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DOI: 10.1055/s-0040-1718737
Differential Role of Glycoprotein VI in Mouse and Human Thrombus Progression and Stability
Introduction
Platelet glycoprotein (GP) VI is a promising, safe, antithrombotic target as its absence or blockade prevents in vitro thrombus formation and experimental thrombosis in various animal models without impacting the tail-bleeding time.[1] In addition, patients with a mutation in the GPVI gene exhibit only a mild bleeding diathesis,[2] further suggesting that GPVI does not play a critical role in hemostasis. GPVI is the main platelet activation receptor for collagen and viewed as being important in the initiation of thrombus formation.[1] In addition to collagen, GPVI interacts physically or functionally with other adhesive proteins including laminins, fibrin, and fibrinogen.[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. It is important to appreciate species difference between humans and mice as the latter represent the most broadly used animal model to study experimental thrombosis and that a significant part of our current understanding of the molecular mechanism of thrombosis relies on experiments performed with these animals.
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Results and Discussion
To investigate the role of GPVI in mouse thrombus build-up, beyond its role as a collagen receptor, we used a flow-based assay. We preformed thrombi by perfusing hirudinated whole blood of wild-type (WT) mice over immobilized type-I fibrillar collagen for 1 minute at 300 s−1 and stained platelet aggregates with DiOC6 (green). We next perfused hirudinated blood from WT or GPVI−/− mice for 6 minutes at 300 s−1 which were stained with the anti-GPIbβ antibody RAM.1-A647 (red) to visualize thrombus progression. Three-dimensional reconstructed confocal microscopy images showed that the second population of red platelets formed thrombi on the top of the first population of green aggregates similarly with GPVI−/− and WT blood ([Fig. 1A]). This was confirmed by measuring thrombus volume, which indicated that the aggregates formed with GPVI−/− mouse blood presented a similar volume to WT thrombi (WT: 5.7 ± 0.9 µm3/µm2; GPVI−/−: 5.1 ± 0.6 µm3/µm2, p > 0.05; [Fig. 1B]). This result demonstrates that mouse GPVI does not play a critical role in thrombus build-up beyond its role as an initial collagen receptor and is in sharp contrast with the key role played by human GPVI.[7] Moreover, this result also implies that murine thrombus progression in the absence of thrombin in mice primarily occurs via GPVI-independent activation pathways, notably those relying on soluble agonists such as thromboxane A2 and adenosine diphosphate (ADP; [Fig. 1C]). These in vitro observations are consistent with in vivo results showing that following FeCl3 injury of the carotid artery, which does not expose subendothelial proteins such as collagen, thrombus build-up is largely independent of GPVI.[9]
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We next studied the role of GPVI in thrombus stability and compared this process in humans and mice. Therefore, we preformed aggregates in a similar way to that described for [Fig. 1(A, B)], before testing the thrombus stability by perfusing PBS over the surface. We observed that mouse platelet aggregates were much less stable when compared to human thrombi ([Fig. 1D]). Indeed, a reduction in thrombus stability in mouse versus human platelet thrombi was already observed at wall shear rates of 300 s−1. This difference became very obvious at 600 s−1 where almost all mouse thrombi disaggregated while more than 80% of human platelet aggregates were still stable ([Fig. 1D]). These results indicate that mouse platelet aggregates are much less stable than their human counterpart in such an experimental setting. In parallel, we observed that the blocking anti-GPVI agent ACT017 reduced the stability of human platelet aggregates to the level of mouse thrombi ([Fig. 1D]). This result confirms that GPVI-fibrinogen mediated platelet activation in humans markedly increases thrombus stability. In contrast, when we perfused GPVI−/− or WT blood over aggregates formed with WT blood as described for the data in [Fig. 1](A, B), we observed that GPVI−/− platelet aggregates were as stable as WT platelet thrombi ([Fig. 1E]). Quantification confirmed no difference in the number of stable platelet aggregates between GPVI−/− and WT thrombi (WT: 100 ± 0%; GPVI−/−: 90 ± 10%; [Fig. 1F]). As a positive control, we used ARC69931MX (10 µM), an antagonist of the ADP receptor P2Y12, which efficiently destabilized mouse platelet aggregates ([Fig. 1E, F]). Together, these results indicate that mouse platelet thrombi are less stable than human thrombi and that their stability at low shear does not critically rely on GPVI, whereas GPVI is critical for the stability of human platelet thrombi. We propose that this species difference does limit the relevance of using standard mouse models to study the thrombus stabilizing action of GPVI and limits extrapolation of data obtained on the progression and stability of thrombi in mice to the human system with regard to GPVI.
In conclusion, this study highlights a significant species difference between the human and mouse hemostatic systems with regard to the contribution of GPVI in thrombus progression and stability beyond GPVI's role as a collagen receptor ([Table 1]). The reason why mouse GPVI does not contribute to thrombus build-up and stability most likely results from an inability to promote platelet activation on fibrinogen. This adhesive protein, found in every layer of a growing thrombus, is very well known to support platelet aggregation through its interaction with integrin αIIbβ3, but also through its interplay with human GPVI to promote and maintain platelet activation. This species difference is critical when judging the importance of GPVI in thrombosis as most of our knowledge is acquired from in vivo experiments performed in mice. This study suggests that the importance of GPVI in cardiovascular events in humans might be more important than previously anticipated based on experiments in mice. This is particularly important to highlight since anti-GPVI agents are currently under evaluation in phase II studies in the setting of acute coronary syndromes and ischemic stroke.
Abbreviations: ADP, adenosine diphosphate; CRP, C-reactive protein; GPVI, glycoprotein VI.
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Conflict of Interest
M.J.-P.: founder of Acticor Biotech. All other authors have declared that they have no conflict of interest.
Authors' Contributions
E.J.-B., M.U.A., and N.R. acquired, analyzed, and interpreted the data, and wrote the manuscript; C.M. acquired and analyzed the data; B.N. provided essential tools and contributed to the writing of the manuscript; C.G. and E.E.G. contributed to the writing of the manuscript; M.J.-P. and P.H.M. conceived and designed the research, interpreted the data, wrote the manuscript, and handled funding and supervision.
* These authors contributed equally to this work.
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References
- 1 Zahid M, Mangin P, Loyau S. et al. The future of glycoprotein VI as an antithrombotic target. J Thromb Haemost 2012; 10 (12) 2418-2427
- 2 Jandrot-Perrus M, Hermans C, Mezzano D. Platelet glycoprotein VI genetic quantitative and qualitative defects. Platelets 2019; 30 (06) 708-713
- 3 Inoue O, Suzuki-Inoue K, McCarty OJT. et al. Laminin stimulates spreading of platelets through integrin alpha6beta1-dependent activation of GPVI. Blood 2006; 107 (04) 1405-1412
- 4 Schaff M, Tang C, Maurer E. et al. Integrin α6β1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis. Circulation 2013; 128 (05) 541-552
- 5 Mammadova-Bach E, Ollivier V, Loyau S. et al. Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation. Blood 2015; 126 (05) 683-691
- 6 Alshehri OM, Hughes CE, Montague S. et al. Fibrin activates GPVI in human and mouse platelets. Blood 2015; 126 (13) 1601-1608
- 7 Mangin PH, Onselaer M-B, Receveur N. et al. Immobilized fibrinogen activates human platelets through glycoprotein VI. Haematologica 2018; 103 (05) 898-907
- 8 Ahmed MU, Kaneva V, Loyau S. et al. Pharmacological blockade of glycoprotein VI promotes thrombus disaggregation in the absence of thrombin. Arterioscler Thromb Vasc Biol 2020; 40 (09) 2127-2142
- 9 Eckly A, Hechler B, Freund M. et al. Mechanisms underlying FeCl3-induced arterial thrombosis. J Thromb Haemost 2011; 9 (04) 779-789
Address for correspondence
Publication History
Article published online:
29 October 2020
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References
- 1 Zahid M, Mangin P, Loyau S. et al. The future of glycoprotein VI as an antithrombotic target. J Thromb Haemost 2012; 10 (12) 2418-2427
- 2 Jandrot-Perrus M, Hermans C, Mezzano D. Platelet glycoprotein VI genetic quantitative and qualitative defects. Platelets 2019; 30 (06) 708-713
- 3 Inoue O, Suzuki-Inoue K, McCarty OJT. et al. Laminin stimulates spreading of platelets through integrin alpha6beta1-dependent activation of GPVI. Blood 2006; 107 (04) 1405-1412
- 4 Schaff M, Tang C, Maurer E. et al. Integrin α6β1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis. Circulation 2013; 128 (05) 541-552
- 5 Mammadova-Bach E, Ollivier V, Loyau S. et al. Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation. Blood 2015; 126 (05) 683-691
- 6 Alshehri OM, Hughes CE, Montague S. et al. Fibrin activates GPVI in human and mouse platelets. Blood 2015; 126 (13) 1601-1608
- 7 Mangin PH, Onselaer M-B, Receveur N. et al. Immobilized fibrinogen activates human platelets through glycoprotein VI. Haematologica 2018; 103 (05) 898-907
- 8 Ahmed MU, Kaneva V, Loyau S. et al. Pharmacological blockade of glycoprotein VI promotes thrombus disaggregation in the absence of thrombin. Arterioscler Thromb Vasc Biol 2020; 40 (09) 2127-2142
- 9 Eckly A, Hechler B, Freund M. et al. Mechanisms underlying FeCl3-induced arterial thrombosis. J Thromb Haemost 2011; 9 (04) 779-789
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