Potentiating role of Gas6 and Tyro3, Axl and Mer (TAM) receptors in human and murine platelet activation and thrombus stabilization

Abstract

Summary. Background: Interaction of murine Gas6 with the platelet Gas6 receptors Tyro3, Axl and Mer (TAM) plays an important role in arterial thrombus formation. However, a role for Gas6 in human platelet activation has been questioned. Objective: To determine the role of Gas6 in human and murine platelet activation and thrombus formation. Methods and Results: Gas6 levels appeared to be 20-fold higher in human plasma than in platelets, suggesting a predominant role of plasma-derived Gas6. Human Gas6 synergizes with ADP-P2Y 12 by enhancing and prolonging the phosphorylation of Akt. Removal of Gas6 from plasma impaired ADP-induced platelet aggregation. Under flow conditions, absence of human Gas6 provoked gradual platelet disaggregation and integrin a IIb b 3 inactivation. Recombinant human Gas6 reversed the effects of Gas6 removal. In mouse blood, deficiency in Gas6 or in one of the TAM receptors led to reduced thrombus formation and increased disaggregation, which was completely antagonized by external ADP. In contrast, collagen-induced platelet responses were unchanged by the absence of Gas6 in both human and mouse systems. Conclusions: The ADP-P2Y 12 and Gas6-TAM activation pathways synergize to achieve persistent a IIb b 3 activation and platelet aggregation. We postulate a model of thrombus stabilization in which plasma Gas6, by signaling via the TAM receptors, extends and enhances the platelet-stabilizing effect of autocrine ADP, particularly when secretion becomes limited.

Supporting Roles of Platelet Thrombospondin-1 and CD36 in Thrombus Formation on Collagen

“…Together, this pointed to a platelet-activating effect of immobilized TSP1 via CD36, thus supporting collagendependent thrombus formation and procoagulant activity. Considering that paracrine platelet agents such as ADP, thromboxane, and Gas6 contribute to thrombus stabilization, 30,31 we investigated the stability of thrombi formed with Cd36…”

Dual Mechanism of Integrin αIIbβ3 Closure in Procoagulant Platelets

“…Platelet samples were fixed and stained for transmission electron microscopy, as described before (30). Calpain activity in platelets was assessed using a calpain activity assay kit according to the manufacturer's instructions (Abcam).…”

“…Stained blots were visualized with an ECL system. Blot quantification was by densitometric analysis, as before (30).…”

New Fundamentals in Hemostasis

“…These secreted proteins trigger additional feed-forward loops that appear to be required for stable and tight platelet-platelet contacts. For instance, activated platelets express on their surface members of the TAM family of Gas6 receptors which, by interacting with plasma Gas6, support integrin activation and thrombus formation (57). Tight intercellular contacts of platelets in a thrombus are established by a number of signaling interactions: 1) the EphA4 receptor kinase with ephrinB1, 2) the receptor semaphorin 4D (CD100) with plexin (36), 3) P-selectin (CD62P, in ␣-granules) with its ligand PSGL-1 (292), and 4) the TNFfamily receptor CD40 with CD40L (161).…”

“…The external part consists of two fibronectin-type III domains, each with an extracellular disulfide bond (Cys 49 -Cys 57 and Cys…”