Suppressive Effect Of CORM-2 On Lps-induced Plateletá-granule Exocytosis And Molecular Mechanisms

Background: Sepsis is a systemic inflammatory response syndrome caused by a severe systemic infection, and further develops to multiple organ dysfunction syndrome(MODS),accounting for the major cause of death in clinical. 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. Our previous studies suggest that carbon monoxide-releasing molecules-2(CORM-2) attenuated the activation of LPS-induced platelets such as spreading, aggregation, and granules release. However, little was known whether CORM-2 released CO could decrease platelet α-granule exocytosis in sepsis.Method: Collecting the blood from healthy volunteers, establishing the LPS-induced platelet activation moldel, administering CORM-2. Platelet α-granule contents(e.g. PDGF-BB,MMP-2) were detected by using standard ELISA kits. The expressions of P-selectin and integrin αIIbβ3 were detected by flow cytometer. Immunofluorescence microscope and transmission electron microscope(TEM) were used to observe distribution of plateletα-granules in LPS-induced platelet. Expressions of PKCθ and Munc18 a phosphorylation were detected by Western boltting. To identify association of Munc18 a with SNARE proteins in platelets, the method of immunoprecipitation was used..Results: 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 phosphorylation of PKCθ and Munc18 a, SNARE complex assembly and subsequently platelet α-granule exocytosis.Conclution: We suggested that the potential mechanism of suppressive effect of exogenous carbon monoxide, carbon monoxide-releasing molecules II(CORM-2)-liberated CO, on LPS-induced platelet SNAREs complex assembly and α-granule exocytosis might involve integrin αIIbβ3-mediated PKCθ/Munc18 a pathway activation.