Eliminate dark side from antiplatelet therapy

In this issue of Blood, Stefanini and colleagues report a new way to regulate platelet function. Mice expressing a talin-1 mutant (W359A) that disrupts its binding to the NPxY motif in β₃ were protected from experimental thrombosis without undermining hemostasis by decelerating αIIbβ3 activation.¹ 

In addition to their essential role in hemostasis, platelets play a crucial role in a pathological thrombus formation, particularly within atherosclerotic arteries subjected to high shear stress.²  As an initial step in thrombogenesis (or hemostasis), platelets adhere to altered vascular surfaces or exposed subendothelial matrices, then become activated and aggregate each other to grow thrombus (or platelet plug) volume. These processes are primarily mediated by platelet surface glycoproteins: GPIb-IX-V, integrin α₂β₁ (also known as GPIa-IIa), GPVI, and integrin αIIbβ3 (GPIIb-IIIa).³  Integrins are a family of αβ heterodimeric adhesion receptors that mediate cellular attachment to the extracellular matrix and cell cohesion.⁴  Among integrins, platelet αIIbβ3 is a prototypic non-I domain integrin and plays an essential role in platelet aggregation and thrombus growth as a physiological receptor for fibrinogen and von Willebrand factor, as evidenced by the clinical features of congenital bleeding disorder—Glanzmann thrombasthenia.³  After exposure to subendothelial matrix including collagen and von Willebrand factor and/or several mediators (agonists) including adenosine 5′-diphosphate (ADP), thromboxane A₂, and thrombin, platelets become activated and a series of intracellular signaling events (“inside-out” signaling) that rapidly induce a high-affinity state of αIIbβ3 from its low-affinity state for soluble ligands (αIIbβ3 activation) are generated. In fact, αIIbβ3 activation is indispensable for platelet aggregation, and specific binding of the cytoskeletal proteins, talin and kindlin-3, to the cytoplasmic tail of the β₃ subunit is the final common step in αIIbβ3 activation.⁵  Although αIIbβ3 represents a rationale target for antithrombotic therapy, strong inhibition of αIIbβ3 increased bleeding complication. This is also true in the case of P2Y₁₂ inhibitors.⁶ 

Investigators are still searching for the “magic bullet” that selectively targets pathological thrombus formation without undermining hemostasis.⁷  In this issue of Blood, Stefanini et al report a new strategy to segregate antithrombotic capacity from bleeding complications by modifying αIIbβ3 activation via disrupting of talin-1 interaction with β₃ cytoplasmic tail. Talin-1 is a large protein (∼270 kDa) that consists of a ∼50 kDa globular head domain and an elongated flexible 220-kDa rod domain. Talin head domain contains 4 subdomains: F0, F1, F2, and F3, and recent structural and biochemical studies have established that αIIbβ3 activation requires interaction of talin F3 domain with 2 distinct binding sites in the β₃ cytoplasmic tail: a membrane distal region (a proximal NPxY motif) and a membrane proximal region (MPR) of β₃. “Inside-out” signaling first induces the talin F3 domain binding to the NPxY motif, which stabilizes additional interactions between talin F3 and MPR.^8,9  Unlike talin-1, kindlin-3 binds to the membrane-distal NxxY motif of the β₃ cytoplasmic tail⁹  (see figure). Both talin L325R and W359A mutants that selectively disrupt talin binding to the membrane proximal region and to the NPxY motif, respectively, have been reported to abolish integrin activation in Chinese hamster ovary cells.⁸  In contrast, it has been reported that in a human megakaryocytic cell line, CMK, all talin mutants (L325R, S365D, S379R, and Q381V) selectively disrupting the binding to MPR completely lost their ability to activate αIIbβ3, whereas talin W359A retained some ability to activate αIIbβ3.¹⁰  To further elucidate the functional differences between talin L325R and talin W359A in vivo, the authors generate a series of platelet-specific talin-1 mutant knock-in mice. Because homozygotes for either talin-1 L325R or talin-1 W359A were embryonic lethal, talin-1(L325R/wt) or talin-1(W359A/wt) mice were crossed with platelet-specific talin-1 knockout (talin-1flox/flox Pf4-Cre⁺) mice to generate compound heterozygous (talin-1 L325R/flox Pf4-Cre⁺ and talin-1W359A/flox Pf4-Cre⁺) and control mice (talin-1wt/flox Pf4-Cre⁺). Thus, as compared with wild-type mice, these mice express only 50% of talin-1 in platelets, even in control mice (talin-1 wt/flox PF4-Cre⁺). The phenotype of talin-1(L325R) mice was very similar to that of talin1-deficient mice. These mice were protected from FeCl₃-induced thrombosis model in the carotid artery, whereas tail-bleeding time was markedly increased as expected. Compared with wild-type mice, the 50% reduction of talin-1 expression in platelets in control mice (talin-1wt/flox Pf4-Cre⁺) used in this report had no effect on occlusion times in the thrombosis experiments. It is especially noteworthy that talin-1 (W359A) mice were protected from the experimental thrombosis without pathological bleeding. Moreover, in vitro experiments revealed that talin-1 (W359A) head domain bound to β₃ tail with 2.9-fold lower affinity compared with wild-type talin-1 head domain, which is likely responsible for slower αIIbβ3 activation, delayed platelet aggregation and markedly reduced ex vivo thrombus formation under high sear rate. However, under low shear rate, talin-1 (W359A) platelets had a capacity to form a small 3-dimensional thrombus.

This report reveals that the interaction of talin-1 with the MPR of β₃ tail plays a more critical role in αIIbβ3 activation than with the NPxY motif in vivo. The disruption of the interaction with the NPxY motif did not abolish, but reduced, the affinity of talin-1 for β₃ tail, thereby decelerating αIIbβ3 activation. It is likely that rapid αIIbβ3 activation on platelets is a prerequisite for pathological thrombus formation, particularly within atherosclerotic arteries subjected to high shear stress. This report newly proposes that deceleration of αIIbβ3 activation could be a smart way to prevent thrombosis without increasing bleeding tendency. Further studies are warranted to find the “magic bullet.”