Tissue Factor Is Internalized by Platelets Through Activation of RhoA and PI₃-Kinase Promoting Reversible Cytoskeletal Assembly,

Abstract 3264

TF associated with platelets may play a critical role in the development of ischemic complications at sites of vascular damage. Platelets can internalize and store tissue factor-rich microvesicles from human origin (hTF), a process that can be potentiated by serotonin (5-HT) (Galan et al, Thromb Haemost. 102:511, 2009). There is limited knowledge on the mechanisms involved in the uptake, redistribution and functional expression of TF by platelets. We have investigated the mechanisms implied in the uptake and traffic of hTF with especial attention to CD36 and the 5HT transporter receptor (SERT), but also to the involvement of PI3-kinase, small GTPases (RhoA) and modifications of cytoskeletal organization during vesicular trafficking. Isolated platelets were exposed to hTF for up to 10 min. Cytoskeletal assembly and activation of RhoA were analyzed by electrophoresis and ELISA, respectively. Aggregometry and ultrastructural microscopy were used to assess platelet activation and hTF uptake. Protein arrays were used to evaluate changes in signaling molecules induced by hTF. In addition, inhibitory strategies to block GPIIbIIIa (Reopro), the scavenger receptor GPIV (anti-CD36), the SERT (S-Citalopram, SCit), and PI3-kinase (Wortmannin, Wo) were applied to platelets prior exposure to hTF. Internalization of hTF resulted in reversible actin polymerization and association of contractile proteins (alpha-actinin, actin binding protein and myosin) to the cytoskeleton, being maximal at 1 min. Maximal activation of RhoA occurred at 1 min, decreasing gradually afterwards, while activation of PI3-kinase occurred later (at 5 min). Screening of signaling proteins revealed activation of Akt, ERK1/2, and SNAP-25, among others. All inhibitory strategies fully prevented platelet aggregation to hTF. Uptake of hTF was slightly prevented by Reopro, while both anti-CD36 and Wo partially inhibited hTF uptake and, interestingly, SCit was the most efficient. We conclude that uptake of hTF by platelets promotes activation of specific signaling molecules with reversible cytoskeletal assembly and membrane fusion. While it may be difficult to design specific strategies to prevent the activation of small GTPases, cytoskeletal arrangements or membrane fusion specifically for platelets, our results indicate that the CD36 scavenger receptor and the 5-HT transporter (SERT) may become realistic therapeutic targets to prevent or reduce the association of TF to platelets and hence its possible prothrombotic action.

Grants: SAF2009-10365, Red HERACLES RD06/0009/1003