Thrombus formation: stability matters

Comment on Cosemans et al, page 3045

In this issue, Cosemans and colleagues demonstrate that ADP-dependent signaling via PI3K promotes sustained integrin activation and thrombus stability, as well as contributing to the onset of platelet aggregation.

Much of the effort to understand platelet activation at sites of vascular injury has focused on the signaling events that lead to integrin activation and the onset of aggregation. Relatively less attention has been given to the events that occur after aggregation has begun—the events that help to maintain platelets in stable contact with each other. ADP and TxA₂ have long-established roles as recruiting agents that promote thrombus formation by activating nearby platelets and potentiating the effects of other agonists, including thrombin and collagen. The effects of ADP on platelets are mediated by 2 G protein–coupled receptors, P2Y₁ and the platelet target for clopidogrel, P2Y₁₂. In this issue of Blood, Cosemans and colleagues provide new evidence that ADP plays a role in the maintenance of thrombus stability and that phosphatidylinositol 3-kinase (PI3K)–dependent signaling downstream of P2Y₁₂ is important for this effect. In 2 different model systems, P2Y₁₂ antagonists and PI3K inhibitors destabilized platelet-platelet contacts, leading to disaggregation and, ultimately, microembolization and macroembolization. That these effects are due to destabilization rather than to a failure to form an adequate thrombus is shown by adding the inhibitors after platelet aggregation had occurred.

Although elegantly demonstrated here by Cosemans et al, the concept that continued signaling within platelets is needed to help maintain thrombus stability is not new. Accumulated evidence from a number of laboratories has provided an increasingly detailed picture of events following the onset of aggregation (recently including Andre et al¹  and Goto et al² ). Stable contacts between platelets occur when activated α_IIbβ₃ on the platelet surface engages multivalent ligands such as fibrinogen and von Willebrand factor. Once engaged, the integrin serves as the nidus for signaling complexes that help to maintain the platelet in an activated state.³  Platelet-platelet contacts allow the binding of ligands on the surface of one platelet with receptors on the surface of nearby platelets, and the narrow gaps between platelets provide a protected microenvironment in which molecules that are secreted, released, or proteolytically shed by activated platelets can accumulate.⁴ FIG1 

The study by Cosemans et al focuses on the contribution of ADP and PI3K but brings out several general points. First, platelet thrombi do not appear to be intrinsically stable. Continued signaling is required to keep platelets in contact with each other, at the very least until the development of a sufficiently large cross-linked fibrin meshwork that can hold them in place. Interruption of required signaling pathways causes platelets to let go of each other, tilting the balance of platelet accumulation and loss in favor of the loss of individual platelets or small clusters of platelets. Second, α_IIbβ₃ activation is not irreversible. The transition from an inactive conformation of the integrin to an active, ligand-binding conformation can be reversed by removing ADP, blocking ADP receptors, or inhibiting PI3K. This implies that α_IIbβ₃ is not locked in the “on” position and calls for further investigation of the molecular basis of integrin activation. PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP₂) to form PI-3.4.5-P₃. How this contributes to either integrin activation or stabilization remains to be determined.

Finally, an extended model for thrombus formation that incorporates thrombus stability suggests that targeting postaggregation events can be a fruitful strategy for the development of new antiplatelet agents, as well as providing an additional mechanism of action for existing drugs (see figure). Clopidogrel is a case in point. It and other P2Y₁₂ antagonists block one of 2 known ADP receptors on platelets. As is reemphasized by the present report, P2Y₁₂ antagonists work in part by impairing platelet activation and in part by impairing thrombus stability. Events downstream of P2Y₁₂ are worth further consideration as drug targets. Isoform-selective inhibitors of PI3K have been developed and proposed as a new strategy for inhibiting thrombus formation.⁵  Other strategies may exist as well. ▪