Eph Kinases and Ephrins Support Thrombus Growth and Stability by Regulating Integrin Outside-In Signaling in Platelets

The ability of activated platelets to adhere to each other at sites of vascular injury depends on the integrin α IIbβ 3. However, as aggregation continues, other signaling and adhesion molecules can contribute as well. We have previously shown that human platelets express on their surface the Eph receptor kinases EphA4 and EphB1 and the Eph kinase ligand ephrinB1. We now show that EphA4 is physically associated with α IIbβ 3 in resting platelets, increases its surface expression when platelets are activated, and colocalizes with α IIbβ 3 at sites of contact between platelets. We also show that Eph/ephrin interactions can support the stable accumulation of platelets on collagen under flow and contribute to postengagement "outside-in" signaling through α IIbβ 3 by stabilizing platelet aggregates and facilitating tyrosine phosphorylation of the β 3 cytoplasmic domain. β 3 phosphorylation allows myosin to bind to α IIbβ 3 and clot retraction to occur. The data support a model in which the onset of aggregation permits Eph/ephrin interactions to occur, after which signaling downstream from ephrinB1 and its receptors favors continued growth and stability of the thrombus by several mechanisms, including positive effects on outside-in signaling through α IIbβ 3.