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Updated: Jun 13, 2025
Purification of Platelets from Mouse Blood
Published on: May 7, 2019
[Platelet-specific Rictor knockout inhibits platelet production and activation and reduces thrombosis in mice]
1Department of Biochemistry and Molecular Biology, Southern Medical University, Guangzhou 510515, China.
Objective:
To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice.
Methods:
PF4-Cre and Rictorfl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout (Rictor-KO) mice and wild-type mice (n=65), whose expression levels of Rictor, protein kinase B (AKT) and p-AKT were detected using Western blotting. Platelet counts of the mice were determined using routine blood tests, and hemostatic function was assessed by tail vein hemorrhage test. Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis. Platelet aggregation induced by ADP and thrombin was observed in Rictor-KO and wild-type mice, and flow cytometry was used to analyze the expression levels of integrin αIIbβ3 and CD62P in resting and activated platelets. Plasma PF4 levels were determined with ELISA. Megakaryocytes from Rictor-KO and wild-type mice were incubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes, respectively. Platelet elongation on collagen surface was observed with scanning electron microscopy.
Results:
Compared with the wild-type mice, Rictor-KO mice showed significantly decreased AKT phosphorylation, decreased platelet production, reduced thrombosis, and decreased platelet activation in response to ADP and thrombin stimulation. The Rictor-KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbβ3 with suppression of platelet extension, reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow. The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor-KO mice.
Conclusion:
Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice, suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.
Insights
Platelet-specific Rictor knockout in mice reduced platelet production and activation, leading to decreased thrombus formation. This suggests inhibiting mTORC2 activity may be an effective antithrombotic strategy.
Area of Science:
- Molecular Biology
- Hematology
- Biochemistry
Context:
- The mammalian target of rapamycin complex 2 (mTORC2) pathway plays a crucial role in cell signaling.
- Rictor is a key component of mTORC2, and its function in platelets is not fully understood.
- Understanding Rictor's role in platelets is essential for developing targeted therapies.
Purpose:
- To investigate the impact of platelet-specific Rictor knockout on platelet activation and thrombus formation in a mouse model.
- To elucidate the downstream signaling pathways affected by Rictor deficiency in platelets.
- To assess the potential of targeting Rictor as an antithrombotic strategy.
Summary:
- Platelet-specific Rictor knockout mice exhibited significantly reduced AKT phosphorylation, platelet production, and thrombus formation compared to wild-type controls.
- Rictor deficiency impaired platelet activation in response to ADP and thrombin, evidenced by decreased integrin αIIbβ3 activation and P-selectin expression.
- Megakaryocyte number and ploidy were reduced in Rictor-KO mice, impacting overall platelet generation.
Impact:
- This study demonstrates that Rictor is critical for platelet generation and function.
- The findings suggest that inhibiting mTORC2 activity via Rictor could be a viable therapeutic approach for preventing thrombosis.
- This research opens new avenues for antithrombotic drug development targeting the mTORC2 pathway.
