Manipulating integrin signaling for anti-thrombotic benefits

Blood platelets play an important role during physiological hemostasis and pathological thrombosis.¹ On unactivated platelets, integrin α_IIbβ₃, the platelet fibrinogen (Fg) receptor, is in a low-affinity state, unable to bind soluble Fg. When platelet activation by physiological agonists such as collagen, thrombin, or ADP, occurs, a cascade of signaling events induces a conformational change in the extracellular domains of α_IIbβ₃. This converts it into a high-affinity state capable of binding Fg, through a process known as inside-out signaling.² Once a ligand binds to an integrin receptor, a signal is once again transmitted, this time from the extracellular domain of α_IIbβ₃ through the transmembrane domain to the cytoplasmic domain, a phenomenon known as outside-in signaling.² The outside-in signaling controls the process of platelet spreading and clot retraction, which requires a significant contractile force between the platelet cytoskeleton and fibrin through integrin α_IIbβ₃.³

Most platelet agonists such as thrombin, ADP, epinephrine, and thromboxane A2 induce inside-out signaling through guanine nucleotide-binding protein (G-protein) coupled receptors (GPCRs), which activate heterotrimetric G-proteins consisting of Gα, Gβ and Gγ subunits.⁴ Platelets express 9 Gα subunits; among them G_i, G_q, G₁₂, G₁₃, and G_s are known to be essential mediators of hemostasis and thrombosis.⁵ Activation of platelets through these GPCRs leads to activation of platelet integrin α_IIbβ₃, an essential common step in platelet aggregation leading to hemostasis and thrombosis. Antagonists of platelet integrin α_IIbβ₃ are potent anti-thrombotic drugs, but they can cause bleeding in patients, which can be life threatening.^6,7 Recently it has been shown that Gα₁₃ is essential for outside-in signaling through integrin α_IIbβ₃.^8,9 Shen et al, in a series of outstanding experiments, now describe the discovery of a novel anti-thrombotic drug that does not cause bleeding.¹⁰

Building on their previous finding that Gα₁₃ is an essential regulator of integrin outside-in signaling,⁹ Shen et al found that Gα₁₃ binds to a conserved EXE motif of the β subunit of major platelet integrins, as indicated by the ability of Gα₁₃ to bind integrin β₁, β₂, and β₃, but not β₈, which lacks this motif.¹⁰ The first and third Glu in the EXE motif are important for Gα₁₃-binding as indicated by testing with various mutants and wild-type β₃. Since the EXE motif is located in a talin-binding region of β₃, using overexpression of the talin head domain (THD), which is sufficient to bind integrin, or Gα₁₃, and co-immunoprecipitation with β₃, it was shown that they are mutually exclusive in β₃ binding. Additionally, involvement of a distinct temporal factor to the binding of talin and Gα₁₃ to integrin signaling was unveiled. It was determined that the transition from the talin-bound to Gα₁₃-bound state in α_IIbβ₃ is initiated by macromolecular ligand binding to the integrin. These studies suggested that talin and Gα₁₃ selectively mediate inside-out and outside-in signaling respectively, due to their opposing waves of binding to β₃. This was confirmed using talin knockout platelets, which demonstrate defective aggregation to ADP, but could be corrected with manganese or integrin-activating antibody LIBS6, both of which independently activate integrins in the absence of talin-dependent inside-out signaling. When Fg changes conformation, whether due to immobilization or converting to fibrin, it is currently believed that it can interact with integrin independently of inside-out signaling. This is due to ligand-induced integrin activation caused by the contact of the integrin recognition sequence RGD of the ligand with the integrin. Interestingly, it was noted that there was defective adhesion of resting talin-knockout or -knockdown platelets to immobilized Fg, which was rescued fully by adding manganese or LIBS6. This indicates that the importance of talin in resting platelet adhesion to fibrinogen is due to its role in integrin activation induced by ligands. Since platelet spreading occurs in the absence of talin after artificial activation of integrin, it indicates that talin is not required during outside-in signaling once its role in integrin activation is no longer a factor.

Shen et al next evaluated whether Gα₁₃ binding to the EXE motif of β₃ selectively mediates outside-in signaling without perturbing talin-dependent integrin activation function.¹⁰ Wild-type and AAA mutant β₃-transfected β₃ knockout mouse bone marrow stem cells were transplanted into irradiated mice from the same background. Recipient mouse platelets expressed similar levels of the wild-type or AAA mutant. The β₃ interaction with Gα₁₃ was inhibited by the AAA mutation, but the interaction with talin was unaffected, as was the agonist-induced soluble Fg binding. This shows that the EXE is not necessary for talin-dependent inside-out signaling. On the other hand, β₃-AAA mutant platelets showed defects when spreading on immobilized Fg. The results suggest that Gα₁₃-binding deficiency in β₃ causes a selective defect in platelet spreading and integrin outside-in signaling. Similarly, Gα₁₃ and fibrinogen binding defects were also noted in β₃-AAA, -DED, and -QSE mutants expressed in CHO cells. However, there was no observed negative effect on THD binding. Additionally, cells expressing β₃-AAA mutant demonstrated defects in integrin-dependent activation of c-Src as seen by Y⁴¹⁶ phosphorylation, as well as transient inhibition of Rho-A during cell spreading, both of which are important elements of outside-in signaling. This data in conjunction with previous studies identifying β₃ sequences that mediate talin binding, suggest that Gα₁₃ and talin serve as a molecular switch temporally controlling the direction of integrin signaling by interacting with distinct recognition sequences in the cytoplasmic domain of β₃.

The particular role that the EXE motif seems to play in outside-in signaling led to a designing of selective inhibitors of such, including several myristoylated EXE motif-containing β₃ peptides: mP5, mP6, and mP13.¹⁰ The inhibition of co-immunoprecipitation of Gα₁₃ and β₃ indicated that only the minimal EEERA sequence is necessary to bind Gα₁₃, whereas mP13 inhibited both talin and Gα₁₃ binding to the integrin β₃. mP6 had no observed effect on agonist-induced Fg binding to platelets or adhesion of resting platelets to immobilized Fg. Platelet-dependent clot retraction, however, was accelerated by mP6. It can be inferred from these data that mP6, which is an EXE-based inhibitor, inhibits the early phase of outside-in signaling selectively without any effect on integrin activation or clot retraction, which are both associated with talin. On the other hand, mP13 showed inhibition of both outside-in and inside-out signaling, as well as platelet adhesion, clot retraction, and Fg binding. Thus, while mP6 interferes selectively with the early phase of outside-in signaling, mP13 affects all integrin signaling phases. Interestingly, the second wave of thrombin-induced platelet aggregation in vitro was inhibited by mP6. It would have been interesting to see the effect of mP13 on platelet aggregation induced by thrombin. Would it actually block platelet aggregation completely as envisioned? When injected into mice, mP6 proved to be an effective inhibitor of thrombosis as assessed by laser-induced cremaster arteriole injury as well as FeCl₃-induced carotid artery injury, 2 well-established assays for in vivo thrombosis. Interestingly, mP6 had no effect on tail-bleeding time, a measure of hemostatic functions. When compared to integrilin, a commonly-used integrin antagonist, it appears that mP6 is a potent anti-thrombotic agent with fewer adverse effects.¹⁰

In summation, the authors unveil a novel molecular switch affecting integrin signaling. It is mediated by talin and Gα₁₃ binding in opposing waves to nearby sequences in the cytoplasmic domain of β₃. The authors noted that it is possible to selectively inhibit outside-in signaling without disrupting integrin ligand binding. They made use of this observation to design a potent integrin antagonist that can prevent thrombosis without the bleeding complications present in current anti-thrombotics (Fig. 1). There exists a potential for this knowledge to be used in clinical settings in the realm of anti-integrin and -thrombotic therapies.

Figure 1.

Schematic representation of thrombus growth after vascular injury. Circulating platelets adhere to the exposed subendothelial matrix proteins and initiate inside-out signaling leading to integrin α_IIbβ₃ activation. Activated integrin binds soluble bivalent Fg to form an aggregate, which is stabilized by outside-in signaling, thus forming a large thrombus capable of vessel occlusion. Inhibition of integrin activation as in current integrin antagonists, such as integrilin, blocks platelet adhesion as well as aggregation potentially causing bleeding. On the other hand, mP6 through virtue of selectively inhibiting outside-in signaling, allows initial platelet adhesion and activation, but blocks thrombus growth, thus allowing hemostasis to occur, but protecting from thrombosis.