Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRγ deficiency

Mangin, P; Yap, Cindy L; Nonne, Christelle; Sturgeon, Sharelle Anne; Goncalves, Isaac; Yao, Yuan; Schoenwaelder, Simone M.; Wright, Christine E.; Lanza, François; Jackson, Shaun P.

doi:10.1182/blood-2005-10-4244

Cited by 138 publications

(148 citation statements)

References 39 publications

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“…This notion is supported by recent elegant studies indicating that in models that favor thrombin-driven thrombosis, the antithrombotic effect of GPVI deficiency is reduced or overcome. 41,42 This agrees with the observation that GPVI inhibition or loss has a relatively mild effect on normal hemostasis in humans and mice, 4,13-17 a process also known to be highly thrombin dependent. Thus, anti-GPVI treatment could be preferable to inhibitors of pathways that are crucial for thrombus formation, irrespective of the initial stimulus, and are also powerful antithrombotics (but by definition also bear a bleeding risk).…”

supporting

confidence: 80%

Diverging signaling events control the pathway of GPVI down-regulation in vivo

Rabie

Varga-Szabó

Bender

et al. 2007

Blood

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Coronary artery thrombosis is often initiated by platelet activation on collagenrich subendothelial layers in the disrupted atherosclerotic plaque. The activating platelet collagen receptor glycoprotein VI (GPVI) noncovalently associates with the Fc receptor ␥-chain (FcR␥), which signals through its immunoreceptor-tyrosine-based activation motif (ITAM) via the adaptor LAT leading to the activation of phospholipase C␥2 (PLC␥2). GPVI is a promising antithrombotic target as anti-GPVI antibodies induce the irreversible loss of the receptor from circulating platelets by yet undefined mechanisms in humans and mice and long-term antithrombotic protection in the latter. However, the treatment is associated with transient but severe thrombocytopenia and reduced platelet reactivity to thrombin questioning its clinical usefulness. Here we show that GPVI down-regulation occurs through 2 distinct pathways, namely ectodomain shedding or internalization/intracellular clearing, and that both processes are abrogated in mice carrying a point mutation in the FcR␥-associated ITAM. In mice lacking LAT or PLC␥2, GPVI shedding is IntroductionArterial thrombosis is often initiated by abrupt disruption of the atherosclerotic plaque and deposition and activation of platelets on the subendothelial layers that are enriched in highly thrombogenic collagens. [1][2][3] Glycoprotein VI (GPVI) 4-6 is an essential receptor for collagen-induced platelet activation, adhesion, and thrombus growth. 7 It noncovalently associates with the Fc receptor ␥-chain (FcR␥-chain) 8,9 and the complex signals through sequential activation of Src and Syk family tyrosine kinases. The Src kinases Fyn and Lyn stimulate tyrosine phosphorylation of 2 conserved tyrosines in the FcR␥-chain immunoreceptor tyrosine-based activation motif (ITAM). 10,11 This leads to engagement of Syk via its 2 SH2 domains and its subsequent activation. Syk orchestrates a downstream signaling cascade that is regulated through the interaction of several adaptor proteins, including LAT, and SLP-76, and leads to activation of effector enzymes, including phosphatidylinositol 3-kinases (PI3-kinases) and phospholipase C␥2 (PLC␥2). 12 GPVI-deficient patients suffer from a mild bleeding diathesis and their platelets show severely impaired responses to collagen. 4,13,14 Similarly, platelets from GPVI-deficient mice 15,16 or FcR␥-chain-deficient mice, which also lack GPVI, 17 do not aggregate to collagen, but no major bleeding has been observed in those animals, making GPVI a potential target for safe antithrombotic therapy. In vivo treatment of mice with the anti-GPVI antibody JAQ1 induces the rapid and quantitative down-regulation of the receptor in circulating platelets resulting in a prolonged "GPVI knockout"-like phenotype 18 and profound antithrombotic protection in various thrombosis models. 19,20 Antibody-induced loss of GPVI has also been reported in autoimmune patients who had developed anti-GPVI antibodies resulting in clearing of the receptor from their platelets. 4,14 Furthermore, it ha...

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supporting

confidence: 80%

Diverging signaling events control the pathway of GPVI down-regulation in vivo

Rabie

Varga-Szabó

Bender

et al. 2007

Blood

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“…Furthermore, inhibition of thrombin resulted in diminished platelet adhesion to collagen. Mangin et al (40) reported that thrombin was able to compensate for the effect of GPVI/FcR␥ deficiency in mouse platelets in in vivo models of thrombosis. The mechanism we describe, selective enhancement of the adhesive activity of the ␣ 2 ␤ 1 integrin via PAR4 activation, is the likely explanation for the observations of van der Meijden et al (39) and Mangin et al (40).…”

Section: Discussionmentioning

confidence: 99%

Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen

Marjoram

Voss

Pan

et al. 2009

Journal of Biological Chemistry

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Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the ␣ 2 ␤ 1 integrin (␣ 2 ␤ 1 ) and the glycoprotein VI (GPVI)/FcR␥ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (␣2-CRP) containing the ␣ 2 ␤ 1 -specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to ␣2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was ␣ 2 ␤ 1 -dependent and GPVI/FcR␥-independent as revealed in experiments with ␣ 2 ␤ 1 -or FcR␥-deficient mouse platelets. We further show that suboptimal activation of other platelet G q -linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to ␣2-CRP. The enhanced ␣ 2 ␤ 1 -mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of ␣ IIb ␤ 3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet G q -linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced ␣ 2 ␤ 1 binding to collagens containing GFOGER sites.Platelets are small anuclear cellular elements that play a central role in hemostasis and contribute to vascular pathology. At sites of intravascular injury, platelets are exposed to multiple prothrombotic factors that promote thrombus formation and trigger firm adhesion of platelets to the subendothelial extracellular matrix. Thrombin and fibrillar collagen are two of the more potent stimuli (1, 2).Thrombin is an essential serine protease in the coagulation cascade that ultimately converts fibrinogen to fibrin. Thrombin also has a direct signaling effect on cells through protease-activated receptors (PARs) 2 , which are G protein-coupled receptors (GPCRs) that are activated by enzymatic cleavage of the N terminus of the receptor to produce a tethered ligand (3). Of the four known PAR isoforms, human platelets express PAR1 and PAR4. In platelets, these receptors show different signaling kinetics; PAR1 is activated at low thrombin concentrations with a quick signal shutoff, whereas, PAR4 is activated at higher thrombin concentrations with a slow signal shut off (4 -6). Thrombin-induced signaling has been shown to modulate ligand affinity of the platelet membrane ␣ IIb ...

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“…In the case of GPVI, the absence of this platelet receptor does not create an inherent bleeding phenotype in vivo, and defects in thrombus formation or prolonged bleeding in vivo are dependent upon the genetic background at Mh. Thus, conclusions from previously established murine models of GPVI deficiency in animals with mixed backgrounds, whether induced by immune-mediated down-regulation 9,10 or targeted gene disruption of related molecules, such as FcR␥, 11,20 or even direct targeted disruption of Gp6 itself, 21 need to be reevaluated. Moreover, the indication of GPVI as a target for antithrombotic intervention must also be reconsidered, because its potential efficacy as well as side effects may be influenced by putative human equivalents of Mh genes.…”

Section: Discussionmentioning

confidence: 99%

“…GPVI is considered a major receptor that mediates collagendependent platelet function, an assertion that finds support in the complete lack of collagen-initiated platelet responses in vitro in mice that are genetically FcR␥ deficient, 9,20 that are temporarily GPVI-depleted by administration of JAQ1, 10 and that are Gp6 Ϫ/Ϫ . 8 All of the Gp6 Ϫ/Ϫ mice in our current study exhibit markedly deficient platelet responses in ex vivo perfusion studies on fibrillar type I collagen and a virtual absence of in vitro collagen-induced platelet aggregation, as expected.…”

Section: Discussionmentioning

confidence: 99%

The Modifier of hemostasis (Mh) locus on chromosome 4 controls in vivo hemostasis of Gp6−/− mice

Chéli

Jensen

Marchese

et al. 2008

Blood

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Platelet glycoprotein VI (GPVI) is a key receptor for collagens that mediates the propagation of platelet attachment and activation. Targeted disruption of the murine gene Gp6 on a mixed 129 ؋ 1/ SvJ ؋ C57BL/6J background causes the expected defects in collagen-dependent platelet responses in vitro. The extent of this dysfunction in all Gp6 ؊/؊ mice is uniform and is not affected by genetic background. However, the same Gp6 ؊/؊ mice exhibit 2 diametrically opposed phenotypes in vivo. In some mice, tail bleeding times are extremely prolonged, and thrombus formation in an in vivo carotid artery ferric chloride-injury model is significantly impaired. In other littermates, tail bleeding times are within the range of wild-type mice, and in vivo thrombus formation is indistinguishable from that of control mice. Directed intercrosses revealed that these phenotypes are heritable, and a genome-wide singlenucleotide polymorphism scan revealed the most significant linkage to a single locus (8 megabases) on chromosome 4 (logarithm of the odds [LOD] score ‫؍‬ 6.9, P < .0001) that we designate Modifier of hemostasis (Mh) . IntroductionGlycoprotein VI (GPVI), which is restricted in expression to megakaryocytes and platelets, is an important receptor for collagens. GPVI (60-65 kDa) is noncovalently associated with the fragment crystallizable receptor ␥ (FcR␥) subunit, an immunoreceptor tyrosine-based activation motif (ITAM)-based signaling coreceptor, 1,2 and in the mouse, surface GPVI expression requires coexpression of FcR␥. 3 Upon engagement of collagens, the GPVI-FcR␥ complex transduces outside-in signals that involve spleen tyrosine kinase (Syk), linker for the activation of T cells (LAT), and Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), resulting in phospholipase ␥2 and phosphatidylinositol 3-kinase activation, leading to release of granule contents, prothrombinase activity, and platelet aggregation. [4][5][6][7] Targeted gene deletion in the mouse is one strategy to generate experimental models to assess the role of membrane receptors in hemostasis and thrombosis. We were the first to report the targeted disruption of murine Gp6 8 and demonstrated, using functional in vitro assays, that platelets from homozygous Gp6 Ϫ/Ϫ mice fail to aggregate upon stimulation with type I fibrillar collagen and fail to form thrombi upon perfusion over collagen-coated surfaces. However, the in vivo tail bleeding times for the same Gp6 Ϫ/Ϫ mice were more often within the range seen with wild-type littermates (45-140 seconds), and only a minority of the Gp6 Ϫ/Ϫ F2 littermates (3/13) exhibited an abnormal, extremely prolonged tail bleeding time (Ͼ 600 seconds). Similar proportions of normal and abnormal mice have been observed in studies of genetically engineered FcR␥ Ϫ/Ϫ mice, in which platelet surface GPVI expression is ablated, or wild-type mice made GPVI-deficient after 7 days by intraperitoneal injection of rat anti-mouse GPVI monoclonal antibody JAQ1. [9][10][11] On repeated examination, we dete...

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Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRγ deficiency

Cited by 138 publications

References 39 publications

Diverging signaling events control the pathway of GPVI down-regulation in vivo

Diverging signaling events control the pathway of GPVI down-regulation in vivo

Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen

The Modifier of hemostasis (Mh) locus on chromosome 4 controls in vivo hemostasis of Gp6−/− mice

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