Tst Positive Staphylococcus Aureus Induces Platelet Apoptosis Through Mitochondrial Pathway

[Objective]Staphylococcus aureus is one of the most important bacterial pathogens,which can cause a wide range of clinical diseases,including skin and soft tissue infections,bacteriaemia,sepsis and toxic shock syndrome(TSS).Studies have shown that the excessive inflammatory response and coagulation dysfunction of sepsis are the pathophysiological basis of the occurrence and development of multiple organ dysfunction syndrome(MODS),and the activated platelets are the bridge between thrombosis and inflammation.Platelet plays an important role in the occurrence and development of sepsis.S.aureus can secrete toxic shock syndrome toxin 1(TSST-1).In clinical cases,TSST-1 contributes to the extensive T-lymphocytes activation anddamage Kupffer cells directly.These changes in the body are believed to be responsible for the pathogenesis of hypotension and shock,which finally leads to death.It is not clear whether TSST-1 can induce platelet apoptosis and activation.So we explore the difference between tst positive S.aureus and t.st negative S.aureus on platelets.[Methods]This experiment is divided into two parts:in vitro experiment and in vivo experiment.Platelets used in vitro are washed platelets.Fresh blood from health volunteers were collected and centrifugated,then washed platelets and platelet rich plasma(PRP)were prepared,and the platelet concentration was adjusted to 3×108/ml.Washed platelets were incubated with tst positive and tst negative Staphylococcus aureus in ratio of 1:20,at 37?for 1.5 hours respectively.Flow cytometry was used to detect changes in platelet mitochondrial transmembrane potential(??m),the phosphatidylserine(PS)externalization,platelet membrane glycoprotein ? b/?a complex(PAC-1)receptor exposure and expression of CD62p(P-selectin).The expression of apoptotic protein(Bak,Bcl-XL and caspase-3)in platelets was detectedby Western blot.The other part of washed platelets were incubated with the same amount of two kinds of Staphylococcus aureus culture medium at 37? for 1.5 hours respectively.??m,PS externalization,PAC-1 receptor exposure and expression of CD62p in platelets were detected by flow cytometry.Two kinds of Staphylococcus aureus were injected into 6-7 weeks old female wild mice.Blood samples were collected at different time points,and blood routine values such as platelet count,white blood cells count,red blood cells count and hemoglobin were measured by blood cell analyser.And the numbers of colonies changes over time were counted at each time point.After statistical analysis,a significant time point was obtained,flow cytometry was used to detect changes in platelet ??m,PS,PAC-1 and CD62p.The changes of inflammatory factors(TNF-a,IL-6,MCP-1 and IL-10)were detected in the remaining serum.The survival rate of mice was calculated in three days after injection of bacteria.[Results]Our data showed that either tst positive S.aureus or the supernatant can induce more platelets apoptosis and activation in vitro,compared with tst negative S.aureus and the supernatant of the tst negative S.aureus.Meanwhile,tst positive S.aureus induced up-regulation of Bak and down-regulation of Bcl-XL in addition to the activation of casepase-3.Compared with tst negative group,the platelet count,white blood cell count,red blood cell count and hemoglobin showed significant decline in mice injected with tst positive S.aureus at 6 hours,together with higher mortality andplatelet activation level.The levels of TNF-?,IL-6,and MCP-1 increased in different degrees,but there was no significant difference between bacteria groups.[Conclusions]These data indicate that tst positive Staphylococcus aureus and its toxin protein(TSST-1)can make platelet apoptosis and activation more obvious,and the mortality is higher.These findings provide new ideas for diagnosis and treatment of patients with sepsis and thrombocytopenia.