Anti‐GPVI nanobody blocks collagen‐ and atherosclerotic plaque–induced GPVI clustering, signaling, and thrombus formation

Abstract

Background:The collagen receptor glycoprotein VI (GPVI) is an attractive antiplatelet target due to its critical role in thrombosis but minor involvement in hemostasis.Objective: To investigate GPVI receptor involvement in platelet activation by collagen-I and atherosclerotic plaque using novel blocking and non-blocking anti-GPVI nanobodies (Nbs).Methods: Nb effects on GPVI-mediated signaling and function were assessed by western blot and whole blood thrombus formation under flow. GPVI clustering was visualized in thrombi using fluorescently labeled Nb28.Results: Under arterial shear, inhibitory Nb2 blocks thrombus formation and platelet activation on collagen and plaque, but only reduces adhesion on plaque. In contrast, adhesion on collagen, but not plaque, is decreased by blocking integrin α2β1.Adhesion on plaque is maintained despite inhibition of integrins αvβ3, α5β1, α6β1, and αIIbβ3. Only combined αIIbβ3 and α2β1 blockade inhibits adhesion and thrombus formation to the same extent as Nb2 alone. Nb2 prevents GPVI signaling, with loss of Syk, Lat, and PLCɣ2 phosphorylation, especially to plaque stimulation. Non-blocking fluorescently labeled Nb28 reveals distinct GPVI distribution patterns on collagen and plaque, with GPVI clustering clearly apparent on collagen fibers and less frequent on plaque. Clustering on collagen fibers is lost in the presence of Nb2.Conclusions: This work emphasizes the critical difference in GPVI-mediated platelet activation by plaque and collagen; it highlights the importance of GPVI clustering for downstream signaling and thrombus formation. Labeled Nb28 is a novel tool for providing mechanistic insight into this process and the data suggest Nb2 warrants further investigation as a potential anti-thrombotic agent.

Pharmacological Inhibition of Glycoprotein VI- and Integrin α2β1-Induced Thrombus Formation Modulated by the Collagen Type

“…39 After labeling, the nanobody appeared to be suitable for the imaging of platelet GPVI clusters. 49 After whole-blood perfusion over collagen-H, we observed that the adhered platelets showed a high Nb28-AF488 signal. The AF488 fluorescence was concentrated at contact sites of the platelets with collagen ( Fig.…”

Modelling arterial thrombus formation in vitro

“…The isolation of plaque material from carotid endarterectomy and the homogenisation process, may affect the structural arrangement of the different extracellular matrix proteins, altering the function compared to the atherosclerotic lesion in situ . A lack of fibrillar collagen has been reported in homogenates [ 49 ], which may in part explain recent findings demonstrating differential GPVI activation on fibrillar collagen compared to plaque homogenate [ 48 , 52 ]. Our own studies, however, have also demonstrated a significant difference in thrombus formation on Type 1 Horm collagen compared to native extracellular matrix proteins produced by human coronary artery endothelial cells (Drysdale and Jones, unpublished data).…”

Platelet biology and function: plaque erosion vs. rupture

“…Inhibition of α2β1, however, was without effects. 67–70 Notably, plaque composition and structure are not taken into account in these experimental set-ups.…”