1. Platelet MEKK3 regulates arterial thrombosis and myocardial infarct expansion in mice
by Xuemei Fan, Conghui Wang, Panlai Shi, Wen Gao, Jianmin Gu, Yan Geng, Wenlong Yang, Ningbo Wu, Yang Wang, Yanyan Xu, Xue Chen, Lin Zhang, Kemin Wang, Bing Su, and Junling Liu
BloodAdv
Volume 2(12):
June 26, 2018
© 2018 by The American Society of Hematology

2. Xuemei Fan et al. Blood Adv 2018;2:1439-1448
© 2018 by The American Society of Hematology

3. MAP3K expression profiles in human and mouse platelets.
MAP3K expression profiles in human and mouse platelets. Relative mRNA levels of MAP3Ks in human platelets (H-plt) (n = 3) (A) and in mouse platelets (M-plt) (n = 3) (B). Data are mean ± SD. (C) Expression of MEKK3, MEKK5, MLK3, A-Raf, B-Raf, and C-Raf in H-plt and M-plt was detected by western blot. Proteins extracted from HeLa cells were used as positive controls for human MAP3K detection. NIH 3T3 cells were used as positive controls for mouse MLK3, A-Raf, and C-Raf detection; mouse heart tissue was used as positive controls for MEKK3 and MEKK5 detection; and mouse leukocytes were used as positive controls for B-Raf detection. GAPDH was used to verify equal loading.
Xuemei Fan et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

4. MEKK3 deficiency delayed arterial thrombosis but had no effect on platelet counts or hemostasis.
MEKK3 deficiency delayed arterial thrombosis but had no effect on platelet counts or hemostasis. (A) Washed platelets were prepared from MEKK3f/f and MEKK3−/− mice. Western blot results show that MEKK3 was depleted in MEKK3−/− platelets. (B) Platelet counts in the peripheral blood of MEKK3f/f and MEKK3−/− mice (n = 10; mean ± SD; P > .05). (C) The mouse carotid artery was treated with 10% FeCl3 for 3 minutes, and blood flow traces were monitored (n = 10, mean ± SD). (D) Mouse tail-bleeding time was determined as the time to visual cessation of bleeding (n = 10, mean ± SD; P > .05). ***P < ns, not significant.
Xuemei Fan et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

5. MEKK3 regulates platelet activation through integrin αIIbβ3–mediated inside-out signaling.
MEKK3 regulates platelet activation through integrin αIIbβ3–mediated inside-out signaling. Aggregation traces of washed MEKK3f/f and MEKK3−/− platelets treated with thrombin (A), ADP (B), collagen (C), or U46619 (D) were measured by aggregometry. (E) ATP secretion by MEKK3f/f and MEKK3−/− platelets induced by 0.06 U/mL thrombin, 10 µmol/L ADP, 2 µmol/L U46619, or 1.5 µg/mL collagen (n = 3, mean ± SD). (F) Expression of P-selectin in MEKK3f/f and MEKK3−/− platelets stimulated with 0.05 U/mL thrombin, 10 µmol/L ADP, 5 µmol/L U46619 or 2 µg/mL collagen was analyzed by flow cytometry. P-selectin expression was presented as mean fluorescence density (n = 3, mean ± SD). (G) Binding of Alexa Fluor 647–conjugated Fg to washed MEKK3f/f and MEKK3−/− platelets stimulated with 0.05 U/mL thrombin, 10 µmol/L ADP, 5 µmol/L U46619, or 2 µg/mL collagen. The results are expressed as mean fluorescence intensity (n = 3, mean ± SD). (H) Representative phalloidin staining and quantification of washed MEKK3f/f and MEKK3−/− platelets spreading on immobilized Fg for 90 minutes. (I) Quantification of the areas of spreading platelets in (H) (n = 4, mean ± SD; P > .05). (J) Representative pictures of the clot retraction of washed MEKK3f/f and MEKK3−/− platelets stimulated by 1 U/mL thrombin. (K) The 2-dimensional area of clots in panel J. Data are expressed as retraction ratios (n = 3, mean ± SD; P > .05). *P < .05, **P < .01, ***P < .001.
Xuemei Fan et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

6. MEKK3 regulates platelet activation through ERK1/2 and JNK2.
MEKK3 regulates platelet activation through ERK1/2 and JNK2. (A) Expression levels of β3, GPIbα, GPVI, α2b, and α2 proteins in MEKK3f/f and MEKK3−/− platelets were detected using flow cytometry. The results are expressed as mean fluorescence intensity (n = 3, mean ± SD; P > .05). (B) Lysates of washed MEKK3f/f and MEKK3−/− platelets were probed for the expression of p38, ERK1/2, JNK, and ERK5 proteins. Washed MEKK3f/f and MEKK3−/− platelets were stimulated with 0.06 U/mL thrombin or 10 µmol/L ADP (C) or with 2 µmol/L U46619 or 1.5 μg/mL collagen (D) for 3 minutes, and the lysates were probed with phospho-specific antibodies for p38, ERK1/2, JNK, or ERK5 to quantify MAPK activation. GAPDH was used to verify equal loading. Aggregation of washed MEKK3f/f and MEKK3−/− mouse platelets in response to 0.05 U/mL thrombin (E), 10 µM ADP (F), 1.5 µg/mL collagen (G), and 2 µM U46619 (H) in the presence of 5 µM JNK inhibitor SP or 5 µM MEK1/2 inhibitor U0126. At least 3 independent experiments were performed.
Xuemei Fan et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

7. Platelet MEKK3 deficiency reduced microvascular obstruction and infarct expansion post-MI.
Platelet MEKK3 deficiency reduced microvascular obstruction and infarct expansion post-MI. (A) CD42C staining of hearts from MEKK3f/f and MEKK3−/− mice on day 3 following ligation of the LAD artery (original magnification ×400). Relative platelet (plt) number (pixels) in 4 random fields per experiment was quantified (mean ± SD). (B) Masson’s trichrome staining in parasternal short-axis sections of hearts from MEKK3f/f and MEKK3−/− mice on day 7 post-LAD coronary artery ligation (original magnification ×20). Masson’s trichrome–stained areas were measured. Infarct size is expressed as a percentage of the LV area (n = 3, mean ± SD). (C) Representative M-mode echocardiograms for MEKK3f/f and MEKK3−/− mice on day 7 post–LAD coronary artery ligation. (D) Echocardiography quantification of LVEF, LVFS, LVESV, LVEDV, LVIDs, and LVIDd in MEKK3f/f and MEKK3−/− mice on day 7 post–LAD coronary artery ligation (n = 8-10, mean ± SD). *P < .05, **P < .01, ***P < IVS, interventricular septum; PW, posterior wall.
Xuemei Fan et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology