Long-Term Antithrombotic Protection by in Vivo Depletion of Platelet Glycoprotein VI in Mice

Nieswandt, Bernhard; Schulte, Valerie; Bergmeier, Wolfgang; Mokhtari-Nejad, Rabée; Rackebrandt, Kirsten; Cazenave, J.‐P.; Ohlmann, Philippe; Gachet, Christian; Zirngibl, Hubert

doi:10.1084/jem.193.4.459

Cited by 326 publications

(418 citation statements)

References 61 publications

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“…These results indicated that the Cvx-binding sites of GPVI are completely different from the collagen-binding sites of GPVI. This is further supported by the results of Nieswandt et al (24) who found that the anti-GPVI antibody JAQ-1 inhibits the collageninduced aggregation but not the Cvx-induced one.…”

Section: Fig 1 Sds-page Of Recombinant Gpvi Proteinssupporting

confidence: 79%

Analysis of the Interaction of Platelet Collagen Receptor Glycoprotein VI (GPVI) with Collagen

Miura¹,

Takahashi

Jung³

et al. 2002

Journal of Biological Chemistry

150

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Glycoprotein VI (GPVI) is a platelet-specific glycoprotein that has been indicated to react with collagen and activate platelets. Its structure was recently identified by cDNA cloning (Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1999) J. Biol. Chem. 274, 29019 -29024). However, the mechanism of the interaction between collagen and GPVI has not been analyzed in detail because both collagen and GPVI are insoluble molecules. In this study, we expressed the extracellular domain of GPVI as soluble forms as follows: the monomeric form (GPVIex) and the dimeric form of GPVI fused with the human immunoglobulin Fc domain (GPVI-Fc 2 ). Purified GPVIex strongly inhibited convulxin (Cvx)-induced platelet aggregation but only weakly inhibited that induced by collagen-related peptide. However, only GPVI-Fc 2 , and not GPVIex, inhibited collagen-induced platelet aggregation. The dimeric form of GPVI exhibits high affinity for collagen, as concluded from measurements of GPVI binding to immobilized collagen by both the enzyme-linked immunosorbent assay and surface plasmon resonance methods. GPVI-Fc 2 bound to the surface of immobilized collagen with a dissociation constant (K D ) of 5.76 ؋ 10 ؊7 M, but the binding of GPVIex was too weak to allow estimation of this parameter. Cvx did not inhibit the binding of dimeric GPVI to collagen, indicating that the binding site of GPVI to collagen was different from that to Cvx. Taken together, our data indicate that the high affinity binding site for collagen is composed from two chains of GPVI. Furthermore, they suggest that the binding sites for Cvx are different from the collagen-binding sites and do not need to be formed by two GPVI molecules. Because dimeric GPVI is the only form that shows high affinity to fibrous collagen, our results indicate that GPVI would be present as a dimeric form on the platelet. Moreover, surface plasmon resonance indicated that there is no detectable interaction between soluble collagen and GPVI, supporting our previous observation that GPVI only reacts with fibrous collagen.Collagen is one of the major components of the vessel wall. When the vessel wall becomes damaged, platelets adhere to and are activated on the exposed collagen surface, leading to thrombus formation. Many proteins on the platelet surface were reported to be putative collagen receptors, but among them, only two glycoproteins have properties consistent with them being relevant collagen receptors under normal physiological conditions: one is glycoprotein (GP) 1 VI and the other is integrin ␣ 2 ␤ 1 (GPIa/IIa). Platelets deficient in either integrin ␣ 2 ␤ 1 (1, 2) or GPVI (3, 4) show loss of reactivity toward collagen, and antibodies against integrin ␣ 2 ␤ 1 , such as 6F1 (5) and P1E6 (6), and the Fab fragment of an anti-GPVI antibody (7) inhibited collagen-induced platelet aggregation. Snake venom convulxin (Cvx) (8) and collagen-related peptide (CRP), which mimic the collagen triple helix (9), can each activate platelets by binding specifically to GPVI,...

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Section: Fig 1 Sds-page Of Recombinant Gpvi Proteinssupporting

confidence: 79%

Analysis of the Interaction of Platelet Collagen Receptor Glycoprotein VI (GPVI) with Collagen

Miura¹,

Takahashi

Jung³

et al. 2002

Journal of Biological Chemistry

150

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“…Blocking of GPVI with JAQ1 Fab fragments abolished platelet adhesion and aggregation ( Figure 1A), also confirming earlier results. 4,15 The absence of FcR␥ or treatment with Src kinase inhibitor resulted in a moderately reduced deposition of platelets on collagen; these platelets remained single and showed little tendency to aggregate. Similar results were obtained with blood from LAT-null or PLC␥2-null mice ( Figure 1A).…”

mentioning

confidence: 99%

“…15,23 After 10 minutes, this injection resulted in a greatly reduced platelet concentration (20% of normal platelet count), and a specific disappearance of GPVI on the platelet surface with other platelet glycoproteins remaining unchanged (data not shown). 15 At 5 days after injection, however, the platelet count was normalized, whereas GPVI expression on platelets was still completely absent.In venules from wild-type mice, thrombus formation started after seconds, whereas in arterioles there was a lag time of several minutes. Thrombus size, quantified as thrombus height perpendicular to the vessel wall, increased with time in the vessels from wild-type mice ( Figure V, available online at http://atvb.ahajournals.org).…”

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confidence: 99%

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The Glycoprotein VI-Phospholipase Cγ2 Signaling Pathway Controls Thrombus Formation Induced by Collagen and Tissue Factor In Vitro and In Vivo

Munnix

Strehl

Kuijpers

et al. 2005

ATVB

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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 ␥-chain, Src kinases, adaptor protein LAT, and phospholipase C␥2 (PLC␥2). 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 ␥-chain, Src kinase, LAT, and PLC␥2. 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 ␥-chain, and Src kinases enhanced the formation of phosphatidylserine-exposing and fibrin-rich thrombi. Key Words: glycoprotein VI Ⅲ LAT Ⅲ platelets Ⅲ Src kinase Ⅲ thrombin T hrombus formation can be initiated by both platelet-and coagulation-activating factors. Collagens in the extracellular matrix and other vascular layers are thought to act as principal platelet-activating components of the damaged vessel wall; they also provide a surface for von Willebrand factor adhesion. 1 Tissue factor, also exposed in damaged vessels, is a key trigger of the coagulation process. 2 Because of the proposed major role of platelets in arterial thrombosis and the importance of coagulation in venous thrombosis, the current understanding is that collagen/von Willebrand factor-mediated events are more important in arteries, whereas tissue factor plays a more prominent role in venous thrombus formation. Conclusions-TheFlow studies with human and mouse blood have established that the signaling receptor glycoprotein VI (GPVI) exclusively mediates collagen-induced platelet procoagulant activity, thus linking the processes of platelet activation and coagulation. [3][4][5] This procoagulant platelet response is mediated by a prolonged and potent rise in cytosolic [Ca 2ϩ ] i , which results in exposure of procoagulant phosphatidylserine (PS) at the platelet outer surface. 6 PS exposure is a key regulating factor in the coagulation process. For instance, PS-containing membrane surfaces dramatically increase the formation of factor Xa and thrombin. 7 However, several authors have argued that this platelet response has only an assistant role in coagulation and that, in vivo, other platelet reactions may play important roles as well. 2,8 Thus, although there is no doubt that platelets enhance thrombin generation (coagulation) in plasma or whole blood, the precise mechanism is still a matter of debate. The platelet immunoreceptor GPVI is coexpressed with the Fc receptor (FcR) ␥-chain, although the latter also interacts with other plat...

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“…And even though GPVI has adapted to meet the accelerated temporal needs of hemostasis[30], the ITAM signaling response is still slower compared to the GPCR response. Consistently, the mild bleeding phenotype of patients [31,32] or mice[33,34] lacking GPVI suggests that this receptor has a minor role in classical hemostasis when compared to the major GPCRs[15]. Surprisingly, GPVI was shown to be more important for thrombus stabilization than for thrombus initiation[35], suggesting the existence of ligands other than collagen.…”

Section: Classical Hemostasis – Balancing Platelet Adhesiveness In CImentioning

confidence: 96%

Platelets at the vascular interface

Bergmeier

Stefanini

2018

Research and Practice in Thrombosis and Haemostasis

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In this brief review paper, we will summarize the State-of-the-Art on how platelet reactivity is regulated in circulation and at sites of vascular injury. Our review discusses recent and ongoing work, presented at this year’s International Society on Thrombosis and Haemostasis (ISTH) meeting, on the role of platelets in (1) classical hemostasis at sites of mechanical injury, and (2) the maintenance of vascular integrity at sites of inflammation.

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Long-Term Antithrombotic Protection by in Vivo Depletion of Platelet Glycoprotein VI in Mice

Cited by 326 publications

References 61 publications

Analysis of the Interaction of Platelet Collagen Receptor Glycoprotein VI (GPVI) with Collagen

Analysis of the Interaction of Platelet Collagen Receptor Glycoprotein VI (GPVI) with Collagen

The Glycoprotein VI-Phospholipase Cγ2 Signaling Pathway Controls Thrombus Formation Induced by Collagen and Tissue Factor In Vitro and In Vivo

Platelets at the vascular interface

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