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Phycoerythrin conjugated Hamster anti Mouse CD61 antibody, clone HM beta 3.1 (MCA2299PE) used to identify platelets in conjunction with thiazole orange for reticulated platelets by flow cytometry
Insulin acting at IR enhances mouse platelet function. (A) Immunoblotting confirmed ablation of IR protein expression in platelets (i) but not in liver (ii) or skeletal muscle (iii) from IRflox/flox; Pf4-Cre+ (KO) mice. KO platelet expression of IGF1R, IRS-1, 2, 3, and Akt protein were comparable to WT platelets (i, n = 3). (B) For evaluation of megakaryocyte number and morphology sections of bone marrow were imaged with a transmission electron microscope. The number of megakaryocytes per section was quantified manually using ImageJ, and expressed as the number of megakaryocytes/section corrected for total area of the section. Bars represent means ± SE (n = 3). Data were analysed by Student's unpaired t-test, and there was no significant difference in the number of megakaryocytes in WT and KO mice. Subjectively, there was no difference in the subcellular morphology of WT megakaryocytes compared with those lacking IR. Scale bars = 5 and 1 μm. (C) For evaluation of reticulated platelets by flow cytometry, whole blood from WT and KO mice was fixed, stained for CD61-PE, and incubated with thiazole orange (Retic-count™, BD Bioscience). Reticulated platelets are expressed as a percentage of thiazole orange and CD61-PE double-positive cells in 10 000 identified platelets. Bars represent mean ± SE (n = 3). Data were analysed by Student's unpaired t-test, and there was no significant difference in the number of reticulated platelets in WT and KO mice. (D and E) Monitoring of platelet aggregation using a Chronolog 590-2A aggregometer demonstrated that pre-incubation of platelets (2 × 108/mL) with insulin (20 nM, 5 min) enhances PAR-4-mediated platelet aggregation in WT (D), but not in IR KO (E) platelets (n = 3). (F and G) In agreement with platelet aggregation, PAR-4-mediated integrin αIIbβ3 activation, as measured by FACS using the JON/A antibody against the high affinity integrin αIIbβ3 conformation, was enhanced by insulin (20 nM, 5 min) in WT (F) but not in IR KO platelets (G). FACS results are expressed as mean ± SE of the percentage of maximal fluorescence of vehicle-treated platelets (n = 13). Data were analysed by two-way ANOVA (P-value), followed by Bonferroni's post-test (asterisks). A value of P < 0.05 indicates that insulin significantly altered PAR-4-mediated integrin activation in WT platelets. Asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001) indicate a significant difference induced by insulin from vehicle at the matching PAR4-AP concentration. (H) Immunoprecipitation of IRS-1 demonstrates that IGF-1 (100 nM, 5 min), but not insulin (20 nM, 5 min), stimulates tyrosine phosphorylation (pY) of IRS-1 in IR KO platelets (n = 3). Membranes were reprobed to confirm equal loading.
From: Moore SF, Williams CM, Brown E, Blair TA, Harper MT, Coward RJ, Poole AW, Hers I. Loss of the insulin receptor in murine megakaryocytes/platelets causes thrombocytosis and alterations in IGF signalling.
Cardiovasc Res. 2015 Jul 1;107(1):9-19.