血小板特异性Rictor敲除抑制小鼠血小板生成和活化及血栓形成

Platelet-specific Rictor knockout inhibits platelet production and activation and reduces thrombosis in mice

目的探究Rictor对血小板活化和血栓形成的影响。方法利用PF4-Cre、Rictorfl/fl转基因小鼠,构建血小板特异性Rictor敲除小鼠和野生型小鼠,分别设为敲除组和对照组(总实验小鼠n=65),用于后续实验。Western blotting检测小鼠Rictor蛋白、蛋白激酶B(AKT)、p-AKT的表达水平。血常规检测敲除和对照组小鼠血小板数目。尾静脉出血实验检测敲除和对照小鼠止血功能。构建静脉血栓模型检测Rictor敲除对血栓形成的影响。使用ADP和凝血酶刺激血小板,检测Rictor敲除对血小板聚集的影响。流式细胞术分析敲除组和对照组血小板在静息态和活化态整合素αIIbβ3与CD62P的表达水平。检测血浆中PF4水平。vWF免疫组化标记巨核细胞。用APC-CD41抗体孵育敲除和对照小鼠的巨核细胞,流式细胞术检测细胞倍体。扫描电镜检测敲除和对照组血小板在胶原表面的伸展变化。结果与对照小鼠相比,血小板Rictor特异性敲除小鼠AKT磷酸化降低(P<0.001),血小板生成减少(P<0.05),血栓形成受到抑制(P<0.05),血小板对ADP和凝血酶刺激的活化水平降低(P<0.01),P-选择素蛋白表达水平(P<0.05)、整合素αIIbβ3活化程度(P<0.01)受到抑制,血小板伸展受到抑制(P<0.01),PF4的血浆含量降低(P<0.05),骨髓中巨核细胞数量减少(P<0.01),倍体降低(P<0.05),前血小板平均面积减少(P<0.01)。结论血小板特异性敲除Rictor抑制血小板生成和活化,进而减少血栓形成。抑制mTORC2活性可能是一种潜在的抗血栓靶标。

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 integrinαIIbβ3 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 integrinαIIbβ3 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.