Full clot retraction: running on mTOR

In this issue, Weyrich and colleagues define a novel role for B-cell lymphoma-3 (Bcl-3). The anticancer drug rapamycin, acting at mammalian target of rapamycin (mTOR), impairs de novo synthesis of Bcl-3 in activated human platelets and also blocks clot contraction. Clot contraction is inhibited in Bcl-3–deficient mice and enhanced by Bcl-3–overexpressing cell lines.

Bcl-3, a member of the Ikβα family of regulators of NF-κB, is poorly expressed in human and mouse platelets, if at all. On platelet activation induced by thrombin, de novo protein synthesis from constitutive message occurs within minutes, with major expression within 1 to 2 hours.¹ Expression of Bcl-3 requires outside-in signals by ligand-engaged α_IIbβ₃ and a downstream signaling pathway involving mTOR, a phosphatidylinositol kinase–related kinase.^1,2  In activated platelets, another downstream consequence of α_IIbβ₃ engagement by fibrinogen/fibrin is clot retraction, a process that also requires 1 to 2 hours for completion and involves the actin-rich cytoskeleton. In this issue, Weyrich and colleagues demonstrate that Bcl-3 synthesis and clot retraction are intimately linked, with Bcl-3 levels regulating the capacity of platelets and other cell types to mediate fibrin clot retraction (see figure). The clue to these findings came from the observation that rapamycin, which inhibits the platelet activation–dependent expression of Bcl-3, also inhibits clot retraction. Consistently, clot retraction is severely impaired in Bcl-3 knock-out mice, but is enhanced by overexpression of Bcl-3 in α_IIbβ₃-expressing CHO cell lines. Of interest, Weyrich and colleagues found that megakaryocytes can specifically target mTOR and downstream components of the mTOR signaling pathway, such as S6 kinase 1 (S6K1), to developing proplatelets. In megakaryocytes, mTOR has a punctate distribution in the cytoplasm and is also localized in the perinuclear region. In contrast, during proplatelet formation, mTOR is localized to the proplatelet shafts and to the tips (the developing proplatelet). S6K1 and ribosomal S6 are similarly colocalized to the proplatelet tips, and all 3 components of the mTOR signaling pathway are found in mature platelets. In thrombin-activated platelets, both S6K1 and eukaryotic initiation factor 4E–binding protein (4EBP1), another downstream translation regulator in the mTOR pathway,¹  are phosphorylated, events blocked by rapamycin, suggesting that both mTOR downstream targets may regulate Bcl-3 message translation.

A key question not specifically addressed in the present study is how Bcl-3 is involved in α_IIbβ₃-dependent fibrin clot retraction. Rapamycin was found not to block activation-dependent changes in α_IIbβ₃ conformation, thrombin-induced platelet aggregation, or platelet adhesion to fibrinogen, suggesting the effect of Bcl-3 on fibrin clot retraction is not directly at the level of α_IIbβ₃. Rather, expressed Bcl-3 was found to colocalize with the platelet cytoskeleton. Since Bcl-3 is known to bind the SH3 domain of the Src family kinase, Fyn,¹  the role of cytoskeletal-associated Bcl-3 may be to localize and activate Fyn, with Fyn substrates involved in coordinating downstream events key to clot retraction. In this regard, tyrosine kinase inhibitors have been reported to inhibit fibrin clot retraction.³  Future studies are needed to clarify the precise mechanism of action of Bcl-3 and whether its de novo mTOR-dependent synthesis regulates the time course of fibrin clot retraction.