Differential effects of reduced glycoprotein VI levels on activation of murine platelets by glycoprotein VI ligands

Snell, Daniel C.; Schulte, Valerie; Jarvis, Gavin E.; Arase, Kanako; Sakurai, Daiju; Saito, Takashi; Watson, Steve P.; Nieswandt, Bernhard

doi:10.1042/bj20020335

Cited by 41 publications

(43 citation statements)

References 32 publications

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“…Low-GPVI platelets expressing as few as 30 to 40 receptors per platelet (6) exhibited relatively preserved aggregation responses to collagen despite the loss of aggregation responses to the high-affinity GPVI-specific ligands CVX or CRP. Unlike wild-type, but similar to GPVIblocked human platelets, low-GPVI platelet collagen responses were ␣2␤1 dependent, a result supported by a recent study of platelets with a less drastic reduction in GPVI receptor density (37). Thus, reducing GPVI-mediated collagen signals either through pharmacologic blockade or through genetic reduction in receptor number results in collagen signals that are supported by the integrin ␣2␤1.…”

Section: Discussionsupporting

confidence: 61%

Reciprocal Signaling by Integrin and Nonintegrin Receptors during Collagen Activation of Platelets

Chen

Kahn

2003

Molecular and Cellular Biology

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Activation of platelets by exposed collagen after vessel wall injury is a primary event in the pathogenesis of stroke and myocardial infarction. Two collagen receptors, integrin ␣2␤1 and glycoprotein VI (GPVI), are expressed at similar levels on human and mouse platelets, but their individual roles during collagen activation remain poorly defined. Recent genetic and pharmacologic experiments have revealed an essential role for GPVI but have failed to define the role of ␣2␤1 or explain how two structurally distinct collagen receptors might function together to mediate platelet collagen responses. Discriminating the roles of these two collagen receptors is complicated by evidence suggesting that GPVI and platelet integrins may activate a common intracellular signaling pathway. To determine how ␣2␤1 and GPVI activate platelets in response to collagen, we have (i) examined collagen signaling conferred by expression of these receptors in hematopoietic cell lines; (ii) determined the effect of blocking each receptor on the activation of human platelets by collagen; (iii) generated low-GPVI mice in which the ␣2␤1/GPVI receptor ratio has been altered from 1:1 to 50:1 to expose ␣2␤1 function; (iv) studied the collagen responses of mouse platelets lacking LAT, an adaptor protein critical for GPVI but not integrin signaling; and (v) addressed the mechanism by which soluble collagens activate wild-type platelets. These studies demonstrate that ␣2␤1 requires inside-out signals to participate in collagen signaling and that ␣2␤1 is required for collagen activation of platelets when GPVI signals are reduced by blocking anti-GPVI antibody, low receptor number, specific disruption of the GPVI signaling pathway, or forms of collagen that bind weakly to GPVI relative to ␣2␤1. We propose a reciprocal two-receptor model of collagen signaling in platelets in which the nonintegrin receptor GPVI provides the primary collagen signal that activates and recruits the integrin receptor ␣2␤1 to further amplify collagen signals and fully activate platelets through a common intracellular signaling pathway. This model explains many of the genetic and pharmacologic observations regarding collagen signaling in platelets and demonstrates a novel mechanism by which hematopoietic cells integrate signaling by structurally distinct receptors that share a common ligand.Platelet activation in response to vessel wall injury is an initiating event in atherothrombotic diseases such as stroke and myocardial infarction (22). Collagen is a vessel wall protein known to directly activate platelets (38), and platelet activation by exposed collagen is believed to be an early and important step in the pathogenesis of these diseases. The molecular basis of platelet activation by collagen has been studied for more than 15 years with the identification of two major collagen receptors on mouse and human platelets: the integrin ␣2␤1 (34) and glycoprotein VI (GPVI), a receptor homologous to immune receptors that signals through the transmembrane signaling adaptor Fc gamm...

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Section: Discussionsupporting

confidence: 61%

Reciprocal Signaling by Integrin and Nonintegrin Receptors during Collagen Activation of Platelets

Chen

Kahn

2003

Molecular and Cellular Biology

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“…Blood from control mice exhibited robust thrombus formation at 800 s Ϫ1 , which equates to 11.6 Ϯ 1.6 g protein deposited on the collagen-coated surface during 2 minutes ( Figure 5A). Thrombus formation in FcR ␥-chain heterozygote blood, which expresses 50% of control levels of GPVI, 27 was not significantly different than the control with protein levels of 9.8 Ϯ 0.9 g deposited during 2 minutes. Quantitation of percent of surface coverage of the collagen-coated microslide gave results that are consistent with the protein assay, with control platelets giving 70 Ϯ 4% coverage compared with 72 Ϯ 5% for the heterozygotes.…”

Section: Platelet Adhesion and Thrombus Formation Is Dependent On Gpvmentioning

confidence: 91%

“…FcR ␥-Tg mice express a transgene for FcR ␥-chain in an FcR ␥-chain-null background and were crossed with FcR ␥ (Ϫ/Ϫ) to reduce the level of expression of the GPVI-FcR ␥-chain complex to approximately 20% of that of controls. 27 The level of GPVI in the FcR ␥-chain ITAM mutant was approximately 40% of controls. The generation of these mice will be described (T.S., manuscript in preparation).…”

Section: Genetically Modified Micementioning

confidence: 99%

GPVI levels in platelets: relationship to platelet function at high shear

Best¹

2003

Blood

120

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We have investigated the density of the collagen receptors glycoprotein VI (GPVI) and ␣ 2 ␤ 1 on human platelets and their relationship to polymorphisms within the GPVI gene. GPVI levels varied 1.5-fold and showed a weak correlation (r ‫؍‬ 0.35) with the levels of ␣ 2 ␤ 1 , which varied 3-fold. GPVI genotype had a significant effect on receptor levels with carriers of the proline 219 allele (approximately 22% of the population) having 10% lower GPVI levels than the more common serine homozygotes. GPVI and ␣ 2 ␤ 1 levels were found to be significantly decreased on platelets from patients with myeloproliferative disorders (MPDs). In both the MPD and the control group, GPVI levels were found not to affect platelet function under high shear in whole blood. Similarly murine platelets that express up to 5-fold lower levels of GPVI showed no significant difference than controls in thrombus formation on a high-density collagen-coated surface.

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“…21 Nonetheless, genetic manipulation in mice associated with major reductions in GPVI levels severely modifies collageninduced platelet responses. 21,43 The characteristics from our patient SDS-soluble extracts of platelets from the patient (p) and a control donor (c) were separated by SDS-PAGE without disulfide reduction and transferred to nitrocellulose membrane. The membranes were probed for GPIb (Bx-1), ␣IIb (SZ22), ␤3 (XIIF9), and GPVI using the MoAbs F5 (A,B), 3J24.2 (C), and 9E18.2 (D).…”

Section: Discussionmentioning

confidence: 99%

Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome

Nurden

Jandrot‐Perrus

Combrié

et al. 2004

Blood

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We report a novel case of gray platelet syndrome (GPS) where a severe deficiency of the platelet collagen receptor, glycoprotein (GP) VI, accompanies classical symptoms of a low platelet count and platelets lacking ␣-granules. Dense granules were normally present. Platelet aggregation with collagen was severely decreased, as was the response to convulxin (Cvx), a GPVI agonist. Quantitative analysis of GPVI using fluorescein isothiocyanate (FITC)-Cvx in flow cytometry showed its virtual absence on the patient's platelets. The GPVI deficiency was confirmed using monoclonal antibodies in Western blotting and in immunogold labeling on frozen thin sections where internal pools of GPVI were confirmed for normal platelets. The Fc receptor ␥-chain, constitutively associated with GPVI in normal platelets, was present in subnormal amounts, and the phospholipase C␥2-dependent activation pathway appeared to function normally. No autoantibodies to GPVI were found in the patient's serum using monoclonal antibody immobilization of platelet antigen (MAIPA). Sequencing of coding regions of the GPVI gene failed to show abnormalities, and mRNA for GPVI was present in the patient's platelets, pointing to a probable acquired defect in GPVI expression. Our results may provide a molecular explanation for the subgroup of patients with severely deficient collagen-induced platelet aggregation as previously described for GPS in the

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Differential effects of reduced glycoprotein VI levels on activation of murine platelets by glycoprotein VI ligands

Cited by 41 publications

References 32 publications

Reciprocal Signaling by Integrin and Nonintegrin Receptors during Collagen Activation of Platelets

Reciprocal Signaling by Integrin and Nonintegrin Receptors during Collagen Activation of Platelets

GPVI levels in platelets: relationship to platelet function at high shear

Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome

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