Effects of CORM-2 on platelet HS1 expression and HS1 tyrosine phosphorylation.

<p>The platelets were stimulated by LPS and co-incubated with CORM-2 for 30 min. The platelets were lysed in RIPA buffer that contained protease and phosphatase inhibitor cocktails. Platelet HS1 expression and HS1 tyrosine phosphorylation were detected by SDS- polyacrylamide gel electrophoresis and Western blotting. Representative experiments are shown in A and B. The average ratio of HS1/beta-actin and p-HS1/t-HS1 are shown in C and D. Results are described as mean ± SE of three experiments, *P<0.01 as compared to control; #P<0.05 as compared to LPS.</p

Effect of CORM-2 preconditioning on platelet HS1 expression and HS1 tyrosine phosphorylation.

<p>The platelets were pretreated with CORM-2 for 30 min followed by LPS stimulation for an additional 30 min. The platelets were lysed in RIPA supplemented with protease and phosphatase inhibitor cocktails. Platelet HS1 expression and HS1 tyrosine phosphorylation status were detected using SDS-polyacrylamide gel electrophoresis and Western immunoblotting. The representative experiments are shown in A and B. The average ratio of HS1/beta-actin and p-HS1/t-HS1 are shown in C and D. Results are described as mean ± SE of three independent experiments, *P<0.01 as compared to control; #P<0.05 as compared to LPS.</p

Effects of CORM-2 on aggregation in LPS-stimulated platelets.

<p>Platelets were stimulated and intervened as described in Fig. 1. LPS stimulation resulted in a significant increase in platelet aggregation, and treatment of platelets with CORM-2 (10, 50 µM) for 30 min significantly reduced this increase in response to LPS stimulation (A, B). Similar results were also shown in CORM-2 pre-conditioning (C, D) and CORM-2 delayed treatment groups (E, F). The representative images are shown in A, C, and E; the average platelet aggregation ratio of five experiments are shown in B, D and F, respectively. Results are mean ± SE, *P<0.01 as compared to control; #P<0.05 as compared to LPS. Note that LPS-induced platelet aggregation was inhibited by CORM-2 in a dose-dependent manner.</p

Effects of CORM-2 on platelet spreading following LPS stimulation.

<p>Platelets were stimulated by LPS (10 µg/ml) for 30 min in the presence or absence of CORM-2 (10, 50 µM). In addition, CORM-2 preconditioning and delayed treatment were also investigated as described in “Platelet stimulation model” section above. LPS stimulation significantly increased the spreading of platelet numbers as compared to the control groups, and treatment of platelets with CORM-2 (10, 50 µM) for 30 min significantly reduced platelet spreading in response to LPS stimulation (A, D). Similar results were also shown in CORM-2 pre-conditioning (B, E) and CORM-2 delayed treatment groups (C, F). Results are presented as mean ± SE of five experiments, *P<0.01 as compared to control; #P<0.05 as compared to LPS. Note that LPS-induced platelet spreading was inhibited by CORM-2 in a dose-dependent manner.</p

Effects of CORM-2 on expression of platelet membrane glycoprotein GPIbα.

<p>The platelets were stimulated by LPS and treated with CORM-2 as described in Fig. 1. Samples were collected and fixed in 1% paraformaldehyde for 15 min at room temperature and incubated with FITC-labeled CD41 (CD41-FITC). GPIbα-PE was added to the samples. All samples were incubated in the dark for 30 min, washed three times and analyzed by flow cytometry. GPIbα expression in LPS-stimulated platelets that were co-incubated with CORM-2 is shown in A. Similar results were also shown in CORM-2 pre-conditioning (B) and CORM-2 delayed treatment groups (C). The average expression rates of GPIbα of five experiments are shown in D, E and F, respectively. Results are described as mean ± SE, *P<0.01 as compared to control; #P<0.05 as compared to LPS. Note that LPS-induced GPIbα expression was down-regulated by CORM-2 in a dose-dependent manner.</p

Effects of CORM-2 on expression of platelet membrane glycoprotein GPVI.

<p>The platelets were stimulated by LPS and treated with CORM-2 as described in Fig. 1. Samples were collected and fixed in 1% paraformaldehyde for 15 min at room temperature and incubated with FITC-labeled CD41 (CD41-FITC). The GPVI -eFluor 660 was added to the samples. All samples were incubated in the dark for 30 min, washed three times and analyzed by flow cytometry. The GPVI expression in LPS-stimulated platelets that were co-incubated with CORM-2 is shown in A. Similar results were shown in CORM-2 pre-conditioning (B) and CORM-2 delayed treatment groups (C). The average expression rates of GPVI of five experiments are shown in D, E and F, respectively. Results are described as mean ± SE, *P<0.01 as compared to control; #P<0.05 as compared to LPS. Note that LPS-induced GPVI expression was down-regulated by CORM-2 in a dose-dependent manner.</p

Effects of CORM-2 delayed treatment on platelet HS1 expression and HS1 tyrosine phosphorylation.

<p>LPS stimulated platelets (30 min) were treated with CORM-2 for an additional 30 min. Next, the platelets were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails. Platelet HS1 expression and HS1 tyrosine phosphorylation status were detected by SDS-polyacrylamide gel electrophoresis and Western immunoblotting. The representative experiments are shown in A and B. The average ratio of HS1/beta-actin and p-HS1/t-HS1 are shown in C and D. Results are described as mean ± SE of three independent experiments. *P<0.01 as compared to control; #P<0.05 as compared to LPS.</p

Effects of CORM-2 on granule secretion and intracellular ATP in LPS-stimulated platelets.

<p>The platelets were stimulated by LPS and treated with CORM-2 as described in Fig. 1. ATP release in the supernatant (A), and intracellular ATP levels (B) were measured. Results are described as mean ± SE of five experiments, *P<0.01 as compared to control; #P<0.05 as compared to LPS. Note that LPS-induced ATP release was inhibited by CORM-2 in a dose-dependent manner.</p

Characteristics of survivors and non-survivors in hospital.

Characteristics of survivors and non-survivors in hospital.</p

Characteristics of survivors and non-survivors within ICU.

Characteristics of survivors and non-survivors within ICU.</p