Role of Rubicon in platelets: a promoter of thrombosis but not an autophagy repressor.

Abstract

Rubicon (RUN domain Beclin-1-interacting and cysteine-rich-containing protein) is a negative regulator of autophagy in nucleated cells; however, its role in platelets is unstudied. Here, we identify an autophagy-independent role for Rubicon in arterial thrombosis. Mice with a platelet/megakaryocyte-specific deletion of Rubicon (RUBCN-/-) showed normal circulating platelet counts, with a slight increase in platelet size. The basal levels of autophagy-related, vesicle-elongation proteins (ie, ATG5, ATG7, syntaxin 17, LC3, and Rab7) were unaffected, and there was no disruption in canonical platelet signaling pathways upon activation. Platelet secretion from all 3 granule classes remained intact. Aggregation/Agglutination in response to thrombin, convulxin, or botrocetin was unaffected. There was no overt defect in activation-dependent autophagic flux (ie, loss of LC3 or p62). However, RUBCN-/- mice had a significant thrombosis defect in the FeCl3-induced, carotid artery injury model, but no defect in the tail-transection hemostasis model. An intrinsic platelet defect was confirmed using microfluidics, in which RUBCN-/- platelets had reduced collagen binding under flow at high shear flow but not low shear. RUBCN-/- platelets also showed impaired surface exposure of phosphatidylserine (PS) after thrombin and convulxin activation, though there was no effect on total anoctamin 6, which contributes to PS exposure. There was no effect on platelet endocytosis, and only modest reductions in mitochondrial membrane potential with a slight delay in clot contraction, suggesting that Rubicon contributes to late stages of platelet function. Collectively, our findings demonstrate that Rubicon contributes to thrombosis and procoagulant platelet formation in a manner that appears independent of classical autophagy.