Effects Of CD154 On Platelets On The Expression Of Tissue Factor And Tissue Factor Pathway Inhibitor In Endothelial Cells

Objective: To investigate the effects of activated platelets'CD154on expression of tissue factor (TF) and tissue factor pathway inhibitor(TFPI) in human umbilical vein endothelial cells (HUVECs) fromseveral aspects such as molecular and protein level by co-incubatingumbilical vein endothelial cells and platelets.Methods: Platelets were isolated from healthy volunteers(n=3) byisodensity centrifugation and induced by ADP in vitro. Flow cytometrywas used to detect the expression of CD154 on the platelets which wereinduced before or after. Meanwhile, platelets (either induced ornon-induced) were co-incubated with cultured human umbilical veinendothelial cells (HUVECs). unstimulated HUVECs were negtive controland HUVECs stimulated by LPS (5μg/ml) were positive control. Themembrane-bound tissue factor and tissue factor inhibitor expression inHUVECs were analyzed by flow cytometry and reverse transcription-polymerase chain reaction.To further validate the effect level of theinteraction between platelets'CD154 and endothelial cells'CD40 on theTF and TFPI in HUVECs, specific monoconal antibody to CD154 orCD40 was respective added to activated platelets suspection andHUVECs culture medium in order to stop the function of activated platelets'CD154 or endothelial cells'CD40. All statistical analyses wereperformed with the SPSS13.0 for Windows statistical package (SPSSInc).Result:①After interacted with ADP, the expression of CD154 onplatelet membrane surface increased significantly (t=11.1, P＜0.01).②The expression of TF mRNA (t=32.3, P＜0.01) and protein TF(t=13.76,P＜0.01) in HUVECs were markedly increased after co-incubated withADP-activated platelets. But these effects were partly blocked by CD40(F=124.415,P＜0.01) or CD154 (F=144.134,P＜0.01 ) specificmonoclonal antibody. Compared with the activated-platelets group andthe negative control respectively,the three groups were significantlydifferent. Unactivated platelets (t=2.27,P=0.08) or simplex ADP (t=0.23,P=0.83) had not the effect to promote the expression of TF.③They hadno any effects on TFPI (F=1.36,P=0.295),which refer to resting oractivated platelets, CD154 or CD40 monoclonal antibody and ADP oralone.Conclusion:①In vitro ADP-activated platelets were able toexpress inflammatory mediator CD154,but CD154 was expresseddeficiently in unactivated platelets,so CD154 was a marker for plateletactivation.②Activated platelets could up-regulate the expression ofTF in HUVECs through CD40-CD154 signal pathway, and the effectcould be suppressed by monoconal antibody to CD154 or CD40.③ CD40-CD154 signal pathway had no effect on the expression of TFPI.This implied that activated platelets may play a important role ininitiating the extrinsic coagulation pathway through interaction of itsCD154 with CD40 in HUVECs,and participate unstable plaque ruptureand thrombosis in the atherosclerosis.