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

doi:10.12122/j.issn.1673-4254.2024.08.20

摘要/Abstract

摘要：

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、Rictor^fl/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。

关键词: Rictor, 血小板, 血栓形成

Abstract:

Objective To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice. Methods PF4-Cre and Rictor^fl/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 wereincubated 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.

Key words: Rictor, platelets, thrombosis

龙清, 杨峻, 刘安玲. 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[J]. 南方医科大学学报, 2024, 44(8): 1605-1611.

Qing LONG, Jun YANG, Anling LIU. Platelet-specific Rictor knockout inhibits platelet production and activation and reduces thrombosis in mice[J]. Journal of Southern Medical University, 2024, 44(8): 1605-1611.

图/表 4

图1 血小板特异性Rictor敲除小鼠模型的生成

Fig.1 Generation of platelet-specific Rictor knockout (KO) mouse models. A: Genotyping results. B: Immunoblotting for Rictor, Akt, and p-Akt (S473) in the platelets. C, D: Gray scale of Rictor (C) and p-Akt (D) measured by Image J. E: Platelet counts of control and KO mice. *P<0.05,***P<0.001.

图2 Rictor敲除降低小鼠对血栓形成的易感性

Fig.2 Rictor deletion reduces susceptibility to thrombosis in mice. A: Average tail bleeding time of Con and KO mice (n=10). B-D: Images (B), lengths (C), and weights (D) of thrombus from Con and KO mice. *P<0.05.

图3 PF4-Rictor小鼠血小板活化功能缺陷

Fig3 PF4-Rictor mice exhibit severe deficiency in platelet function. A, B: Platelet aggregation induced by ADP (A) and thrombin (B) in PRP. C-D: FACS analysis of αIIbβ3 (C) and CD62P (D) in resting platelets. E, F: FACS analysis of αIIbβ3 (E) and CD62P (F) in platelets pretreated with thrombin. G: Platelet spreading on fibrinogen. H: ELISA analysis of plasma PF4 levels. *P<0.05, **P<0.01.

图4 Rictor缺失抑制巨核细胞成熟

Fig.4 Rictor deletion inhibits megakaryocyte maturation. A: vWF staining of femoral bone sections from Con and Rictor-KO mice (Scale bar=200 μm). B: Ploidy formation of 3-month-old Con and KO mice. C: Ultrastructure and proplatelet formation of megakaryocytes from Con and KO mice (Scale bar=2 μm). *P<0.05, **P<0.01.

参考文献 30

1	Næss IA, Christiansen SC, Romundstad P, et al. Incidence and mortality of venous thrombosis: a population-based study[J]. J Thromb Haemost, 2007, 5(4): 692-9.
2	Camerer E, Kolstø AB, Prydz H. Cell biology of tissue factor, the principal initiator of blood coagulation[J]. Thromb Res, 1996, 81(1): 1-41.
3	Rojnuckarin P, Kaushansky K. Actin reorganization and proplatelet formation in murine megakaryocytes: the role of protein kinase calpha[J]. Blood, 2001, 97(1): 154-61.
4	Cramer EM, Norol F, Guichard J, et al. Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand[J]. Blood, 1997, 89(7): 2336-46.
5	Flaumenhaft R. A new story ARC for α‑granule formation[J]. Blood, 2015, 126(2): 123-4.
6	Jr IJE. Unraveling mechanisms that control platelet production[J]. Semin Thromb Hemost, 2013, 39(1): 15-24.
7	Junt T, Schulze H, Chen Z, et al. Dynamic visualization of thrombopoiesis within bone marrow[J]. Science, 2007, 317(5845): 1767-70.
8	Cleator JH, Zhu WQ, Vaughan DE, et al. Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP[J]. Blood, 2006, 107(7): 2736-44.
9	Choi ES, Nichol JL, Hokom MM, et al. Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional[J]. Blood, 1995, 85(2): 402-13.
10	Brown EJ, Beal PA, Keith CT, et al. Control of p70 s6 kinase by kinase activity of FRAP in vivo [J]. Nature, 1995, 377(6548): 441-6.
11	Burnett PE, Barrow RK, Cohen NA, et al. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1[J]. Proc Natl Acad Sci USA, 1998, 95(4): 1432-7.
12	Sarbassov DD, Ali SM, Kim DH, et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton[J]. Curr Biol, 2004, 14(14): 1296-302.
13	Dada S, Demartines N, Dormond O. mTORC2 regulates PGE2-mediated endothelial cell survival and migration[J]. Biochem Biophys Res Commun, 2008, 372(4): 875-9.
14	Albert V, Hall MN. mTOR signaling in cellular and organismal energetics[J]. Curr Opin Cell Biol, 2015, 33: 55-66.
15	Wang SC, Wang JL, Wang SW, et al. mTOR signaling pathway in bone diseases associated with hyperglycemia[J]. Int J Mol Sci, 2023, 24(11): 9198.
16	Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex[J]. Science, 2005, 307(5712): 1098-101.
17	Jacinto E, Loewith R, Schmidt A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive[J]. Nat Cell Biol, 2004, 6(11): 1122-8.
18	尹承龙, 劳学军. PI3K-AKT-mTOR信号通路的研究进展[J]. 中国医学创新, 2016, 13(1): 145-8.
19	宋晓红, 刘明明. mTOR信号通路与相关疾病的研究进展[J]. 微循环学杂志, 2018, 28(3): 64-70.
20	Fuhler GM, Tyl MR, Olthof SG, et al. Distinct roles of the mTOR components Rictor and Raptor in MO7e megakaryocytic cells[J]. Eur J Haematol, 2009, 83(3): 235-45.
21	Pleines I, Dütting S, Cherpokova D, et al. Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42[J]. Blood, 2013, 122(18): 3178-87.
22	Zhang XY, Evans TD, Chen S, et al. Loss of macrophage mTORC2 drives atherosclerosis via FoxO1 and IL-1β signaling[J]. Circ Res, 2023, 133(3): 200-19.
23	张浩, 包新杰, 代从新, 等. PI3K/Akt/mTOR信号通路在无功能垂体腺瘤中的研究进展[J]. 中华医学杂志, 2015, 95(47): 3874-6.
24	Chen X, Zhang Y, Wang YH, et al. PDK1 regulates platelet activation and arterial thrombosis[J]. Blood, 2013, 121(18): 3718-26.
25	Etienne-Manneville S, Hall A. Rho GTPases in cell biology[J]. Nature, 2002, 420(6916): 629-35.
26	Hervieu A, Heuss SF, Zhang C, et al. A PI3K- and GTPase-independent Rac1-mTOR mechanism mediates MET-driven anchorage-independent cell growth but not migration[J]. Sci Signal, 2020, 13(637): eaba8627.
27	Vidal C, Geny B, Melle J, et al. Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin[J]. Blood, 2002, 100(13): 4462-9.
28	Aslan JE, McCarty OJT. Rho GTPases in platelet function[J]. J Thromb Haemost, 2013, 11(1): 35-46.
29	Joo SJ. Mechanisms of platelet activation and integrin αIIβ3[J]. Korean Circ J, 2012, 42(5): 295-301.
30	Tang ZH, Shi H, Chen CM, et al. Activation of platelet mTORC2/akt pathway by anti‑β2GP1 antibody promotes thrombosis in antiphospholipid syndrome[J]. Arterioscler Thromb Vasc Biol, 2023, 43(10): 1818-32.

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