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In vivo role and mechanism of platelet migfilin
Figure 6. A model for the role of the migfilin in regulating filamin A-β3 interaction during platelet activation. In resting platelets, filamin A binding to the β3 tail pre- vents interactions among talin, kindlin, and integrin. When a platelet is activated, inside-out signals, through displacement of filamin A and enabling subsequent talin and kindlin binding to β3, culminate in the high affinity conformational change of aIIbβ3. Upon binding of activated aIIbβ3 to fibrinogen, migfilin (or WT-migfilin-CCR7 peptide) prevents the reassociation of filamin A to β3 and supports a sustained outside-in signaling. Without migfilin, filamin A binds back to β3 prematurely and inhibits further transmission of integrin outside-in signals.
ing associated with aIIbβ3. However, supported by the functional measurements and phosphorylation events rep- resentative of inside-out and outside-in signaling, our cur- rent study demonstrates that the involvement of migfilin is restricted to outside-in signaling of aIIbβ3. In particular, using migfilin peptide synthesized in the same way as described in the previous study,13 the current study demonstrate that platelet aggregation is only induced by the peptide at a concentration above 10 mM. At a lower concentration (5 mM), the migfilin peptide does not induce platelet aggregation (Online Supplementary Figure S5), yet this concentration of migfilin peptide fully rescues the impaired function and aIIbβ3 outside-in signaling in migfilin-/- platelets. These results not only support that the observed platelet phenotypes in the current study are bona fide migfilin-dependent, but also reveal that platelets are sensitive to the amount of intracellular migfilin. Hence, combining the observations obtained in migfilin-/- platelets and those using migfilin peptides, it is possible to envisage that the natural amount of migfilin participates in outside- in signaling, while excessive migfilin might influence inside-out signaling. This may also justify the extremely low expression of migfilin in platelets. Intriguingly, the use of migfilin peptide could be further translated into a res- cued hemostatic and thrombotic function in vivo in migfilin-/- mice.
Migfilin-/- platelets exhibit a reduced phosphorylation level of the molecules corresponding to early outside-in signaling such as β3, Src, and Syk, as well as a hampered ability to spread on immobilized fibrinogen. On the con- trary, the late outside-in signaling events, such as phos- phorylation of ERK, and p38,24 were not drastically changed, nor did migfilin-deficiency critically influence clot retraction. Corroborating these findings, migfilin pep- tides harboring the filamin A binding sequence rescue the phosphorylation events and sequential functions associated with early but not late outside-in signaling. Our results may have suggested that filamin A constitute the
major functional partner of migfilin in platelets. Yet the inability to visualize migfilin protein in platelets has pre- sented itself as a large hurdle to further dissection of the mechanism of migfilin. Particularly, one question persists: how can migfilin have such an important role when the protein is present in such a low amount? Indeed, based on the published literature,13 stoichiometry of migfilin bind- ing to filamin A (IgFLNa21 domain) is either 1:1 or 1:2. However, since migfilin-filamin A interaction is much stronger than filamin A-integrin interaction,29,14 the amount of migfilin required to trigger integrin activation is con- ceivably much lower than the proposed stoichiometry of migfilin-filamin A binding. Alternatively, migfilin may be enriched spatially to the integrin adhesion sites during activation,30,31 which eliminates the needs of high expres- sion in the cytoplasm. Moreover, migfilin interacts with multiple important proteins such as kindlin, Src and VASP,30-33 the confluence of synergistic effects executed by these important platelet regulators may result in an ampli- fied platelet activation.7,34,35 We acknowledge that the mechanism of migfilin in platelet is only partially unveiled by the current study. A further elaboration of the migfilin interaction network in platelets is warranted.
A previous study has elegantly unveiled the important role of filamin A-aIIbβ3 interaction in pro-platelet formation and explained the pathogenesis of macrothrombocytopenia in filaminopathies (conditions connected to heterozygous filamin A mutations).36 Intriguingly, although the current study has shown that migfilin regulates filamin A-aIIbβ3 interaction in platelets, deletion of migfilin seems to neither influence the platelet count nor the platelet morphology. It may be explained by the fact that loss of migfilin essentially enhances the interaction between filamin A and aIIbβ3, which con- trasts the phenotype caused by filamin A mutations, whose ability to interact with aIIbβ3 is reduced. The het- erogenous phenotypes of platelets and megakaryocytes associated with filamin A mutations indicate that com-
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