Targeting of a Conserved Epitope in Mouse and Human GPVI Differently Affects Receptor Function

Navarro, Stefano; Starke, Andreas; Heemskerk, Johan W. M.; Kuijpers, Marijke J. E.; Stegner, David; Nieswandt, Bernhard

doi:10.3390/ijms23158610

Cited by 6 publications

(3 citation statements)

References 47 publications

(47 reference statements)

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“…JAQ-1 is an anti-mouse GPVI monoclonal antibody, which inhibits the major collagen and CRP-binding site in platelets [41]. Interestingly, JAQ-1 showed specific interaction with mouse and human GPVI, but, unexpectedly, it cannot bind platelets isolated from different mammalian species, such as rat, rabbit, guinea pig, dog, and swine, experimentally supporting our finding that the collagen and CRP-binding surface of GPVI diverged during evolution [42]. Although the JAQ-1 antibody recognized an epitope on hGPVI, it also induced an abnormal aggregation response in human platelets on collagen [42].…”

Section: Discussionsupporting

confidence: 79%

Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets

et al. 2023

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Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix.

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Section: Discussionsupporting

confidence: 79%

Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets

et al. 2023

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“…29 Treatment of whole blood from wildtype mice with JAQ1 reduced thrombus formation on collagen as well but does not avoid thrombus formation using whole blood from human GPVI knock-in mice (hGP6tg/tg), suggesting that binding of JAQ1 to a structurally conserved epitope in GPVI differently affects receptor function in human and murine platelets. 30 JAQ1-mediated effect on thrombus formation under flow was confirmed by the analysis of GPVI-deficient mouse platelets because thrombus formation on collagen was abolished. 31 An adaption of the microfluidic whole blood assay with the flow chamber revealed an early involvement of GPVI in thrombus formation using the newly synthesized antibody EMF-1.…”

Section: Platelet Transmembrane Proteins and Their Regulators: Role O...mentioning

confidence: 84%

Flow Chamber Analyses in Cardiovascular Research: Impact of Platelets and the Intercellular Crosstalk with Endothelial Cells, Leukocytes, and Red Blood Cells

Krott,

Feige,

Elvers

2023

Hamostaseologie

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Platelets are main drivers of thrombus formation. Besides platelet aggregate formation, platelets interact with different blood cells such as red blood and white blood cells (RBCs, WBCs) and endothelial cells (ECs), to promote thrombus formation and inflammation. In the past, the role of different proteins in platelet adhesion, activation, and aggregate formation has been analyzed using platelets/mice with a genetic loss of a certain protein. These knock-out mouse models have been investigated for changes in experimental arterial thrombosis or hemostasis. In this review, we focused on the Maastricht flow chamber, which is a very elegant tool to analyze thrombus formation under flow using whole blood or different blood cell components of genetically modified mice. Besides, the interaction of platelets with RBCs, WBCs, and ECs under flow conditions has been evaluated with regard to thrombus formation and platelet-mediated inflammation. Importantly, alterations in thrombus formation as emerged in the flow chamber frequently reflect arterial thrombosis in different mouse models. Thus, the results of flow chamber experiments in vitro are excellent indicators for differences in arterial thrombosis in vivo. Taken together, the Maastricht flow chamber can be used to (1) determine the severity of platelet alterations in different knock-out mice; (2) analyze differences in platelet adhesion, aggregation, and activation; (3) investigate collagen and non–collagen-dependent alterations of thrombus formation; and (4) highlight differences in the interaction of platelets with different blood/ECs. Thus, this experimental approach is a useful tool to increase our understanding of signaling mechanisms that drive arterial thrombosis and hemostasis.

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“…The F(ab’) 2 fragment of the mAb to FcγRIIA is widely used to block the Fc receptor in human platelets and has not been reported to induce activation. The mAb JAQ1 which binds to human and mouse GPVI is unable to activate human platelets on its own 51 but potentiates activation of mouse platelets 52 . We speculate that the presence of FcγRIIA on human platelets may be one reason why they are less sensitive than mouse platelets to divalent ligands, as this will counter activation by circulating IgG and small immune complexes in the blood.…”

Section: Discussionmentioning

confidence: 99%

Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists

et al. 2023

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CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.

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Targeting of a Conserved Epitope in Mouse and Human GPVI Differently Affects Receptor Function

Cited by 6 publications

References 47 publications

Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets

Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets

Flow Chamber Analyses in Cardiovascular Research: Impact of Platelets and the Intercellular Crosstalk with Endothelial Cells, Leukocytes, and Red Blood Cells

Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists

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