title: [Suppressive effect of exogenous carbon monoxide on abnormal platelet exocytosis and its molecular mechanism in sepsis].
abstract: OBJECTIVE
  To investigate the suppressive effect of exogenous carbon monoxide (CO) on abnormal platelet exocytosis and its possible molecular mechanism.
  
  
  METHODS
  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.
  
  
  RESULTS
  (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 P < 0.05]. 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 P < 0.05]. (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 P < 0.05]. 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 P < 0.05]. (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 P < 0.05]. 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 P < 0.05]. There was no significant difference in the above mentioned parameters between iCORM-2 group and LPS group.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Critical Care Medicine, the Affiliated Hospital of Jiangsu University, Zhenjiang 212001, Jiangsu, China (Liu DD); Department of Burns and Plastic Surgery, the Affiliated Hospital of Jiangsu University, Zhenjiang 212001, Jiangsu, China (Xu XH, Zhuang MF, Song MM, Qin WT, Wang X, Sun BW). Corresponding author: Sun Bingwei, Email: sunbinwe@hotmail.com.
authors.forename: Dadong
authors.initials: D
authors.lastname: Liu
authors.forename: Xiaohan
authors.initials: X
authors.lastname: Xu
authors.forename: Mingfeng
authors.initials: M
authors.lastname: Zhuang
authors.forename: Mingming
authors.initials: M
authors.lastname: Song
authors.forename: Weiting
authors.initials: W
authors.lastname: Qin
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.forename: Bingwei
authors.initials: B
authors.lastname: Sun
mesh_terms: D000077214:Becaplermin; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects*; D002248:Carbon Monoxide / Q000494:pharmacology*; D005089:Exocytosis; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008070:Lipopolysaccharides; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D051938:Munc18 Proteins / Q000378:metabolism; D009942:Organometallic Compounds / Q000494:pharmacology*; D019007:P-Selectin / Q000378:metabolism; D010766:Phosphorylation; D053657:Platelet-Rich Plasma; D011493:Protein Kinase C / Q000378:metabolism; D000074121:Protein Kinase C-theta; D020574:Proto-Oncogene Proteins c-sis / Q000378:metabolism; D011897:Random Allocation; D050600:SNARE Proteins / Q000378:metabolism; D018805:Sepsis; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D008070:Lipopolysaccharides; D051938:Munc18 Proteins; D009942:Organometallic Compounds; D019007:P-Selectin; D020574:Proto-Oncogene Proteins c-sis; C546937:SELP protein, human; D050600:SNARE Proteins; C497937:STXBP1 protein, human; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C447082:tricarbonyldichlororuthenium (II) dimer; D000077214:Becaplermin; D002248:Carbon Monoxide; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2