Pharmacological Blockade of Glycoprotein VI Promotes Thrombus Disaggregation in the Absence of Thrombin

Abstract

Objective: Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kβ, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue-type plasminogen activator). Conclusions: This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.

Differential Role of Glycoprotein VI in Mouse and Human Thrombus Progression and Stability

“…[3][4][5][6][7] We have shown that human GPVI activates platelets on immobilized fibrinogen and that this process is key for the progression and stability of human thrombi. 7,8 In sharp contrast, we observed that mouse GPVI does not promote such an activation, as platelets deposited on fibrinogen do not fully spread. 7 In this study, we investigated the consequence of absence of GPVI/fibrinogen-mediated platelet activation in mice on the regulation of thrombosis in comparison to the human system.…”

Colchicine inhibits ROS generation in response to glycoprotein VI stimulation

“…Stimulation of the GPVI receptor is distinct from the other platelet activation pathways traditionally targeted by antiplatelet therapy. GPVI inhibitors, including antibodies and GPVI-Fc fusion proteins such as “Revacept”, have demonstrated effectiveness as antiplatelet agents that do not lead to bleeding complications and can assist with disaggregation of thrombi 41 , 44 . Our study indicates that colchicine targets platelet aggregation and platelet activation (ROS generation) through the GPVI pathway, thereby providing an alternate treatment option that is also known for its anti-inflammatory effects.…”

“…GPVI is stimulated by collagen released or exposed from within the subendothelial matrix upon injury to the endothelium and by fibrinogen and fibrin that is present during thrombus formation, among other ligands 39 . Recent studies have identified that anti-GPVI therapy can lead to disaggregation of thrombus in the absence of thrombin 41 .…”

In vitro flow‐based assay: From simple toward more sophisticated models for mimicking hemostasis and thrombosis

“…To allow evaluation of the stability and embolization phase, the experimental setup has to be adapted, notably by replacing perfusion of blood with buffer. 11 At the molecular level, because the surface used to generate platelet aggregation is fibrillar collagen, the most commonly used flow-based assays tend to overemphasize the importance of collagen receptors glycoprotein VI (GPVI) and integrin α2β1 in the process of platelet aggregation and thrombosis. 12,13 This has been nicely illustrated by the group of Zaverio Ruggeri, which confirmed that GPVI plays a central role in thrombus formation on immobilized collagen, but was dispensable in a model of thrombus formation on a fibroblast and endothelial cell matrix, which might better reflect an injured vessel.…”

“…The classical in vitro flow device is best suited to study the early steps involved in thrombus formation and growth. To allow evaluation of the stability and embolization phase, the experimental setup has to be adapted, notably by replacing perfusion of blood with buffer 11 …”