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P.H. Mangin et al.
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Figure 6. Blockade of human glycoprotein VI limits platelet accumulation to a growing aggregate. (A)(i). Hirudinated human whole blood labeled with DIOC6 (1 μmol/L) was perfused over immobilized collagen (200 μg/mL) to preform aggregates for 2 min 30 s, before perfusing hirudinated blood from the same donor in the presence of the Alexa Fluor 647-conjugated monoclonal antibody against GPIbβ (5 μg/mL) and with a Fab control (Control) or the blocking anti-GPVI antibody 9O12 (50 μg/mL). (A)(i). Representative 3D reconstructions from confocal images of aggregates obtained after 7 min 30 s of blood perfusion at 300 s-1. Preformed aggre- gates are represented in gray, aggregates formed in the presence of a Fab control are represented in red and aggregates formed in the presence of the Fab 9O12 are depicted in orange. (A)(ii). Bar graph representing the volume of the platelet aggregates (mean±SEM) in eight random fields, in six separate experiments per- formed with different blood donors (Mann-Whitney test, ***P<0.001). The gray, red and orange colors represent the volume of the preformed aggregates, the aggre- gates formed in the presence of a Fab control and the aggregates formed in the presence of the Fab 9O12, respectively.
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takes place at a critical checkpoint in aggregate consolida- tion and aggregate growth. Thus, GPVI has the potential to both initiate and propagate thrombus formation through interactions with collagen and fibrin and, it appears now, also with fibrinogen. Moreover, while colla- gen and fibrin are localized at the base of the thrombus and in the core, respectively, fibrinogen is found through- out the aggregate. This suggests a model in which throm- bus growth could be sustained by GPVI/fibrinogen, poten- tially in association with other adhesive proteins. Indeed, in addition to fibrinogen other adhesive proteins such as von Willebrand factor and fibronectin have been shown to support thrombus growth.47–50 Whether these proteins par- ticipate in GPVI-mediated platelet aggregation is unclear since von Willebrand factor is not known to be a ligand of GPVI and fibronectin does not directly promote platelet adhesion and activation through GPVI.51 Selective inhibi- tion of the interaction of GPVI with collagen, fibrinogen and fibrin is required to establish their respective contribu- tions to platelet activation in hemostasis and thrombosis.
The discovery that GPVI initiates and propagates platelet aggregation at sites of vessel injury suggests a major role in hemostasis and thrombosis. Paradoxically, however, mice and humans deficient in GPVI only have at most a mild bleeding diathesis, which in the case of humans may be due to additional confounders such as a low platelet count as seen in patients with immune- induced thrombocytopenia caused by antibodies to GPVI. The relatively minor role of GPVI in hemostasis
can be explained by redundancy in pathways of platelet activation, with the GPIb-von Willebrand factor axis ini- tiating hemostasis, and ADP, thromboxane and thrombin inducing powerful activation. Additionally, the reactive fibrillar type I and III collagen present in deeper layers of the vessels would limit the role of GPVI in the hemosta- sic response following superficial injury. On the other hand, the discovery that fibrin and immobilized fibrino- gen activate GPVI may be of significance at sites of fib- rinogen deposition or fibrin formation in diseased vessels following inflammation or loss of vascular integrity. The ability of fibrin and immobilized fibrinogen to activate GPVI may also reflect yet-to-be-discovered new roles for GPVI.
In conclusion, in the present study, we have identified immobilized fibrinogen as a novel activator of human but not mouse GPVI and have shown that this interaction supports platelet aggregation under flow. This further emphasizes the contribution of GPVI to platelet activation in thrombosis.
Acknowledgments
This work was supported by the British Heart Foundation (RG/13/18/30563); SPW holds a BHF Chair (CH03/003) and ATH holds a BHF Studentship (FS/15/71/31677). NLL’s con- tract was funded by the Agence Nationale pour la Recherche ANR-14-CE35-0022-02. The authors would like to thank Victor Tybulewicz and Edina Schweighoffer for providing critical reagents.
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