Pinning a tail on platelet thrombi

In this issue of Blood, Ablooglu and colleagues report that deleting the c-Src binding site at the carboxyl-terminus of the integrin β3 cytoplasmic tail in murine platelets confers protection against arterial thrombosis, suggesting that this site may be an apt target for antithrombotic therapy.

Integrins are transmembrane heterodimeric adhesion receptors interposed between extracellular matrix proteins and intracellular signaling pathways that control cytoskeletal reorganization, cell migration, cell proliferation, and in the case of platelets, granule secretion.¹  The ability of integrins to interact with extracellular ligands is regulated by agonist-induced signals, whereas the intracellular signaling pathways are initiated after ligand binding by activation of integrin-associated kinases. A key structure for all of these events is the integrin β subunit cytoplasmic tail.

One of the best studied integrins is αIIbβ3 (aka, glycoprotein IIb/IIIa), the platelet membrane protein responsible for the formation of hemostatic platelet plugs and pathologic platelet thrombi.²  The cytoplasmic tail of its β subunit consists of 40 residues that fold into 3 helices followed by a short unstructured carboxyl-terminus.³  Remarkably, considering its length, the β3 tail has been found to interact with a substantial number of cytoplasmic proteins.⁴  Notable among these are talin, kindlin-3, and c-Src. Talin, a cytoskeletal protein, interacts via a FERM domain located in its head region with an NPLY motif located in a turn between the second and third β3 helices and with 2 phenylalanines located in the second helix as well.⁵  The interaction of talin with the β3 tail is required for agonist-stimulated αIIbβ3 function because it induces the ligand-binding conformation of αIIbβ3. Kindlin-3, a member of the Kindlin family of focal adhesion proteins whose expression is restricted to hematopoietic cells, interacts with an NITY motif located just after the third β3 helix, as well as with Ser752 located within the helix.⁶  The latter interaction is noteworthy because a naturally-occurring mutation, Ser752Pro, results in Glanzmann thrombasthenia by preventing platelet aggregation. Similarly, kindlin-3 knockout in mice prevents agonist-induced platelet aggregation, despite the presence of talin, implying that a ternary complex consisting of the β3 tail, talin, and kindlin-3 is required for agonist-stimulated αIIbβ3 function. Approximately 3% of the c-Src present in platelets constitutively interacts with the Arg-Ala-Thr (RGT) sequence at the carboxyl-terminus of the β3 tail via its SH3 domain.⁷  Following ligand binding to αIIbβ3, autophosphorylation of β3-associated c-Src initiates a cascade of tyrosine phosphorylation events, including phosphorylation of the β3 tail itself, that have been collectively termed “outside-in” signaling.⁸ 

In this issue of Blood, Ablooglu et al study αIIbβ3-mediated c-Src activity in situ in murine platelets after deleting the RGT sequences at the carboxyl-terminus of the β3 tail (RGT mice).⁹  Thus, the authors were able to assess the function of β3-associated c-Src in a more physiologic setting than could be achieved using pharmacologic c-Src inhibitors or cell-permeable β3 peptides. Although they found that deleting the RGT sequence decreased αIIbβ3 expression to about 60% of normal, like mice who express only one β3 allele, the RGT mice exhibited normal spontaneous hemostasis. RGT mice also exhibited normal clot retraction, but their tail bleeding times were longer than those of wild-type mice and thrombosis was impaired following FeCl₃-induced carotid artery injury. Moreover, as would be predicted from what is already known about c-Src activity in platelets, the authors found that filopodial extension was decreased when the RGT platelets adhered to fibrinogen, as was platelet spreading when the platelets were treated with Mn²⁺, ADP, or subsaturating amounts of the PAR-4 peptide AYPGFK, although spreading equaled that of wild-type platelets at higher AYPGFK concentrations. Similarly, platelet aggregation and fibrinogen binding to αIIbβ3 were impaired in response to ADP but were normal at higher AYPGFK concentrations. There was also decreased ADP-stimulated P-selectin expression and diminished, but not absent, protein tyrosine phosphorylation following incubation of RGT platelets with Mn²⁺ and fibrinogen. Based on these results, the authors conclude that deletion of the c-Src binding site on β3 alters c-Src–mediated αIIbβ3 signaling and confers protection from arterial thrombosis. Thus, c-Src binding to the β3 tail could be considered a target for antithrombotic therapy.

The data in this paper nicely demonstrate that loss of c-Src binding to the β3 tail induces a mild defect in platelet function manifest as perturbed cytoskeletal function, altered platelet secondary responses, such as diminished platelet aggregation and secretion induced by weak agonists like ADP, as well as impaired FeCl₃-induced carotid artery thrombosis. But whether they translate into protection against atherothrombosis in humans is another matter. After all, impairing platelet function with aspirin or clopidogrel produces essentially the same effect.