Rab31 promotes platelet activation and arterial thrombosis via ERK–PLCβ3 signaling and regulation of CD151

Abstract

Background

Ras-related protein in brain (Rab) guanosine triphosphatases regulate vesicular trafficking and membrane dynamics, processes essential for platelet activation. Although several Rab family members are expressed in platelets, the role of Rab31 remains undefined.

Objectives

To determine the role of Rab31 in platelet activation, thrombosis, and hemostasis and to explore the underlying mechanisms.

Methods

Rab31 expression was examined in human and murine platelets. Platelet function was evaluated in Rab31^−/− and wild-type mice using aggregation, adenosine triphosphate release, spreading, and clot retraction assays. In vivo thrombosis and hemostasis were assessed by ferric chloride–induced mesenteric arterial injury, collagen/epinephrine-induced pulmonary embolism, tail-snip bleeding, and platelet depletion and repletion experiments. Quantitative proteomic and phosphoproteomic analyses were performed to identify Rab31-dependent signaling pathways in platelets.

Results

Rab31 was expressed in human and murine platelets at both messenger ribonucleic acid and protein levels. Rab31 deficiency significantly reduced platelet aggregation, adenosine triphosphate secretion, spreading, and clot retraction. In vivo, Rab31^−/− mice showed delayed arterial thrombus formation and reduced pulmonary embolism, whereas tail bleeding time and blood loss remained unchanged. Platelet depletion and repletion experiments confirmed the platelet intrinsic role of Rab31 in thrombosis. Phosphoproteomic analysis revealed decreased extracellular signal-regulated kinase 2 and phospholipase C beta 3 phosphorylation in Rab31-deficient platelets, and quantitative proteomics identified reduced CD151 antigen expression. These findings were validated by immunoblotting.

Conclusion

Rab31 promotes platelet activation and arterial thrombosis without affecting physiological hemostasis, likely through regulation of ERK–phospholipase C beta 3 signaling and CD151 antigen expression.