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1.
Figure 6

Figure 6. From: Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

ROS activate TACE in human platelets. (A) GO (25 mU/mL) was added to washed human platelets, and platelets were incubated for 6 h at 37°C in the presence or absence of TAPI-1 (1 µM) or SB203580 (20 µM). The expression of GPIbα was determined by FACS; n = 5. (B) Human platelets were incubated with GO (25 mU/mL) for 6 h, then Ca2+ (1 mM) was added, and aggregation in the presence of 1 U/mL thrombin was recorded. Representative graph of five experiments is shown.

Alexander Brill, et al. Cardiovasc Res. 2009 October 1;84(1):137-144.
2.
Figure 3

Figure 3. From: Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

H2O2 does not induce platelet activation and shedding of other major platelet receptors. Murine platelets were treated with H2O2 (100 µM, 1 h), and the expression of GPVI, GPIX, αIIbβ3, and α5β1 integrin was determined by flow cytometry (A). To evaluate platelet activation, platelets were stained for surface expression of P-selectin, activated αIIbβ3, or phosphatidylserine (by annexin-V binding) (B). Data represent mean ± SD of four to six independent experiments.

Alexander Brill, et al. Cardiovasc Res. 2009 October 1;84(1):137-144.
3.
Figure 1

Figure 1. From: Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

H2O2 induces loss of GPIbα and GPV from the platelet membrane. Washed murine platelets were treated with H2O2 at increasing concentrations for the indicated periods of time at 37°C. GPIbα and GPV expression was evaluated by FACS. Representative dot plots for GPIbα (A) and for GPV (B) expression on control and H2O2-treated platelets (100 µM, 1 h). Dose dependency (C) and time course (D) of receptor loss determined as percentage mean fluorescence intensity (MFI) of control platelets. Data represent mean ± SD of six to eight independent experiments.

Alexander Brill, et al. Cardiovasc Res. 2009 October 1;84(1):137-144.
4.
Figure 4

Figure 4. From: Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

ROS-treated platelets do not adhere and incorporate into a thrombus in vivo. Washed murine platelets were labelled with calcein-green (control) or calcein-red (H2O2-treated, 100 µM, 1 h) and injected into a wild-type mouse. The amount of injected control and H2O2-treated cells was 2.5 × 107 and 7 × 107, respectively. Thrombosis was induced by the application of FeCl3 on mesenteric arterioles of 90–100 µm in diameter. (A) Number of adherent (transiently and firmly) platelets 6 min after FeCl3 application, counted for 1 min. (B) Representative photograph of an occlusive thrombus, rich with control but not with H2O2-treated platelets. White lines delineate the vessel. Bar 50 µm.

Alexander Brill, et al. Cardiovasc Res. 2009 October 1;84(1):137-144.
5.
Figure 2

Figure 2. From: Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

H2O2- and 12-HpETE-induced shedding of GPIbα and GPV is TACE-dependent. Murine platelets from wild-type (WT) or TACEΔZn/ΔZn chimeras were treated with H2O2 (100 µM, 1 h, 37°C) in the presence or absence of the TACE inhibitor, TAPI-1. Platelet supernatants (A) and lysates (B) were analysed by SDS–PAGE, transferred to a membrane, and blotted with specific anti-GPIbα mAbs. (1) WT, untreated; (2) WT, H2O2; (3) WT/TAPI-1, H2O2; (4) TACEΔZn/ΔZn, untreated; (5) TACEΔZn/ΔZn, H2O2. Platelets from the same samples were analysed by FACS for GPIbα expression. (C) GPIbα and (D) GPV, left panel represents WT platelets and right panel TACEΔZn/ΔZn platelets. The grey-shaded area represents resting platelets; the solid line, H2O2-treated platelets; and the dotted line, H2O2-treated WT platelets in the presence of TAPI-1. (E) Platelets were treated with 12-HpETE, a peroxide naturally produced by platelets, for 1 h in indicated concentrations in the absence or presence of TAPI-1 (1 µM). Surface expression of GPIbα was analysed by FACS, n = 5; Asterisk indicates statistically significant difference compared with untreated cells.

Alexander Brill, et al. Cardiovasc Res. 2009 October 1;84(1):137-144.
6.
Figure 5

Figure 5. From: Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

Signal transduction pathways involved in ROS-induced TACE activation. Murine platelets were pre-treated with the indicated inhibitors for 15 min, and then H2O2 (100 µM) was added for 1 h. GPIbα expression was detected by FACS. The Y axis represents percentage of mean fluorescence intensity (MFI) compared with untreated cells, whose MFI was taken as 100%. #P < 0.05 vs. untreated platelets; *P < 0.05 vs. H2O2 only. (A) EDTA, 2 mM; BAPTA, 50 µM; Cytochalasin D, 5 µg/mL. (B) RO31-8220 (a PKC inhibitor), 2 µg/mL; wortmannin (a PI3 kinase inhibitor), 1 µM; SB203580 (an inhibitor of p38 MAPK), 20 µM; PD98059 (an inhibitor of ERK), 50 µM; PP2 (an Src inhibitor), 20 µM; z-VAD (general caspase inhibitor), 20 µM. (C) KRIBB3 (an HSP27 phosphorylation inhibitor), 10 µg/mL; aristolochic acid (PLA2 inhibitor), 20 µM. (D) Murine platelets were pre-treated with lipoxygenase inhibitors esculetin (40 µM) or CDC (30 µM) for 15 min, followed by incubation with H2O2 (100 µM) for 1 h; n = 6.

Alexander Brill, et al. Cardiovasc Res. 2009 October 1;84(1):137-144.

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