Eph kinases and ephrins support thrombus growth and stability by regulating integrin outside-in signaling in platelets

Abstract

The ability of activated platelets to adhere to each other at sites of vascular injury depends on the integrin alpha(IIb)beta(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 alpha(IIb)beta(3) in resting platelets, increases its surface expression when platelets are activated, and colocalizes with alpha(IIb)beta(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 alpha(IIb)beta(3) by stabilizing platelet aggregates and facilitating tyrosine phosphorylation of the beta(3) cytoplasmic domain. beta(3) phosphorylation allows myosin to bind to alpha(IIb)beta(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 alpha(IIb)beta(3).

Fig. 1.

Inhibition of Eph/ephrin interactions impairs clot retraction and the association of myosin with α_IIbβ₃. (A and B) Platelet-rich plasma was preincubated for 10 min with either PBS, 10 μg/ml His-EphA4 plus His-EphB1 (Monomers), or 10 μg/ml binding-domain-deleted His-ΔEphA4 plus His-ΔEphB1 (ΔMonomers), after which 5–10 units/ml thrombin was added to initiate thrombus formation. The clots were photographed after 120 min, and clot volume was determined at each time indicated. (C) Platelets were incubated for 10 min with thrombin. Proteins were immunoprecipitated (IP) with anti-β₃ or an isotype-matched control (M2) and then immunoblotted (IB) with anti-β₃ plus anti-myosin (My). The experiments shown are representative of three similar studies.

Fig. 2.

Tyr phosphorylation of the β₃ cytoplasmic domain. Except where indicated, platelets were incubated with an agonist for 10 min under aggregating conditions (300 μg/ml fibrinogen plus stirring), then lysed, immunoprecipitated with anti-β₃, and immunoblotted with a phospho-specific antibody to β₃ Tyr-773 (pTyr-773-β₃). (A) ADP (10 μM), thrombin (1 units/ml), or GST-EphB1 (16 μg/ml). (B) GST-EphB1 (16 μg/ml) or collagen (100 μg/ml) with or without the Src family inhibitor PP2 (10 μM). (C) ADP (5 μM), His-EphA4 (20 μg/ml), or GST-EphB1 (16 μg/ml). (D) ADP dose–response curve for β₃ phosphorylation preincubated for 10 min with 10 μg/ml His-EphA4 plus His-EphB1 (denoted Monomers or M) or His-ΔEphA4 plus His-ΔEphB1 (ΔMonomers or ΔM). (E) The aggregation traces for the samples analyzed in D.

Fig. 3.

Association of EphA4 with α_IIbβ₃. (A) Flow cytometry of platelets performed with monoclonal anti-EphA4 (clone 16c11) and anti-β₃ (clone SSA6). The results shown are representative of three similar studies. (B) Platelet lysates were resolved by Percoll gradient centrifugation into granule and membrane fractions. The results shown are representative of two similar studies. (C) Platelets were activated with 100 nM PMA and stained with anti-EphA4 or anti-β₃. The experiment shown is representative of two similar studies. (D and E) Platelets were incubated for 5 min with 20 μM ADP or 2 μM SFLLRN under aggregating conditions in the presence of PBS, 10 μg/ml His-EphA4 plus His-EphB1 (Monomers), or 10 μg/ml binding-domain-deleted His-ΔEphA4 plus His-ΔEphB1 (ΔMonomers), then immunoprecipitated (IP) with anti-β₃, anti-EphA4, or (as a negative control) antibody M2 and immunoblotted (IB). The experiment shown is representative of three similar studies. (F) Lysates were prepared from platelets activated with 100 nM PMA for 30 min, immunoprecipitated with anti-EphA4 (IP#1), and probed for β₃ and EphA4. Afterward, the supernatant from the first immunoprecipitate was reprecipitated with anti-β₃ (IP#2) and probed for β₃, EphA4, and phospho-β₃. The results shown are representative of two similar studies.

Fig. 4.

Thrombus formation under arterial flow conditions. Mepacrine-labeled platelets in whole blood were perfused across a collagen-coated slide at a shear rate of 1,500 s^–1 in the presence of 10 μg/ml His-EphA4 plus His-EphB1 (Monomers) or 10 μg/ml binding-domain-deleted His-ΔEphA4 plus His-ΔEphB1 (ΔMonomers). After 5 min, the cells were fixed, and the slide was examined by confocal microscopy to determine thrombus volume. (A) Representative cross sections of the accumulated platelet thrombi in a series of parallel planes 10, 13, and 16 μm above the slide surface. (B) Average thrombus volume measured at four surface locations in each of four different experiments (mean ± SEM).