RASA3 is a critical inhibitor of RAP1-dependent platelet activation
Lucia Stefanini, … , Luanne L. Peters, Wolfgang Bergmeier
Lucia Stefanini, … , Luanne L. Peters, Wolfgang Bergmeier
Published February 23, 2015
Citation Information: J Clin Invest. 2015;125(4):1419-1432. https://doi.org/10.1172/JCI77993.
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RASA3 is a critical inhibitor of RAP1-dependent platelet activation

Abstract

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Authors

Lucia Stefanini, David S. Paul, Raymond F. Robledo, E. Ricky Chan, Todd M. Getz, Robert A. Campbell, Daniel O. Kechele, Caterina Casari, Raymond Piatt, Kathleen M. Caron, Nigel Mackman, Andrew S. Weyrich, Matthew C. Parrott, Yacine Boulaftali, Mark D. Adams, Luanne L. Peters, Wolfgang Bergmeier

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Figure 4

RASA3 deletion leads to increased αIIbβ3 activation in stimulated platelets.

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RASA3 deletion leads to increased αIIbβ3 activation in stimulated platel...
(A) Normal membrane expression of glycoprotein receptors in WT, Caldaggef1–/– Rasa3+/+, or Caldaggef1–/– Rasa3hlb/hlb platelets. Diluted whole blood was stained for 10 minutes with fluorophore-labeled antibodies (2 μg/ml) to the indicated antigens and analyzed by flow cytometry (n = 6). (B) RASA3 and RAP1 protein level evaluated by immunoblotting in lysates of WT, Caldaggef1–/– Rasa3+/+, or Caldaggef1–/– Rasa3hlb/hlb platelets. Results are representative of 3 independent experiments. (C) Increased integrin αIIbβ3 activation (JON/A-PE binding) in activated Caldaggef1–/– Rasa3hlb/hlb platelets when compared with Caldaggef1–/– controls. Platelets were stimulated for 10 minutes with increasing concentrations of Par4-activating peptide (Par4p) or the GPVI-specific agonist convulxin (Cvx), stained with JON/A-PE, and immediately analyzed by flow cytometry. MFI, mean fluorescence intensity. *P < 0.05, ***P < 0.0001, 2-way ANOVA with Bonferroni post-test (n = 6, 3 independent experiments). (D) Aggregation response of washed WT, Caldaggef1–/– Rasa3+/+, or Caldaggef1–/– Rasa3hlb/hlb platelets stimulated with low (left) or high (right) doses of collagen, thrombin, or the thromboxane analog U46619. Results are representative of 3 independent experiments.