Platelet Rubicon drives thrombosis independent of autophagy

In this issue of Blood Advances, Alfar et al¹ have identified an unexpected, autophagy-independent function for the protein Rubicon (RUN domain Beclin-1–interacting and cysteine-rich–containing protein) in platelets, demonstrating that it is a key promoter of arterial thrombosis. Rubicon is widely recognized in nucleated cells as a negative regulator that actively inhibits autophagy, a critical housekeeping process during which cellular components are recycled.^2,3 The new findings, based on mice with a platelet-specific deletion of Rubicon (Rubcn^−/−), reveal that its role in platelets is different, promoting thrombosis without significantly altering the activation-induced recycling mechanisms. This work fundamentally changes our understanding of how Rubicon controls platelet biology and offers a new pathway to explore in the treatment of cardiovascular disease.

The traditional function of Rubicon is to bind to the protein Beclin-1 and inhibit the formation of the autophagosome, the double-membrane vesicle that surrounds cellular waste for disposal.^2,3 This inhibitory role is why the authors initially hypothesized that deleting Rubicon in platelets would increase their autophagic activity.

The core and most surprising finding of the article was that the hypothesis was nullified. The absence of Rubicon in platelets had virtually no effect on the levels of other key autophagy proteins (such as ATG5, ATG7, and LC3) or on autophagic flux, the measure of the recycling process itself, both at baseline and upon activation.¹ This strongly suggests that, in platelets, Rubicon is largely dispensable for regulating autophagy, indicating that platelets use alternative, dominant control mechanisms over their internal recycling process, a finding that distinguishes platelet biology from that of other cell types.^4,5

Despite the lack of an effect on autophagy, the loss of Rubicon had a major impact on the ability of the blood to clot, or thrombosis. Although the platelet-specific Rubcn^−/− mice showed unaltered hemostasis in the tail bleeding assay, these mice exhibited a significant defect in the FeCl₃-induced carotid artery injury model.¹ Specifically, platelet-specific Rubcn^−/− mice had (1) significantly increased occlusion times, (2) failed to form thrombi, and (3) the carotid artery remained patent for the duration of the assay.

This protection from arterial thrombosis was confirmed ex vivo using microfluidic analysis, a method that simulates blood flow in small channels. Platelets that lacked Rubicon failed to form stable thrombi when exposed to collagen under high shear flow (a condition that matches the fast, high-pressure flow in arteries) but not under low shear (venous) flow.¹ This demonstrates that platelet Rubicon is specifically important for stable clotting in the high-stress environment of the arterial vasculature.

If Rubicon does not affect autophagy or key canonical platelet signaling pathways (such as aggregation or granule secretion), what mechanism drives its role in arterial thrombosis? The authors identified a key change in the platelet membrane, specifically a moderate, but significant, impairment in the surface exposure of phosphatidylserine (PS).¹

Normally, upon strong activation, platelets flip PS from the inner to the outer face of their membrane. This externalized PS is essential because it provides the necessary charged surface for the assembly of coagulation factors, which locally amplify the production of thrombin, the enzyme that converts fibrinogen into fibrin threads to stabilize the clot.^6,7

The reduced PS exposure in the Rubicon deficient platelets, although insufficient to cause defects in in vitro thrombin generation assays, provides a strong candidate for the in vivo defect. The Rubcn^−/− phenotype closely resembles Scott syndrome, a rare, mild bleeding disorder in humans that is caused by mutations in anoctamin 6 (ANO6), the enzyme responsible for flipping PS.⁸ This suggests that Rubicon may function by regulating the activity or localization of ANO6 or another related component in the PS scrambling pathway.

The work by Alfar et al definitively shifts the conversation surrounding Rubicon in platelets from an autophagy repressor to an essential component of arterial thrombosis. In nucleated cells, Rubicon is a negative regulator that actively blocks autophagosome-lysosome fusion; in contrast, in platelets, Rubicon is dispensable for autophagy but plays a key role in the procoagulant surface formation by promoting PS exposure. This functional dichotomy between anucleate platelets and nucleated cells is a crucial insight into cell-specific regulatory systems. The discovery that Rubicon functions independently of autophagy presents a promising therapeutic opportunity. Targeting the Rubicon-mediated thrombosis pathway could reduce the risk of thromboembolic events, such as stroke and myocardial infarction, without disrupting essential autophagic processes in other tissues or increasing the risk of bleeding. Future studies should focus on identifying specific binding partners of Rubicon in the platelet membrane to precisely determine how it regulates PS exposure.

Conflict-of-interest disclosure: The author declares no competing financial interests.