| Platelet transfusions are used primarily to prevent bleeding or potentially severe bleeding due to thrombocytopenia or platelet dysfunction.In general,“cryopreserved platelets”(CPPs)refer to platelets cryopreserved at-65°C to-80°C with dimethyl sulfoxide(DMSO)as the protectant.The risk of bacterial contamination is less than that for platelets stored at 22°C,and the shelf life is ?2 years.Although CPPs cannot increase the platelet count after transfusion,they have the same hemostatic capability as fresh platelets,and are suitable for therapeutic infusion of coagulation factors caused by blood loss.After 2005,the US Military Medical Research Institute and Netherlands Military Blood Station improved the preparation of frozen platelets.Using type-O platelets,DMSO was added and centrifuged to give ?14 m L of concentrated platelets.They were stored at-80°C,thawed before infusion,and resuspended with type-AB fresh-frozen plasma.The advantages of this method are that:(1)the volume of frozen platelets of one unit are concentrated from the original 300 m L to ?14 m L,and the volume is reduced greatly,which facilitates storage and transport;(2)AB-type plasma does not need to be washed and cross-matched after resuspension,and is infused directly to the wounded patient to realize rapid rescue.Thus,the immediate hemostatic efficacy of CPPs and the advantage of facilitating transportation and storage are valued by the military of several countries.The Netherlands and Belgium have applied CPPs during military operations.The US army has included CPPs in its wartime blood-reserve program,and it was submitted to the US Food and Drug Administration for approval in 2009(a phase-I clinical study has been completed).Although CPPs have attracted the attention of the militaries of several countries,they have not been approved as official blood products in any single country.Safety issues have been the main reason for the delay in their clinical application.Apart from their hemostatic function,platelets also mediate inflammation and immune regulation.Therefore,besides paying attention to their hemostatic effect upon transfusion,we must also clarify their possible adverse reactions.Recently,clinical trials on the feasibility and safety of CPPs were started in Australia,the Czech Republic,and the United States,but no clear conclusions have been reached.Our research team has carried out animal studies on the safety of CPP infusion.We used a mouse model of blood loss to compare the effects of fresh platelets,stored platelets at room temperature,and CPP infusion on tissue damage.We found that the number of Kupffer cells(KCs)and release of pro-inflammatory factors in the CPP group were significantly higher than those of the other two groups.In a hepatic ischemia–reperfusion study,platelet transfusions increased KC damage.Therefore,we speculated that the interaction between frozen platelets and KCs was an important part of promoting liver injury.In this study,we used human apheresis platelets and phorbol myristate acetate(PMA)-induced differentiation of THP-1 monocytic cells.We studied the activation of THP-1 cells by CPPs in vitro and explored the possible mechanism of action.Our study was divided into three parts.First,we compared the changes of surfactant molecules and release of pro-inflammatory factors during platelet storage at 22°C,4°C and-80°C,and postulated the molecules that mediate the interaction between CPPs and THP-1 cells.Second,we compared the interaction between platelets and THP-1 at 22°C,4°C,and-80°C,and specified the activation effect of CPPs on THP-1 cells.Finally,we studied the effect of phosphatidylserine(PS)on CPP surfaces on activation of THP-1 cells,and explored the interaction between CPPs and THP-1 cells.Part I: Comparative study of platelet injury at different storage temperaturesStudies have suggested that room temperature(22°C)preservation of platelet transfusions exacerbates hepatic ischemia–reperfusion injury.In addition,platelets stored at 4°C,through clustering of glycoprotein(GP)Ib?,promote binding to the KC surface receptor Mac-1,resulting in the rapid clearance of KCs.However,studies on the interaction between platelets and KCs at-80°C have not been reported.We compared the effects of changes of surfactant molecules on the storage of platelets at 22°C,4°C,and-80°C,release of pro-inflammatory factors,and ascertained the molecules that mediate the interaction between CPPs and THP-1 cells.Methods: Each unit was divided into three parts and stored,respectively,at 22°C or 4°C,or cryopreserved at-80°C,in accordance with methods set by the military of the Netherlands.Mean platelet volume and breadth of distribution were measured at different storage times.The contents of lactate and lactate dehydrogenase(LDH)were measured using commercial kits.Flow cytometry was used to measure expression of GPIb?,P-selectin,and PS on platelet surfaces.Enzyme-linked immunosorbent assays(ELISAs)were used to determine plasma levels of soluble cluster of differentiation(s CD)40L,and regulated on activation,normal T cell expressed and secreted(RANTES).Results showed no significant differences in macrophage morphology after induction of differentiation with different PMA concentrations,but 5 ng/m L macrophages did not adhere firmly.With increasing PMA concentration,the phagocytic capacity of macrophages showed a downward trend,and the fluorescence intensity of CD11 b and CD14 expression decreased.The fluorescence intensity at 50,100 and 200 ng/m L was significantly lower than that at 5 and 25 ng/m L.However,there was no significant difference in fluorescence intensity between 50,100,and 200 ng/m L concentrations.Expression of CD11 b and CD14 at these five concentrations was close to 100%.With increasing PMA concentration,reactive oxygen species(ROS)increased gradually,and their concentration at 25 ng/m L was significantly higher than that at 5 ng/m L.There was no significant difference in ROS concentration between 25,50,100,and 200 ng/m L concentrations.Summary: Compared with platelets preserved at 22°C,platelets preserved at 4°C did not aggravate membrane damage,but instead promoted the activation and apoptosis of platelets.Platelets preserved at-80°C had severely damaged membranes,significantly reduced GPIb? expression,increased PS expression,as well as inhibited metabolism and release of pro-inflammatory factors.Expression of s CD40 L,RANTES,P-selectin and GPIb? in platelets at-80°C was significantly lower than that at 4°C,but PS expression was significantly higher than that at 4°C.We speculated that PS may be the main molecule mediating the interaction between frozen platelets and KCs.Part II: THP-1 activation in frozen plateletsPreviously,we infused fresh,normal temperature-stored,and frozen platelets into mice that had suffered severe hemorrhage.We found that transfusion of frozen platelets exacerbated liver injury,and that the number of liver macrophages and expression of pro-inflammatory factors were significantly higher than those of fresh and normal temperature-stored platelets.To further clarify the interaction between frozen platelets and macrophages,PMA-induced THP-1 cells were used to detect phagocytosis and activation between THP-1 cells and cryopreserved leuko-reduced platelets of humans in vitro.Methods: THP-1 cells were induced by PMA.The induction conditions of PMA were determined by morphologic analyses,phagocytosis,and expression of the surface markers CD11 b and CD14.Different concentrations of lipopolysaccharide(LPS)were used to pre-activate THP-1 cells and detect expression of tumor necrosis factor(TNF)-?,interleukin(IL)-6 and IL-1? after co-incubation with frozen platelets.No co-incubation of platelets with THP-1 was used as a control to clarify the appropriate conditions for LPS pre-activation.Platelets stored for 3 days at 22°C,4°C,or-80°C were co-incubated with THP-1 cells.Phagocytosis of platelets was detected by confocal fluorescence microscopy and flow cytometry.ELISAs were used to detect expression of TNF-?,IL-6,IL-1? and transforming growth factor(TGF)-? after co-incubation of platelets with THP-1 cells.Results: Induction of THP-1 cells was achieved by 25 ng/ml of PMA for 24 h;PMA culture was not continued after 48 h.Under this condition,THP-1 cells adhered firmly,phagocytosis was 80%,and CD11 b and CD14 were expressed.Some THP-1 cells had long fusiforms and protruding pseudopods.With increasing LPS concentration,expression of TNF-?,IL-6 and IL-1? increased gradually in experimental and control groups.When THP-1 cells were stimulated with 0.5 ng/m L LPS for 3 h,expression of TNF-?,IL-6,and IL-1? in the experimental group was significantly higher than that in the control group.The adhesion and phagocytosis between THP-1 cells and platelets preserved at-80°C were significantly higher than those at 22°C and 4°C(P<0.05).Expression of TNF-?,IL-1,IL-6 and TGF-? was significantly higher in platelets preserved at-80°C incubated with THP-1 than that of platelets preserved at 22°C and 4°C(P<0.05).When platelets were co-incubated with THP-1,expression of TNF-?,IL-1?,IL-6 and TGF-? with LPS pre-activation was significantly higher than that with no LPS pre-activation(P<0.05).Summary: We confirmed the induction conditions for THP-1 cells.Under optimized LPS pre-activation conditions,THP-1 cells were activated mildly by platelets.Compared with platelets stored at 22°C and 4°C,platelets preserved at-80°C were more likely to be phagocytosed by THP-1 cells and produce higher levels of pro-inflammatory factors.LPS pre-activation of THP-1 cells promotes platelet activation of THP-1 cells.Part III: How frozen platelets promote activation of THP-1 cellsFrom the experimental results shown above,we hypothesized that increased expression of PS in cryopreserved platelets mediated the phagocytosis of platelets by THP-1 cells,and that this was an important way to promote activation of THP-1 cells.Annexin-V was used to block PS on platelet surfaces and to detect the phagocytosis of frozen platelets by THP-1 cells.We also studied the effect of release of pro-inflammatory factors after incubation of frozen platelets with THP-1 cells.Methods: Annexin-V(0,5,10,20,40 ?g/ml)was used to add pre-blocked platelet PS receptors to frozen platelets,followed by co-incubation with THP-1 cells.Then,confocal fluorescence microscopy and flow cytometry were used to detect the phagocytosis of platelets by THP-1 cells.Expression of TNF-?,IL-6,IL-1?,and TGF-? after incubation of frozen platelets with THP-1 cells was detected by ELISAs.Results: With increasing annexin-V concentration,the phagocytosis of frozen platelets by THP-1 cells was reduced gradually by ?50%;20 ?g/m L of annexin-V could significantly inhibit expression of the pro-inflammatory cytokines TNF-?,IL-1?,and IL-6 from frozen platelets co-incubated with THP-1 cells,and could promote expression of the inhibitory factor TGF-?.Summary: Our preliminary study showed that PS in frozen platelets mediates platelet phagocytosis by THP-1 cells and promotes the release of pro-inflammatory factors in THP-1 cells.Through the studies stated above,we showed that frozen platelets are more easily phagocytosed by macrophages than platelets preserved at 22°C and 4°C.This mechanism of action is different from platelets stored at 4°C phagocytosed by macrophages through GPIb? clustering,and a large number of PS molecules on the surface of frozen platelets are the main reason why platelets are phagocytosed by macrophages.Compared with platelets stored at 22°C and 4°C,frozen platelets helped macrophages to release more pro-inflammatory factors.Massive phagocytosis of frozen platelets by macrophages promoted macrophage activation,and the large number of PS molecules on platelet surfaces had an important role in this process. |
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