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.

Formation of the platelet plug represents a primary response to the vessel wall injury, but may also result in vessel occlusion. The decrease of the local blood flow due to platelet thrombus formation may lead to serious complications, such as ischemic stroke and myocardial infarction. However, mechanisms responsible for regulation of thrombus dynamics are not clear. In order to get a deeper insight into the role of blood flow and platelet interactions in the formation of the primary platelet plug we developed a particle-based model of microvascular thrombosis using quasisteady flow approximation. In order to simulate thrombus dynamics at physiologically relevant timescales of several minutes, we took advantage of the supercomputer technologies. Our in silico analysis revealed the importance of platelet size heterogeneity for describing experimental data on microvascular thrombus formation. Thus, our model represents a useful tool for the supercomputeraided computational analysis of thrombus dynamics in the microvessels on physiologically relevant timescales.