Generation of Tissue factor-rich microparticles in an ex vivo whole blood model

The aim of this study was to investigate the role of blood cells in the expression of tissue factor (TF) and P-selectin in platelets and microparticles from blood stimulated with lipopolysaccharide (LIPS), with or without the further addition of phorbol myristyl acetate (PMA). TF activity was found to be associated with platelets after 2 h incubation of whole blood with LPS or LIPS + PMA, while no TF activity was detected in microparticles from blood subjected to such stimulation. In blood stimulated for 6 and 24 h, addition of PMA to the samples led to a substantial increase in TF activity associated with microparticles, compared with stimulation with LPS alone. Addition of PMA to blood samples also led to a three-fold increase in the amount of P-selectin found in the isolated microparticle fraction, and a 50% reduction in P-selectin measured in platelets, compared with LPS alone used for stimulation. In a different experiment TF-rich microparticles were shown to be absorbed very efficiently by neutrophils in a calcium-independent reaction. Our results imply that LIDS stimulation of whole blood is associated with a direct transfer of TF from monocytes to platelets in the absence of free TF-rich microparticles, which probably is accounted for by the fusion of TF-rich microparticles with activated platelets exposing P-selectin. Further addition of PMA to samples generates both free TF-rich microparticles as well as enhanced transfer of TF from monocytes to platelets.