Page 173 - Haematologica-5

haematologica | 2018; 103(5)
Fibrinogen, GPVI and platelet activation
grow in the presence of a Fab control but was dramatically inhibited in the presence of Fab 9O12 (Figure 6A and Online Supplementary Figure S2). In contrast, and as previ- ously shown, blockade of GPVI did not impair aggrega- tion measured by light transmission aggregometry in response to ADP, U46619 and thrombin.35 These results demonstrate a critical role for GPVI in aggregate growth under flow when the roles of collagen and fibrin are negat- ed. In contrast, fibrinogen does not induce activation of platelets in suspension either because the interaction is dependent on activation of integrin aIIbβ3 or because it cannot crosslink GPVI.
Discussion
In this study we show that human GPVI binds to immo- bilized fibrinogen and that this leads to intracellular sig- nals, which drive the formation of lamellipodial sheets and stress fibers in human platelets and in human GPVI- expressing mouse platelets. This explains the previously paradoxical observation that only human platelets form lamellipodial sheets and stress fibers on a fibrinogen sur- face, despite mouse platelets being able to form both actin structures in the presence of G protein-coupled receptor agonists such as thrombin. We also show that the interac- tion of fibrinogen with GPVI is important for aggregate growth providing a new understanding of hemostasis and thrombosis.
We recently identified fibrin as a novel ligand for GPVI7,8,42 and have shown that binding resides in the D- dimer region.30 The observation that fibrinogen also acti- vates GPVI should not, therefore, be a surprise. Nevertheless, this was unexpected and came from the observation that human platelets deficient in GPVI adhere to but do not spread on fibrinogen. This raises the ques- tion as to why this has been previously overlooked. One reason is that mouse platelets do not spread on fibrinogen and thus there is no defect in the absence of GPVI. A sec- ond reason is the low level of phosphorylation of the FcR γ-chain induced by fibrinogen in human platelets relative to that by collagen and other GPVI-agonists. This may reflect the extent to which each ligand is able to cluster GPVI and, in the case, of fibrinogen, the dependency on the interaction with integrin aIIbβ3. A third reason is that fibrinogen binds selectively to monomeric GPVI whereas the original binding studies were performed with dimeric GPVI.7,8 It is worth noting that we have reported a reduced number of dimers on immobilized fibrinogen relative to collagen.42
Adhesion of human platelets to fibrinogen is dependent on integrin aIIbβ3. At present, it is not known whether binding to integrin aIIbβ3 is critical for activation of GPVI or simply to promote adhesion such that activation of GPVI can occur. As a dimer, fibrinogen should be able to bind two GPVI monomers, but alternatively the interac- tion with integrin aIIbβ3 may be required to support acti- vation of monomeric GPVI. A similar role for an integrin in the activation of an ITAM receptor has been reported in other hematopoietic cells with the postulate that the inte- grin and the ITAM receptor would be associated via a link- er protein.43
Fibrinogen is present in whole blood at a concentration of 2 - 4 mg/mL but does not induce platelet activation. This may be explained by an inability of soluble fibrino-
gen to bind GPVI in suspension due to conformational dif- ferences between circulating and immobilized fibrinogen. Alternatively it may be due to the inability of the dimeric fibrinogen to cluster GPVI on the platelet surface in sus- pension or because of a dependency on binding to integrin aIIbβ3. While the affinity of fibrinogen for GPVI is in the range of that for collagen for GPVI,44,45 we have shown that fibrinogen (and fibrin) bind selectively to monomeric GPVI and this would not be sufficient to induce activation because of the absence of crosslinking. The reason why human platelets, but not mouse platelets, spread on fib- rinogen is unclear. Based on the fact that human and mouse GPVI share 64% homology,33 one could imagine that only human GPVI binds to fibrinogen or that both bind to this adhesive protein but only human GPVI is able to promote activation.
GPVI is primarily known as the major signaling receptor for collagen. However, in recent years, GPVI has been shown to bind to other ligands including laminin, the transmembrane protein emmprin, adiponectin, histones and fibrin.6-8,10,47 The physiological significance of many of these interactions is uncertain, in part because of their low affinity or because of whether they acutally occur in vivo. The interaction that has received the greatest attention is that with fibrin which lies at the interface of the core and shell of the growing platelet aggregate.7,8 This interaction
A
2+
Figure 5. Human glycoprotein VI, but not integrin aIIbβ3 plays a major role in the regulation of the calcium signaling after platelet adhesion to fibrinogen. Washed platelets from wild-type (WT) or mice expressing human GPVI (hGPVI) were loaded with Oregon-green Bapta-1-AM and Calcein red orange and deposited on immobilized fibrinogen (100 μg/mL). Modifications in fluores- cence of individual adherent platelets were monitored for 7 min by confocal microscopy and the Ca2+ concentrations were determined as detailed in the
profile of one representative platelet adhering to fibrinogen. (A)(ii). Dot plot representing the number of cal- cium spikes over a period of 5 min. Each point represents an individual platelet. The results are presented as the mean±SEM of five independent
Methods section. (A)(i). Typical time-course Ca
experiments (one-way ANOVA, Bonferroni post-hoc test, ***P<0.001).
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