Platelet Clearance By Endothelium Decreases Platelet Procoagulant Activity In Sepsis

Background and PurposeThe coagulation disorder is common in sepsis patients,which is present as acute septic coagulopathy and even disseminated intravascular coagulation(DIC).Recently,studies show that it is associated with the release of procoagulant substances and injury of endothelial cells,however,the mechanism is not clear.Under normal condition,phosphatidylserine(PS)is usually limited on the inner side of cell membranes.When cells become activated or undergo apoptosis,phosphatidylserine is expressed to the outer leaflet of cells.Platelets are activated even apoptotic in sepsis with phosphatidylserine exposure.Exposed phosphatidylserine on cells not only provides a catalytic surface for the assembly of tenase and prothrombinase complexes but also binds the receptors on phagocytes mediating the clearance of the apoptotic cells.Macrophages are professional phagocytes and play important roles in recognizing and engulfing bacteria and senescent cells when inflammation occurs.However,a large number of bacterial and toxins are released into blood in during sepsis,macrophage may be overwhelmed and form aggregates with platelets aggregating inflammation.Thus non-professional phagocytes like endothelial cells will function as well.Lactadherin,secreted by macrophages and endothelial cells,is a milk fat globule membrane glycoprotein which has a domain structure of EGF1-EGF2-C1-C2.The C-terminus can anchor phosphatidylserine-externalized cells and the EGF2 domain links to phagocyte ?v?3/5 integrins for engulfment via its RGD motif,and thus lactadherin acts as a bridge enhancing the clearance of apoptotic cells by phagocytes.The aim of this study was to investigate the alternative pathway of platelet phagocytosis in sepsis and the role of platelet clearance by endothelial cells in procoagulant activity.MethodsPhosphatidylserine exposure of platelets of patients and healthy controls was evaluated by confocal laser scanning microscopy and flow cytometry.Platelet morphology and platelet-leukocyte aggregates in sepsis were measured with light microscopy,flow cytometry,transmission electron microscopy or scanning electron microscopy.Phagocytosis of activated platelets by endothelial cells which is mediated by lactadherin or not was assayed by flow cytometry,transmission electron microscopy,scanning electron microscopy or immunofluorescence microscopy.Then,clotting time of platelets from patients and healthy controls was examined by coagulometer.Eventually,intrinsic or extrinsic FXa,thrombin and fibrin formation of platelets from patients and healthy controls were measured by microplate reader.ResultsWe found about 9% of platelets from sepsis patients were positive for phosphatidylserine probe,which was 3 fold higher than those in healthy subjects.Activated platelets in sepsis extended pseudopods,and some were even apoptotic forming microparticles.Platelets of healthy controls were mainly quiescent and sphere without pseudopods.Platelets formed aggregates with leukocytes and erythrocytes in blood,indicating comprehensive platelet activation.Compared with healthy controls,septic patients had 3 fold,5.3 fold and 2.5 fold platelet-neutrophil aggregates,platelet-lymphocyte aggregates and platelet-monocyte aggregates respectively.Human umbilical vein endothelial cells bound,engulfed and subsequently degraded activated platelets,but had little interactions with quiescent platelets of healthy controls.Compared with platelets of healthy controls,septic platelets had markedly shortened coagulation time and enhanced procoagulant activity(PCA),which is present with increased generation of intrinsic FXa and thrombin.Platelets in septic patients formed fibrin network on the cell membrane,and even formed platelet-fibrin clots.After incubation of human umbilical vein endothelial cells,FXa,prothrombinase activity and fibrin of phosphatidylserine-exposed platelets were significantly decreased,and the coagulation time of septic platelets markedly increased.After incubation with human umbilical vein endothelial cells,the number of activated platelets was significantly reduced,which formed less platelet-leukocyte aggregates.Inhibiting phosphatidylserine with annexin V,the phagocytosis was nearly reduced by 80%,and blocking P-selectin on platelets also significantly downregulated the platelet clearance by human umbilical vein endothelial cells.While the inhibition of GPIb and GPIV on platelets had little effect on platelet engulfment in sepsis.Furthermore,inhibiting ?v?3 integrin on human umbilical vein endothelial cells,platelet clearance was markedly reduced and prothrombinase complexes increased.Lactadherin promoted the clearance of platelets by 2 fold,and cooperated with phagocytosis to reduce 75% thrombin generation and about 50% fibrin formation,make the coagulation time nearly restored.Compared with onset,patients in remission had significantly reduced procoagulant activity: phosphatidylserine expression on platelets decreased 40%,the number of platelet-leukocyte aggregates reduced more than 50%,coagulation time markedly prolonged,and the level of thrombin in peripheral blood downregulated to 40%,which may be closely related with the clearance of activated platelets in circulation.ConclusionIncreased phosphatidylserine expression on platelets in septic patients contributes to the high procoagulant activity and effective phagocytosis of activated platelets.Engulfment by endothelial cells reduces the number of phosphatidylserine-exposing platelets,which directly downregulated the platelet-leukocyte aggregates,procoagulant activity as well as fibrin formation of platelets in sepsis.Lactadherin and phagocytosis could cooperatively clear the activated platelets and better ameliorate the clotting disorders in sepsis.