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Activation of coagulation occurs in sepsis and contributes to the development of thrombosis. Platelet α-granule exocytosis plays an important role in septic coagulation abnormalities. The present study aimed to investigate the effects and the underlying mechanisms of exogenous carbon monoxide, carbon monoxide-releasing molecules II (CORM-2)-liberated CO, on suppressing platelet α-granule exocytosis in sepsis. It was shown that CORM-2 weakened α-granule membrane fusion with platelet plasma membrane and attenuated α-granule contents exocytosis in LPS-Induced platelet. Further studies revealed that CORM-2 suppressed the expression of integrin αIIbβ3 in platelets stimulated by LPS. This was accompanied by a decrease in production and phosphorylation of PKCθ and Munc18a, SNARE complex assembly and subsequently platelet α-granule exocytosis. Taken together, we suggested that the potential mechanism of suppressive effect of CORM-2 on LPS-induced platelet SNAREs complex assembly and α-Granule Exocytosis might involve integrin αIIbβ3-mediated PKCθ/Munc18a pathway activation.

To investigate the suppressive effect of carbon monoxide-releasing molecule II (CORM-2) on LPS induced platelet α-granule exocytosis in sepsis via soluble N-ethylmaleimide-sensitive factor attached protein receptor/mammalian uncoordinated 18b (SNARE/Munc18b) complex formation.Blood was collected from healthy volunteers' cubital vein, then platelets were isolated by differential centrifugation. Platelets were randomly divided into 5 groups. The control group did not undergo any treatment, the LPS group received 10 mg/L LPS simulation, the CORM-2 group and iCORM-2 group underwent LPS simulation and immediate administration of CORM-2 (10 μmol/L and 50 μmol/L) or iCORM-2 (50 μmol/L), respectively. Samples were incubated in a COCompared with the control group, levels of PF4, PDGF-BB, MMP-2 and P-selectin in LPS induced platelets were found to markedly elevated, while CORM-2 (10 μmol/L and 50 μmol/L) could decrease platelet α-granule contents exocytosis: [PF4 (μg/L): 7.69±0.58, 6.03±0.71 vs. 10.13±0.82; PDGF-BB (μg/L): 112.71±1.79, 102.91±5.86 vs. 128.78±1.39; MMP-2 (ng/L): 32.94±2.73, 27.58±3.36 vs. 53.26±1.21; P-selectin: (17.14±0.57)%, (15.35±0.68)% vs. (23.78±0.62)%; all P0.01]. Transmission electron microscope and immunofluorescence microscope showed that the extent of platelet α-granules assembled to platelet plasma membrane was significantly decreased following CORM-2 treatment. Compared with the control group, the expressions of Munc18b and SNARE proteins and SNARE/Munc18b complex formation in LPS-stimulated platelets were significantly increased, while CORM-2 (10 μmol/L and 50 μmol/L) inhibited these elevations (Munc18b/GAPDH: 0.80±0.08, 0.69±0.01 vs. 0.99±0.09; VAMP-8/GAPDH: 0.72±0.09, 0.50±0.12 vs. 1.18±0.14; SNAP-23/GAPDH: 1.18±0.22, 0.63±0.10 vs. 1.90±0.08; STX-11/GAPDH: 0.76±0.02, 0.57±0.08 vs. 1.16±0.23; VAMP-8/Munc18b: 0.65±0.09, 0.53±0.07 vs. 1.21±0.20; SNAP-23/Munc18b: 0.85±0.07, 0.55±0.09 vs. 1.26±0.08; STX-11/Munc18b: 0.78±0.05, 0.61±0.10 vs. 1.39±0.16; all P0.01). Above all, the data showed a dose dependent change.We could suggest that CORM-2 suppressed α-granule exocytosis in LPS-stimulated platelets and the potential mechanisms might involve SNARE/Munc18b complex formation.

Platelet activation is an important event involved in the pathophysiological processes of the coagulation system. Clinical evidence has shown that platelets undergo distinctive pathological processes during sepsis. Unfortunately, how platelets physiologically respond to inflammation or sepsis is not well understood. In this study, we used a lipopolysaccharide (LPS)-stimulated platelet model to systemically investigate alterations in membrane glycoprotein expression, molecular signaling, morphology and critical functions of platelets. We found that platelet adhesion, aggregation, secretion and spreading on immobilized fibrinogen and the expression of platelet membrane glycoproteins were significantly increased by LPS stimulation and these changes were accompanied by a significant decrease in cGMP levels and an abnormal distribution of platelet α-granules. Exogenous CO reversed these alterations. Profound morphological changes in LPS-stimulated platelets were observed using atomic force microscopy and phase microscopy. Furthermore, the elevated activities of PI3Ks, AKt and GSK-3β were effectively suppressed by exogenous CO, leading to the improvement of platelet function. Together, these results provide evidence that platelet over-activation persists under LPS-stimulation and that exogenous CO plays an important role in suppressing platelet activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway.

To investigate the suppressive effect of exogenous carbon monoxide (CO) on abnormal platelet exocytosis and its possible molecular mechanism.Venous blood was collected from healthy volunteers. Platelet-rich plasma (PRP) was isolated from the blood by differential centrifugation. The PRP was randomly divided into five groups by random number table, namely normal control group, lipopolysaccharide (LPS) group (challenged with 10 mg/L LPS), inactively exogenous carbon monoxide releasing molecule 2 (iCORM-2) group (given 10 mg/L LPS + 50 μmol/L iCORM-2 for intervention), exogenous carbon monoxide releasing molecule 2 (CORM-2) 10 μmol/L and 50 μmol/L groups (given 10 mg/L LPS + CORM-2 10 μmol/L or 50 μmol/L for intervention). After 30 minutes, enzyme linked immunosorbent assay (ELISA) was used to determine the platelet-derived growth factor BB (PDGF-BB) and matrix metalloproteinase 2 (MMP-2). Chemical fluorescein method was used to determine the platelet adenosine triphosphate (ATP). Flow cytometer was used to determine the expression of P-selectin. The expressions of Toll-like receptor 4 (TLR4), phosphorylation of protein kinase Cθ (PKCθ) and syntaxin binding protein 1 (STXBP-1) were determined by Western Bolt. The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) complex formation [syntaxin 2-synaptosomal-associated protein 23-vesicle associated membrane protein 8 (STX2-SNAP23-VAMP8)] mediated by STXBP-1 was determined by immunoprecipitation.(1) Compared with normal control group, the platelet release of PDGF-BB, MMP-2 and ATP was significantly increased after LPS challenge, and the P-selectin expression of platelet was also obviously up-regulated [PDGF-BB (μg/L): 127.53±1.78 vs. 94.35±5.84, MMP-2 (ng/L): 51.87±9.20 vs. 35.83±3.17, ATP (μmol/L): 1.288±0.056 vs. 0.975±0.010, P-selectin: (3.93±0.19)% vs. (0.44±0.10)%, all P0.05]. The increases in platelet release of PDGF-BB, MMP-2 and ATP were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration, as well as high-expression of P-selectin in a dose-dependent manner [PDGF-BB (μg/L): 114.68±1.35, 97.08±6.14 vs. 127.53±1.78, MMP-2 (ng/L): 32.67±8.00, 24.63±1.63 vs. 51.87±9.20, ATP (μmol/L): 0.999±0.015, 0.965±0.008 vs. 1.288±0.056, P-selectin: (1.95±0.27)%, (0.94±0.11)% vs. (3.93±0.19)%, all P0.05]. (2) Compared with normal control group, LPS challenge resulted in a significant increase in the expression of TLR4 and the phosphorylation of PKCθ and STXBP-1 [TLR4 (gray value): 1.21±0.38 vs. 0.67±0.06, p-PKCθ (gray value): 1.36±0.20 vs. 0.44±0.03, p-STXBP-1 (gray value): 1.13±0.06 vs. 0.59±0.04, all P0.05]. The increases in above parameters were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [TLR4 (gray value): 0.76±0.05, 0.65±0.04 vs. 1.21±0.38; p-PKCθ (gray value): 0.71±0.07, 0.47±0.10 vs. 1.36±0.20; p-STXBP-1 (gray value): 0.56±0.02, 0.48±0.01 vs. 1.13±0.06, all P0.05]. (3) Compared with normal control group, the SNAREs proteins in platelet that combined with STXBP-1, including STX2, SNAP23 and VAMP8, were obviously increased after LPS challenge [STX2 (gray value): 1.35±0.06 vs. 0.57±0.04, SNAP23 (gray value): 0.97±0.04 vs. 0.30±0.12, VAMP8 (gray value): 1.37±0.12 vs. 0.77±0.10, all P0.05]. The increases in SNAREs complex formation were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [STX2 (gray value): 0.77±0.02, 0.39±0.03 vs. 1.35±0.06, SNAP23 (gray value): 0.41±0.03, 0.22±0.08 vs. 0.97±0.04, VAMP8 (gray value): 0.85±0.07, 0.66±0.07 vs. 1.37±0.12, all P0.05]. There was no significant difference in the above mentioned parameters between iCORM-2 group and LPS group.LPS-induced abnormal secretion of platelet was suppressed by CORM-2 administration. The mechanism may involve the TLR4/PKCθ/STXBP-1 signaling pathway activation and the SNAREs complex formation.

To explore the effects of exogenous carbon monoxide-releasing molecule 2 (CORM-2) on formation of human neutrophil extracellular traps (NETs) stimulated by endotoxin/lipopolysaccharide (LPS) and its relevant mechanism.Venous blood samples were collected from a healthy adult volunteer to isolate neutrophils. The neutrophils were divided into normal control (NC) group, LPS group, LPS+ 10 μmol/L CORM-2 group, LPS+ 50 μmol/L CORM-2 group, and LPS+ inactive CORM-2 (iCORM-2) group according to the random number table. No treatment was given to the neutrophils in NC group. The neutrophils in LPS group underwent LPS stimulation (1 μL, 1 μg/mL). The neutrophils in LPS+ 10 μmol/L CORM-2 group, LPS+ 50 μmol/L CORM-2 group, and LPS+ iCORM-2 group underwent the same LPS stimulation as that in LPS group and treatment of 10 μmol/L CORM-2, 50 μmol/L CORM-2, and 50 μmol/L iCORM-2, respectively, with the volune of 1 μL. After conventional culture for 1 h, the number of NETs was determined with propidium iodide staining method; the early cell apoptosis rate was determined with flow cytometer; the generation level of reactive oxygen species (ROS) was assessed with dihydrogenrhodamine 123 fluorescent probe staining method (denoted as mean fluorescence intensity); the expression level of phosphorylated extracellular regulated kinase 1/2 (p-ERK1/2) was determined by Western blotting. The sample numbers of each group in the 4 experiments were all 5. Data were processed with one-way analysis of variance and SNK test.(1) The numbers of NETs per 400-time visual field in cells of LPS and LPS+ iCORM-2 groups were close to the number in NC group (with P values above 0.05). The number of NETs per 400-time visual field was significantly larger in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in NC and LPS groups (with P values below 0.05). The number of NETs per 400-time visual field in cells of LPS+ iCORM-2 group was close to that of LPS group (P0.05). (2) The early cell apoptosis rate was significantly increased in LPS, LPS+ 10 μmol/L CORM-2, LPS+ 50 μmol/L CORM-2, and LPS+ iCORM-2 groups than in NC group (with P values below 0.05). The early cell apoptosis rates in LPS+ 10 μmol/L CORM-2, LPS+ 50 μmol/L CORM-2, and LPS+ iCORM-2 groups were close to the rate in LPS group (with P values above 0.05). (3) The generation level of ROS was significantly higher in cells of LPS, LPS+ 10 μmol/L CORM-2, and LPS+ iCORM-2 groups than in NC group (with P values below 0.05). The generation level of ROS in cells of LPS+ 50 μmol/L CORM-2 group was close to that of NC group (P0.05). The generation level of ROS was lower in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in LPS group (with P values below 0.05), while the generation level of ROS in cells of LPS+ iCORM-2 group was close to that of LPS group (P0.05). (4) The expression levels of p-ERK1/2 in cells of LPS and LPS+ iCORM-2 groups (respectively 0.0311±0.001 and 0.0309±0.0018) were close to the level in NC group (0.0304±0.0046, with P values above 0.05). The expression level of p-ERK1/2 was significantly higher in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups (respectively 0.7891±0.0201 and 1.2970±0.0056) than in NC group (with P values below 0.05). The expression level of p-ERK1/2 was significantly higher in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in LPS group (with P values below 0.05). The expression level of p-ERK1/2 in cells of LPS+ iCORM-2 group was close to that of LPS group (P0.05).CORM-2 can obviously increase the production of NETs in LPS-induced neutrophils, and it might be attributable to the promotion of inhibition of ROS generation and phosphorylation of ERK1/2.

To explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on LPS-induced abnormal activation of platelets in peripheral blood of healthy human donors and its possible molecular mechanism.Venous blood samples were collected from a healthy volunteer, and platelet-rich plasma (PRP) from the blood were isolated by differential centrifugation. The PRP was subpackaged into siliconized test tubes and then divided into control group, LPS group, inactive CORM-2 (iCORM-2) group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group according to the random number table, with 3 tubes in each group. The PRP in control group did not receive any treatment. The PRP in LPS group received LPS (20 mL, 10 µg/mL) stimulation, and the PRP in iCORM-2 group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group underwent the same LPS stimulation and treatment of 50 µmol/L iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2, respectively, with the dosage of 20 mL. After being cultured for 30 min, the platelet adhesion rate was determined by glass bottle method, the number of platelet spreading on fibrinogen was determined with immunofluorescent method, and the platelet aggregation rate was measured by turbidimetric method. The platelet poor plasma (PPP) was prepared from PRP, the levels of ATP in PPP and platelets were determined by chemical fluorescein method. The expressions of platelet glycoprotein I bα (GPIbα) and GPVI were analyzed by flow cytometer. The expressions of glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β were determined by Western blotting and immunoprecipitation, respectively. Measurement of the above indices was repeated for 3 times. Data were processed with one-way analysis of variance and SNK test.Compared with those in control group, the platelet adhesion rates, numbers of platelets spreading on fibrinogen, platelet aggregation rates, expressions of GPIbα and GPVI in PRP, levels of ATP in PPP in LPS and iCORM-2 groups were significantly increased, while levels of ATP in platelets were significantly decreased (with P values below 0.05). Compared with those in LPS group, the former 7 indices in iCORM-2 group showed no significant differences (with P values above 0.05), while the levels of ATP in platelets in the 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly increased, and the other 6 indices in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased (with P values below 0.05). The expression levels of GSK-3β of the platelets in PRP in control, LPS, iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2 groups were 0.550 ± 0.060, 1.437 ± 0.214, 1.210 ± 0.108, 0.720 ± 0.010, and 0.670 ± 0.010, respectively, and the expression levels of the phosphorylated GSK-3β of the platelets in PRP in the above 5 groups were 0.950 ± 0.070, 1.607 ± 0.121, 1.420 ± 0.040, 1.167 ± 0.015, and 0.513 ± 0.122, respectively. Compared with those in control group, both the expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in LPS and iCORM-2 groups were significantly increased (with P values below 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP between LPS group and iCORM-2 group were similar (with P values above 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased compared with those in LPS group (with P values below 0.05).LPS stimulation can abnormally activate the platelets in peripheral blood of healthy human, but the abnormal activation can be inhibited by CORM-2 intervention, and the mechanism of the latter may involve the phosphorylation of GSK-3β mediated by GP.