GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism

Lockyer, Simon; Okuyama, Keiji; Begum, Shahinoor; Shi, Le; Sun, Bing; Watanabe, Takeshi; Matsumoto, Yutaka; Yoshitake, Masuhiro; Kambayashi, Jun-ichi; Tandon, Narendra N.

doi:10.1016/j.thromres.2005.08.001

Cited by 91 publications

(73 citation statements)

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“…Lockyer et al 21 reported that some Gp6 Ϫ/Ϫ mice (proportion not stipulated) exhibited prolonged bleeding times, whereas most had an average bleeding time not significantly different from Gp6 ϩ/ϩ mice. Moreover, only 10 of 18 Gp6 Ϫ/Ϫ mice (55%) survived an otherwise lethal thromboembolic challenge of intravenous collagen plus epinephrine infusion.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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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Section: Discussionmentioning

confidence: 99%

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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“…It has been reported that GPVIdeficient mice are protected against thrombus formation in vitro and in vivo, but do not show severely increased tail bleeding time in vivo. 53,54 We, therefore, hypothesize that increased bleeding time in pdk1 −/− mice may be a result of additionally impaired GPCR-dependent signaling in these mice.…”

Section: Pdk1mentioning

confidence: 99%

PDK1 Determines Collagen-Dependent Platelet Ca ²⁺ Signaling and Is Critical to Development of Ischemic Stroke In Vivo

Münzer

Walker-Allgaier

Geue

et al. 2016

ATVB

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Objective-Activation of platelets by subendothelial collagen results in an increase of cytosolic Ca 2+ concentration ([Ca 2+ ] i ) and is followed by platelet activation and thrombus formation that may lead to vascular occlusion. The present study determined the role of phosphoinositide-dependent protein kinase 1 (PDK1) in collagen-dependent platelet Ca 2+ signaling and ischemic stroke in vivo. Approach and Results-Platelet activation with collagen receptor glycoprotein VI agonists collagen-related peptide or convulxin resulted in a significant increase in PDK1 activity independent of second-wave signaling. PDK1 deficiency was associated with reduced platelet phospholipase Cγ2-dependent inositol-1,4,5-trisphosphate production and intracellular [Ca 2+ ] i in response to stimulation with collagen-related peptide or convulxin. The defective increase of [Ca 2+ ] i resulted in a substantial defect in activation-dependent platelet secretion and aggregation on collagen-related peptide stimulation. Furthermore, Rac1 activation and spreading, adhesion to collagen, and thrombus formation under high arterial shear rates were significantly diminished in PDK1-deficient platelets. Mice with PDK1-deficient platelets were protected against arterial thrombotic occlusion after FeCl 3 -induced mesenteric arterioles injury and ischemic stroke in vivo. These mice had significantly reduced brain infarct volumes, with a significantly increased survival of 7 days after transient middle cerebral artery occlusion without increase of intracerebral hemorrhage. Tail bleeding time was prolonged in pdk1 −/− mice, reflecting an important role of PDK1 in primary hemostasis. Conclusions-PDK1 is required for Ca

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“…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.…”

mentioning

confidence: 99%

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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GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism

Cited by 91 publications

References 27 publications

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

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

PDK1 Determines Collagen-Dependent Platelet Ca ²⁺ Signaling and Is Critical to Development of Ischemic Stroke In Vivo

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

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GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism

Cited by 91 publications

References 27 publications

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

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

PDK1 Determines Collagen-Dependent Platelet Ca 2+ Signaling and Is Critical to Development of Ischemic Stroke In Vivo

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

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PDK1 Determines Collagen-Dependent Platelet Ca ²⁺ Signaling and Is Critical to Development of Ischemic Stroke In Vivo