developed a novel bead-based sandwich immunoassay to determine soluble GPVI (sGPVI), which has been validated in ACS patients. This study aimed to evaluate the plasma levels of sGPVI and pGPVI expression in patients with suspected stroke. We consecutively evaluated 176 patients, who were admitted to the stroke unit. Surface expression of pGPVI was determined by flow cytometry, sGPVI concentrations were determined using our sandwich immunoassay. Unlike patients with TIA, patients with stroke showed significantly decreased plasma levels of sGPVI compared to patients with non-ischemic (NI) events (TIA: mean [µg/L] ± standard deVIation): 6.1 ± 2.1 vs. NI: 8 ± 4; p = 0.192; stroke: 5.9 ± 2.3 vs. NI; p = 0.013), whereas for pGPVI, patients with TIA and ischemic stroke revealed a significantly increased platelet surface expression compared to NI patients (TIA: mean fluorescence intensity [MFI] ± standard deVIation): 20.9 ± 5.4 vs. NI: 17.6 ± 5.2; p = 0.021; stroke: 20.3 ± 6.2 vs. NI; p = 0.016). Using logistic regression analysis, both sGPVI (p = 0.002) and pGPVI (p = 0.012) are independently associated with ischemic stroke compared to other laboratory markers. To predict the indiVIdual risk for ischemic stroke using the plasma levels of sGPVI, receiver operating characteristic (ROC) analysis determined an optimal cutoff value of sGPVI at 6.5 µg/l, thus, patients with decreased plasma levels (<6.5 µg/l) have a 1.5-fold adjusted odds ratio (95%confidence interval, 1.4-2.7). Lower plasma levels of sGPVI are associated with the slightly elevated risk of stroke and may be a promising novel biomarker.

  • sandwich immunoassay for soluble Glycoprotein VI in patients with symptomatic coronary artery disease
    Clinical Chemistry, 2011
    Co-Authors: Boris Bigalke, Tobias Geisler, Peter Seizer, Elisabeth Kremmer, Oliver Potz, Valentina O Puntmann, Alkystis Phinikaridou, Amedeo Chiribiri, Eike Nagel
    Abstract:

    BACKGROUND: Platelet Glycoprotein VI (pGPVI) expression is increased in acute coronary syndrome (ACS), reflecting platelet activation. There is no reliable method available to measure pGPVI. Our aim was to develop a bead-based sandwich immunoassay to measure soluble GPVI (sGPVI). METHODS: Based on antibodies for sGPVI developed earlier, we established and validated a bead-based sandwich immunoassay in 2438 consecutive patients with stable angina pectoris (SAP; n = 1371), non–ST-elevation myocardial infarction (NSTEMI; n = 724), and ST-elevation MI (STEMI; n = 343). In a subgroup (n = 1011), we measured surface expression of pGPVI using flow cytometry. RESULTS: The assay revealed a working range of 8–500 ng/L. Intra- and interassay imprecision was <7% and <14%, respectively. Patients with NSTEMI and STEMI showed significantly lower mean sGPVI concentrations than patients with SAP [mean (SD), 8.4 (3.6) μ g/L and 8.6 (4.1) μ g/L vs 9.8 (4.8) μ g/L; P = 0.002], whereas subgroup analysis revealed significantly enhanced pGPVI in NSTEMI (n = 276) and STEMI (n = 80) patients compared with SAP (n = 655) [mean fluorescence intensity (SD), 21.2 (8.1) and 19.8 (6.8) vs 18.5 (7.7); P = 0.002 and P = 0.018]. pGPVI and sGPVI were inversely correlated ( r = −0.076; P = 0.023). Area under the ROC curve was 0.716, 95% CI 0.681–0.751, for sGPVI, distinguishing patients with SAP from those with ACS, and was superior ( P = 0.044) to the curve of subgroup analysis for pGPVI (0.624, 95% CI 0.586–0.662). sGPVI ( P = 0.023) and pGPVI ( P = 0.028) had better association with the development of ACS than troponin I ( P = 0.055) in the very early stage of disease, based on logistic regression analysis. CONCLUSIONS: This sandwich immunoassay reliably measures sGPVI and may help to identify patients with ACS earlier than other laboratory markers.

  • expression of platelet Glycoprotein VI is associated with transient ischemic attack and stroke
    European Journal of Neurology, 2010
    Co-Authors: Boris Bigalke, Konstantinos Stellos, Tobias Geisler, Peter Seizer, Elisabeth Kremmer, Andreas E May, Stephan Lindemann, Arthur Melms, Andreas R Luft, Meinrad Gawaz
    Abstract:

    Background and purpose:  Platelet collagen receptor Glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation. We aimed to investigate GPVI in patients presenting with symptoms of acute cerebrovascular disease and to define GPVI as biomarker for acute stroke. Methods:  We consecutively evaluated 205 patients, who admitted the stroke unit with symptoms for stroke. Surface expression of the platelet activation markers (GPVI, CD62P, GPIb) was determined by two-color whole blood flow cytometry. Results:  Patients with transient ischemic attack (TIA) (n = 18; 8.8%) as well as with stroke (n = 133; 64.9%) showed a significantly enhanced GPVI expression (mean fluorescence intensity ± SD) on admission compared to patients with non-ischemic (NI) events (n = 54; 26.3%) (TIA: 20.9 ± 7.1 vs. NI: 16.2 ± 3.9; P = 0.002; stroke: 20.4 ± 5.7 vs. NI; P = 0.002). Neither CD62P nor GPIb surface expression showed a significant difference. Logistic regression analysis revealed that on admission GPVI was associated with stroke independent of conventional laboratory markers such as C-reactive protein, blood glucose, and creatine kinase. Using a receiver operating characteristic curve on GPVI, we have determined the cut off value of 18.2 for stroke. Thus, patients with enhanced GPVI expression levels (≥18.2) had a 2.4-fold relative risk for stroke. Patients with elevated platelet GPVI expression level had a poorer clinical outcome in cumulative event-free surVIval for stroke, myocardial infarction, and cerebro-/cardiovascular death at 3-month follow-up (log rank; P = 0.045). Conclusions:  These findings indicate that platelet GPVI surface expression is significantly enhanced in patients with TIA and stroke compared to patients with NI events. Determination of platelet-specific GPVI may be useful as an early biomarker for cerebral ischemia.

  • influence of platelet count on the expression of platelet collagen receptor Glycoprotein VI gpVI in patients with acute coronary syndrome
    Thrombosis and Haemostasis, 2009
    Co-Authors: Boris Bigalke, Konstantinos Stellos, Tobias Geisler, Elisabeth Kremmer, Oliver Potz, Thomas O Joos, Dimitrios Stakos, Christian Bischofs, Bjorn F Kramer, Peter Seizer
    Abstract:

    Platelets play a key role in the development of an acute coronary syndrome (ACS) and contribute to cardiovascular events. Platelet collagen receptor Glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation in ACS. We consecutively investigated both the platelet count and the platelet surface expression of GPVI in 843 patients with a symptomatic coronary artery disease verified by coronary angiography. Four hundred fourteen patients presented with stable angina pectoris and 429 patients with ACS. Platelet surface expression of GPVI and CD62P was determined by flow cytometry and platelet count with a coulter counter, plasmatic soluble GPVI was measured by ELISA. Platelet GPVI expression in patients with ACS was compared to platelet count. Patients with ACS showed significantly elevated GPVI expression levels in the first and second quartiles of platelet count compared to patients with higher platelet count [mean fluorescence intensity (MFI) ± standard deVIation): 1st vs. 4th: 20.44 ± 6.1 vs. 18.62 ± 3.7; p=0.012; 2ndvs.3rd:21.2±8.5vs.18.76±3.7;P=0.03; 2ndvs.4th: 21.2±8.5vs.18.62±3.7;P=0.004], which was paralleled in trend for the CD62P expression [MFI: 1st vs. 4th: 11.2 ± 6.8 vs. 12.3 ± 9; p=0.057; 2nd vs. 3rd: 16.3 ± 16 vs.12.7 ± 5.3; p=0.138; 2nd vs. 4th: 16.3 ± 16 vs.11 ± 4.4; p=0.043]. In a subgroup of 48 patients with ACS, determination of soluble GPVI showed similar results [plasma GPVI (ng/ml): 1stvs.4th: 1.6 ± 0.6 vs. 1.2 ± 0.4; p=0.046; 1st vs. 3rd: 1.6 ± 0.6 vs. 1.1 ± 0.5; p=0.038; 2nd vs. 3rd: 1.9 ± 0.8 vs. 1.1 ± 0.5; p=0.04; 2nd vs. 4th: 1.9 ± 0.8 vs. 1.2 ± 0.4; p=0.056]. Thus, a lower platelet count comes along with a higher GPVI surface expression and plasma concentration in patients with ACS, which potentially reflects increased activation and enhanced recruitment of platelets to the site of vascular injury.

  • regulation of platelet Glycoprotein VI gpVI surface expression and of soluble gpVI in patients with atrial fibrillation af and acute coronary syndrome acs
    Basic Research in Cardiology, 2009
    Co-Authors: Boris Bigalke, Konstantinos Stellos, H J Weig, Tobias Geisler, Peter Seizer, Elisabeth Kremmer, Oliver Potz, Thomas O Joos, Andreas E May, Stephan Lindemann
    Abstract:

    Background The platelet collagen receptor Glycoprotein VI (GPVI) mediates platelet adhesion to subendothelial matrix and thrombus formation in acute coronary syndrome (ACS). This study examined patients with both ACS and stable coronary artery disease (CAD), which presented with atrial fibrillation (AF) and sinus rhythm (SR).

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  • gpVI Glycoprotein VI interaction with fibrinogen is mediated by aVIdity and the fibrinogen αc region
    Arteriosclerosis Thrombosis and Vascular Biology, 2021
    Co-Authors: Julia S Gauer, Steve P. Watson, Arkadiusz Bonna, Stephen Baker, Alexandre Slater, Eleyna M Martin, Helen R Mcpherson, Cedric Duval, Iain W Manfield, Robert A S Ariens
    Abstract:

    Objective: GPVI (Glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent eVIdence indicates that it also plays important role in platelet aggregati...

  • Dual role of collagen in factor XII-dependent thrombus formation
    Blood, 2009
    Co-Authors: Paola E. J. Van Der Meijden, Thomas Renné, Imke C. A. Munnix, Jocelyn M. Auger, José W. P. Govers-riemslag, Judith M.e.m. Cosemans, Marijke J. E. Kuijpers, Henri M. H. Spronk, Steve P. Watson, Johan W. M. Heemskerk
    Abstract:

    In VIvo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma VIa activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets VIa Glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective Glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cγ2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of Glycoprotein VI signaling VIa LAT and PLCγ2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

  • g6b b inhibits constitutive and agonist induced signaling by Glycoprotein VI and clec 2
    Journal of Biological Chemistry, 2008
    Co-Authors: Jun Mori, Johannes A Eble, Andrew C Pearce, Jennifer C Spalton, Beata Grygielska, Michael G Tomlinson, Yotis A Senis, Steve P. Watson
    Abstract:

    Platelets play an essential role in wound healing by forming thrombi that plug holes in the walls of damaged blood vessels. To achieve this, platelets express a diverse array of cell surface receptors and signaling proteins that induce rapid platelet activation. In this study we show that two platelet Glycoprotein receptors that signal VIa an immunoreceptor tyrosine-based activation motif (ITAM) or an ITAM-like domain, namely the collagen receptor complex Glycoprotein VI (GPVI)-FcR γ-chain and the C-type lectin-like receptor 2 (CLEC-2), respectively, support constitutive (i.e. agonist-independent) signaling in a cell line model using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay that can detect low level activation of phospholipase Cγ (PLCγ). Constitutive and agonist signaling by both receptors is dependent on Src and Syk family kinases, and is inhibited by G6b-B, a platelet immunoglobulin receptor that has two immunoreceptor tyrosine-based inhibitory motifs in its cytosolic tail. Mutation of the conserved tyrosines in the two immunoreceptor tyrosine-based inhibitory motifs prevents the inhibitory action of G6b-B. Interestingly, the inhibitory actiVIty of G6b-B is independent of the Src homology 2 (SH2)-domain containing tyrosine phosphatases, SHP1 and SHP2, and the inositol 5′-phosphatase, SHIP. Constitutive signaling VIa Src and Syk tyrosine kinases is observed in platelets and is associated with tyrosine phosphorylation of GPVI-FcR γ-chain and CLEC-2. We speculate that inhibition of constitutive signaling through Src and Syk tyrosine kinases by G6b-B may help to prevent unwanted platelet activation.

  • delineation of the region in the Glycoprotein VI tail required for association with the fc receptor γ chain
    Journal of Biological Chemistry, 2003
    Co-Authors: Teresa Borisanz, Steve P. Watson, Michael C Berndt, Katsue Suzuki Inoue, David Tulasne
    Abstract:

    The Glycoprotein VI (GPVI).Fc receptor gamma-chain (FcRgamma-chain) complex is the major activation receptor for collagen on platelets. GPVI cross-linking mediates activation through tyrosine phosphorylation of an ITAM (immunoreceptor tyrosine-based activation motif) in the FcR gamma-chain by Src family kinases. It has been preVIously shown that a transmembrane arginine and the cytoplasmic domain of GPVI are required for association with the FcR gamma-chain in immortalized cell lines. In this study, we have delineated the regions in the GPVI tail that promote binding to FcR gamma-chain and mediate functional responses to the snake venom convulxin by reconstitution of mutant forms of GPVI in RBL-2H3 cells. Sequential truncation of the cytoplasmic tail of GPVI revealed a major role for the basic region and a minor role for the juxtamembrane six amino acids in the association with FcR gamma-chain and functional responses to convulxin. Analysis of selective deletions in the GPVI tail supported this conclusion. In addition, we show that the proline-rich domain is required for optimal Ca2+ release, whereas it is dispensable for FcR gamma-chain association.

  • association of fyn and lyn with the proline rich domain of Glycoprotein VI regulates intracellular signaling
    Journal of Biological Chemistry, 2002
    Co-Authors: Katsue Suzukiinoue, Masaaki Moroi, Stephanie M Jung, David Tulasne, Yang Shen, Teresa Borisanz, Osamu Inoue, Robert K Andrews, Michael C Berndt, Steve P. Watson
    Abstract:

    The Glycoprotein VI (GPVI)–Fc receptor (FcR) γ-chain complex, a key activatory receptor for collagen on platelet surface membranes, is constitutively associated with the Src family kinases Fyn and Lyn. Molecular cloning of GPVI has revealed the presence of a proline-rich domain in the sequence of GPVI cytoplasmic tail which has the consensus for interaction with the Src homology 3 (SH3) domains of Fyn and Lyn. A series of in VItroexperiments demonstrated the ability of the SH3 domains of both Src kinases to bind the proline-rich domain of GPVI. Furthermore, depletion of the proline-rich domain in GPVI (Pro(−)-GPVI) prevented binding of Fyn and Lyn and markedly reduced phosphorylation of FcR γ-chain in transiently transfected COS-7 cells, but did not affect the association of the γ-chain with GPVI. Jurkat cells stably transfected with wild type GPVI show robust increases in tyrosine phosphorylation and intracellular Ca2+ in response to the snake venom convulxin that targets GPVI. Importantly, convulxin is not able to activate cells transfected with Pro(−)-GPVI, even though the association with the immunoreceptor tyrosine-based activation motif-containing chains is maintained. These findings demonstrate that the proline-rich domain of GPVI mediates the association with Fyn/Lyn VIa their SH3 domain and that this interaction initiates activation signals through GPVI.